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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 hydrolysis of peptide bonds and in the diagnosis,
treatment, and prevention 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.
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 active
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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 (I994) 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
fihree 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 (S 1) 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. I~ringles 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-XII, 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 Barren,
su ra). The prolyl oligopeptidase (S9) family includes enzymes from
prokaryotes and eukaryotes with
greatly differing specificities. Dipeptidyl peptidase IV (DPP-IV) is identical
to CD26 and is implicated
in the inactivation of peptide hormones, as well as in regulating T-cell
growth (reviewed in Kahne, T. et
al. (1999) Int. J. Mol. Med. 4:3-15; Mentlein, R. (1999) Regul. Pept. 85:9-
24): Inhibition of DPP-IV
has been suggested as a treatment for type 2 diabetes (Holst, J.J. and C.F.
Deacon (1998) Diabetes
47;1663-1670), and lowered serum DPP-IV activity has been measured in anorexia
and bulimia
patients (van West, D. et al. (2000) Eur. Arch. Psych. Clin. Neurosci. 250:86-
92).
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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
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 cell
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 cells 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 phosphoxylation,
activate the protein. Signal peptidases exist as multi-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
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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
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, su
ra). 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).
C~steine 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
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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
(Karxer, 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 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, su ra).
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
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of the cell. Two active site residues, a cysteine and a histidine, have been
implicated in the catalytic
mechanism. Caspases are 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 signal causes
autoproteolytic cleavage of a specifc aspartate residue (D297 in the caspase-1
numbering convention)
and removal of the spacer and prodomain, leaving a p10/p20 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, su ra; 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-
1b and possibly other inflammatory cytokines (Chan 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, s-u~ra;
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 pol
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 Al's 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
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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. 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
ischemialreperfusion 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 aminopeptidases B and 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
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can bind to substrate molecules in the ECM or to inhibitors produced by the
tissue (TIMPs, 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+2 ion in the active site interacts with a cysteine
in the pro-sequence. Activating
factors disrupt the Zn+2-cysteine interaction, or "cysteine switch," exposing
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).
Ehlers-Danlos syndrome type VII C is caused by mutations in the procollagen I
N-proteinase gene
(Colige, A. et al. (1999) Am. J. Hum. Genet. 65:308-317).
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 TIMP-1.
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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 itselfj,
activating the program for lateral inhibition in Drosophila 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 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 proinflammatory
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/hester/adamts.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., sera) andlor procollagen processing
(Colige, A. et al. (1997)
Proc. Natl. Acad. Sci. USA 94:2374).
The discovery of new proteases and the polynucleotides encoding them satisfies
a need in the
art by providing new compositions which are useful in hydrolysis of peptide
bonds and 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-1," "PRTS-2," "PRTS-3," "PRTS-4," "PRTS-5," "PRTS-6,"
"PRTS-7,"
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"PRTS-8," "PRTS-9," "PRTS-10," "PRTS-I1," "PRTS-12," "PRTS-13," "PRTS-14,"
"PRTS-15,"
"PRTS-16," "PRTS-17," "PRTS-18," "PRTS-19," "PRTS-20," and "PRTS-21." 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 ID
N0:1-21, 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-21, c) a
biologically active fragment
of a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID N0:1-
21, and d) an immunogenic fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-21. In one alternative, the invention provides
an isolated polypeptide
comprising the amino acid sequence of SEQ ID N0:1-21.
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 ID N0:1-21, 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-
21, c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-21, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID N0:1-21. In
one alternative, the
polynucleotide encodes a polypeptide selected from the group consisting of SEQ
ID N0:1-21. In
another alternative, the polynucleotide is selected from the group consisting
of SEQ ID NO:22-42.
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:1-21, 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-
21, c) a biologically
active fragment of a polypeptide having an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-21, and d) an immunogenic fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-21. 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 N0:1-21, 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:1-21, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
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consisting of SEQ ID NO:1-21, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-21. 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 NO:1-21, 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 N0:1-21, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-21, and d) an
immunogenic fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-21.
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 ID
N0:22-42, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at Least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:22-42, 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
N0:22-42, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID NO:22-42, 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) 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
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polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:22-42, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:22-42, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
S 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:I-21, 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 N0:1-21, c) a biologically active fragment of a polypeptide having an amino
acid sequence selected
from the group consisting of SEQ ID NO:l-21, and d) an immunogenic fragment of
a polypeptide
1S having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-21, and a
pharmaceutically acceptable excipient In one embodiment, the composition
comprises an amino acid
sequence selected from the group consisting of SEQ ID NO:I-21. 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 ID N0:1-21, 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:1-21, c) a biologically active fragment of a
polypeptide having an
2S amino acid sequence selected from the group consisting of SEQ ID NO:1-21,
and d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ ID
N0:1-21. 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
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amino acid sequence selected from the group consisting of SEQ ID N0:1-21, 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 N0:1-21, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-21, and
d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-21. 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:l-22, 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-21, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID NO:1-21, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ ID
NO:1-21. 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:1-21, 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 N0:1-21, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID NO:1-21, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ ID
N0:1-21. 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.
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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
sequence selected from the group consisting of SEQ ID N0:22-42, 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
ID N0:22-42, 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 NO:22-42,
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
N0:22-42, 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 N0:22-42, 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.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide arid
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability score for the match
between each
polypeptide and its GenBank homolog is also shown.
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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 the cell fines,
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.
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DEFINITIONS
"PRTS" refers to the amino acid sequences of substantially purified PRTS
obtained from any
species, particularly a mammalian species, including bovine, ovine, porcine,
murine, 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.
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
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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 ca~.Tied out using polymerise 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')2, 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 (KLH). 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 "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. Andsense
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
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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
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCl), 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 which
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
WI) 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 Ieast
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
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Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, GIn, GIu
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 Ile, Leu, Thr
Conservative amino acid substitufiions 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.
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.
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,
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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 ID N0:22-42 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:22-42, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:22-42 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:22-42 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:22-42 and the region of SEQ ID N0:22-42 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 fragment of SEQ ID N0:1-21 is encoded by a fragment of SEQ ID NO:22-42, A
fragment
of SEQ ID N0:1-21 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:I-21. For example, a fragment of SEQ ID N0:1-21 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID N0:1-21.
The precise length of
a fragment of SEQ ID N0:1-21 and the region of SEQ ID NO:I-21 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
CA 02411971 2002-12-10
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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 alignments of polynucleotide sequences, the default parameters
are set as follows:
Ktuple=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
20 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. 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 mateh: 1
Penalty fog mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off:' S0
Expect: l0
Word Size: 11
Filter.' on
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.
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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 "% 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 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 parametexs. Such default parameters
may be, for example:
Matrix: BLOSUM62
Operz Gap: 11 arid Extension Gap: 1 penalties
Gap x drop-off.' S0
Expect: 10
Word Size: 3
Filter: orr
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.
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"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. 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 ~~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 (T~ for the
specific sequence at a defined ionic
strength and pH. The Tin 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
Cloning: A LaboratorYManual, 2nd 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 ~ g/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
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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 amino 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 ax immune response when introduced into a living
organism, for example, a
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
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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 S nucleotides in length linked
to a peptide backbone of
S 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
1S 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
DNA strand by a DNA
polymerise enzyme. Primer pairs can be used for amplification (and
identification) of a nucleic acid
sequence, e.g., by the polymerise chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 1S 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, 2S, 30, 40,
S0, 60, 70, 80, 90, 100,
or at least 1S0 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
2S 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, 2nd
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo , 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 O.S, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
2S
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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 InstitutelMIT Center for
Genome Research,
IO 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
UK) 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 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, su ra. 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.
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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 chromogenic 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 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.
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A "transcript image" 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 cell. 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
20 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
into such organisms are widely known and provided in references such as
Sambrook et al. ( 1989),
supra.
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 ox lesser
number of polynucleotides
due to alternative splicing of exons during mRNA processing. The corresponding
polypeptide may
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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
single Incyte project identification number (Incyte Project ID). 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 ID 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 ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) 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 score for the match between each polypeptide
and its GenBank
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homolog. Column S shows the annotation of the GenBank homolog 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
IO shows analytical methods for protein structurelfunction 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 ID N0:1 is a
ubiquitin carboxyl terminal hydrolase. SEQ ID N0:1 is 48% identical, from
residue M1 to residue
6225, to human ubiquitin-specific processing protease (GenBank ID g9971757) as
determined by the
Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 1.00e-
49, which indicates the probability of ortaining the observed polypeptide
sequence alignment by chance.
SEQ ID N0:1 contains a ubiquitin carboxyl terminal hydrolase catalytic site
domain as determined by
searching for statistically significant matches in the hidden Markov model
(HMM)-based~PFAM
database of conserved protein family domains. The score is 53.4 bits and the E-
value is 4.9e-12, which
indicates the probability of obtaining the observed structural motif by
chance. The presence of this
motif was corroborated by BLIMPS (probability score=2.6e-4) and MOTIFS
analyses. This provides
further evidence that SEQ ID N0:1 is a ubiquitin carboxyl-terminal hydrolase.
In an alternative
example, SEQ ID N0:2 is 45 % identical to amino acids 15-235 of human
prostasin, a serine protease
(GenBank ID g1 143194) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 1.3e-46, which indicates the
probability of obtaining the
observed polypeptide sequence alignment by chance. SEQ ID NO:2 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. This
match has a probability score of 2.7e-58. BLIMPS, MOTIFS, and PROFILESCAN
analyses confirm
the presence of this domain. (See Table 3.) BLIMPS analysis also reveals a
kringle domain, providing
further corroborative evidence that SEQ ID N0:2 is a serine protease of the
trypsin family. In an
alternative example, SEQ ID N0:7 is a dipeptidase which hydrolyses a variety
of peptides (Kozak, E.
and S. Tate (1982) J. Biol. Chem. 257:6322-6327), and is responsible for the
hydrolysis of the beta
CA 02411971 2002-12-10
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lactam rings of antibiotics such as penem and carbapenem (Campbell et al.,
(1984) J. Biol. Chem.
259:14586-14590). SEQ ID N0:7 shows 48% amino acid sequence identity over 377
amino acids
(total length equals 411 amino acids) to human dipeptidase precursor (GenBank
ID g219600) as
determined by Basic Local Alignment Search Tool (BLAST). The BLAST probability
score is 1.1e-
88, which indicates the probability of obtaining the observed polypeptide
sequence alignment by chance.
Additionally, the protease of the invention demonstrates a renal dipeptidase
domain as determined by
searching for statistically significant matches in the hidden Markov model
(HMM)-based PFAM
database of conserved protein family domains. The HMM score for the renal
dipeptidase PFAM hit is
412.7. Data from BLIMPS, MOTIFS, BLAST-DOMO, and BLAST-PRODOM analyses provide
further corroborative evidence that SEQ ID N0:7 is a renal dipeptidase. The
BLIMPS-BLOCKS hit
scores for localized regions range from 1040-1537. The BLAST-DOMO hit
probability score is 5.2e-
85. The BLAST-PRODOM hit probability score is 4.7e-73. In an alternative
example, SEQ ID N0:8
is 86% identical to human transmembrane tryptase (GenBank ID g6103629) as
determined by the Basic
Local Alignment. Search Tool (BLAST). (See Table 2.) The BLAST probability
score is 3.9e-166,
which indicates the probability of obtaining the observed polypeptide sequence
alignment by chance.
SEQ ID NO:B contains a trypsin family protease active site domain with a
probability score of 5.3e-89
as determined by searching for matches in the hidden Markov model (HMM)-based
PFAM database of
conserved protein family domains. BLIMPS, MOTIFS, and PROFILESCAN analyses
confirm the
presence of this motif. BLIMPS analysis also shows that SEQ ID N0:8 contains a
kringle domain and
a type I fibronectin domain. HMMER-based analysis reveals the presence of a
transmembrane domain
(See Table 3.). Taken together, these analyses show that SEQ ID N0:8 is a
transmembrane member of
the trypsin family of serine proteases. In an alternative example, SEQ ID
N0:17 shares 44% local
identity with human membrane-type serine protease 1 (MT-SP1, GenBank ID
g6002714) as determined
by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is
5.1e-94, which indicates the probability of obtaining the observed polypeptide
sequence alignment by
chance. SEQ ID N0:17 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.) HMM-based
analysis also reveals a
transmembrane domain near the N-terminus of SEQ ID N0:17. A domain found in
the low-density
lipoprotein receptor and other proteins, including MT-SP 1 (PDOC00929) was
also identified in this
way. The presence of the trypsin active site motif is confirmed by
PROFILESCAN, BLIMPS, and
MOTIFS analyses. BLIMPS analysis revealed the presence of kringle and type I
fibronectin domains.
Taken togefiher, these data provide further corroborative evidence that SEQ ID
N0:17 is a
transmembrane member of the trypsin family of serine proteases. SEQ ID N0:3-6,
SEQ ID N0:9-16,
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and SEQ ID N0:18-21 were analyzed and annotated in a similar manner. The
algorithms and
parameters for the analysis of SEQ ID NO:1-21 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 ID N0:22-42 or that distinguish between SEQ ID
N0:22-42 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')
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,
724646778 is the
identification number of an Incyte cDNA sequence, and PROSTMY01 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.8., 71041539V1). Alternatively, the identification numbers
in column 5 may refer to
GenBank cDNAs or ESTs (e.8., 85745066) which contributed to the assembly of
the full length
polynucleotide sequences. Alternatively, the identification numbers in column
5 may refer to coding
regions predicted by Genscan analysis of genomic DNA. For example,
GNN.g7208751 000002 002.edit is the identification number of a Genscan-
predicted coding sequence,
with 87208751 being the GenBank identification number of the sequence to which
Genscan was
applied. The Genscan-predicted coding sequences may have been edited prior to
assembly. (See
Example IV.) Alternatively, the identification numbers in column 5 may refer
to assembla8es of both
cDNA and Genscan-predicted exons brought together by an "exon stitching"
algorithm. For example,
FL1389845 00001 represents a "stitched" sequence in which 1389845 is the
identification number of
the cluster of sequences to which the algorithm was applied, and 00001 is the
number of the prediction
generated by the algorithm. (See Example V.) Alternatively, the identification
numbers in column 5
may refer to assemblages of both cDNA and Genscan-predicted exons brought
together by an "exon-
stretching" algorithm. For example, FL2256251~7708357_000002_86103629 is the
identification
32
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WO 01/98468 PCT/USO1/19178
number of a "stretched" sequence, with 2256251 being the Incyte project
identification number,
g7708357 being the GenBank identification number of the human genomic sequence
to which the
"exon-stretching" algorithm was applied, and g6103629 being the GenBank
identification number of
the nearest GenBank protein homolog. (See Example V.) 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 cDNA 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 N0:22-42, which encodes PRTS. The
polynucleotide sequences of
SEQ ID N0:22-42, 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 ID
N0:22-42 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 of SEQ ID N0:22-42.
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.
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
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WO 01/98468 PCT/USO1/19178
by selecting combinations based on possible colon 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 occurring PRTS under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding PRTS or its
derivatives possessing a substantially different colon usage, e.g., inclusion
of non-naturally occurring
colons. Colons 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 colons
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 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
N0:22-42 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 N~,
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,
34
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
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 Biolo~y, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
(1995) Molecular
Biolo~y and Biotechnolo~y, 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 unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
IO 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
legations 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
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 intxon/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 lengthy to have a
GC content of about 50% or more, and to anneal to the template at temperatures
of about 68°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 S'
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
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
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
known 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.
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
Number
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.
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WO 01/98468 PCT/USO1/19178
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 Properties, 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 conf'2rmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, supra, 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
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 eases 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
translatronal control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
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CA 02411971 2002-12-10
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vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, ALaboratorv
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, su ra; 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~y (I992) McGraw
Hill, New
York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and
Harrington, 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.
(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 PBLUESCRIPT (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.
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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
nastoris. ~ 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, supra;
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; Brogue, 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.
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 (uposomes,
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, Bells 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
39
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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 hisD, 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 f3-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
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 andlor 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
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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 Immunolo~y, 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 xetained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors 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, and/or
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
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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-t~iyc, 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
20 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, su ra, ch. 20). 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.
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
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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 fine 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.
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 Number 5,175,383 and U.S. Patent Number
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
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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
PRTS are useful for hydrolyzing peptide bonds. 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 heroic, neurological,
reproductive, endocrine,
urogenital, diseased, teratocarcinoma, and tumorous tissues,. Therefore, PRTS
appears to play a role
in gastrointestinal, cardiovascular, autoimmune/intlarnmatory, cell
proliferative, 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-
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Weirs syndrome, colonic carcinoma, colonic obstruction, irritable bowel
syndrome, short bowel
syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired
immunodeficiency syndrome
(AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome,
hepatic steatosis,
hemochromatosis, Wilson's disease, alphas-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, mural 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 autoimmuneJinflammatory
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 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,
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 extxacorporeal
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
CA 02411971 2002-12-10
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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, spina bifida, anencephaly,
craniorachischisis, congenital
glaucoma, cataract, age-related macular degeneration, and sensorineural
hearing loss; an epithelial
disorder, such as dyshidr otic 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 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
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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.
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
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CA 02411971 2002-12-10
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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 shill 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 lmown 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 gels
such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, I~LH, and dinitrophenol. Among adjuvants
used in humans, BCG
(bacilli Calmette-Guerin) and Corynebacterium~arvum 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
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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.
Immunol. 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 fox 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.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities axe 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
49
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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 L/mole 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' Llmole 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 Approach, 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. su ra.)
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 can be
designed from various locations along the coding or control regions of
sequences encoding PRTS. (See,
e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, 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 Cli. 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
CA 02411971 2002-12-10
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al. (1998) J. Pharm. Sci. 87(1I):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 immunodeficiency (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) Cel175:207-216; 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 falci~ and
Tryoanosoma 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 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 vectors (Invitrogen,
Carlsbad CA),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON, PTRE2, PTRE2-LUC, PTI~-HYG (Clontech, Palo Alto CA). PRTS may be
expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV),
Rous sarcoma virus
51
CA 02411971 2002-12-10
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(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
89:5S47-5551; 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 PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M. V.
and Blau, H.M. supra)), 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 HIT, 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), or 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. Refrovirus 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
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 Number 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
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CA 02411971 2002-12-10
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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 Number 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 Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is
hereby incorporated by reference. U.S. Patent Number 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.
For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and
Xu, H. et aI. (1994) Dev.
Biol. 163:152-161, hereby incorporated by reference. The manipulation of
cloned herpesvirus
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
known to those of ordinary
skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
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
53
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
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~ic 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.
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
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CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
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-, thio-, 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
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 and/or structural properties of the target polynucleotide;
and selection from a
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
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) Bioehem.
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
using methods which are well known in the art. (See, e.g., Goldman, C.I~. 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.
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CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
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, fransdermal, subcutaneous, intraperitoneal,
infranasal, 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 for 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-I 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, 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 LDSa/EDSO ratio. Compositions
which exhibit large
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CA 02411971 2002-12-10
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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 0.1 ~cg to 100,000 ~cg, 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
PRTS 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 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 PRTS
expressed in subject,
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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
N0:22-42 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 35S, or by
enzymatic labels, such as
alkaline phosphatase coupled to the probe via avidin/biotin coupling systems,
and the like.
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,
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Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irritable
bowel syndrome, short
bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired
immunodeficiency
syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal
syndrome, hepatic
steatosis, hemochromatosis, Wilson's disease, alphas-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
autoimmuneJinflammatory 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 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;
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
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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, 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, 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 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
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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 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
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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
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
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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. Immunol. 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.
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
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given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number
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 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
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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, su ra). 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 aI. (1999) Anal.
Biochem. 270:103-111;
Mendoze, L. G, et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a 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
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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 axe separated so that the amount of each protein can be quantified. The
amount of each protein
is compaxed 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 axe 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. Acid. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acid. 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
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 multi-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., Harrington,
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
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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, Larder, 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.
In 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 mammalian 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 l 1q22-23, 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.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding afFnity to the protein of interest. (See, e.g.,
Geyser, 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
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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
S properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the txiplet 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. 60/212,336, U.S. Ser. No. 60/213,995, U.S. Ser. No.
60/215,396, U.S. Ser.
No. 60/216,821, and U.S. Ser. No. 60/218,946, are hereby expressly
incorporated by reference.
1S 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 S. Some tissues
were homogenized
and lysed in guanidinium isothiocyanate, while others were homogenized and
Iysed 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 CsCl
cushions or extracted
with chloroform. RNA was precipitated from the lysates with either isopropanol
or sodium acetate and
ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
2S 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, supra, units
S.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
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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), PBK-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo Alto
CA), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli cells
including XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DHSa, DH10B, or
ElectroMAX
DH10B 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 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 II 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
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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, 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 HMMER. 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 Conned, and
cDNA assemblages
were screened for open reading frames using programs based on GeneMark, BLAST,
and FASTA.
20. The full length polynucleotide sequences were translated to derive the
corresponding full length
polypeptide 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,
BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model (HMM)-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
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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
N0:22-42. 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.
Marlin (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
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 find
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 III. 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
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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 analysis
to either Incyte cDNA sequences or GenScan exon predicted sequences described
in Example IV. 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 N0:22-42 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
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SEQ ID N0:22-42 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 ID 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.nlm.nih.govlgenemap/), can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
In this manner, SEQ ID N0:25 was mapped to chromosome 5 within the interval
from 69.60
to 76.50 centiMorgans. SEQ ID N0:28 Was mapped to chromosome 16 within the
interval from
81.80 to 84.40 centiMorgans.
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 sequenee 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)
su ra, 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
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 Identi~
5 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
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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 III). 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;
heroic 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
diseaseJcondition 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 libraryltissue information are found in
the LIFESEQ
GOLD database (Incyte Genomics, Palo Alto CA).
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.
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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 Mg2+, (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 1: 94 ° C, 3 min; Step 2: 94 ° C, 1 S
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 SK+ 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 ~1
PICOGREEN
quantitation reagent (0.25 % (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~1 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 ~l to 10 /x1 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
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
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using the same conditions as described above. Samples were diluted with 20%
dimethysulfoxide (1:2,
vlv), 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 ID N0:22-42 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 ,uCi 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 dextrin 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 RI, 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
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
microspotting 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
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WO 01/98468 PCT/USO1/19178
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 are 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
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/iQ oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/i~l RNase inhibitor, 500 ~M dATP, 500 iiM dGTP, 500
~M dTTP, 40 ~M
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 O.SM 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
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100~o
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 ~.~1 SX 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
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CA 02411971 2002-12-10
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amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
fig. 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 US
Patent No. 5,807,522, incorporated herein by reference. 1 p1 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 microaxrays 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 Eil of sample mixture consisting of 0.2 ~g
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX 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 E.il of SX 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.
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.
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CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
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 N~ 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 crosstalk
(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).
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.
CA 02411971 2002-12-10
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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 tr p-lac (tac)
hybrid promoter and the
TS 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 eukaryotic cells is achieved by infecting insect or
mammalian cell lines with
recombinant Auto~raphica californica nuclear polyhedrosis virus (AcMNPV),
commonly known 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
fru~~erda (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 japonicum, 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 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, su ra,
ch. 10 and 16). Purified PRTS obtained by these methods can be used directly
in the assays shown in
Examples XVI, XVII, XVIII, and XIX, 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
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CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
contain the cytomegalovirus promoter. 5-10 ~cg of recombinant vector are
transiently transfected into a
human cell line, foi example, an endothelial or hematopoietic cell line, using
either liposome
formulations or electroporation. 1-2 /cg 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
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, supra, ch. 11.)
82
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
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-KLH 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 for 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 lzsl 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 multi-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.
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 aI. (2000) U.S. Patent
No. 6,057,101).
XVII. Demonstration of PRTS Activity
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WO 01/98468 PCT/USO1/19178
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) Proteol~tic 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. For example,
arginine-(3-napthylamide can be used as a substrate for SEQ ID N0:3 (Fukasawa,
K.M. et al. (1996) J.
Biol. Chem. 271:30731-30735) and 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-
D-Arg can be
used as a substrate for SEQ ID N0:4. In an alternative example, a substrate
for SEQ ID N0:9 would
be 7-amino-4-trifluoromethyl coumarin-Phe-Pro-AFC. 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 increase/decrease in absorbance of
the chromogen released
during hydrolysis of the peptide substrate is measured. The change in
absorbance 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. For SEQ ID N0:1, 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).
Alternatively, the ubiquitin protease activity of SEQ ID NO:S can be measured
using the
method of Sloper-Mould et al. ((1999) J. Biol. Chem. 274:26878-26884).
Aliquots of PRTS are
incubated with 5 ~1 [35S]-labeled ubiquitin-GST fusion substrate for 1 hour at
37 °C in an appropriate
buffer. Samples are resolved by electrophoresis on a 12% SDS-PAGE gel.
Ubiquitin cleavage is
monitored by fluorography of the gel.
Alternatively, the activity of SEQ ID NO:10, for example, can be measured by
the method of
Cofige et al. (1999, J. Biol. Chem. 270:16724-16730). An aliquot of PRTS is
incubated with amino
procollagen type I substrate radioactively labeled only in the propeptide in a
250 p1 reaction containing
50 mM sodium cacodylate, pH 7.5, 200 mM KCl, 2 mM CaCl, 2.5 mM NEM, 0.5 mM
PMSF, and
0.02% Brij (standard assay buffer). After 16 h at 26 °C, the reaction
is stopped by adding 50 ~l of
EDTA solution (0.2 M EDTA, pH 8, 0.5% SDS, 0.5 M DTT) and 300 ~1 of 99%
ethanol. The
samples axe kept for 30 min at 4 °C and centrifuged for 30 min at 9500
g. Collagen and uncleaved
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CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
radioactive pN-collagen substrate are pelleted, whereas the freed amino
propeptides remained in
solution. An aliquot of the supernatant is assayed by liquid scintillation
spectrometry.
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
tramfer 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 (Mitra, R.l~. 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 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).
In yet another alternative, an assay for PRTS dipeptidase activity measures
the hydrolysis
activity of PRTS on a variety of dipeptides such as leukotriene D4 (Kozak, E.
and S. Tate (1982) J.
Biol. Chem. 257:6322-6327), or hydrolysis of the beta-lactam ring of
antibiotics such as penum and
carbapenem (Campbell et al., (1984) J. Biol. Chem. 259:14586-14590).
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 WI) 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.
CA 02411971 2002-12-10
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XIX. Identification of PRTS Inhibitors
Compounds to be tested are arrayed in the wells of a multi-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, multi-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 are tested for
PRTS inhibitory activity using an assay described in Example XVII.
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 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.
86
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
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CA 02411971 2002-12-10
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<110> INCYTE GENOMICS, INC.
YUE, Henry
ELLIOTT, Vicki
GANDHI, Ameena R.
LAL, Preeti
AU-YOUNG, Janice
TRIBOULEY, Catherine M.
DELEGEANE, Angelo M.
BAUGHN, Mariah R.
NGUYEN, Danniel B.
LEE, Ernestine A.
HAFALIA, April
KHAN, Farrah A.
WALIA, Narinder It.
YAO, Monique G.
LU, Dyung Aina M.
PATTERSON, Chandra
TANG, Y. Tom
WALSH, Roderick T.
AZIMZAI, Yalda
LU, Yan
RAMKUMAR, Jayalaximi
XU, Yuming
REDDY, Roopa
DAS, Depopriya
KEARNEY, Liam
KALLICK, Deborah A.
<120> Proteases
<130> PI-0123 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/212,336; 60/213,955; 60/215,396; 60/216,821; 60/218,946
<151> 2000-06-16; 2000-06-22; 2000-06-29; 2000-07-07; 2000-07-14
<160> 42
<170> PERL Program
<210> 1
<211> 232
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 275791CD1
<400> 1
Met Pro G1u Asn Pro Asp Thr Met Glu Thr Glu Lys Pro Lys Thr
1 5 10 15
Ile Thr Glu Leu Asp Pro A1a Ser Phe Thr Glu Ile Thr Lys Asp
20 25 30
Cys Asp Glu Asn Lys Glu Asn Lys Thr Pro G1u Gly Ser Gln Gly
35 40 45
Glu Val Asp Trp Leu Gln Gln Tyr Asp Met G1u Arg Glu Arg Glu
50 55 60
Glu Gln Glu Leu Gln Gln Ala Leu Ala Gln Ser Leu Gln Glu G1n
65 70 75
Glu Ala Trp Glu Gln Lys Glu Asp Asp Asp Leu Lys Arg Ala Thr
80 85 90
Glu Leu Ser Leu Gln Glu Phe Asn Asn Ser Phe Val Asp Ala Leu
95 100 105
G1y Ser Asp Glu Asp Ser Gly Asn Glu Asp Val Phe Asp Met Glu
110 115 120
1/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Tyr Thr Glu Ala Glu Ala Glu Glu Leu Lys Arg Asn Ala Glu Thr
125 130 135
Gly Asn Leu Pro His Ser Tyr Arg Leu Ile Ser Val Val Ser His
140 145 150
Ile Gly Ser Thr Ser Ser Ser Gly His Tyr Ile Ser Asp Val Tyr
155 160 165
Asp Ile Lys Lys Gln Ala Trp Phe Thr Tyr Asn Asp Leu Glu Val
170 175 180
Ser Lys Ile Gln Glu Ala Ala Val Gln Ser Asp Arg Asp Arg Ser
185 190 195
Gly Tyr Ile Phe Phe Tyr Met His Lys Glu Ile Phe Asp Glu Leu
200 205 210
Leu Glu Thr Glu Lys Asn Ser G1n Ser Leu Ser Thr Glu Val Gly
215 220 225
Lys Thr Thr Arg Gln Ala Ser
230
<210> 2
<211> 365
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1389845CD1
<400> 2
Met Pro Lys Tyr Leu Gly Gly Gly Cys Cys Ile Pro Gly Pro Trp
1 5 10 15
Ala Glu Arg Arg Val Tyr Ser Leu G1y His Gln Asp Lys Sex Arg
20 25 30
Thr His Gln Glu Leu Arg Thr Asp Arg Arg Thr Thr Glu Gly Val
35 40 45
Thr G1y Trp Cys Glu Asp Trp Cys Pro Trp Ala Arg Thr Leu Leu
50 55 60
Ser Ser Pro Cys Trp Leu Gln Thr Arg Val G1n Ala Leu Gly Ser
65 70 75
Ala Thr Leu Thr Gln Pro Ser Leu Glu Asp Arg Met Arg Gly Val
80 85 90
Ser Cys Leu Gln Val Leu Leu Leu Leu Val Leu Gly Ala Ala Gly
95 100 105
Thr Gln Gly Arg Lys Ser Ala Ala Cys Gly Gln Pro Arg Met Ser
110 115 120
Ser Arg Ile Val Gly Gly Arg Asp Gly Arg Asp Gly Glu Trp Pro
125 130 135
Trp Gln Ala Ser Ile Gln His Arg Gly Ala His Val Cys G1y Gly
140 145 150
Ser Leu Ile Ala Pro Gln Trp Val Leu Thr Ala Ala His Cys Phe
155 160 165
Pro Arg Arg Ala Leu Pro Ala Glu Tyr Arg Val Arg Leu Gly A1a
170 175 180
Leu Arg Leu G1y Ser Thr Ser Pro Arg Thr Leu Ser Val Pro Val
185 190 195
Arg Arg Val Leu Leu Pro Pro Asp Tyr Ser Glu Asp Gly Ala Arg
200 205 210
G1y Asp Leu Ala Leu Leu Gln Leu Arg Arg Pro Val Pro Leu Ser
215 220 225
Ala Arg Val Gln Pro Val Cys Leu Pro Val Pro Gly Ala Arg Pro
230 235 240
Pro Pro Gly Thr Pro Cys Arg Val Thr Gly Trp Gly Ser Leu Arg
245 250 255
Pro G1y Val Pro Leu Pro Glu Trp Arg Pro Leu Gln Gly Val Arg
260 265 270
Val Pro Leu Leu Asp Ser Arg Thr Cys Asp Gly Leu Tyr His Val
275 280 285
Gly A1a Asp Val Pro Gln Ala Glu Arg Ile Val Leu Pro Gly Ser
290 295 300
2/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Leu Cys Ala Gly Tyr Pro Gln Gly His Lys Asp Ala Cys Gln Val
305 310 315
Cys Thr Gln Pro Pro Gln Pro Pro Glu Ser Pro Pro Cys Ala Gln
320 325 330
His Pro Pro Ser Leu Asn Ser Arg Thr Gln Asp Ile Pro Thr Gln
335 340 345
Ala Gln Asp Pro Gly Leu Gln Pro Arg Gly Thr Thr Pro Gly Val
350 355 360
Trp Asn Pro Glu Asn
365
<210> 3
<211> 416
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 1726609CD1
<400> 3
Met Trp Gly Arg Tyr Asp Tle Val Phe Leu Pro Pro Ser Phe Pro
1 5 10 15
Ile Val Ala Met Glu Asn Pro Cys Leu Thr Phe Ile Ile Ser Ser
20 25 30
Ile Leu Glu Ser Asp Glu Phe Leu Val Ile Asp Val Ile His Glu
35 40 45
Val Ala His Ser Trp Phe Gly Asn Ala Val Thr Asn Ala Thr Trp
50 55 60
Glu Glu Met Trp Leu Ser Glu Gly Leu Ala Thr Tyr Ala Gln Arg
65 70 75
Arg Ile Thr Thr Glu Thr Tyr Gly Ala Ala Phe Thr Cys Leu Glu
80 85 90
Thr Ala Phe Arg Leu Asp Ala Leu His Arg Gln Met Lys Leu Leu
95 100 105
Gly Glu Asp Ser Pro Val Ser Lys Leu Gln Val Lys Leu Glu Pro
110 115 120
Gly Val Asn Pro Ser His Leu Met Asn Leu Phe Thr Tyr Glu Lys
125 130 135
G1y Tyr Cys Phe Val Tyr Tyr Leu Ser Gln Leu Cys Gly Asp Pro
140 145 150
Gln Arg Phe Asp Asp Phe Leu Arg Ala Tyr Val Glu Lys Tyr Lys
155 160 165
Phe Thr Ser Val Val Ala Gln Asp Leu Leu Asp Ser Phe Leu Ser
170 175 180
Phe Phe Pro Glu Leu Lys Glu Gln Ser Va1 Asp Cys Arg Ala G1y
185 190 195
Leu Glu Phe Glu Arg Trp Leu Asn Ala Thr Gly Pro Pro Leu Ala
200 205 210
Glu Pro Asp Leu Ser Gln Gly Ser Ser Leu Thr Arg Pro Val Glu
215 220 225
Ala Leu Phe Gln Leu Trp Thr Ala Glu Pro Leu Asp Gln Ala Ala
230 235 240
Ala Ser Ala Ser Ala Ile Asp Ile Ser Lys Trp Arg Thr Phe G1n
245 250 255
Thr A1a Leu Phe Leu Asp Arg Leu Leu Asp Gly Ser Pro Leu Pro
260 265 270
G1n Glu Val Val Met Ser Leu Ser Lys Cys Tyr Ser Ser Leu Leu
275 280 285
Asp Ser Met Asn Ala Glu Ile Arg I1e Arg Trp Leu Gln Ile Val
290 295 300
Val Arg Asn Asp Tyr Tyr Pro Asp Leu His Arg Val Arg Arg Phe
305 310 315
Leu G1u Ser Gln Met Ser Arg Met Tyr Thr Ile Pro Leu Tyr Glu
320 325 330
Asp Leu Cys Thr Gly Ala Leu Lys Ser Phe Ala Leu Glu Val Phe
335 340 345
3/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Tyr Gln Thr Gln Gly Arg Leu His Pro Asn Leu Arg Arg Ala Ile
350 355 360
Gln Gln Ile Leu Ser Gln Gly Leu Gly Ser Ser Thr Glu Pro Ala
365 370 375
Ser Glu Pro Ser Thr Glu Leu Gly Lys Ala Glu Ala Asp Thr Asp
380 385 390
Ser Asp Ala Gln Ala Leu Leu Leu Gly Asp Glu Ala Pro Ser Ser
395 400 405
Ala Ile Ser Leu Arg Asp Val Asn Val Ser Ala
410 415
<210> 4
<211> 714
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4503848CD1
<400> 4
Met His Ile His Met Leu Thr Leu Asp Gln Gln Lys Ser Leu Ile
1 5 10 15
Leu Ile Leu Phe Leu Ile Leu Phe Arg Val Gly Gly Ser Arg Ile
20 25 30
Leu Leu Arg Met Thr Leu Gly Arg Glu Val Met Ser Pro Leu Gln
35 40 45
Ala Met Ser Ser Tyr Thr Val Ala Gly Arg Asn Val Leu Arg Trp
50 55 60
Asp Leu Ser Pro Glu Gln Ile Lys Thr Arg Thr Glu Glu Leu Ile
65 70 75
Val Gln Thr Lys Gln Val Tyr Asp Ala Val Gly Met Leu Gly I1e
80 85 90
G1u Glu Val Thr Tyr Glu Asn Cys Leu Gln Ala Leu A1a Asp Val
95 100 105
Glu Val Lys Tyr Ile Val Glu Arg Thr Met Leu Asp Phe Pro G1n
110 115 120
His Val Ser Ser Asp Lys Glu Val Arg Ala Ala Ser Thr Glu Ala
125 13 0 135
Asp Lys Arg Leu Ser Arg Phe Asp I1e Glu Met Ser Met Arg Gly
140 145 150
Asp Ile Phe Glu Arg Ile Val His Leu Gln Glu Thr Cys Asp Leu
155 160 165
Gly Lys Ile Lys Pro Glu Ala Arg Arg Tyr Leu Glu Lys Ser Ile
170 175 180
Lys Met G1y Lys Arg Asn Gly Leu His Leu Pro Glu Gln Val Gln
l85 190 195
Asn Glu Ile Lys Ser Met Lys Lys Arg Met Ser Glu Leu Cys Ile
200 205 210
Asp Phe Asn Lys Asn Leu Asn Glu Asp Asp Thr Phe Leu Va1 Phe
215 220 225
Ser Lys Ala Glu Leu Gly Ala Leu Pro Asp Asp Phe Ile Asp Ser
230 235 240
Leu Glu Lys Thr Asp Asp Asp Lys Tyr Lys Ile Thr Leu Lys Tyr
245 250 255
Pro His Tyr Phe Pro Val Met Lys Lys Cys Cys Ile Pro Glu Thr
260 265 270
Arg Arg Arg Met Glu Met Ala Phe Asn Thr Arg Cys Lys Glu Glu
275 280 285
Asn Thr Ile I1e Leu Gln Gln Leu Leu Pro Leu Arg Thr Lys Val
290 295 300
Ala Lys Leu Leu Gly Tyr Ser Thr His Ala Asp Phe Val Leu Glu
305 310 315
Met Asn Thr A1a Lys Ser Thr Ser Arg Val Thr Ala Phe Leu Asp
320 325 330
Asp Leu Ser G1n Lys Leu Lys Pro Leu Gly Glu Ala Glu Arg Glu
335 340 345
4/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Phe Ile Leu Asn Leu Lys Lys Lys Glu Cys Lys Asp Arg Gly Phe
350 355 360
Glu Tyr Asp Gly Lys Ile Asn Ala Trp Asp Leu Tyr Tyr Tyr Met
365 370 375
Thr Gln Thr Glu Glu Leu Lys Tyr Ser Ile Asp Gln Glu Phe Leu
380 385 390
Lys Glu Tyr Phe Pro Ile Glu Val Val Thr Glu Gly Leu Leu Asn
395 400 405
Thr Tyr G1n Glu Leu Leu Gly Leu Ser Phe Glu Gln Met Thr Asp
410 415 420
Ala His Val Trp Asn Lys Ser Val Thr Leu Tyr Thr Val Lys Asp
425 430 435
Lys Ala Thr Gly Glu Val Leu Gly Gln Phe Tyr Leu Asp Leu Tyr
440 445 450
Pro Arg Glu Gly Lys Tyr Asn His Ala Ala Cys Phe Gly Leu Gln
455 460 465
Pro Gly Cys Leu Leu Pro Asp Gly Ser Arg Met Met Ala Val Ala
470 475 480
Ala Leu Val Val Asn Phe Ser G1n Pro Val Ala Gly Arg Pro Ser
485 490 495
Leu Leu Arg His Asp Glu Val Arg Thr Tyr Phe His Glu Phe Gly
500 505 510
His Val Met His G1n Ile Cys Ala Gln Thr Asp Phe Ala Arg Phe
515 520 525
Ser Gly Thr Asn Val Glu Thr Asp Phe Val Glu Val Pro Ser Gln
530 535 540
Met Leu Glu Asn Trp Val Trp Asp Va1 Asp Ser Leu Arg Arg Leu
545 550 555
Ser Lys His Tyr Lys Asp Gly Ser Pro Ile Ala Asp Asp Leu Leu
560 565 570
Glu Lys Leu Val Ala Ser Arg Leu Val Asn Thr Gly Leu Leu Thr
575 580 585
Leu Arg Gln Ile Val Leu Ser Lys Val Asp Gln Ser Leu His Thr
590 595 600
Asn Thr Ser Leu Asp Ala Ala Ser Glu Tyr Ala Lys Tyr Cys Ser
605 610 615
Glu Ile Leu Gly Val Ala Ala Thr Pro Gly Thr Asn Met Pro Ala
62 0 625 63 0
Thr Phe Gly His Leu Ala Gly Gly Tyr Asp Gly Gln Tyr Tyr Gly
635 640 645
Tyr Leu Trp Ser Glu Val Phe Ser Met Asp Met Phe Tyr Ser Cys
650 655 660
Phe Lys Lys Glu Gly Ile Met Asn Pro Glu Val Gly Met Lys Tyr
665 670 675
Arg Asn Leu Ile Leu Lys Pro Gly Gly Ser Leu Asp G1y Met Asp
680 685 690
Met Leu His Asn Phe Leu Lys Arg Glu Pro Asn Gln Lys Ala Phe
695 700 705
Leu Met Ser Arg Gly Leu His Ala Pro
710
<210> 5
<211> 3 67
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5544089CD1
<400> 5
Met Phe A1a Pro Ser Val Leu Ser Ser Gly Leu Ser Gly G1y Ala
1 5 10 15
Ser Lys Gly Arg Lys Met Glu Leu I1e Gln Pro Lys Glu Pro Thr
20 25 30
Ser Gln Tyr Ile Ser Leu Cys His Glu Leu His Thr Leu Phe Gln
35 40 45
5/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Val Met Trp Ser Gly Lys Trp Ala Leu Val Ser Pro Phe Ala Met
50 55 60
Leu His Ser Val Trp Arg Leu Ile Pro Ala Phe Arg Gly Tyr Ala
65 70 75
Gln Gln Asp Ala G1n Glu Phe Leu Cys Glu Leu Leu Asp Lys Ile
80 85 90
Gln Arg Glu Leu G1u Thr Thr Gly Thr Ser Leu Pro Ala Leu Ile
95 100 105
Pro Thr Ser Gln Arg Lys Leu Ile Lys Gln Val Leu Asn Val Val
110 115 120
Asn Asn Ile Phe His Gly Gln Leu Leu Ser Gln Val Thr Cys Leu
125 130 135
Ala Cys Asp Asn Lys Ser Asn Thr Ile Glu Pro Phe Trp Asp Leu
140 145 150
Ser Leu Glu Phe Pro Glu Arg Tyr Gln Cys Ser Gly Lys Asp Ile
155 160 165
Ala Sex Gln Pro Cys Leu Val Thr Glu Met Leu Ala Lys Phe Thr
170 l75 180
Glu Thr Glu Ala Leu Glu Gly Lys Ile Tyr Val Cys Asp Gln Cys
185 190 195
Asn Ser Lys Arg Arg Arg Phe Ser Ser Lys Pro Val Val Leu Thr
200 205 210
Glu Ala Gln Lys Gln Leu Met Ile Cys His Leu Pro Gln Val Leu
215 220 225
Arg Leu His Leu Lys Arg Phe Arg Trp Ser Gly Arg Asn Asn Arg
230 235 240
Glu Lys Ile Gly Val His Val Gly Phe Glu Glu Ile Leu Asn Met
245 250 255
Glu Pro Tyr Cys Cys Arg Glu Thr Leu Lys Ser Leu Arg Pro Glu
260 265 270
Cys Phe Ile Tyr Asp Leu Ser Ala Val Val Met His His Gly Lys
275 280 285
Gly Phe Gly Ser Gly His Tyr Thr Ala Tyr Cys Tyr Asn Ser Glu
290 295 300
Gly Gly Phe Trp Val His Cys Asn Asp Ser Lys Leu Ser Met Cys
305 310 315
Thr Met Asp Glu Val Cys Lys Ala Gln Ala Tyr Ile Leu Phe Tyr
320 325 330
Thr Gln Arg Val Thr Glu Asn Gly His Ser Lys Leu Leu Pro Pro
335 340 345
Glu Leu Leu Leu Gly Ser Gln His Pro Asn Glu Asp A1a Asp Thr
350 355 360
Ser Ser Asn Glu Ile Leu Ser
365
<210> 6
<211> 235
<212> PRT
<213> Homa sapiens
<220>
<221> misc_feature
<223> Incyte ID Na: 7474081CD1
<400> 6
Met Lys Tyr Val Phe Tyr Leu Gly Val Leu Ala G1y Thr Phe Phe
1 5 10 15
Phe Ala Asp Ser Ser Val Gln Lys Glu Asp Pro Ala Pro Tyr Leu
20 25 30
Va1 Tyr Leu Lys Ser His Phe Asn Pro Cys Val G1y Val Leu Ile
35 40 45
Lys Pro Ser Trp Val Leu Ala Pro Ala His Cys Tyr Leu Pro Asn
50 55 60
Leu Lys Val Met Leu Gly Asn Phe Lys Ser Arg Val Arg Asp Gly
65 70 75
Thr Glu Gln Thr Ile Asn Pro Ile Gln Ile Val Arg Tyr Trp Asn
80 85 90
6/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Tyr Ser His Ser Ala Pro G1n Asp Asp Leu Met Leu Ile Lys Leu
95 100 105
Ala Lys Pro Ala Met Leu Asn Pro Lys Val Gln Pro Leu Thr Leu
110 115 120
Ala Thr Thr Asn Val Arg Pro Gly Thr Val Cys Leu Leu Ser Gly
125 130 135
Leu Asp Trp Ser Gln Glu Asn Ser Gly Arg His Pro Asp Leu Arg
140 145 150
Gln Asn Leu Glu Ala Pro Val Met Ser Asp Arg Glu Cys Gln Lys
155 160 165
Thr Glu G1n Gly Lys Ser His Arg Asn Ser Leu Cys Val Lys Phe
170 175 180
Val Lys Val Phe Ser Arg Ile Phe Gly Glu Val Ala Val Ala Thr
185 190 195
Val Ile Cys Lys Asp Lys Leu Gln Gly Ile Glu Val Gly His Phe
200 205 210
Met Gly G1y Asp Val Gly Ile Tyr Thr Asn Val Tyr Lys Tyr Val
215 220 225
Ser Trp Ile Glu Asn Thr Ala Lys Asp Lys
230 235
<210> 7
<211> 488
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5281209CD1
<400> 7
Met Gln Pro Thr Gly Arg Glu Gly Ser Arg A1a Leu Ser Arg Arg
1 5 10 15
Tyr Leu Arg Arg Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Arg
20 25 30
G1n Pro Val Thr Arg Ala Glu Thr Thr Pro Gly Ala Pro Arg A1a
35 40 45
Leu Ser Thr Leu Gly Ser Pro Ser Leu Phe Thr Thr Pro Gly Val
50 55 60
Pro Ser Ala Leu Thr Thr Pro Gly Leu Thr Thr Pro Gly Thr Pro
65 70 75
Lys Thr Leu Asp Leu Arg Gly Arg Ala Gln Ala Leu Met Arg Ser
80 85 90
Phe Pro Leu Val Asp Gly His Asn Asp Leu Pro Gln Val Leu Arg
95 100 105
Gln Arg Tyr Lys Asn Val Leu G1n Asp Va1 Asn Leu Arg Asn Phe
110 115 120
Ser His Gly Gln Thr Ser Leu Asp Arg Leu Arg Asp Gly Leu Val
125 130 135
Gly Ala Gln Phe Trp Ser Ala Ser Va1 Ser Cys Gln Ser Gln Asp
140 145 150
G1n Thr Ala Val Arg Leu Ala Leu Glu Gln Ile Asp Leu Ile His
155 160 165
Arg Met Cys Ala Ser Tyr Ser G1u Leu Glu Leu Val Thr Ser Ala
170 175 180
Glu Gly Leu Asn Ser Ser Gln Lys Leu Ala Cys Leu Ile Gly Val
185 190 195
Glu Gly Gly His Ser Leu Asp Ser Ser Leu Ser Val Leu Arg Ser
200 205 210
Phe Tyr Val Leu Gly Val Arg Tyr Leu Thr Leu Thr Phe Thr Cys
215 220 225
Ser Thr Pro Trp Ala Glu Ser Ser Thr Lys Phe Arg His His Met
230 235 240
Tyr Thr Asn Val Ser Gly Leu Thr Ser Phe G1y Glu Lys Val Val
245 250 255
Glu Glu Leu Asn Arg Leu Gly Met Met Ile Asp Leu Ser Tyr Ala
260 265 270
7/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Ser Asp Thr Leu Ile Arg Arg Val Leu Glu Va1 Ser Gln Ala Pro
275 280 285
Val Ile Phe Ser His Ser Ala Ala Arg Ala Val Cys Asp Asn Leu
290 295 300
Leu Asn Val Pro Asp Asp Ile Leu Gln Leu Leu Lys Lys Asn Gly
305 310 315
Gly Ile Va1 Met Val Thr Leu Ser Met Gly Val Leu Gln Cys Asn
320 325 330
Leu Leu Ala Asn Val Ser Thr Val Ala Asp His Phe Asp His Ile
335 340 345
Arg Ala Val Ile Gly Ser Glu Phe Ile Gly Ile Gly Gly Asn Tyr
350 355 360
Asp Gly Thr Gly Arg Phe Pro Gln Gly Leu Glu Asp Val Ser Thr
365 370 375
Tyr Pro Val Leu Ile Glu Glu Leu Leu Ser Arg Ser Trp Ser Glu
380 385 390
Glu Glu Leu Gln Gly Va1 Leu Arg Gly Asn Leu Leu Arg Val Phe
395 400 405
Arg Gln Val Glu Lys Val Arg Glu Glu Ser Arg Ala Gln Ser Pro
410 415 420
Val Glu Ala Glu Phe Pro Tyr Gly G1n Leu Ser Thr Ser Cys His
425 430 435
Ser His Leu Val Pro Gln Asn Gly His Gln Ala Thr His Leu Glu
440 445 450
Val Thr Lys Gln Pro Thr Asn Arg Val Pro Trp Arg Ser Ser Asn
455 460 465
Ala Ser Pro Tyr Leu Val Pro Gly Leu Val Ala Ala Ala Thr Ile
470 ~ 475 480
Pro Thr Phe Thr Gln Trp Leu Cys
485
<210> 8
<211> 346
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2256251CD1
<400> 8
Met Leu Leu Gly Arg Va1 Trp Gln Thr Arg Glu Leu Lys Ser Lys
1 5 l0 15
Val Pro Lys Lys Ala Gly Arg Cys Gly Gln Gly Arg Leu His Gly
20 25 30
Gly Ser Ala Val Gly Phe Leu Gly Ser Pro Pro G1y Thr Pro Ser
35 40 45
Ser Phe Asp Leu Gly Cys G1y Arg Pro Gln Val Ser Asp Ala Gly
50 55 60
Gly Arg Ile Val Gly Gly His Ala Ala Pro Ala Gly Ala Trp Pro
65 70 75
Trp Gln Ala Ser Leu Arg Leu Arg Arg Val His Val Cys Gly G1y
80 85 90
Ser Leu Leu Ser Pro Gln Trp Val Leu Thr Ala A1a His Cys Phe
95 100 105
Ser Gly Ser Leu Asn Ser Ser Asp Tyr G1n Val His Leu Gly Glu
110 115 120
Leu Glu Ile Thr Leu Ser Pro His Phe Ser Thr Val Arg Gln" Ile
12 5 13 0 13 5
Ile Leu His Ser Ser Pro Ser Gly Gln Pro Gly Thr Ser Gly Asp
140 145 150
Ile Ala Leu Val Glu Leu Ser Val Pro Val Thr Leu Phe Ser Arg
155 160 165
Ile Leu Pro Val Cys Leu Pro Glu Ala Ser Asp Asp Phe Cys Pro
170 175 180
G1y Ile Arg Cys Trp Val Thr Gly Trp Gly Tyr Thr Arg G1u Gly
185 190 195
8/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Glu Pro Leu Pro Pro Pro Tyr Ser Leu Arg Glu Val Lys Val Ser
200 205 210
Val Val Asp Thr Glu Thr Cys Arg Arg Asp Tyr Pro Gly Pro Gly
215 220 225
Gly Ser Ile Leu Gln Pro Asp Met Leu Cys Ala Arg Gly Pro Gly
230 235 240
Asp Ala Cys Gln Asp Asp Ser Gly Gly Pro Leu Val Cys Gln Val
245 250 255
Asn Gly Ala Trp Val Gln Ala Gly Ile Val Ser Trp Gly Glu Gly
260 265 270
Cys Gly Arg Pro Asn Arg Pro Gly Val Tyr Thr Arg Val Pro Ala
275 280 285
Tyr Val Asn Trp Ile Arg Arg His Ile Thr Ala Ser Gly Gly Ser
290 295 300
G1u Ser G1y Tyr Pro Arg Leu Pro Leu Leu Ala Gly Leu Phe Leu
305 310 315
Pro Gly Leu Phe Leu Leu Leu Val Ser Cys Val Leu Leu Ala Lys
320 325 330
Cys Leu Leu His Pro Ser Ala Asp Gly Thr Pro Phe Pro Ala Pro
335 340 345
Asp
<210> 9
<211> 882
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7160544CD1
<400> 9
Met Ala Ala Ala Met Glu Thr Glu G1n Leu Gly Val Glu Ile Phe
1 5 10 15
Glu Thr Ala Asp Cys Glu Glu Asn Ile Glu Ser Gln Asp Arg Pro
20 25 30
Lys Leu Glu Pro Phe Tyr Val Glu Arg Tyr Ser Trp Ser Gln Leu
35 40 45
Lys Lys Leu Leu Ala Asp Thr Arg Lys Tyr His Gly Tyr Met Met
50 55 60
Ala Lys Ala Pro His Asp Phe Met Phe Val Lys Arg Asn Asp Pro
65 70 75
Asp Gly Pro His Ser Asp Arg Ile Tyr Tyr Leu Ala Met Ser Gly
80 85 . 90
Glu Asn Arg Glu Asn Thr Leu Phe Tyr Ser Glu Ile Pro Lys Thr
95 100 105
Ile Asn Arg Ala Ala Val Leu Met Leu Ser Trp Lys Pro Leu Leu
110 115 120
Asp Leu Phe Gln Ala Thr Leu Asp Tyr Gly Met Tyr Ser Arg Glu
125 130 135
Glu Glu Leu Leu Arg Glu Arg Lys Arg Ile Gly Thr Val Gly Ile
140 145 150
Ala Ser Tyr Asp Tyr His Gln Gly Ser Gly Thr Phe Leu Phe Gln
155 160 165
Ala Gly Ser Gly Ile Tyr His Val Lys Asp G1y Gly Pro Gln Gly
170 175 180
Phe Thr Gln Gln Pro Leu Arg Pro Asn Leu Val Glu Thr Ser Cys
185 190 195
Pro Asn Ile Arg Met Asp Pro Lys Leu Cys Pro Ala Asp Pro Asp
200 205 210
Trp Ile Ala Phe Ile His Ser Asn Asp I1e Trp Ile Ser Asn Ile
215 220 225
Val Thr Arg Glu Glu Arg Arg Leu Thr Tyr Val His Asn Glu Leu
230 235 240
Ala Asn Met Glu Glu Asp Ala Arg Ser A1a Gly Val Ala Thr Phe
245 250 255
9/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Val Leu Gln Glu Glu Phe Asp Arg Tyr Ser Gly Tyr Trp Trp Cys
260 265 270
Pro Lys Ala Glu Thr Thr Pro Ser Gly Gly Lys Ile Leu Arg Ile
275 280 285
Leu Tyr Glu Glu Asn Asp Glu Ser Glu Val Glu Ile Ile His Val
290 295 300
Thr Ser Pro Met Leu Glu Thr Arg Arg Ala Asp Ser Phe Arg Tyr
305 310 315
Pro Lys Thr Gly Thr Ala Asn Pro Lys Val Thr Phe Lys Met Ser
320 325 330
Glu Ile Met Ile Asp Ala Glu Gly Arg Ile Tle Asp Val Ile Asp
335 340 345
Lys Glu Leu Ile Gln Pro Phe Glu Ile Leu Phe Glu G1y Va1 Glu
350 355 360
Tyr Ile Ala Arg Ala Gly Trp Thr Pro Glu Gly Lys Tyr Ala Trp
365 370 375
Ser Ile Leu Leu Asp Arg Ser Gln Thr Arg Leu Gln Ile Val Leu
380 385 390
Ile Ser Pro Glu Leu Phe Ile Pro Val Glu Asp Asp Va1 Met Glu
395 400 405
Arg Gln Arg Leu Ile Glu Ser Val Pro Asp Ser Val Thr Pro Leu
410 415 420
Ile Ile Tyr Glu G1u Thr Thr Asp Ile Trp Ile Asn Ile His Asp
425 430 435
Tle Phe His Val Phe Pro Gln Ser His Glu Glu Glu Tle Glu Phe
440 445 450
Ile Phe Ala Ser Glu Cys Lys Thr Gly Phe Arg His Leu Tyr Lys
455 460 465
21e Thr Ser Ile Leu Lys G1u Ser Lys Tyr Lys Arg Ser Ser Gly
470 475 480
G1y Leu Pro Ala Pro Ser Asp Phe Lys Cys Pro Tle Lys Glu Glu
485 490 495
Ile Ala Ile Thr Ser Gly Glu Trp Glu Val Leu Gly Arg His Gly
500 505 510
Ser Asn Ile Gln Val Asp Glu Val Arg Arg Leu Val Tyr Phe Glu
515 520 525
Gly Thr Lys Asp Ser Pro Leu Glu His His Leu Tyr Val Val Ser
530 535 540
Tyr Val Asn Pro Gly G1u Val Thr Arg Leu Thr Asp Arg Gly Tyr
545 550 555
Ser His Ser Cys Cys Ile Ser Gln His Cys Asp Phe Phe Ile Ser
560 565 570
Lys Tyr Ser Asn Gln Lys Asn Pro His Cys Val Ser Leu Tyr Lys
575 580 585
Leu Ser Ser Pro Glu Asp Asp Pro Thr Cys Lys Thr Lys Glu Phe
590 595 600
Trp Ala Thr Ile Leu Asp Ser Ala Gly Pro Leu Pro Asp Tyr Thr
605 610 615
Pro Pro G1u Ile Phe Ser Phe Glu Ser Thr Thr Gly Phe Thr Leu
620 625 630
Tyr Gly Met Leu Tyr Lys Pro His Asp Leu Gln Pro Gly Lys Lys
635 640 645
Tyr Pro Thr Val Leu Phe I1e Tyr Gly Gly Pro Gln Val Gln Leu
650 655 660
Val Asn Asn Arg Phe Lys Gly Val Lys Tyr Phe Arg Leu Asn Thr
665 670 675
Leu Ala Ser Leu Gly Tyr Val Val Va1 Va1 Ile Asp Asn Arg Gly
680 685 690
Ser Cys His Arg Gly Leu Lys Phe Glu Gly Ala Phe Lys Tyr Lys
695 700 705
Met G1y Gln Ile Glu Ile Asp Asp Gln Val Glu Gly Leu Gln Tyr
710 715 720
Leu Ala Ser Arg Tyr Asp Phe Ile Asp Leu Asp Arg Val Gly Ile
725 730 735
His Gly Trp Ser Tyr Gly Gly Tyr Leu Ser Leu Met Ala Leu Met
740 745 750
Gln Arg Ser Asp Ile Phe Arg Val Ala Ile A1a Gly Ala Pro Val
10145
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
755 760 765
Thr Leu Trp Ile Phe Tyr Asp Thr Gly Tyr Thr Glu Arg Tyr Met
770 775 780
Gly His Pro Asp Gln Asn Glu Gln Gly Tyr Tyr Leu Gly Ser Val
785 790 795
Ala Met Gln Ala Glu Lys Phe Pro Ser Glu Pro Asn Arg Leu Leu
800 805 810
Leu Leu His Gly Phe Leu Asp Glu Asn Val His Phe Ala His Thr
815 820 825
Ser Ile Leu Leu Ser Phe Leu Val Arg Ala Gly Lys Pro Tyr Asp
830 835 840
Leu Gln Ile Tyr Pro Gln Glu Arg His Ser Ile Arg Val Pro Glu
845 850 855
Ser Gly Glu His Tyr G1u Leu His Leu Leu His Tyr Leu Gln Glu
860 865 870
Asn Leu Gly Ser Arg Ile Ala Ala Leu Lys Va1 Ile
875 880
<210> 10
<211> 1189
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 7477386CD1
<400> 10
Met Ala Pro Leu Arg Ala Leu Leu Ser Tyr Leu Leu Pro Leu His
1 5 10 15
Cys Ala Leu Cys Ala Ala A1a Gly Ser Arg Thr Pro Glu Leu His
20 25 30
Leu Ser Gly Lys Leu Ser Asp Tyr G1y Val Thr Val Pro Cys Ser
35 40 ' 45
Thr Asp Phe Arg Gly Arg Phe Leu Ser His Va1 Val Ser Gly Pro
50 55 60
Ala Ala Ala Ser Ala Gly Ser Met Val Val Asp Thr Pro Pro Thr
65 70 75
Leu Pro Arg His Ser Ser His Leu Arg Val Ala Arg Ser Pro Leu
80 85 90
His Pro G1y Gly Thr Leu Trp Pro Gly Arg Val Gly Arg His Ser
95 100 105
Leu Tyr Phe Asn Va1 Thr Val Phe Gly Lys Glu Leu His Leu Arg
110 115 120
Leu Arg Pro Asn Arg Arg Leu Val Val Pro G1y Ser Ser Val Glu
125 13 0 135
Trp Gln Glu Asp Phe Arg Glu Leu Phe Arg Gln Pro Leu Arg Gln
140 145 150
Glu Cys Val Tyr Thr Gly Gly Val Thr Gly Met Pro Gly Ala Ala
155 160 165
Val A1a Ile Ser Asn Cys Asp Gly Leu Ala Gly Leu Ile Arg Thr
170 175 180
Asp Ser Thr Asp Phe Phe Ile Glu Pro Leu Glu Arg Gly Gln G1n
185 190 195
Glu Lys G1u Ala Ser Gly Arg Thr His Val Val Tyr Arg Arg Glu
200 205 210
Ala Val Gln Gln Glu Trp Ala Glu Pro Asp Gly Asp Leu His Asn
215 220 225
Glu Ala Phe Gly Leu Gly Asp Leu Pro Asn Leu Leu Gly Leu Val
230 235 240
Gly Asp G1n Leu Gly Asp Thr G1u Arg Lys Arg Arg His Ala Lys
245 250 255
Pro Gly Ser Tyr Ser Ile Glu Val Leu Leu Val Val Asp Asp Ser
260 265 270
Val Va1 Arg Phe His Gly Lys Glu His Val Gln Asn Tyr Val Leu
275 280 285
Thr Leu Met Asn Ile Val Val Asp Glu Ile Tyr His Asp Glu Ser
11145
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
290 295 300
Leu Gly Val His Ile Asn Ile Ala Leu Val Arg Leu Ile Met Val
305 310 315
Gly Tyr Arg Gln Gln Ser Leu Ser Leu Ile Glu Arg Gly Asn Pro
320 325 330
Ser Arg Ser Leu Glu Gln Val Cys Arg Trp Ala His Ser Gln Gln
335 340 345
Arg Gln Asp Pro Ser His Ala Glu His His Asp His Val Val Phe
350 355 360
Leu Thr Arg Gln Asp Phe Gly Pro Ser Gly Gly Tyr Ala Pro Val
365 370 375
Thr Gly Met Cys His Pro Leu Arg Ser Cys Ala Leu Asn His Glu
380 385 390
Asp Gly Phe Ser Ser Ala Phe Val Ile Ala His Glu Thr Gly His
395 400 405
Val Leu Gly Met Glu His Asp Gly Gln Gly Asn Gly Cys Ala Asp
410 415 420
Glu Thr Ser Leu Gly Ser Val Met Ala Pro Leu Val Gln Ala Ala
425 430 435
Phe His Arg Phe His Trp Ser Arg Cys Ser Lys Leu Glu Leu Ser
440 445 450
Arg Tyr Leu Pro Ser Tyr Asp Cys Leu Leu Asp Asp Pro Phe Asp
455 460 465
Pro Ala Trp Pro Gln Pro Pro Glu Leu Pro Gly Ile Asn Tyr Ser
470 475 480
Met Asp Glu Gln Cys Arg Phe Asp Phe Gly Ser Gly Tyr Gln Thr
485 490 495
Cys Leu Ala Phe Arg Thr Phe Glu Pro Cys Lys Gln Leu Trp Cys
500 505 510
Ser His Pro Asp Asn Pro Tyr Phe Cys Lys Thr Lys Lys Gly Pro
515 520 525
Pro Leu Asp Gly Thr Glu Cys Ala Pro G1y Lys Trp Cys Phe Lys
530 535 540
Gly His Cys Ile Trp Lys Ser Pro Glu Gln Thr Tyr Gly Gln Asp
545 550 555
Gly Gly Trp Ser Ser Trp Thr Lys Phe Gly Ser Cys Ser Arg Ser
560 565 570
Cys Gly Gly Gly Val Arg Ser Arg Ser Arg Ser Cys Asn Asn Pro
575 580 585
Ser Pro Ala Tyr Gly Gly Arg Leu Cys Leu Gly Pro Met Phe Glu
590 595 600
Tyr Gln Val Cys Asn Ser G1u Glu Cys Pro Gly Thr Tyr Glu Asp
605 610 615
Phe Arg Ala Gln Gln Cys Ala Lys Arg Asn Ser Tyr Tyr Va1 His
620 625 630
Gln Asn Ala Lys His Ser Trp Val Pro Tyr Glu Pro Asp Asp Asp
635 640 645
Ala Gln Lys Cys G1u Leu Ile Cys Gln Ser Ala Asp Thr Gly Asp
650 655 660
Va1 Val Phe Met Asn Gln Val Val His Asp Gly Thr Arg Cys Ser
665 670 675
Tyr Arg Asp Pro Tyr Ser Val Cys Ala Arg Gly Glu Cys Val Pro
680 685 690
Val Gly Cys Asp Lys Glu Val Gly Ser Met Lys Ala Asp Asp Lys
695 700 705
Cys Gly Val Cys Gly Gly Asp Asn Ser His Cys Arg Thr Val Lys
710 715 720
Gly Thr Leu Gly Lys Ala Ser Lys Gln Ala Gly Ala Leu Lys Leu
725 730 735
Val Gln Ile Pro A1a Gly Ala Arg His Ile Gln Ile Glu Ala Leu
740 745 750
Glu Lys Ser Pro His Arg Ile Val Va1 Lys Asn Gln Val Thr Gly
755 760 765
Ser Phe Ile Leu Asn Pro Lys Gly Lys Glu Ala Thr Ser Arg Thr
770 775 780
Phe Thr Ala Met Gly Leu Glu Trp Glu Asp A1a Val G1u Asp Ala
785 790 795
12/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Lys Glu Ser Leu Lys Thr Ser Gly Pro Leu Pro Glu Ala Ile Ala
800 805 810
Ile Leu Ala Leu Pro Pro Thr Glu Gly Gly Pro Arg Ser Ser Leu
815 820 825
Ala Tyr Lys Tyr Val Ile His Glu Asp Leu Leu Pro Leu Ile Gly
830 835 840
Ser Asn Asn Val Leu Leu Glu Glu Met Asp Thr Tyr Glu Trp Ala
845 850 855
Leu Lys Ser Trp Ala Pro Cys Ser Lys Ala Cys Gly Gly Gly Ile
860 865 870
Gln Phe Thr Lys Tyr Gly Cys Arg Arg Arg Arg Asp His His Met
875 880 885
Val Gln Arg His Leu Cys Asp His Lys Lys Arg Pro Lys Pro Ile
890 ~ 895 900
Arg Arg Arg Cys Asn Gln His Pro Cys Ser Gln Pro Val Trp Va1
905 910 915
Thr Glu Glu Trp Gly Ala Cys Ser Arg Ser Cys Gly Lys Leu Gly
920 925 930
Val Gln Thr Arg Gly Ile Gln Cys Leu Leu Pro Leu Ser Asn Gly
935 940 945
Thr His Lys Val Met Pro Ala Lys Ala Cys Ala Gly Asp Arg Pro
950 955 960
Glu Ala Arg Arg Pro Cys Leu Arg Val Pro Cys Pro A1a Gln Trp
965 970 975
Arg Leu Gly Ala Trp Ser Gln Cys Ser Ala Thr Cys Gly Glu Gly
980 985 990
Ile Gln Gln Arg Gln Val Val Cys Arg Thr Asn Ala Asn Ser Leu
995 1000 1005
Gly His Cys Glu Gly Asp Arg Pro Asp Thr Val Gln Va1 Cys Ser
1010 1015 1020
Leu Pro A1a Cys Gly Ala Glu Pro Cys Thr Gly Asp Arg Ser Va1
1025 1030 1035
Phe Cys Gln Met Glu Val Leu Asp Arg Tyr Cys Ser Ile Pro Gly
1040 1045 1050
Tyr His Arg Leu Cys Cys Val Ser Cys Ile Lys Lys Ala Ser Gly
1055 1060 1065
Pro Asn Pro Gly Pro Asp Pro Gly Pro Thr Ser Leu Pro Pro Phe
1070 1075 1080
Ser Thr Pro Gly Ser Pro Leu Pro Gly Pro Gln Asp Pro Ala Asp
1085 1090 1095
Ala Ala Glu Pro Pro G1y Lys Pro Thr Gly Ser Glu Asp His Gln
1100 1105 1110
His Gly Arg Ala Thr Gln Leu Pro Gly A1a Leu Asp Thr Ser Ser
1115 1120 1125
Pro G1y Thr Gln His Pro Phe Ala Pro Glu Thr Pro Ile Pro Gly
1130 1135 1140
Ala Ser Trp Ser Ile Ser Pro Thr Thr Pro G1y Gly Leu Pro Trp
1145 1150 1155
Gly Trp Thr Gln Thr Pro Thr Pro Va1 Pro Glu Asp Lys Gly Gln
1160 1165 1170
Pro Gly Glu Asp Leu Arg His Pro Gly Thr Ser Leu Pro Ala A1a
1175 1180 1185
Ser Pro Val Thr
<210> 11
<211> 952
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473089CD1
<400> 11
Met Leu Leu Leu Gly Ile Leu Thr Leu Ala Phe Ala Gly Arg Thr
1 5 10 15
13145
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Ala Gly Gly Ser Glu Pro Glu Arg Glu Val Val Val Pro Ile Arg
20 25 30
Leu Asp Pro Asp Ile Asn Gly Arg Arg Tyr Tyr Trp Arg Gly Pro
35 40 45
Glu Asp Ser Gly Asp Gln Gly Leu Ile Phe Gln Ile Thr Ala Phe
50 55 60
Gln Glu Asp Phe Tyr Leu His Leu Thr Pro Asp Ala Gln Phe Leu
65 70 75
Ala Pro Ala Phe Ser Thr Glu His Leu Gly Val Pro Leu Gln Gly
80 85 90
Leu Thr Gly Gly Ser Ser Asp Leu Arg Arg Cys Phe Tyr Ser Gly
95 100 105
Asp Val Asn Ala Glu Pro Asp Ser Phe Ala Ala Val Ser Leu Cys
110 115 120
Gly Gly Leu Arg Gly Ala Phe Gly Tyr Arg Gly Ala Glu Tyr Val
125 130 135
Tle Ser Pro Leu Pro Asn Ala Ser Ala Pro Ala Ala Gln Arg Asn
140 145 150
Ser Gln Gly Ala His Leu Leu Gln Arg Arg Gly Val Pro Gly Gly
155 160 165
Pro Ser Gly Asp Pro Thr Ser Arg Cys Gly Val Ala Ser Gly Trp
170 175 180
Asn Pro A1a Ile Leu Arg Ala Leu Asp Pro Tyr Lys Pro Arg Arg
185 190 195
Ala Gly Phe Gly Glu Ser Arg Ser Arg Arg Arg Ser Gly Arg Ala
200 205 210
Lys Arg Phe Val Ser Ile Pro Arg Tyr Val Glu Thr Leu Val Va1
215 220 225
A1a Asp Glu Ser Met Val Lys Phe H1s Gly Ala Asp Leu Glu His
230 235 240
Tyr Leu Leu Thr Leu Leu Ala Thr Ala Ala Arg Leu Tyr Arg His
245 250 255
Pro Ser Ile Leu Asn Pro Ile Asn Ile Val Val Val Lys Val Leu
260 265 270
Leu Leu Arg Asp Arg Asp Ser Gly Pro Lys Val Thr Gly Asn Ala
275 280 285
Ala Leu Thr Leu Arg Asn Phe Cys Ala Trp Gln Lys Lys Leu Asn
290 295 300
Lys Val Ser Asp Lys His Pro Glu Tyr Trp Asp Thr Ala Ile Leu
305 310 315
Phe Thr Arg Gln Asp Leu Cys Gly Ala Thr Thr Cys Asp Thr Leu
320 325 330
Gly Met Ala Asp Val Gly Thr Met Cys Asp Pro Lys Arg Ser Cys
335 340 345
Ser Val Ile Glu Asp Asp Gly Leu Pro Ser A1a Phe Thr Thr Ala
350 355 360
His Glu Leu Gly His Val Phe Asn Met Pro His Asp Asn Va1 Lys
365 370 375
Val Cys Glu Glu Val Phe Gly Lys Leu Arg Ala Asn His Met Met
380 385 390
Ser Pro Thr Leu Ile Gln Ile Asp Arg Ala Asn Pro Trp Ser Ala
395 400 405
Cys Ser AIa Ala Ile Ile Thr Asp Phe Leu Asp Ser Gly His Gly
410 415 420
Asp Cys Leu Leu Asp Gln Pro Ser Lys Pro Ile Sex Leu Pro Glu
425 430 435
Asp Leu Pro Gly Ala Ser Tyr Thr Leu Ser G1n Gln Cys Glu Leu
440 445 450
Ala Phe Gly Val Gly Ser Lys Pro Cys Pro Tyr Met Gln Tyr Cys
455 460 465
Thr Lys Leu Trp Cys Thr Gly Lys A1a Lys G1y Gln Met Val Cys
470 475 480
Gln Thr Arg His Phe Pro Trp Ala Asp Gly Thr Ser Cys Gly Glu
485 490 495
Gly Lys Leu Cys Leu Lys Gly Ala Cys Val Glu Arg His Asn Leu
500 505 510
Asn Lys His Arg Val Asp Gly Ser Trp Ala Lys Trp Asp Pro Tyr
14/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
515 520 525
G1y Pro Cys Ser Arg Thr Cys Gly G1y Gly Val Gln Leu Ala Arg
530 535 540
Arg Gln Cys Thr Asn Pro Thr Pro Ala Asn Gly Gly Lys Tyr Cys
545 550 555
Glu Gly Val Arg Val Lys Tyr Arg Ser Cys Asn Leu Glu Pro Cys
560 565 570
Pro Ser Ser Ala Ser Gly Lys Ser Phe Arg Glu Glu Gln Cys Glu
575 580 585
Ala Phe Asn Gly Tyr Asn His Ser Thr Asn Arg Leu Thr Leu A1a
590 595 600
Val Ala Trp Val Pro Lys Tyr Ser Gly Val Ser Pro Arg Asp Lys
605 610 615
Cys Lys Leu Ile Cys Arg Ala Asn Gly Thr Gly Tyr Phe Tyr Val
62 0 625 63 0
Leu Ala Pro Lys Va1 Val Val Asp Gly Thr Leu Cys Ser Pro Asp
635 640 645
Ser Thr Ser Val Cys Val Gln Gly Lys Cys Ile Lys A1a Gly Cys
650 655 660
Asp Gly Asn Leu Gly Ser Lys Lys Arg Phe Asp Lys Cys G1y Val
665 670 675
Cys Gly Gly Asp Asn Lys Ser Cys Lys Lys Val Thr Gly Leu Phe
680 685 690
Thr Lys Pro Met His Gly Tyr Asn Phe Val Val Ala I1e Pro Ala
695 700 705
Gly Ala Ser Ser Ile Asp Ile Arg Gln Arg Gly Tyr Lys G1y Leu
710 715 720
Ile Gly Asp Asp Asn Tyr Leu A1a Leu Lys Asn Ser Gln Gly Lys
725 730 735
Tyr Leu Leu Asn Gly His Phe Val Val Ser Ala Val Glu Arg Asp
740 745 750
Leu Val Val Lys Gly Ser Leu Leu Arg Tyr Ser Gly Thr Gly Thr
755 760 765
Ala Val Glu Ser Leu Gln Ala Ser Arg Pro Ile Leu Glu Pro Leu
770 775 780
Thr Val Glu Val Leu Ser Val Gly Lys Met Thr Pro Pro Arg Va1
785 790 795
Arg Tyr Ser Phe Tyr Leu Pro Lys Glu Pro Arg Glu Asp Lys Ser
800 805 810
Ser His Pro Pro His Pro Arg Gly Gly Pro Ser Va1 Leu His Asn
815 820 825
Ser Val Leu Ser Leu Ser Asn Gln Val Glu G1n Pro Asp Asp Arg
830 835 840
Pro Pro Ala Arg Trp Val Ala Gly Ser Trp Gly Pro Cys Ser Ala
845 850 855
Ser Cys Gly Ser Gly Leu Gln Lys Arg Ala Va1 Asp Trp Arg Gly
860 865 870
Ser Ala Gly Gln Arg Thr Val Pro Ala Cys Asp Ala Ala His Arg
875 880 885
Pro Val Glu Thr Gln Ala Cys Gly G1u Pro Cys Pro Thr Trp G1u
890 895 900
Leu Ser Ala Trp Ser Pro Cys Ser Lys Ser Cys Gly Arg Gly Phe
905 910 915
Gln Arg Arg Ser Leu Lys Cys Val G1y His Gly Gly Arg Leu Leu
920 925 930
Ala Arg Asp Gln Cys Asn Leu His Arg Lys Pro G1n G1u Leu Asp
935 940 945
Phe Cys Val Leu Arg Pro Cys
950
<210> 12
<211> 898
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
15/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
<223> Incyte ID No: 7604035CD1
<400> 12
Met Glu Asn Trp Thr Gly Arg Pro Trp Leu Tyr Leu Leu Leu Leu
1 5 ' 10 15
Leu Ser Leu Pro Gln Leu Cys Leu Asp Gln Glu Val Leu Ser Gly
20 25 30
His Ser Leu Gln Thr Pro Thr Glu Glu G1y Gln Gly Pro Glu Gly
35 40 45
Val Trp Gly Pro Trp Val Gln Trp Ala Ser Cys Ser Gln Pro Cys
50 55 60
Gly Val Gly Val Gln Arg Arg Ser Arg Thr Cys Gln Leu Pro Thr
65 70 75
Val Gln Leu His Pro Ser Leu Pro Leu Pro Pro Arg Pro Pro Arg
80 85 90
His Pro Glu Ala Leu Leu Pro Arg Gly Gln Gly Pro Arg Pro Gln
95 100 105
Thr Ser Pro Glu Thr Leu Pro Leu Tyr Arg Thr Gln Ser Arg Gly
110 115 120
Arg Gly Gly Pro Leu Arg Gly Pro Ala Ser His Leu Gly Arg Glu
125 130 135
Glu Thr Gln Glu Ile Arg Ala Ala Arg Arg Ser Arg Leu Arg Asp
140 145 150
Pro Ile Lys Pro Gly Met Phe G1y Tyr Gly Arg Val Pro Phe Ala
155 160 165
Leu Pro Leu His Arg Asn Arg Arg His Pro Arg Ser Pro Pro Arg
170 175 180
Ser Glu Leu Ser Leu Ile Ser Ser Arg Gly Glu Glu Pro Ile Pro
185 190 195
Ser Pro Thr Pro Arg Ala Glu Pro Phe Ser Ala Asn Gly Ser Pro
200 205 210
Gln Thr Glu Leu Pro Pro Thr Glu Leu Ser Val His Thr Pro Ser
215 220 225
Pro Gln Ala Glu Pro Leu Ser Pro Glu Thr Ala Gln Thr Glu Val
230 235 240
A1a Pro Arg Thr Arg Pro Ala Pro Leu Arg His His Pro Arg Ala
245 250 255
Gln Ala Ser Gly Thr Glu Pro Pro Ser Pro Thr His Ser Leu G1y
260 265 270
Glu Gly Gly Phe Phe Arg Ala Ser Pro Gln Pro Arg Arg Pro Ser
275 280 285
Ser Gln Gly Trp A1a Ser Pro G1n Val Ala Gly Arg Arg Pro Asp
290 295 300
Pro Phe Pro Ser Val Pro Arg Gly Arg Gly Gln Gln Gly Gln Gly
305 310 315
Pro Trp G1y Thr Gly Gly Thr Pro His Gly Pro Arg Leu Glu Pro
320 325 330
Asp Pro Gln His Pro Gly Ala Trp Leu Pro Leu Leu Ser Asn G1y
335 340 345
Pro His Ala Ser Ser Leu Trp Ser Leu Phe Ala Pro Ser Ser Pro
350 355 360
Ile Pro Arg Cys Ser Gly Glu Ser Glu Gln Leu Arg A1a Cys Ser
365 370 375
Gln Ala Pro Cys Pro Pro Glu Gln Pro Asp Pro Arg Ala Leu Gln
380 385 390
Cys Ala Ala Phe Asn Ser Gln Glu Phe Met Gly Gln Leu Tyr G1n
395 400 405
Trp Glu Pro Phe Thr Glu Val Gln Gly Ser G1n Arg Cys Glu Leu
410 415 420
Asn Cys Arg Pro Arg Gly Phe Arg Phe Tyr Val Arg His Thr G1u
425 430 435
Lys Val Gln Asp Gly Thr Leu Cys Gln Pro Gly Ala Pro Asp Ile
440 445 450
Cys Val Ala Gly Arg Cys Leu Ser Pro Gly Cys Asp G1y Ile Leu
455 460 465
Gly Ser Gly Arg Arg Pro Asp Gly Cys Gly Val Cys G1y Gly Asp
470 475 480
16/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Asp Ser Thr Cys Arg Leu Val Ser Gly Asn Leu Thr Asp Arg Gly
485 490 495
Gly Pro Leu Gly Tyr Gln Lys Ile Leu Trp Ile Pro Ala Gly Ala
500 505 510
Leu Arg Leu Gln Ile Ala Gln Leu Arg Pro Ser Ser Asn Tyr Leu
515 520 525
Ala Leu Arg Gly Pro Gly Gly Arg Ser Ile Ile Asn Gly Asn Trp
530 535 540
Ala Val Asp Pro Pro Gly Ser Tyr Arg Ala Gly Gly Thr Val Phe
545 550 555
Arg Tyr Asn Arg Pro Pro Arg Glu Glu G1y Lys Gly Glu Ser Leu
560 565 570
Ser Ala G1u G1y Pro Thr Thr G1n Pro Val Asp Val Tyr Met Ile
575 580 585
Phe Gln Glu Glu Asn Pro Gly Val Phe Tyr Gln Tyr Val Ile Ser
590 595 600
Ser Pro Pro Pro Ile Leu Glu Asn Pro Thr Pro Glu Pro Pro Val
605 610 615
Pro Gln Leu Gln Pro Glu Ile Leu Arg Val Glu Pro Pro Leu Ala
620 625 630
Pro Ala Pro Arg Pro Ala Arg Thr Pro Gly Thr Leu Gln Arg Gln
635 640 645
Val Arg Ile Pro Gln Met Pro Ala Pro Pro His Pro Arg Thr Pro
650 655 660
Leu Gly Ser Pro Ala Ala Tyr Trp Lys Arg Val Gly His Ser Ala
665 670 675
Cys Ser Ala Ser Cys Gly Lys Gly Val Trp Arg Pro Ile Phe Leu
680 685 690
Cys Ile Ser Arg Glu Ser Gly G1u Glu Leu Asp Glu Arg Ser Cys
695 700 705
Ala Ala Gly A1a Arg Pro Pro Ala Ser Pro Glu Pro Cys His Gly
710 715 720
Thr Pro Cys Pro Pro Tyr Trp Glu Ala Gly Glu Trp Thr Ser Cys
725 730 735
Ser Arg Ser Cys Gly Pro Gly Thr Gln His Arg Gln Leu Gln Cys
740 745 750
Arg Gln Glu Phe G1y Gly Gly Gly Ser Ser Val Pro Pro Glu Arg
755 760 765
Cys Gly His Leu Pro Arg Pro Asn Ile Thr Gln Ser Cys Gln Leu
770 775 780
Arg Leu Cys Gly His Trp Glu Val Gly Ser Pro Trp Ser Gln Cys
785 790 795
Ser Val Arg Cys Gly Arg Gly Gln Arg Ser Arg Gln Val Arg Cys
800 805 810
Val Gly Asn Asn Gly Asp Glu Val Ser Glu Gln Glu Cys Ala Ser
815 820 825
Gly Pro Pro Gln Pro Pro Ser Arg G1u Ala Cys Asp Met Gly Pro
830 835 840
Cys Thr Thr A1a Trp Phe His Ser Asp Trp Ser Ser Lys Cys Ser
845 850 855
Ala Glu Cys Gly Thr G1y Ile Gln Arg Arg Ser Val Val Cys Leu
860 865 870
Gly Ser Gly Ala Ala Thr Arg Ala Arg Pro Gly Gly Ser Arg Ser
875 880 885
Arg Asn Trp Ala Glu Leu Ser Asn Arg Lys Pro Ala Pro
890 895
<210> 13
<211> 631
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3473847CD1
<400> 13
17/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Met Phe Leu Leu Ala Trp Gly Gln Asp Pro Trp Arg Leu Pro Gly
1 5 10 15
Thr Tyr Val Val Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser
20 25 30
Glu Arg Thr Ala Arg Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly
35 40 45
Tyr Leu Thr Lys Ile Leu His Val Phe His Gly Leu Leu Pro Gly
50 55 ' 60
Phe Leu Val Lys Met Ser Gly Asp Leu Leu Glu Leu Ala Leu Lys
65 70 75
Leu Pro His Val Asp Tyr Ile Glu Glu Asp Ser Ser Val Phe Ala
80 85 90
Gln Ser Ile Pro Trp Asn Leu Glu Arg Ile Thr Pro Pro Arg Tyr
95 100 105
Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly Gly Ser Leu Val Glu
110 115 120
Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp His Arg Glu Ile
125 130 135
Glu G1y Arg Val Met Val Thr Asp Phe Glu Asn Val Pro Glu Glu
140 145 150
Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp Ser His
155 160 165
Gly Thr His Leu Ala G1y Val Val Ser Gly Arg Asp Ala Gly Val
170 175 180
Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Va1 Leu Asn Cys Gln
185 190 195
Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile
200 205 210
Arg Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu
215 220 225
Leu Pro Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys
230 235 240
Gln Arg Leu Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly
245 250 255
Asn Phe Arg Asp Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro
260 265 270
Glu Val Ile Thr Val G1y Ala Thr Asn Ala Gln Asp G1n Pro Val
275 280 285
Thr Leu Gly Thr Leu Gly Thr Asn Phe Gly Arg Cys Val Asp Leu
290 295 300
Phe Ala Pro Gly Glu Asp Ile Ile Gly Ala Ser Ser Asp Cys Ser
305 310 315
Thr Cys Phe Val Ser Gln Ser Gly Thr Ser G1n Ala Ala Ala His
320 325 330
Val Ala Gly Tle Ala Ala Met Met Leu Ser Ala Glu Pro Glu Leu
335 340 345
Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile His Phe Ser A1a Lys
350 355 360
Asp Val Ile Asn G1u Ala Trp Phe Pro Glu Asp Gln Arg Val Leu
365 370 375
Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr His Gly Ala
380 385 390
Gly Trp Gln Leu Phe Cys Arg.Thr Val Trp Ser Ala His Ser Gly
395 400 405
Pro Thr Arg Met Ala Thr Ala Ile Ala Arg Cys Ala Pro Asp Glu
410 415 420
Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg
425 430 435
Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala
440 445 450
His Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys
455 460 465
Cys Leu Leu Pro G1n Ala Asn Cys Ser Val His Thr Ala Pro Pro
470 475 480
Ala Glu A1a Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly
485 490 495
His Val Leu Thr Gly Cys Ser Ser His Trp Glu Va1 Glu Asp Leu
18/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
500 505 510
G1y Thr His Lys Pro Pro Val Leu Arg Pro Arg Gly Gln Pro Asn
515 520 525
Gln Cys Val Gly His Arg G1u Ala Ser Ile His Ala Ser Cys Cys
530 535 540
His Ala Pro Gly Leu Glu Cys Lys Val Lys Glu His Gly Ile Pro
545 550 555
Ala Pro Gln Glu Gln Val Thr Val Ala Cys Glu Glu Gly Trp Thr
560 565 570
Leu Thr Gly Cys Ser Ala Leu Pro Gly Thr Ser His Val Leu Gly
575 580 585
Ala Tyr A1a Val Asp Asn Thr Cys Val Val Arg Ser Arg Asp Val
590 595 600
Ser Thr Thr Gly Ser Thr Ser Glu Glu Ala Val Thr Ala Val Ala
605 610 615
Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser Gln Glu Leu
620 625 630
Gln
<210> 14
<211> 470
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3750004CD1
<400> 14
Met Arg His Arg Thr Asp Leu Gly Gln Asn Leu Leu Leu Phe Leu
1 5 10 15
Trp Ala Leu Leu Asn Cys Gly Leu Gly Val Ser Ala Gln Gly Pro
20 25 30
G1y Glu Trp Thr Pro Trp Val Ser Trp Thr Arg Cys Ser Ser Ser
35 40 45
Cys Gly Arg Gly Val Ser Val Arg Ser Arg Arg Cys Leu Arg Leu
50 55 60
Pro Gly Glu Glu Pro Cys Trp Gly Asp Ser His Glu Tyr Arg Leu
65 70 75
Cys Gln Leu Pro Asp Cys Pro Pro Gly Ala Val Pro Phe Arg Asp
80 85 90
Leu Gln Cys Ala Leu Tyr Asn Gly Arg Pro Val Leu Gly Thr Gln
95 100 105
Lys Thr Tyr Gln Trp Val Pro Phe His Gly Ala Pro Asn Gln Cys
110 115 120
Asp Leu Asn Cys Leu Ala Glu Gly His Ala Phe Tyr His Ser Phe
125 130 135
Gly Arg Val Leu Asp Gly Thr Ala Cys Ser Pro Gly Ala G1n Gly
140 145 150
Val Cys Val Ala Gly Arg Cys Leu Ser Ala Gly Cys Asp G1y Leu
155 160 165
Leu G1y Ser Gly Ala Leu Glu Asp Arg Cys Gly Arg Cys G1y Gly
170 175 180
Ala Asn Asp Ser Cys Leu Phe Val Gln Arg Va1 Phe Arg Asp Ala
185 190 195
Gly Ala Phe Ala Gly Tyr Trp Asn Val Thr Leu Ile Pro Glu Gly
200 205 210
Ala Arg His Ile Arg Val Glu His Arg Ser Arg Asn His Leu Gly
215 220 225
Ile Leu Gly Ser Leu Met Gly Gly Asp Gly Arg Tyr Val Leu Asn
230 235 240
G1y His Trp Va1 Val Ser Pro Pro Gly Thr Tyr Glu Ala Ala Gly
245 250 255
Thr His Val Val Tyr Thr Arg Asp Thr Gly Pro Gln Glu Thr Leu
260 265 270
Gln Ala Ala Gly Pro Thr Ser His Asp Leu Leu Leu Gln Va1 Leu
19/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
275 280 285
Leu Gln Glu Pro Asn Pro Gly Ile Glu Phe Glu Phe Trp Leu Pro
290 295 300
Arg Glu Arg Tyr Ser Pro Phe Gln Ala Arg Val Gln Ala Leu Gly
305 310 315
Trp Pro Leu Arg Gln Pro G1n Pro Arg Gly Va1 Glu Pro Gln Pro
320 325 330
Pro Ala Ala Pro Ala Val Thr Pro Ala Gln Thr Pro Thr Leu Ala
335 340 345
Pro Asp Pro Cys Pro Pro Cys Pro Asp Thr Arg Gly Arg A1a His
350 355 360
Arg Leu Leu His Tyr Cys Gly Ser Asp Phe Val Phe Gln Ala Arg
365 370 375
Val Leu Gly His His His Gln Ala Gln Glu Thr Arg Tyr Glu Val
380 385 390
Arg Ile Gln Leu Val Tyr Lys Asn Arg Ser Pro Leu Arg Ala Arg
395 400 405
Glu Tyr Val Trp Ala Pro Gly His Cys Pro Cys Pro Met Leu Ala
410 415 420
Pro His Arg Asp Tyr Leu Met Ala Val Gln Arg Leu Val Ser Pro
425 430 435
Asp Gly Thr Gln Asp Gln Leu Leu Leu Pro His A1a Gly Tyr Ala
440 445 450
Arg Pro Trp Ser Pro A1a Glu Asp Ser Arg Ile Arg Leu Thr Ala
455 460 465
Arg Arg Cys Pro Gly
470
<210> l5
<211> 110
<212> PRT
<213> Homo Sapiens
<220>
<22l> misc_feature
<223> Incyte ID No: 4904126CD1
<400> 15
Met Ala Asp Lys Val Leu Lys Glu Lys Arg Lys Gln Phe Ile Arg
1 5 10 15
Ser Val Gly Glu Gly Thr Ile Asn Gly Leu Leu Gly Glu Leu Leu
20 25 30
Glu Thr Arg Val Leu Ser Gln Glu Glu Ile Glu Ile Val Lys Cys
35 40 45
Glu Asn Ala Thr Val Met Asp Lys Ala Arg A1a Leu Leu Asp Ser
50 55 60
Val Ile Arg Lys Gly Ala Pro Ala Cys Gln Ile Cys Ile Thr Tyr
65 70 75
Ile Cys Glu Glu Asp Ser His Leu Ala Gly Thr Leu Gly Leu Ser
80 85 90
Ala Gly Pro Thr Ser G1y Asn His Leu Thr Thr Gln Asp Ser Gln
95 100 105
Ile Val Leu Pro Ser
110
<210> 16
<211> 879
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 71268415CD1
<400> 16
Met Ser Leu Phe Ile Phe Cys Arg Gln Leu Phe Ala Pro Ser Tyr
1 5 10 15
20/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Thr Glu Thr His Tyr Thr Ser Ser Gly Asn Pro Gln Thr Thr Thr
20 25 30
Arg Lys Leu Glu Asp His Cys Phe Tyr His Gly Thr Val Arg G1u
35 40 45
Thr Glu Leu Ser Ser Val Thr Leu Ser Thr Cys Arg Gly Ile Arg
50 55 60
Gly Leu Ile Thr Val Ser Ser Asn Leu Ser Tyr Val Ile Glu Pro
65 70 75
Leu Pro Asp Ser Lys G1y Gln His Leu Ile Tyr Arg Ser Glu His
80 85 90
Leu Lys Pro Pro Pro Gly Asn Cys Gly Phe Glu His Ser Lys Pro
95 100 105
Thr Thr Arg Asp Trp Ala Leu Gln Phe Thr Gln Gln Thr Lys Lys
110 115 120
Arg Pro Arg Arg Met Lys Arg Glu Asp Leu Asn Ser Met Lys Tyr
125 130 135
Val Glu Leu Tyr Leu Val Ala Asp Tyr Leu Glu Phe Gln Lys Asn
140 145 150
Arg Arg Asp Gln Asp Ala Thr Lys His Lys Leu Ile G1u Ile A1a
155 160 165
Asn Tyr Val Asp Lys Phe Tyr Arg Ser Leu Asn Ile Arg Ile Ala
170 175 180
Leu Val Gly Leu Glu Val Trp Thr His Gly Asn Met Cys Glu Val
185 190 195
Ser Glu Asn Pro Tyr Ser Thr Leu Trp Ser Phe Leu Ser Trp Arg
200 205 210
Arg Lys Leu Leu Ala Gln Lys Tyr His Asp Asn Ala Gln Leu Ile
215 220 225
Thr Gly Met Ser Phe His Gly Thr Thr Ile Gly Leu Ala Pro Leu
230 235 240
Met Ala Met Cys Ser Val Tyr Gln Ser Gly Gly Val Asn Met Asp
245 250 255
His Ser Glu Asn Ala Ile Gly Va1 A1a Ala Thr Met Ala His G1u
260 265 270
Met Gly His Asn Phe Gly Met Thr His Asp Ser Ala Asp Cys Cys
275 280 285
Ser Ala Ser Ala A1a Asp Gly Gly Cys I1e Met Ala A1a Ala Thr
290 295 300
Gly His Pro Phe Pro Lys Val Phe Asn Gly Cys Asn Arg Arg G1u
305 310 315
Leu Asp Arg Tyr Leu Gln Ser Gly Gly Gly Met Cys Leu Ser Asn
320 325 330
Met Pro Asp Thr Arg Met Leu Tyr Gly Gly Arg Arg Cys Gly Asn
335 340 . 345
Gly Tyr Leu Glu Asp Gly Glu Glu Cys Asp Cys Gly Glu Glu Glu
350 355 360
Glu Cys Asn Asn Pro Cys Cys Asn Ala Ser Asn Cys Thr Leu Arg
365 370 375
Pro Gly Ala Glu Cys Ala His Gly Ser Cys Cys His Gln Cys Lys
380 385 390
Leu Leu Ala Pro Gly Thr Leu Cys Arg Glu Gln Ala Arg Gln Cys
395 400 405
Asp Leu Pro Glu Phe Cys Thr Gly Lys Ser Pro His Cys Pro Thr
410 415 420
Asn Phe Tyr Gln Met Asp Gly Thr Pro Cys Glu Gly Gly Gln Ala
425 430 435
Tyr Cys Tyr Asn Gly Met Cys Leu Thr Tyr Gln Glu Gln Cys Gln
440 445 450
Gln Leu Trp Gly Pro Gly Ala Arg Pro Ala Pro Asp Leu Cys Phe
455 460 465
Glu Lys Val Asn Val Ala G1y Asp Thr Phe Gly Asn Cys Gly Lys
470 475 480
Asp Met Asn Gly Glu His Arg Lys Cys Asn Met Arg Asp Ala Lys
485 490 495
Cys Gly Lys Ile Gln Cys Gln Ser Ser Glu Ala Arg Pro Leu Glu
500 505 510
Ser Asn Ala Val Pro Ile Asp Thr Thr Ile 21e Met Asn Gly Arg
21/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
515 520 525
Gln Ile Gln Cys Arg Gly Thr His Val Tyr Arg Gly Pro Glu Glu
530 535 540
Glu Gly Asp Met Leu Asp Pro Gly Leu Val Met Thr Gly Thr Lys
545 550 555
Cys Gly Tyr Asn His Ile Cys Phe G1u Gly Gln Cys Arg Asn Thr
560 565 570
Ser Phe Phe Glu Thr Glu Gly Cys Gly Lys Lys Cys Asn Gly His
575 580 585
Gly Val Cys Asn Asn Asn Gln Asn Cys His Cys Leu Pro Gly Trp
590 595 600
Ala Pro Pro Phe Cys Asn Thr Pro Gly His Gly Gly Ser Ile Asp
605 610 615
Ser Gly Pro Met Pro Pro Glu Ser Val Gly Pro Val Val Ala Gly
620 625 630
Val Leu Val Ala Ile Leu Val Leu Ala Val Leu Met Leu Met Tyr
635 640 645
Tyr Cys Cys Arg Gln Asn Asn Lys Leu Gly Gln Leu Lys Pro Ser
650 655 660
Ala Leu Pro Ser Lys Leu Arg Gln Gln Phe Ser Cys Pro Phe Arg
665 670 675
Val Ser Gln Asn Ser Gly Thr Gly His Ala Asn Pro Thr Phe Lys
680 685 690
Leu Gln Thr Pro Gln Gly Lys Arg Lys Val Ile Asn Thr Pro Glu
' 695 700 705
I1e Leu Arg Lys Pro Ser Gln Pro Pro Pro Arg Pro Pro Pro Asp
710 715 720
Tyr Leu Arg Gly Gly Ser Pro Pro Ala Pro Leu Pro Ala His Leu
725 730 735
Ser Arg Ala Ala Arg Asn Ser Pro Gly Pro Gly Ser Gln Ile Glu
740 745 750
Arg Thr Glu Ser Ser Arg Arg Pro Pro Pro Ser Arg Pro Ile Pro
755 760 765
Pro Ala Pro Asn Cys I1e Val Ser Gln Asp Phe Ser Arg Pro Arg
770 775 780
Pro Pro Gln Lys Ala Leu Pro Ala Asn Pro Val Pro Gly Arg Arg
785 790 795
Ser Leu Pro Arg Pro Gly Gly Ala Ser Pro Leu Arg Pro Pro Gly
800 805 810
Ala Gly Pro Gln Gln Ser Arg Pro Leu Ala Ala Leu A1a Pro Lys
815 820 825
Val Ser Pro Arg Glu Ala Leu Lys Val Lys Ala Gly Thr Arg Gly
830 835 840
Leu Gln Gly Gly Arg Cys Arg Va1 Glu Lys Thr Lys Gln Phe Met
845 850 . 855
Leu Leu Val Val Trp Thr G1u Leu Pro Glu Gln Lys Pro Arg Ala
860 865 870
Lys His Ser Cys Phe Leu Val Pro A1a
875
<210> 17
<211> 850
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473301CD1
<400> 17
Met Asp Lys Glu Asn Ser Asp Val Ser Ala Ala Pro Ala Asp Leu
1 5 10 15
Lys Ile Ser Asn Ile Ser Val Gln Val Va1 Ser Ala Gln Lys Lys
20 25 30
Leu Pro Val Arg Arg Pro Pro Leu Pro Gly Arg Arg Leu Pro Leu
35 40 45
Pro Gly Arg Arg Pro Pro Gln Arg Pro Ile Gly Lys Ala Lys Pro
22/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
50 55 60
Lys Lys Gln Ser Lys Lys Lys Val Pro Phe Trp Asn Val Gln Asn
65 70 75
Lys Ile Ile Leu Phe Thr Val Phe Leu Phe Ile Leu Ala Val Ile
80 85 90
Ala Trp Thr Leu Leu Trp Leu Tyr Ile Ser Lys Thr Glu Ser Lys
95 100 105
Asp A1a Phe Tyr Phe Ala Gly Met Phe Arg Ile Thr Asn Ile Glu
110 115 120
Phe Leu Pro Glu Tyr Arg Gln Lys Glu Ser Arg Glu Phe Leu Ser
125 130 135
Val Ser Arg Thr Val Gln Gln Val Ile Asn Leu Val Tyr Thr Thr
140 145 150
Ser A1a Phe Ser Lys Phe Tyr Glu Gln Ser Val Val Ala Asp Val
155 160 165
Ser Ser Asn Asn Lys Gly Gly Leu Leu Val His Phe Trp Ile Val
170 175 180
Phe Val Met Pro Arg Ala Lys Gly His Ile Phe Cys Glu Asp Cys
185 190 195
Val Ala Ala Tle Leu Lys Asp Ser Ile Gln Thr Ser Ile Ile Asn
200 205 210
Arg Thr Ser Val Gly Ser Leu Gln Gly Leu Ala Val Asp Met Asp
215 220 225
Ser Val Val Leu Asn Gly Asp Cys Trp Ser Phe Leu Lys Lys Lys
230 235 240
Lys Arg Lys Glu Asn Gly Ala Val Ser Thr Asp Lys Gly Cys Ser
245 250 255
Gln Tyr Phe Tyr Ala Glu His Leu Ser Leu His Tyr Pro Leu Glu
260 265 270
Ile Ser Ala Ala Ser Gly Arg Leu Met Cys His Phe Lys Leu Val
275 280 285
Ala Ile Val Gly Tyr Lew Ile Arg Leu Ser Ile Lys Ser Ile G1n
290 295 300
Ile Glu Ala Asp Asn Cys Val Thr Asp Ser Leu Thr Ile Tyr Asp
305 310 315
Ser Leu Leu Pro Ile Arg Ser Ser Tle Leu Tyr Arg Ile Cys Glu
320 325 330
Pro Thr Arg Thr Leu Met Ser Phe Val Ser Thr Asn Asn Leu Met
335 340 345
Leu Val Thr Phe Lys Ser Pro His Ile Arg Arg Leu Ser Gly Ile
350 355 360
Arg Ala Tyr Phe Glu Val Ile Pro Glu Gln Lys Cys Glu Asn Thr
365 370 375
Val Leu Val Lys Asp Ile Thr Gly Phe Glu Gly Lys Ile Ser Ser
380 385 390
Pro Tyr Tyr Pro Ser Tyr Tyr Pro Pro Lys Cys Lys Cys Thr Trp
395 400 405
Lys Phe Gln Thr Ser Leu Ser Thr Leu Gly Ile Ala Leu Lys Phe
410 41S 420
Tyr Asn Tyr Ser Ile Thr Lys Lys Ser Met Lys Gly Cys G1u His
425 430 . 435
Gly Trp Trp Glu I1e Tyr G1u His Met Tyr Cys Gly Ser Tyr Met
440 445 450
Asp His Gln Thr Ile Phe Arg Va1 Pro Ser Pro Leu Val His Ile
455 460 465
Gln Leu Gln Cys Ser Ser Arg Leu Ser Gly Lys Pro Leu Leu Ala
470 475 480
Glu Tyr Gly Ser Tyr Asn Ile Ser Gln Pro Cys Pro Val Gly Ser
485 490 495
Phe Arg Cys Ser Ser G1y Leu Cys Val Pro Gln A1a Gln Arg Gly
500 505 510
Asp Gly Val Asn Asp Cys Phe Asp Glu Ser Asp Glu Leu Phe Cys
515 520 525
Val Ser Pro Gln Pro Ala Cys Asn Thr Ser Ser Phe Arg Gln His
530 535 540
Gly Pro Leu Ile Cys Asp Gly Phe Arg Asp Cys Glu Asn Gly Arg
545 550 555
23/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Asp Glu Gln Asn Cys Thr Gln Ser Ile Pro Cys Asn Asn Arg Thr
560 565 570
Phe Lys Cys Gly Asn Asp Ile Cys Phe Arg Lys Gln Asn Ala Lys
575 580 585
Cys Asp Gly Thr Val Asp Cys Pro Asp Gly Ser Asp Glu Glu GIy
590 595 600
Cys Thr Cys Ser Arg Ser Ser Ser Ala Leu His Arg Ile Ile Gly
605 610 615
Gly Thr Asp Thr Leu G1u Gly Gly Trp Pro Trp Gln Va1 Ser Leu
620 625 630
His Phe Val Gly Ser Ala Tyr Cys Gly Ala Ser Val Ile Ser Arg
635 640 645
Glu Trp Leu Leu Ser A1a Ala His Cys Phe His Gly Asn Arg Leu
650 655 660
Ser Asp Pro Thr Pro Trp Thr Ala His Leu Gly Met Tyr Val Gln
665 670 675
Gly Asn Ala Lys Phe Val Ser Pro Val Arg Arg Tle Val Val His
680 685 690
Glu Tyr Tyr Asn Ser Gln Thr Phe Asp Tyr Asp Ile Ala Leu Leu
695 700 705
Gln Leu Ser Ile Ala Trp Pro Glu Thr Leu Lys Gln Leu Ile Gln
710 715 720
Pro Tle Cys Ile Pro Pro Thr Gly Gln Arg Val Arg Ser Gly G1u
725 730 735
Lys Cys Trp Val Thr Gly Trp Gly Arg Arg His Glu Ala Asp Asn
740 745 750
Lys Gly Ser Leu Val Leu Gln Gln Ala Glu Val Glu Leu Ile Asp
755 760 765
Gln Thr Leu Cys Val Ser Thr Tyr Gly Ile Ile Thr Ser Arg Met
770 775 780
Leu Cys Ala Gly Ile Met Ser Gly Lys Arg Asp Ala Cys Lys G1y
785 790 795
Asp Ser Gly Gly Pro Leu Ser Cys Arg Arg Lys Ser Asp Gly Lys
800 805 810
Trp I1e Leu Thr Gly I1e Val Ser Trp Gly His Gly Cys Gly Arg
815 820 825
Pro Asn Phe Pro Gly Val Tyr Thr Arg Val Ser Asn Phe Val Pro
830 835 840
Trp Ile His Lys Tyr Val Pro Ser Leu Leu
845 850
<210> 18
<211> 254
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473308CD1
<400> 18
Met Gln Asp His Arg Lys Gly Lys Ala A1a Val Gly Val Ser Phe
1 5 10 15
Asp Asp Asp Asp Lys Ile Val Gly Gly Tyr Asn Cys Glu Glu Asn
20 25 30
Ser Val Pro Tyr Gln Val Ser Leu Asn Ser Gly Tyr His Phe Cys
35 40 45
Val Gly Ser Leu Asn Arg G1u Tyr Cys Ile Gln Val Arg Leu Gly
50 55 60
G1u His Asn Ile G1u Val Leu Glu Gly Asn Glu Gln Phe Ile Tyr
65 70 75
Ala Val Lys I1e Ile Arg His Pro Lys Tyr Asn Ser Trp Thr Leu
80 85 90
Asp Asn Asp Ile Leu Leu Ile Lys Leu Ser Thr Pro Ala I1e Ile
95 100 105
Asn A1a His Val Ser Thr Ile Ser Leu Pro Thr Thr Pro Pro Ala
110 115 120
24/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Ala Gly Thr Glu Cys Leu Ile Ser Gly Trp Gly Asn Thr Leu Ser
125 13 0 135
Ser Gly Ala Asp Tyr Pro Asp Glu Leu Gln Cys Leu Asp Ala Pro
140 145 150
Val Leu Ser Gln Ala Glu Tyr Glu Ala Ser Tyr Pro Gly Lys Ile
155 160 165
Thr Asn Asn Val Phe Cys Va1 Gly Phe Leu Glu Gly Gly Lys Asp
170 175 180
Ser Cys Gln Ile Ile Pro I1e Lys Val Gln Gln Leu Val Thr Ser
185 190 195
Ser Gln Glu Thr Asp Ile Arg Ile Pro Met Ala Leu Gln Thr Ala
200 205 210
Ala Ser Thr Ser Tyr Leu Gly Pro Leu Asp Ser Leu His Arg Lys
215 220 225
Val Ser His Pro Thr Glu Lys Arg Cys Gln Gln Lys Gln G1y Met
230 235 240
Lys Ile Thr Asp Asn His Gly Ile Thr Ser Lys Trp Ser Val
245 250
<210> 19
<211> 568
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7478021CD1
<400> 19
Met Leu Ala Ala Ser Ile Phe Arg Pro Thr Leu Leu Leu Cys Trp
1 5 10 15
Leu Ala Ala Pro Trp Pro Thr Gln Pro Glu Ser Leu Phe His Ser
20 25 30
Arg Asp Arg Ser Asp Leu Glu Pro Ser Pro Leu Arg Gln Ala Lys
35 40 45
Pro Ile Ala Asp Leu His Ala Ala Gln Arg Phe Leu Ser Arg Tyr
50 55 60
Gly Trp Ser Gly Val Trp Ala Ala Trp Gly Pro Ser Pro Glu Gly
65 70 75
Pro Pro Glu Thr Pro Lys Gly Ala A1a Leu Ala G1u Ala Val Arg
80 85 90
Arg Phe Gln Arg Ala Asn Ala Leu Pro Ala Ser Gly Glu Leu Asp
95 100 105
Ala Ala Thr Leu Ala Ala Met Asn Arg Pro Arg Cys Gly Val Pro
110 115 120
Asp Met Arg Pro Pro Pro Pro Ser Ala Pro Pro Ser Pro Pro G1y
12 5 13 0 13 5
Pro Pro~Pro Arg A1a Arg Ser Arg Arg Ser Pro Arg Ala Pro Leu
140 145 150
Ser Leu Ser Arg Arg Gly Trp Gln Pro Arg Gly Tyr Pro Asp Gly
155 160 165
Gly Ala Ala Gln Ala Phe Ser Lys Arg Thr Leu Ser Trp Arg Leu
170 175 180
Leu Gly G1u Ala Leu Ser Ser Gln Leu Ser Val Ala Asp Gln Arg
185 190 195
Arg Ile Glu Ala Leu Ala Phe Arg Met Trp Ser Glu Val Thr Pro
200 205 210
Leu Asp Phe Arg Glu Asp Leu Ala A1a Pro Gly Ala Ala Val Asp
215 220 225
Ile Lys Leu Gly Phe Gly Arg Arg His Leu Gly Cys Pro Arg Ala
230 235 240
Phe Asp Gly Ser Gly Gln Glu Phe Ala His Ala Trp Arg Leu G1y
245 250 255
Asp Ile His Phe Asp Asp Asp Glu His Phe Thr Pro Pro Thr Ser
260 265 270
Asp Thr Gly Ile Ser Leu Leu Lys Val Ala Val His Glu Ile Gly
275 280 285
5/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
His Val Leu Gly Leu Pro His Thr Tyr Arg Thr Gly Ser Ile Met
290 295 300
Gln Pro Asn Tyr Ile Pro Gln Glu Pro Ala Phe Glu Leu Asp Trp
305 3l0 315
Ser Asp Arg Lys Ala Ile Gln Lys Leu Tyr Gly Ser Cys Glu Gly
320 325 330
Ser Phe Asp Thr A1a Phe Asp Trp Ile Arg Lys Glu Arg Asn Gln
335 340 345
Tyr Gly Glu Val Met Val Arg Phe Ser Thr Tyr Phe Phe Arg Asn
350 355 360
Ser Trp Tyr Trp Leu Tyr Glu Asn Arg Asn Asn Arg Thr Arg Tyr
365 370 375
Gly Asp Pro Ile Gln Ile Leu Thr Gly Trp Pro Gly Ile Pro Thr
380 385 390
His Asn Ile Asp Ala Phe Val His Ile Trp Thr Trp Lys Arg Asp
395 400 405
Glu Arg Tyr Phe Phe Gln Gly Asn Gln Tyr Trp Arg Tyr Asp Ser
410 415 420
Asp Lys Asp Gln Ala Leu Thr Glu Asp G1u Gln Gly Lys Ser Tyr
425 430 435
Pro Lys Leu Ile Ser Glu Gly Phe Pro Gly Ile Pro Ser Pro Leu
440 445 450
Asp Thr Ala Phe Tyr Asp Arg Arg Gln Lys Leu Ile Tyr Phe Phe
455 460 465
Lys Glu Ser Leu Val Phe Ala Phe Asp Val Asn Arg Asn Arg Val
470 475 480
Leu Asn Ser Tyr Pro Lys Arg Ile Thr Glu Val Phe Pro Ala Val
485 490 495
Ile Pro Gln Asn His Pro Phe Arg Asn Ile Asp Ser Ala Tyr Tyr
500 505 5l0
Ser Tyr Ala Tyr Asn Ser Ile Phe Phe Phe Lys Gly Asn Ala Tyr
515 520 525
Trp Lys Val Val Asn Asp Lys Asp Lys Gln Gln Asn Ser Trp Leu
530 535 540
Pro Ala Asn Gly Leu Phe Pro Lys Lys Phe Tle Ser Glu Lys Trp
545 550 555
Phe Asp Val Cys Asp Val His Ile Ser Thr Leu Asn Met
560 565
<210> 20
<221> 306
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4333459CD1
<400> 20
Met Ser Leu Lys Met Leu Ile Ser Arg Asn Lys Leu I1e Leu Leu
1 5 l0 15
Leu Gly Ile Va1 Phe Phe Glu Arg Gly Lys Ser Ala Thr Leu Ser
20 25 30
Leu Pro Lys Ala Pro Ser Cys Gly Gln Ser Leu Val Lys Val Gln
35 40 45
Pro Trp Asn Tyr Phe Asn Ile Phe Ser Arg Ile Leu Gly G1y Ser
50 55 60
Gln Val Glu Lys Gly Ser Tyr Pro Trp G1n Val Ser Leu Lys G1n
65 70 75
Arg G1n Lys His I1e cys Gly Gly Ser Ile Val Ser Pro Gln Trp
80 85 90
Val Ile Thr Ala Ala His Cys Ile Ala Asn Arg Asn Ile Val Ser
95 100 105
Thr Leu Asn Val Thr Ala Gly Glu Tyr Asp Leu Ser Gln Thr Asp
110 115 120
Pro Gly Glu Gln Thr Leu Thr Ile Glu Thr Val Ile Ile His Pro
125 130 135
26/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
His Phe Ser Thr Lys Lys Pro Met Asp Tyr Asp Ile Ala Leu Leu
140 145 150
Lys Met Ala Gly Ala Phe Gln Phe Gly His Phe Val Gly Pro Ile
155 160 165
Cys Leu Pro Glu Leu Arg G1u Gln Phe Glu Ala Gly Phe Ile Cys
170 175 180
Thr Thr Ala Gly Trp Gly Arg Leu Thr Glu Gly Gly Val Leu Ser
185 190 195
Gln Val Leu Gln Glu Val Asn Leu Pro Ile Leu Thr Trp Glu Glu
200 205 210
Cys Val Ala Ala Leu Leu Thr Leu Lys Arg Pro Ile Ser Gly Lys
215 220 '225
Thr Phe Leu Cys Thr Gly Phe Pro Asp Gly Gly Arg Asp Ala Cys
230 235 240
Gln Gly Asp Ser Gly Gly Ser Leu Met Cys Arg Asn Lys Lys Gly
245 250 255
Ala Trp Thr Leu Ala Gly Val Thr Ser Trp Gly Leu Gly Cys Gly
260 265 270
Arg Gly Trp Arg Asn Asn Val Arg Lys Ser Asp Gln Gly Ser Pro
275 280 285
Gly Ile Phe Thr Asp Ile Ser Lys Val Leu Ser Trp Ile His Glu
290 295 300
His I1e Gln Thr Gly Asn
305
<210> 21
<211> 953
<212> PI2T
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6817347CD1
<400> 21
Met Thr Leu Leu Ala Pro Trp Tyr Thr Gly Pro Met Ile Pro Met
1 5 10 15
Asp Val Asn Glu Pro Ser Ser Val Thr Thr Ala Pro Thr Leu Ser
20 25 30
Ser Ser Leu Gln His Ile Ser Ser Phe Leu Ala Thr Gly Lys Lys
35 40 45
Leu Ser Leu His Phe Gly His Pro Arg G1u Cys Glu Val Thr Arg
50 55 60
Ile Asp Asp Lys Asn Arg Arg Gly Leu Glu Asp Ser Glu Pro Gly
65 70 75
Ala Lys Leu Phe Asn Asn Asp Gly Va1 Cys Cys Cys Leu Gln Lys
80 85 90
Arg Gly Pro Val Asn Ile Thr Ser Val Cys Val Ser Pro Arg Thr
95 100 105
Leu Gln Tle Ser Va1 Phe Val Leu Ser Glu Lys Tyr Glu Gly Ile
110 115 120
Val Lys Phe Glu Ser Asp Glu Leu Pro Phe Gly Val Ile Gly Ser
125 13 0 13 5
Asn Ile Gly Asp Ala His Phe Gln Glu Phe Arg Ala Gly Ile Ser
140 145 150
Trp Lys Pro Val Val Asp Pro Asp Asp Pro Ile Pro Gln Phe Pro
155 160 165
Asp Cys Cys Ser Ser Ser Ser Ser Arg I1e Pro Ser Val Ser Val
170 175 180
Leu Val Ala Val Pro Leu Val Ala Gly His Lys Gly Gln Ala Phe
185 190 195
Tle Glu Arg Met Leu Gly Cys Phe Lys Glu Leu Lys Gln G1u Leu
200 205 210
Thr Gln Glu Gly Pro G1y Gly Gly His Pro Arg Ser Ala Trp Pro
225 220 225
Pro Arg Arg His Ala Gln Trp Pro Pro Glu Pro Cys Glu G1n Gly
230 235 240
27/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
Glu Glu Pro Pro Pro Val Glu Ala Glu Glu Val Glu Glu Ala Glu
245 250 255
Thr Ala Glu Lys Ala Glu Arg Lys Val Glu Ala Glu Ala Lys Val
260 265 270
Glu Gly Lys Ala Glu Ala Ala Gly Lys A1a Glu Ala Ala Gly Lys
275 280 285
Val Asp Ala Thr Glu Lys Val Glu Thr Ala Gly Lys Val Asp Ala
290 295 300
Ala Gly Lys Val Glu Thr Ala Glu Gly Pro Gly Arg Arg Ala Glu
305 310 315
Leu Lys Leu Glu Pro Glu Pro Glu Pro Val Arg Glu Ala Glu Gln
320 325 330
G1u Pro Lys Gln Glu Leu Glu Asp Glu Asn Pro Ala Arg Ser Gly
335 340 345
Gly Gly Gly Asn Sex Asp Glu Val Pro Pro Pro Thr Leu Pro Ser
350 355 360
Asp Pro Pro Arg Pro Pro Asp Pro Ser Pro Arg Arg Ser Arg Ala
365 370 375
Pro Arg Arg Arg Pro Arg Pro Arg Pro Gln Thr Arg Leu Arg Thr
380 385 390
Pro Pro Gln Pro Arg Pro Arg Pro Pro Pro Arg Pro Arg Pro Arg
395 400 405
Arg Gly Pro Gly Gly Gly Cys Leu Asp Val Asp Phe Ala Va1 Gly
410 415 420
Pro Pro Gly Cys Ser His Val Asn Ser Phe Lys Va1 Gly Glu Asn
425 430 435
Trp Arg Gln Glu Leu Arg Val Ile Tyr Gln Cys Phe Val Trp Cys
440 445 450
Gly Thr Pro Glu Thr Arg Lys Ser Lys Ala Lys Ser Cys Ile Cys
455 460 465
His Val Cys Gly Thr His Leu Asn Arg Leu His Ser Cys Leu Ser
470 475 480
Cys Val Phe Phe Gly Cys Phe Thr Glu Lys His Ile His Glu His
485 490 495
Ala Glu Thr Lys Gln His Asn Leu Ala Val Asp Leu Tyr Tyr Gly
500 505 510
Gly Ile Tyr Cys Phe Met Cys Lys Asp Tyr Val Tyr Asp Lys Asp
515 520 525
Ile Glu Gln Ile A1a Lys Glu Glu Gln Gly Glu Ala Leu Lys Leu
530 535 540
Gln Ala Ser Thr Ser Thr Glu Val Ser His Gln Gln Cys Ser Val
545 550 555
Pro Gly Leu Gly G1u Lys Phe Pro Thr Trp Glu Thr Thr Lys Pro
560 565 570
G1u Leu Glu Leu Leu Gly His Asn Pro Arg Arg Arg Arg Ile Thr
575 580 585
Ser Ser Phe Thr Ile G1y Leu Arg Gly Leu Ile Asn Leu Gly Asn
590 595 600
Thr Cys Phe Met Asn Cys Ile Val Gln Ala Leu Thr His Thr Pro
605 610 615
Ile Leu Arg Asp Phe Phe Leu Ser Asp Arg His Arg Cys Glu Met
620 625 630
Pro Ser Pro Glu Leu Cys Leu Val Cys Glu Met Ser Ser Leu Phe
635 640 645
Arg Glu Leu Tyr Ser G1y Asn Pro Ser Pro His Val Pro Tyr Lys
650 655 660
Leu Leu His Leu Val Trp Ile His Ala Arg His Leu A1a Gly Tyr
665 670 675
Arg Gln Gln Asp Ala His Glu Phe Leu Ile A1a Ala Leu Asp Val
680 685 690
Leu His Arg His Cys Lys Gly Asp Asp Val Gly Lys Ala Ala Asn
695 700 705
Asn Pro Asn His Cys Asn Cys I1e Ile Asp Gln Ile Phe Thr Gly
710 715 720
G1y Leu Gln Ser Asp Val Thr Cys Gln Ala Cys His Gly Va1 Ser
725 730 735
Thr Thr Ile Asp Pro Cys Trp Asp Ile Ser Leu Asp Leu Pro Gly
28/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
740 745 750
Ser Cys Thr Ser Phe Trp Pro Met Ser Pro Gly Arg Glu Ser Ser
755 760 765
Val Asn Gly Glu Ser His Ile Pro-Gly Ile Thr Thr Leu Thr Asp
770 775 780
Cys Leu Arg Arg Phe Thr Arg Pro Glu His Leu Gly Ser Ser Ala
785 790 795
Lys Ile Lys Cys Gly Ser Cys Gln Ser Tyr Gln Glu Ser Thr Lys
800 805 810
G1n Leu Thr Met Asn Lys Leu Pro Val Val Ala Cys Phe His Phe
815 820 825
Lys Arg Phe Glu His Ser Ala Lys Gln Arg Arg Lys Ile Thr Thr
830 835 840
Tyr Ile Ser Phe Pro Leu Glu Leu Asp Met Thr Pro Phe Met Ala
845 850 855
Ser Ser Lys Glu Ser Arg Met Asn Gly Gln Leu Gln Leu Pro Thr
860 865 870
Asn Ser Gly Asn Asn Glu Asn Lys Tyr Ser Leu Phe Ala Val Val
875 880 885
Asn His Gln Gly Thr Leu Glu Ser Gly His Tyr Thr Ser Phe Ile
890 895 900
Arg His His Lys Asp Gln Trp Phe Lys Cys Asp Asp A1a Val I1e
905 910 915
Thr Lys Ala Ser Ile Lys Asp Val Leu Asp Ser Glu Gly Tyr Leu
920 925 930
Leu Phe Tyr His Lys Gln Val Leu Glu His Glu Ser Glu Lys Val
935 940 945
Lys Glu Met Asn Thr Gln Ala Tyr
950
<210>.22
<211> 2204
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 275791CB1
<400> 22
atatgccaat agacctgaca agtctgaatt ggaaactcag attgacagaa tgacgaagaa 60
gagctttagc agctgtcttg gagataagta agagagatgc ttcaccatct ctgagtcatg 120
aagatgatga taagccaact agcagcccag ataccggatt tgcagaagat gatattcaag 180
aaatgccgga aaatccagac actatggaaa ctgagaagcc caaaacaatc acagagctgg 240
atcctgccag ttttactgag ataactaaag actgtgatga gaataaagaa aacaaaactc 300
cagaaggatc tcagggagaa gttgattggc tccagcagta tgatatggag cgtgaaaggg 360
aagagcaaga gcttcagcag gcactggctc agagccttca agagcaagag gcttgggaac 420
agaaagaaga tgatgacctc aaaagagcta ccgagttaag tcttcaagag tttaacaact 480
cctttgtgga tgcattgggt tctgatgagg actctggaaa tgaggatgtt tttgatatgg 540
agtacacaga agctgaagct gaggaactga aaagaaatgc tgagacagga aatctgcctc 600
attcgtaccg gctcatcagt gttgtcagtc acattggtag cacttcttct tcaggtcatt 660
acattagtga tgtatatgac attaagaagc aagcgtggtt tacttacaat gacctggagg 720
tatcaaaaat ccaagaggct gccgtgcaga gtgatcgaga tcggagtggc tacatcttct 780
tttatatgca caaggagatc tttgatgagc tgctggaaac agaaaagaac tctcagtcac 840
ttagcacgga agtggggaag actacccgtc aggcctcgtg aggaacaaac tcctgggttg 900
gcagcatgca ctgcatattt gttactgctg cccacctcac ctttcctctg ctgaaggaga 960
atttggaatt ctacttgatg cgggagcaac aaacagctca gggccaaacc aaaagacaaa 1020
aattggagta acgtagaatg ctccatgcta ttttatggaa actttggtct cacatccgta 1080
gctgattatc ctctttttct cctatgagtg gcacttcttt tgtcttagga atacatgttg 1140
taaatatata tctgtgtatg tgtgtataca cacacacaga cacacacaca cacacacggg 1200
atgaatggag ccttaaagag ttaggatgag ccaccagaat atgcctgctc aaaattaata 1260
gcacagcagt ttggagaaga aatgaaggtg tcaaagagtc cattcacctg agaaatgtgt 1320
gaagacatac ttatcagttg gcttttagct tttatgttcc ttgagtagtt tcactcaagt 1380
ctgtaacctt ttgtgtttcc ttattagtaa aattcactgg aaagccagct cttcatgtta 1440
cactaatgac agtttgttct ctttgcaaga gaggggcatt actgtcacct gacttgagga 1500
gctgttttgt tgttgttgtt gtctgcaaat ttcatgaatt tgtgatgtct ttgctgttta 1560
catgcagtcc caagaaatgg attgttggtg ctttggaata tgttacagtc ccacatttga 1620
29/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
tatttcttat atactttgtt ttctctaagg agatttcttc acacagtatg ttcatcatat 1680
atcatcatca ttattatggt ggtaaagata gaatcttttt tctttttttg tcattctggc 1740
catggagcag cattacccta atggattgca accaaaactt taaacaagta gaaagataat 1800
atttctccaa ttgggactcc ccagcaggaa tacttaggga taaggaagaa tgctagcatc 1860
tctgtctctc aaacataggg aggataagaa gagtgttctt ctggtaaagc taaaattctg 1920
gaccactgaa gctaaaagcc ctattgcaag tatgaaatta agtacttgag ctataggaca 1980
aaccttgggc atttaaccat ttactgtctg gctttgccct taaaataggg ttgcaattaa 2040
aatgtgattg gcttaggtaa tcccaaaaac taacaaataa caaaggtgca taatttattt 2100
atctactttt taggtgctct gagttgaggc aaagtagagc ggcaacatta agtgctatgc 2160
tagtcactta gctgacgtaa ccagctttgg taagcagctt atga 2204
<210> 23
<211> 2036
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte TD No: 1389845CB1
<400> 23
ccgatggggg ttaggctcca gggcttctgt cgagaccaag gatgcccaaa tatctgggtg 60
gtgggtgctg catacctggg ccctgggcag aacgaagggt atacagcctg ggccaccagg 120
ataagtccag aacccaccag gagctgagga cagacagaag gaccacggag ggggtgacgg 180
gctggtgtga ggattggtgc ccctgggcca ggactctcct ctcttctccc tgctggctcc 240
agaccagagt ccaagcccta ggcagtgcca cccttaccca gcccagcctt gaagacagaa 300
tgagaggggt ttcctgtctc caggtcctgc tccttctggt gctgggagct gctgggactc 360
agggaaggaa gtctgcagcc tgcgggcagc cccgcatgtc cagtcggatc gttgggggcc 420
gggatggccg ggacggagag tggccgtggc aggcgagcat ccagcatcgt ggggcacacg 480
tgtgcggggg gtcgctcatc gccccccagt gggtgctgac agcggcgcac tgcttcccca 540
ggagggcact gccagctgag taccgcgtgc gcctgggggc gctgcgtctg ggctccacct 600
cgccccgcac gctctcggtg cccgtgcgac gggtgctgct gcccccggac tactccgagg 660
acggggcccg cggcgacctg gcactgctgc agctgcgtcg cccggtgccc ctgagcgctc 720
gcgtccaacc cgtctgcctg cccgtgcccg gcgcccgccc gccgcccggc acaccatgcc 780
gggtcaccgg ctggggcagc ctccgcccag gagtgcccct cccagagtgg cgaccgctac 840
aaggagtaag ggtgccgctg ctggactcgc gcacctgcga cggcctctac cacgtgggcg 900
cggacgtgcc ccaggctgag cgcattgtgc tgcctgggag tctgtgtgcc ggctaccccc 960
agggccacaa ggacgcctgc caggtgtgca cccagcctcc ccagcctccg gagtcccctc 1020
cctgtgccca gcaccctccc tccctgaact ccaggaccca ggacatccca actcaggctc 1080
aggatcctgg cctccaacct agaggcacca cgccaggggt ctggaaccct gagaactgaa 1140
gtcctgggag ggctgggact taggctcctc tttctcctgc agggtgattc tgggggacct 1200
ctgacctgcc tgcagtctgg gagctgggtc ctggtgggcg tggtgagctg gggcaagggt 1260
tgtgccctgc ccaaccgtcc aggggtctac accagtgtgg ccacatatag cccctggatt 1320
caggctcgcg tcagcttcta atgctagccg gtgaggctga cctggagcca gctgctgggg 1380
tccctcagcc tcctggttca tccaggcacc tgcctatacc ccacatccct tctgcctcga 1440
ggccaagatg cctaaaaaag ctaaaggcca ccccaccccc cacccaccac ctcctgcctc 1500
ctctcctctt tggggatcac cagctctgac tccaccaacc ctcatccagg aatctgccat. 1560
gagtcccagg gagtcacact ccccactccc ttcctggctt gtatttactt ttcttggccc 1620
tggccagggc tgggcgcaag gcacgcagtg atgggcaaac caattgctgc ccatctggcc 1680
tgtgtgccca tctttttctg~gagaaagtca gattcacagc atgacagaga tttgacacca 1740
gggagatcct ccatagctgg ctttgaggac acggggacca cagccatgag cggcctctaa 1800
gagctgagag acagccggca gggaatcgga accctcagac ccacagccgc aaggcactgg 186.0
attctggcag caccctgaag gagctgggaa gtaagttctt ccccagcctc cagataagag 1920
ccccgccggc caatcccttc atttcaacct aaagagaccc taagcagaga acctagctga 1980
gccactcctg acctacaaag ttgtgactta ataaatgtgt gctttaagct gctcca 2036
<210> 24
<211> 2185
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1726609CB1
<400> 24
gccatgcctc ctgcccacgg ccaccagcaa gctgtcgggc gcagtggagc agtggctgag 60
30/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
tgcagctgag cggctgtatg ggccctacat gtggggcagg tacgacattg tcttcctgcc 120
accctccttc cccatcgtgg ccatggagaa cccctgcctc accttcatca tctcctccat 180
cctggagagc gatgagttcc tggtcatcga tgtcatccac gaggtggccc acagttggtt 240
cggcaacgct gtcaccaacg ccacgtggga agagatgtgg ctgagcgagg gcctggccac 300
ctatgcccag cgccgtatca ccaccgagac ctacggtgct gccttcacct gcctggagac 360
tgccttccgc ctggacgccc tgcaccggca gatgaagctt ctgggagagg acagcccggt 420
cagcaaactg caggtcaagc tggagccagg agtgaatccc agccacctga tgaacctgtt 480
cacctacgag aagggctact gcttcgtgta ctacctgtcc cagctctgcg gagacccaca 540
gcgctttgat gactttctcc gagcctatgt ggagaagtac aagttcacca gcgtggtggc 600
ccaggacctg ctggactcct tcctgagctt cttcccggag ctgaaggagc agagcgtgga 660
ctgccgggca gggctggaat tcgagcgctg gctcaatgcc acaggcccgc cgctggctga 720
gccggacctg tctcagggat ccagcctgac ccggcccgtg gaggcccttt tccagctgtg 780
gaccgcagaa cctctggacc aggcagctgc ctcggccagc gccattgaca tctccaagtg 840
gaggaccttc cagacagcac tcttcctgga ccggctcctg gatgggtccc cgctgccgca 900
ggaggtggtg atgagcctgt ccaagtgcta ctcctccctg ctggactcga tgaacgctga 960
gatccgcatc cgctggctgc agattgtggt ccgcaacgac tactatcctg acctccacag 1020
ggtgcggcgc ttcctggaga gccagatgtc acgcatgtac accatcccgc tgtacgagga 1080
cctctgcacc ggtgccctca agtccttcgc gctggaggtc ttctaccaga cgcagggccg 1140
gctgcacccc aacctgcgca gagccatcca gcagatcctg tcccagggcc tgggctccag 1200
cacagagccc gcctcagagc ccagcacgga gctgggcaag gctgaagcag acacagactc 1260
ggacgcacag gccctgctgc ttggggacga ggcccccagc agtgccatct ctctcaggga 1320
cgtcaatgtg tctgcctagc cctgttggcg ggctgaccct cgacctccca gacaccacaa 1380
ttgtgccttc tgtgggccag gcctgccatg actgcgtctc ggctctggcc atgagctctg 1440
cccaggccca caagcccctc ccctgggctc tcccaggcag ggagaatggg gagagggacc°1500
tccttgtgtc tggcagagac ctgtggacct ggcctcccca ctcccagctc tcttgcactg 1560
caggccctgg ggccagcccg cacacaccat gcctcctgtc tcaacactga cagctgtgcc 1620
tagccccgga tgccagcacc tgccaggtgc cgccccgggg caagggcccc agcagcccta 1680
tggtgaccgc cacactgtgc cttaatgtct gccgggggcc caggctgtgc tgtccctgca 1740
gcacgcctcc ttgcagggat ctgagccacc ctccccgcac agccctgcac cccgccccta 1800
gggttggcag cctcagttgg cccctggcag aggaacaagg acacagacat tccctcagtg 1860
tggggggcag gggacacagg gagaggatgg ttgtccctgg ggagggccct ctggccccag 1920
gcaaccttag cccctcagaa cagggagtcc caggacccag ggagagtgtg gggacaggac 1980
agcctgtctc ttgtagcttc ctggggtggg aggcacaggg gcaaagcaat accccaggga 2040
aagtgggagg tggtgctggt gctctctcca ggcccaccat gctgggagag gcggccagag 2100
cctggggcct ccagcctggg actgctgtga tggggtatca cggtgatggt cccattaaac 2160
ttccactctg caaaaaaaaa aaaaa 2185
<210> 25
<211> 3486
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4503848CB1
<400> 25
ctgtcttaaa aaaagaggga gggaagatta ctgatttaat ttataaagga gaattattat 60
agctccaaca cctgacttta tttatctata tggtttaatt acacaaacaa ttcagtgttt 120
gaattataca aatttcatta aaactatgta attatgcaag aaaaatagga aatacagggg 180
cacttagttt tgtgcatatg tgttcacctg agagtatttg cttgtttttt taaaaaggtt 240
ctttttaatt taatatttaa ttttataatg cacattcata tgttgacttt ggaccaacag 300
aaatccctaa ttcttattct ttttctgatt ctttttagag ttggtggttc caggatttta 360
ctcagaatga cgttaggaag agaagtgatg tctcctcttc aggcaatgtc ttcctatact 420
gtggctggca gaaatgtttt aagatgggat ctttcaccag agcaaattaa aacaagaact 480
gaggagctca ttgtgcagac caaacaggtg tacgatgctg ttggaatgct cggtattgag 540
gaagtaactt acgagaactg tctgcaggca ctggcagatg tagaagtaaa gtatatagtg 600
gaaaggacca tgctagactt tccccagcat gtatcctctg acaaagaagt acgagcagca 660
agtacagaag cagacaaaag actttctcgt tttgatattg agatgagcat gagaggagat 720
atatttgaga gaattgttca tttacaggaa acctgtgatc tggggaagat aaaacctgag 780
gccagacgat acttggaaaa gtcaattaaa atggggaaaa gaaatgggct ccatcttcct 840
gaacaagtac agaatgaaat caaatcaatg aagaaaagaa tgagtgagct atgtattgat 900
tttaacaaaa acctcaatga ggatgatacc ttccttgtat tttccaaggc tgaacttggt 960
gctcttcctg atgatttcat tgacagttta gaaaagacag atgatgacaa gtataaaatt 1020
accttaaaat atccacacta tttccctgtc atgaagaaat gttgtatccc tgaaaccaga 1080
agaaggatgg aaatggcttt taatacaagg tgcaaagagg aaaacaccat aattttgcag 1140
cagctactcc cactgcgaac caaggtggcc aaactactcg gttatagcac acatgctgac 1200
31/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
ttcgtccttg aaatgaacac tgcaaagagc acaagccgcg taacagcctt tctagatgat 1260
ttaagccaga agttaaaacc cttgggtgaa gcagaacgag agtttatttt gaatttgaag 1320
aaaaaggaat gcaaagacag gggttttgaa tatgatggga aaatcaatgc ctgggatcta 1380
tattactaca tgactcagac agaggaactc aagtattcca tagaccaaga gttcctcaag 1440
gaatacttcc caattgaggt ggtcactgaa ggcttgctga acacctacca ggagttgttg 1500
ggactttcat ttgaacaaat gacagatgct catgtttgga acaagagtgt tacactttat 1560
actgtgaagg ataaagctac aggagaagta ttgggacagt tctatttgga cctctatcca 1620
~agggaaggaa aatacaatca tgcggcctgc ttcggtctcc agcctggctg ccttctgcct 1680
gatggaagcc ggatgatggc agtggctgcc ctcgtggtga acttctcaca gccagtggca 1740
ggtcgtccct ctctcctgag acacgacgag gtgaggactt actttcatga gtttggtcac 1800
gtgatgcatc agatttgtgc acagactgat~tttgcacgat ttagcggaac aaatgtggaa 1860
actgactttg tagaggtgcc atcgcaaatg cttgaaaatt gggtgtggga cgtcgattcc 1920
ctccgaagat tgtcaaaaca ttataaagat ggaagcccta ttgcagacga tctgcttgaa 1980
aaacttgttg cttctaggct ggtcaacaca ggtcttctga ccctgcgcca gattgttttg 2040
agcaaagttg atcagtctct tcataccaac acatcgctgg atgctgcaag tgaatatgcc 2100
aaatactgct cagaaatatt aggagttgca gctactccag gcacaaatat gccagctacc 2160
tttggacatt tggcaggggg atacgatggc caatattatg gatatctttg gagtgaagta 2220
ttttccatgg atatgtttta cagctgtttt aaaaaagaag ggataatgaa tccggaggtt 2280
ggaatgaaat acagaaacct aatcctgaaa cctgggggat ctctggacgg catggacatg 2340
ctccacaatt tcttgaaacg tgagccaaac caaaaagcgt tcctaatgag tagaggcctg 2400
catgctccgt gaactgggga tctttggtag ccgtccatgt ctggaggaca agtcgacatc 2460
accatgtgtt actggcctgg aaactgaagg gagttttgca agtgaaaatt tagatttcta 2520
ttgacatcct tttgttttct aattttaaaa attataaaga tgtaaatgga attataaata 2580
ctgtgaccta agaaaagacc cactagaaag taattgtact ataaaatttc ataaaactgg 2640
atttgatttc tttttatgaa agtttcatat gaatgtaact tgatttttta ctattataat 2700
ctagataata tgatataaga gggctaagaa tttttaaatt gaatcatata tatgatataa 2760
tttgatcctt cttgtatctt gaagttttgt acttgggatt tctggactga taaatgaatc 2820
atcacattct tctggtaaat attttcttgg agctctgtgt caactttgat cctttgtctc 2880
ccaggaaggt gtgacctctc ctttgcctgc atacctcaag gccaggggaa tatgcctcag.2940
tgatgcattt atctttgtat atcaggccgc atgattccca actttctgcc acacttaaat 3000
tacgttcctc catttcagtt ttgtcttttc tgtctaaagt tcagtcaaag agtatcaaaa 3060
aattatgttt cagctagact ggtgtaatgt ataagttttt gtatcttgta ttagaggatt 3120
tcgtagcttt tattagaggc tcatttccac ctcagcatac aagatcgtta gtcttttggc 3180
atgtgtgcca attagaatac taaagcaagt ccaagcacat ttttctcttc tcacgtttct 3240
aataagtgtt agggactttg cctcttttac ttaccacgtc cccaaaagtg tcaggtagac 3300
atgtcacaaa tggctctgta gagagccatg ggaagagaga ggaggtggat gtggaacata 3360
aagggttcag aaactccaga agaggagtgg gttttggata gaagcatttg aggacagctg 3420
ctccaaagcc ttatgtgtat gatgaaactt aaccacgggg aagagactct tcagtagcct 3480
gttctg 3486
<210> 26 .
<211> 2847
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5544089CB1
<400> 26
caatgacgct tggacgagga tttatttcta caagctaatt gaatccagga gcagctttaa 60
ttattaacac taacggaaga gaaaaggagt atttccaagg gctcaaatgg aagctgtact 120
cagtccggtg gaggcagggg gaggtaaagt ttctcacact caagtcgtct tcatagttta 180
ctgtcctttt ccaaacaaaa gctaataacg ccatacgcat ccacacactc cctcctggat 240
gaacctaagt ctcgtcccca ctgtcacccc aaggccagtt atcaaaaact gttccttctc 300
tgccctcaaa gactgaagcc gcaggccctg ttctgcctct gctcaggaat ctgattgctc 360
ttaaagtgct cttacaagat tccgtcgatg tttgctccct ctgtcttgtc atcaggacta 420
agtggtggag catcaaaagg tagaaagatg gaacttattc agccaaagga gccaacttca 480
cagtacattt ctctttgtca tgaattgcat actttgttcc aagtcatgtg gtctggaaag 540
tgggcgttgg tctcaccatt tgctatgcta cactcagtgt ggagactcat tcctgccttt 600
cgtggttacg cccaacaaga cgctcaggaa tttctttgtg aacttttaga taaaatacaa 660
cgtgaattag agacaactgg taccagttta ccagctctta tccccacttc tcaaaggaaa 720
ctcatcaaac aagttctgaa tgttgtaaat aacatttttc atggacaact tcttagtcag 780
gttacatgtc ttgcatgtga caacaaatca aataccatag aacctttctg ggacttgtca 840
ttggagtttc cagaaaggta tcaatgcagt ggaaaagata ttgcttccca gccatgtctg 900
gttactgaaa tgttggccaa atttacagaa actgaagctt tagaaggaaa aatctacgta 960
tgtgaccagt gtaactcaaa gcgtagaagg ttttcctcca aaccagttgt actcacagaa 1020
32145
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
gcccagaaac aacttatgat atgccaccta cctcaggttc tcagactgca cctcaaacga 1080
ttcaggtggt caggacgtaa taaccgagag aagattggtg ttcatgttgg ctttgaggaa 1140
atcttaaaca tggagcccta ttgctgcagg gagaccctga aatccctcag accagaatgc 1200
tttatctatg acttgtccgc ggtggtgatg caccatggga aaggatttgg ctcagggcac 1260
tacactgcct actgctataa ttctgaagga gggttctggg tacactgcaa tgattccaaa 1320
ctaagcatgt gcactatgga tgaagtatgc aaggctcaag cttatatctt gttttatacc 1380
caacgagtta ctgagaatgg acattctaaa cttttgcctc cagagctcct gttggggagc 1440
caacatccca atgaagacgc tgatacctcg tctaatgaaa tccttagctg atccaaagac 1500
aatggggttt tcttcctgtg atttatatat atacttttta aaagactgat gtaccatttt 1560
aaacttcatt ttttcttgtg aatcagtgta tactacattt atacatttta tatctaacaa 1620
tttttttttt tacaaagtat aaatgtatat atcaactgaa ggtaactact tttttcatat 1680
ttggagtttt aaacttttgg tgtttacctc agactgatgt tacctctttt atatttttat 1740
gtcttaattg gctcggatga tgaacttgtg caatcttcta ccaacaaagt tcaagtggca 1800
tcattttata tacatgtatc tttttcaggt attttctata caaattctta atagatggaa 1860
aattagactc tactttggtc actaatagtc tttcatttgt atattgaagt taccttgccc 1920
cttggagtta ttgaagtgac atgtcaaggt atcacctaaa tattcttcag tcacactcac 1980
tggtatttct gaggctttgt gtgttaacag gccttgtaat tgacattatt ttggttaatg 2040
taaccccaaa attgctttag taattgctct ttggcatagt caaactataa atgaaaatgg 2100
cagctttaca aatagtatat ttaagtgaac tctggaacta tggacatgaa aaaaatgatg 2160
gctgggattt atgatttttg tctggcagca aacaggtttg tccagaagtc taataattaa 2220
gcagtcataa aaagtctgaa tttagtaaac cagtgtatga tgttattcaa atagtttacc 2280
ttgggtatga gttcatttta taatgtctga tgacattaga tctcttaaaa ctttatgtat 2340
tttttttagt tcaaaggaat agagtcttga agagaaaaaa ttatagggca gaaaagataa 2400
gtgttcaaaa ttggcaactg gactattatt atgtctagca tctcattcta aataactaaa 2460
gcttgattta ctcttgctag gattatgtga ctactaggta ggagcctctt aaaacactgg 2520
ccctgagcat taaaaaaaaa aaaaaaaact aaaagctatc tatctaaact tgcaaaaaaa 2580
aaattccggt gggggtcacc cttttccttc ttctgaaaat ctcacggggt ttctttaaag 2640
ccctgttgct gcaaacttta tcttttttgg ggggggtaga atcacctaat ctctgtagac 2700
cagctatgtt tctaagctct gttaaccacg gggagatctg gtaccccttt tttaaaaggg 2760
ggtttatttg cgggttgaag tcttagtgaa aagtagtccc ctggagaatg cggtccaccc 2820
ctgggggcca tctgttaggt aaaactt 2847
<210> 27
<211> 890
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474081CB1
<400> 27
gaggccaaga attcggcacg aggcacttac tccctgagct aagggggaag agctggatca 60
ccatgaaata tgtcttctat ttgggtgtcc tcgctgggac atttttcttt gctgactcat 120
ctgttcagaa agaagaccct gctccctatt tggtgtacct caagtctcac ttcaacccct 180
gtgtgggcgt cctcatcaaa cccagctggg tgctggcccc agctcactgc tatttaccaa 240
atctgaaagt gatgctggga aatttcaaga gcagagtcag agacggtact gaacagacaa 300
ttaaccccat tcagatcgtc cgctactgga actacagtca tagcgcccca caggatgacc 360
tcatgctcat caagctggct aagcctgcca tgctcaatcc caaagtccag ccccttaccc 420
tcgccaccac caatgtcagg ccaggcactg tctgtctact ctcaggtttg gactggagcc 480
aagaaaacag tggccgacac cctgacttgc ggcagaacct ggaggccccc gtgatgtctg 540
atcgagaatg ccaaaaaaca gaacaaggaa aaagccacag gaattcctta tgtgtgaaat 600
ttgtgaaagt attcagccga atttttgggg aggtggccgt tgctactgtc atctgcaaag 660
acaagctcca gggaatcgag gtggggcact tcatgggagg ggacgtcggc atctacacca 720
atgtttacaa atatgtatcc tggattgaga acactgctaa ggacaagtga gaccctactt 780
ctccctctgc attccactgg ctctgccatg gactatacaa gcagataatt ttccctctat 840
tcaaaataaa atctccaaat gaaaatttgg gaatgtagca aaaaaaaaaa 890
<210> 28
<211> 1577
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5281209CB1
33/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
<400> 28
atgcagccca cgggccgcga gggttcccgc gcgctcagcc ggcggtatct gcggcgtctg 60
ctgctcctgc tactgctgct gctgctgcgg cagcccgtaa cccgcgcgga gaccacgccg 120
ggcgccccca gagccctctc cacgctgggc tcccccagcc tcttcaccac gccgggtgtc 180
cccagcgccc tcactacccc aggcctcact acgccaggca cccccaaaac cctggacctt 240
cggggtcgcg cgcaggccct gatgcggagt ttcccactcg tggacggcca caatgacctg 300
ccccaggtcc tgagacagcg ttacaagaat gtgcttcagg atgttaacct gcgaaatttc 360
agccatggtc agaccagcct ggacaggctt agagacggcc tcgtgggtgc ccagttctgg 420
tcagcctccg tctcatgcca gtcccaggac cagactgccg tgcgcctcgc cctggagcag 480
attgacctca ttcaccgcat gtgtgcctcc tactctgaac tcgagcttgt gacctcagct 540
gaaggtctga acagctctca aaagctggcc tgcctcattg gcgtggaggg tggtcactca 600
ctggacagca gcctctctgt gctgcgcagt ttctatgtgc tgggggtgcg ctacctgaca 660
cttaccttca cctgcagtac accatgggca gagagttcca ccaagttcag acaccacatg 720
tacaccaacg tcagcggatt gacaagcttt ggtgagaaag tagtagagga gttgaaccgc 780
ctgggcatga tgatagattt gtectatgca tcggacacct tgataagaag ggtcctggaa 840
gtgtctcagg ctcctgtgat cttctcccac tcagctgcca gagctgtgtg tgacaatttg 900
ttgaatgttc ccgatgatat cctgcagctt ctgaagaaga acggtggcat cgtgatggtg 960
acactgtcca tgggggtgct gcagtgcaac ctgcttgcta acgtgtccac tgtggcagat 1020
cactttgacc acatcagggc agtcattgga tctgagttca tcgggattgg tggaaattat 1080
gacgggactg gccggttccc tcaggggctg gaggatgtgt ccacataccc agtcctgata 1140
gaggagttgc tgagtcgtag ctggagcgag gaagagcttc aaggtgtcct tcgtggaaac 1200
ctgctgcggg tcttcagaca agtggaaaag gtgagagagg agagcagggc gcagagcccc 1260
gtggaggctg agtttccata tgggcaactg agcacatcct gccactccca cctcgtgcct 1320
cagaatggac accaggctac tcatctggag gtgaccaagc agccaaccaa tcgggtcccc 1380
tggaggtcct caaatgcctc cccatacctt gttccaggcc ttgtggctgc tgccaccatc 1440
ccaaccttca cccagtggct ctgctgacac agtcggtccc cgcagaggtc actgtggcaa 1500
agcctcacaa agccccctct cctagttcat tcacaagcat atgctgagaa taaacatgtt 1560
acacatggaa aaaaaaa 1577
<210> 29
<211> 1958
<212> DNA
<213> Homo saplens
<220>
<221> misc_feature
<223> Incyte ID No: 2256251CB1
<400> 29
aagcggtcga gctcggcatt cattgtaacg gcgccatgtg ctggaaaggt cgtgtggttt 60
ctgctcgcat ctctcggttg agtggggctg gtcggggtgt gctgcagggc tgtctccccc 120
accaccactg tagtcagtct gtaccttggg agatgctctg aggccatgaa acaacctggc 180
cctcctcgaa ctttctcccc acagcgtccc caccgtggcc ctggaaccag ctgggggctt 240
tgccgtgtgg gagagccggt gccccagccc acacccgctg cctatctata aacatctctg 300
tctgtctaca tcccagcttc ccttccattc agcccagtgg gcacactcca tcaccagcac 360
aattatccag ctcaggcaga cccaggtgtg gccagtgggt ctccagctga cttcctctta 420
atttcttctt aacttactga ggtgaagttt acagaagata aagttacatt atcaagcgtc 480
caattcagag gccccgagca ccttggacag tgctgtctgc cccccgcccc ccgaatttta 540
tccagcttca aatatctcca ccacccctga aggaaaaccg gggcccacta ggcagccacc 600
cctagcaccc cgggccttct cgggggctcc accgttctga gcccctttct agccgcctag 660
gggccctctg cagcctttcc accgcctccg ggagccctgg ttagtttgtg gagcatggtg 720
cttagaaaag acgacctgag ccccaggcgc gctcactgct cctgagacgt catttctgct 780
gcacccacga tgcttctggg gagagtctgg cagacgagag agctgaagag caaagtcccc 840
aagaaggcag ggaggtgtgg tcagggaagg cttcatggag gaagtgcagt gggcttcttg 900
ggatccccac caggcacccc ttcctccttc gacttagggt gtggccggcc gcaggtttcg 960
gatgcaggcg gccggatcgt ggggggtcac gctgccccgg ccggcgcatg gccatggcag 1020
gccagcctcc gcctgcggag ggtgcacgtg tgcggcgggt cactgctcag cccccagtgg 1080
gtgctcacag ctgcccactg cttctccggg tccctgaact catccgacta ccaggtgcac 1140
ctgggggaac tggagatcac tctgtctccc cacttctcca ccgtgaggca gatcatcctg 1200
cactccagcc cctcaggaca gccggggacc agcggggaca tcgccctggt ggagctcagt 1260
gtccccgtga ccctcttcag ccggatcctg cccgtctgcc tcccggaggc ctcagatgac 1320
ttctgccctg ggatccggtg ctgggtgacc ggctggggct atacgcggga gggagagcct 1380
ctgccacccc cgtacagcct gcgggaggtg aaagtctccg tggtggacac agagacctgc 1440
cgccgggact atcccggccc cgggggcagc atccttcagc ccgacatgct gtgtgcccgg 1500
ggccccgggg atgcctgcca ggacgactcc ggggggcctc tggtctgcca ggtgaacggt 1560
gcctgggtgc aggctggcat tgtgagctgg ggtgagggct gcggccgccc caacaggccg 1620
ggagtctaca ctcgtgtccc tgcctacgtg aactggatcc gccgccacat cacagcatca 1680
34/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
gggggctcag agtctgggta ccccaggctc cccctcctgg ctggcttatt cctccccggc 1740
ctcttccttc tgctagtctc ctgtgtcctg ctggccaagt gcctgctgca cccatctgcg 1800
gatggtactc ccttccccgc ccctgactga tggcaggaat ccaagtgcat ttcttaaata 1860
agttactatt tattccgctc cgccccctcc ctctcccttg agaagctgag tcttctgcat 1920
cagattattg caacatttaa cctgaattta acgacacc 1958
<210> 30
<211> 3106
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7160544CB1
<400> 30
gctccgaggc caaggccgct gctactgccg ccgctgcttc ttagtgccgc gttcgccgcc 60
tgggttgtca.ccggcgccgc cgctgaggaa gccactgcaa ccaggaccgg agtggaggcg 120
gcgcagcatg aagcggcgca ggcccgctcc atagcgcacg tcgggacggt ccgggcgggg 180
ccggggggaa ggaaaatgca acatggcagc agcaatggaa acagaacagc tgggtgttga 240
gatatttgaa actgcggact gtgaggagaa tattgaatca caggatcggc ctaaattgga 300
gcctttttat gttgagcggt attcctggag tcagcttaaa aagctgcttg ccgataccag 360
aaaatatcat ggctacatga tggctaaggc accacatgat ttcatgtttg tgaagaggaa 420
tgatccagat ggacctcatt cagacagaat ctattacctt gccatgtctg gtgagaacag 480
agaaaataca ctgttttatt ctgaaattcc caaaactatc aatagagcag cagtcttaat 540
gctctcttgg aagcctcttt tggatctttt tcaggcaaca ctggactatg gaatgtattc 600
tcgagaagaa gaactattaa gagaaagaaa acgcattgga acagtcggaa ttgcttctta 660
cgattatcac caaggaagtg gaacatttct gtttcaagcc ggtagtggaa tttatcacgt 720
aaaagatgga gggccacaag gatttacgca acaaccttta aggcccaatc tagtggaaac 780
tagttgtccc aacatacgga tggatccaaa attatgccct gctgatccag actggattgc 840
ttttatacat agcaacgata tttggatatc taacatcgta accagagaag aaaggagact 900
cacttatgtp cacaatgagc tagccaacat ggaagaagat gccagatcag ctggagtcgc 960
tacctttgtt ctccaagaag aatttgatag atattctggc tattggtggt gtccaaaagc 1020
tgaaacaact cccagtggtg gtaaaattct tagaattcta tatgaagaaa atgatgaatc 1080
tgaggtggaa attattcatg ttacatcccc tatgttggaa acaaggaggg cagattcatt 1140
ccgttatcct aaaacaggta cagcaaatcc taaagtcact tttaagatgt cagaaataat 1200
gattgatgct gaaggaagga tcatagatgt catagataag gaactaattc aaccttttga 1260
gattctattt gaaggagttg aatatattgc cagagctgga tggactcctg agggaaaata 1320
tgcttggtcc atcctactag atcgctccca gactcgccta cagatagtgt tgatctcacc 1380
tgaattattt atcccagtag aagatgatgt tatggaaagg cagagactca ttgagtcagt 1440.
gcctgattct gtgacgccac taattatcta tgaagaaaca acagacatct ggataaatat 1500
ccatgacatc tttcatgttt ttccccaaag tcacgaagag gaaattgagt ttatttttgc 1560
ctctgaatgc aaaacaggtt tccgtcattt atacaaaatt acatctattt taaaggaaag 1620
caaatataaa cgatccagtg gtgggctgcc tgctccaagt gatttcaagt gtcctatcaa 1680
agaggagata gcaattacca gtggtgaatg ggaagttctt ggccggcatg gatctaatat 1740
ccaagttgat gaagtcagaa ggctggtata ttttgaaggc accaaagact cccctttaga 1800
gcatcacctg tacgtagtca gttacgtaaa tcctggagag gtgacaaggc tgactgaccg 1860
tggctactca cattcttgct gcatcagtca gcactgtgac ttctttataa gtaagtatag 1920
taaccagaag aatccacact gtgtgtccct ttacaagcta tcaagtcctg aagatgaccc 1980
aacttgcaaa acaaaggaat tttgggccac cattttggat tcagcaggtc ctcttcctga 2040
ctatactcct ccagaaattt tctcttttga aagtactact ggatttacat tgtatgggat 2100
gctctacaag cctcatgatc tacagcctgg aaagaaatat cctactgtgc tgttcatata 2160
tggtggtcct caggtgcagt tggtgaataa tcggtttaaa ggagtcaagt atttccgctt 2220
gaatacccta gcctctctag gttatgtggt tgtagtgata gacaacaggg gatcctgtca 2280
ccgagggctt aaatttgaag gcgcctttaa atataaaatg ggtcaaatag aaattgacga 2340
tcaggtggaa ggactccaat atctagcttc tcgatatgat ttcattgact tagatcgtgt 2400
gggcatccac ggctggtcct atggaggata cctctccctg atggcattaa tgcagaggtc 2460
agatatcttc agggttgcta ttgctggggc cccagtcact ctgtggatct tctatgatac 2520
aggatacacg gaacgttata tgggtcaccc tgaccagaat gaacagggct attacttagg 2580
atctgtggcc atgcaagcag aaaagttecc ctctgaacca aatcgtttac tgctcttaca 2640
tggtttcctg gatgagaatg tccattttgc acataccagt atattactga gttttttagt 2700
gagggctgga aagccatatg atttacagat ctatcctcag gagagacaca gcataagagt 2760
tcctgaatcg ggagaacatt atgaactgca tcttttgcac taccttcaag aaaaccttgg 2820
atcacgtatt gctgctctaa aagtgatata attttgacct gtgtagaact ctctggtata 2880
cactggctat ttaaccaaat gaggaggttt aatcaacaga aaacacagaa ttgatcatca 2940
cattttgata cctgccatgt aacatctact cctgaaaata aatgtggtgc catgcagggg 3000
tctacggttt gtggtagtaa tctaatacct taaccccaca tgctcaaaat caaatgatac 3060
35/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
atattcctga gagacccagc aataccataa gaattactaa aaaaaa 3106
<210> 31
<211> 3567
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477386CB1
<400> 31
atggctccac tccgcgcgct gctgtcctac ctgctgcctt tgcactgtgc gctctgcgcc 60
gccgcgggca gccggacccc agagctgcac ctctctggaa agctcagtga ctatggtgtg 120
acagtgccct gcagcacaga ctttcgggga cgcttcctct cccacgtggt gtctggccca 180
gcagcagcct ctgcagggag catggtagtg gacacgccac ccacactacc acgacactcc 240
agtcacctcc gggtggctcg cagccctctg cacccaggag ggaccctgtg gcctggcagg 300
gtggggcgcc actccctcta cttcaatgtc actgttttcg ggaaggaact gcacttgcgc 360
ctgcggccca atcggaggtt ggtagtgcca ggatcctcag tggagtggca ggaggatttt 420
cgggagctgt tccggcagcc cttacggcag gagtgtgtgt acactggagg tgtcactgga 480
atgcctgggg cagctgttgc catcagcaac tgtgacggat tggcgggcct catccgcaca 540
gacagcaccg acttcttcat tgagcctctg gagcggggcc agcaggagaa ggaggccagc 600
gggaggacac atgtggtgta ccgccgggag gccgtccagc aggagtgggc agaacctgac 660
ggggacctgc acaatgaagc ctttggcctg ggagaccttc ccaacctgct gggcctggtg 720
ggggaccagc tgggcgacac agagcggaag cggcggcatg ccaagccagg cagctacagc 780
atcgaggtgc tgctggtggt ggacgactcg gtggttcgct tccatggcaa ggagcatgtg 840
cagaactatg tcctcaccct catgaatatc gtggtagatg agatttacca cgatgagtcc 900
ctgggggttc atataaatat tgccctcgtc cgcttgatca tggttggcta ccgacagcag 960
tccctgagcc tgatcgagcg cgggaacccc tcacgcagcc tggagcaggt gtgtcgctgg 1020
gcacactccc agcagcgcca ggaccccagc cacgctgagc accatgacca cgttgtgttc 1080
ctcacccggc aggactttgg gccctcaggt gggtatgcac ccgtcactgg catgtgtcac 1140
cccctgagga gctgtgccct caaccatgag gatggcttct cctcagcctt cgtgatagct 1200
catgagaccg gccacgtgct cggcatggag catgacggtc aggggaatgg ctgtgcagat 1260
gagaccagcc tgggcagcgt catggcgccc ctggtgcagg ctgccttcca ccgcttccat 1320
tggtcccgct gcagcaagct ggagctcagc cgctacctcc cgtcctacga ctgcctcctc 1380
gatgacccct ttgatcctgc ctggccccag cccccagagc tgcctgggat caactactca 1440
atggatgagc agtgccgctt tgactttggc agtggctacc agacctgctt ggcattcagg 1500
acctttgagc cctgcaagca gctgtggtgc agccatcctg acaacccgta cttctgcaag 1560
accaagaagg ggcccccgct ggatgggact gagtgtgcac ccggcaagtg gtgcttcaaa 1620
ggtcactgca tctggaagtc gccggagcag acatatggcc aggatggagg ctggagctcc 1680
tggaccaagt ttgggtcatg ttcgcggtca tgtgggggcg gggtgcgatc ccgcagccgg 1740
agctgcaaca acccctcgcc agcctatgga ggccgcctgt gcttagggcc catgttcgag 1800,
taccaggtct gcaacagcga ggagtgccct gggacctacg aggacttccg ggcccagcag 1860
tgtgccaagc gcaactccta ctatgtgcac cagaatgcca agcacagctg ggtgccctac 1920
gagcctgacg atgacgccca gaagtgtgag ctgatctgcc agtcggcgga cacgggggac 1980
gtggtgttca tgaaccaggt ggttcacgat gggacacgct gcagctaccg ggacccatac 2040
agcgtctgtg cgcgtggcga gtgtgtgcct gtcggctgtg acaaggaggt ggggtccatg 2100
aaggcggatg acaagtgtgg agtctgcggg ggtgacaact cccactgcag gactgtgaag 2160
gggacgctgg gcaaggcctc caagcaggca ggtgctctca agctggtgca gatcccagca 2220
ggtgccaggc acatccagat tgaggcactg gagaagtccc cccaccgcat tgtggtgaag 2280
aaccaggtca ccggcagctt catcctcaac cccaagggca aggaagccac aagccggacc 2340
ttcaccgcca tgggcctgga gtgggaggat gcggtggagg atgccaagga aagcctcaag 2400
accagcgggc ccctgcctga agccattgcc atcctggctc tccccccaac tgagggtggc 2460
ccccgcagca gcctggccta caagtacgtc atccatgagg acctgctgcc ccttatcggg 2520
agcaacaatg tgctcctgga ggagatggac acctatgagt gggcgctcaa gagctgggcc 2580
ccctgcagca aggcctgtgg aggaggtatc cagttcacca aatacggctg ccggcgcaga 2640
cgagaccacc acatggtgca gcgacacctg tgtgaccaca agaagaggcc caagcccatc 2700
cgccggcgct gcaaccagca cccgtgctct cagcctgtgt gggtgacgga ggagtggggt 2760
gcctgcagcc ggagctgtgg gaagctgggg gtgcagacac gggggataca gtgcctgctg 2820
cccctctcca atggaaccca caaggtcatg ccggccaaag cctgcgccgg ggaccggcct 2880
gaggcccgac ggccctgtct ccgagtgccc tgcccagccc agtggaggct gggagcctgg 2940
tcccagtgct ctgccacctg tggagagggc atccagcagc ggcaggtggt gtgcaggacc 3000
aacgccaaca gcctcgggca ttgcgagggg gataggccag acactgtcca ggtctgcagc 3060
ctgcccgcct gtggagcgga gccctgcacg ggagacaggt ctgtcttctg ccagatggaa 3120
gtgctcgatc gctactgctc cattcccggc taccaccggc tctgctgtgt gtcctgcatc 3180
aagaaggcct cgggccccaa ccctggccca gaccctggcc caacctcact gccccccttc 3240
tccactcctg gaagcccctt accaggaccc caggaccctg cagatgctgc agagcctcct 3300
36/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
ggaaagccaa cgggatcaga ggaccatcag catggccgag ccacacagct cccaggagct 3360
ctggatacaa gctccccagg gacccagcat ccctttgccc ctgagacacc aatccctgga 3420
gcatcctgga gcatctcccc taccaccccc ggggggctgc cttggggctg gactcagaca 3480
cctacgccag tccctgagga caaagggcaa cctggagaag acctgagaca tcccggcacc 3540
agcctccctg ctgcctcccc ggtgaca 3567
<210> 32
<211> 2930
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473089CB1
<400> 32
cacgcagacc gcggcagcgg ccgagagccc ggcccagccc cttcccacag cgcggcgttg 60
cgctgcccgg cgccatgctt ctgctgggca tcctaaccct ggctttcgcc gggcgaaccg 120
ctggaggctc tgagccagag cgggaggtag tcgttcccat ccgactggac ccggacatta 180
acggccgccg ctactactgg cggggtcccg aggactccgg ggatcaggga ctcatttttc 240
agatcacagc atttcaggag gacttttacc tacacctgac gccggatgct cagttcttgg 300
ctcccgcctt ctccactgag catctgggcg tccccctcca ggggctcacc gggggctctt 360
cagacctgcg acgctgcttc tattctgggg acgtgaacgc cgagccggac tcgttcgctg 420
ctgtgagcct gtgcgggggg ctccgcggag cctttggcta ccgaggcgcc gagtatgtca 480
ttagcccgct gcccaatgct agcgcgccgg cggcgcagcg caacagccag ggcgcacacc 540
ttctccagcg ccggggtgtt ccgggcgggc cttccggaga ccccacctct cgctgcgggg 600
tggcctcggg ctggaacccc gccatcctac gggccctgga cccttacaag ccgcggcggg 660
cgggcttcgg ggagagtcgt agccggcgca ggtctgggcg cgccaagcgt ttcgtgtcta 720
tcccgcggta cgtggagacg ctggtggtcg cggacgagtc aatggtcaag ttccacggcg 780
cggacctgga acattatctg ctgacgctgc tggcaacggc ggcgcgactc taccgccatc 840
ccagcatcct caaccccatc aacatcgttg tggtcaaggt gctgcttctt agagatcgtg 900
actccgggcc caaggtcacc ggcaatgcgg ccctgacgct gcgcaacttc tgtgcctggc 960
agaagaagct gaacaaagtg agtgacaagc accccgagta ctgggacact gccatcctct 1020
tcaccaggca ggacctgtgt ggagccacca cctgtgacac cctgggcatg gctgatgtgg 1080
gtaccatgtg tgaccccaag agaagctgct ctgtcattga ggacgatggg cttccatcag 1140
ccttcaccac tgcccacgag ctgggccacg tgttcaacat gccccatgac aatgtgaaag 1200
tctgtgagga ggtgtttggg aagctccgag ccaaccacat gatgtccccg accctcatcc 1260
agatcgaccg tgccaacccc tggtcagcct gcagtgctgc catcatcacc gacttcctgg 1320
acagcgggca cggtgactgc ctcctggacc aacccagcaa gcccatctcc ctgcccgagg 1380
atctgccggg cgccagctac accctgagcc agcagtgcga gctggctttt ggcgtgggct 1440
ccaagccctg tccttacatg cagtactgca ccaagctgtg gtgcaccggg aaggccaagg 1500
gacagatggt gtgccagacc cgccacttcc cctgggccga tggcaccagc tgtggcgagg 1560
gcaagctctg cctcaaaggg gcctgcgtgg agagacacaa cctcaacaag cacagggtgg 1620
atggttcctg ggccaaatgg gatccctatg gcccctgctc gcgcacatgt ggtgggggcg 1680
tgcagctggc caggaggcag tgcaccaacc ccacccctgc caacgggggc aagtactgcg 1740
agggagtgag ggtgaaatac cgatcctgca atctggagcc ctgccccagc tcagcctccg 1800
gaaagagctt ccgggaggag cagtgtgagg ctttcaacgg ctacaaccac agcaccaacc 1860
ggctcactct cgccgtggca tgggtgccca agtactccgg cgtgtctccc cgggacaagt 1920
gcaagctcat ctgccgagcc aatggcactg gctacttcta tgtgctggca cccaaggtgg 1980
tggtggacgg cacgctgtgc tctcctgact ccacctccgt ctgtgtccaa ggcaagtgca 2040
tcaaggctgg ctgtgatggg aacctgggct ccaagaagag attcgacaag tgtggggtgt 2100
gtgggggaga caataagagc tgcaagaagg tgactggact cttcaccaag cccatgcatg 2160
gctacaattt cgtggtggcc atccccgcag gcgcctcaag catcgacatc cgccagcgcg 2220
gttacaaagg gctgatcggg gatgacaact acctggctct gaagaacagc caaggcaagt 2280
acctgctcaa cgggcatttc gtggtgtcgg cggtggagcg ggacctggtg gtgaagggca 2340
gtctgctgcg gtacagcggc acgggcacag cggtggagag cctgcaggct tcccggccca 2400
tcctggagcc gctgaccgtg gaggtcctct ccgtggggaa gatgacaccg ccccgggtcc 2460
gctactcctt ctatctgccc aaagagcctc gggaggacaa gtcctctcat cccccgcacc 2520
cccggggagg accctctgtc ttgcacaaca gcgtcctcag cctctccaac caggtggagc 2580
agccggacga caggccccct gcacgctggg tggctggcag ctgggggccg tgctccgcga 2640
gctgcggcag tggcctgcag aagcgggcgg tggactggcg gggctccgcc gggcagcgca 2700
cggtccctgc ctgtgatgca gcccatcggc ccgtggagac acaagcctgc ggggagccct 2760
gccccacctg ggagctcagc gcctggtcac cctgctccaa gagctgcggc cggggatttc 2820
agaggcgctc actcaagtgt gtgggccacg gaggccggct gctggcccgg gaccagtgca 2880
acttgcaccg caagccccag gagctggact tctgcgtcct gaggccgtgc 2930
<210> 33
37145
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
<211> 4230
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7604035CB1
<400> 33
agcgaggttg cctggagaga gcgcctgggc gcagaagggt taacgggcca ccgggggctc 60
gcagagcagg agggtgctct cggacggtgt gtcccccact gcactcctga acttggagga 120
cagggtcgcc gcgagggacg cagagagcac cctccacgcc cagatgcctg cgtagttttt 180
gtgaccagtc cgctcctgcc tccccctggg gcagtagagg gggagcgatg gagaactgga 240
ctggcaggcc ctggctgtat ctgctgctgc ttctgtccct ccctcagctc tgcttggatc 300
aggaggtgtt gtccggacac tctcttcaga cacctacaga ggagggccag ggccccgaag 360
gtgtctgggg accttgggtc cagtgggcct cttgctccca gccctgcggg gtgggggtgc 420
agcgcaggag ccggacatgt cagctcccta cagtgcagct ccacccgagt ctgcccctcc 480
ctccccggcc cccaagacat ccagaagccc tcctcccccg gggccagggt cccagacccc 540
agacttctcc agaaaccctc cccttgtaca ggacacagtc tcggggaagg ggtggcccac 600
ttcgaggtcc cgcttcccac ctagggagag aggagaccca ggagattcga gcggccagga 660
ggtcccggct tcgagacccc atcaagccag gaatgttcgg ttatgggaga gtgccctttg 720
cattgccact gcaccggaac cgcaggcacc ctcggagccc acccagatct gagctgtccc 780
tgatctcttc tagaggggaa gagcctattc cgtcccctac tccaagagca gagccattct 840
ccgcaaacgg cagcccccaa actgagctcc ctcccacaga actgtctgtc cacaccccat 900
ccccccaagc agaacctcta agccctgaaa ctgctcagac agaggtggcc cccagaacca 960
ggcctgcccc cctacggcat caccccagag cccaggcctc tggcacagag cccccctcac 1020
ccacgcactc cttaggagaa ggtggcttct tccgtgcatc ccctcagcca cgaaggccaa 1080
gttcccaggg ttgggccagt ccccaggtag cagggagacg ccctgatcct tttccttcgg 1140
tccctcgggg ccgaggccag cagggccaag ggccttgggg aacggggggg actcctcacg 1200
ggccccgcct ggagcctgac cctcagcacc cgggcgcctg gctgcccctg ctgagcaacg 1260
gcccccatgc cagctccctc tggagcctct ttgctcccag tagccctatt ccaagatgtt 1320
ctggggagag tgaacagcta agagcctgca gccaagcgcc ctgcccccct gagcagccag 1380
acccccgggc cctgcagtgc gcagccttta actcccagga attcatgggc cagctgtatc 1440
agtgggagcc cttcactgaa gtccagggct cccagegctg tgaactgaac tgccggcccc 1500
gtggcttccg cttctatgtc cgtcacactg aaaaggtcca ggatgggacc ctgtgtcagc 1560
ctggagcccc tgacatctgt.gtggctggac gctgtctgag ccccggctgt gatgggatcc 1620
ttggctctgg caggcgtcct gatggctgtg gagtctgtgg gggtgatgat tctacctgtc 1680
gccttgtttc ggggaacctc actgaccgag ggggccccct gggctatcag aagatcttgt 1740
ggattccagc gggagccttg cggctccaga ttgcccagct ccggcctagc tccaactacc 1800
tggcacttcg tggccctggg ggccggtcca tcatcaatgg gaactgggct gtggatcccc 1860
ctgggtccta cagggccggc gggaccgtct ttcgatataa ccgtcctccc agggaggagg 1920
gcaaagggga gagtctgtcg gctgaaggcc ccaccaccca gcctgtggat gtctatatga 1980
tctttcagga ggaaaaccca ggcgtttttt atcagtatgt catctcttca cctcctccaa 2040
tccttgagaa ccccacccca gagccccctg tcccccagct tcagccggag attctgaggg 2100
tggagccccc acttgctccg gcaccccgcc cagcccggac cccaggcacc ctccagcgtc 2160
aggtgcggat cccccagatg cccgccccgc cccatcccag gacacccctg gggtctccag 2220
ctgcgtactg gaaacgagtg ggacactctg catgctcagc gtcctgcggg aaaggtgtct 2280
ggcgccccat tttcctctgc atctcccgtg agtcgggaga ggaactggat gaacgcagct 2340
gtgccgcggg tgccaggccc ccagcctccc ctgaaccctg ccacggcacc ccatgccccc 2400
catactggga ggctggcgag tggacatcct gcagccgctc ctgtggcccc ggcacccagc 2460
accgccagct gcagtgccgg caggaatttg gggggggtgg ctcctcggtg cccccggagc 2520
gctgtggaca tctcccccgg cccaacatca cccagtcttg ccagctgcgc ctctgtggcc 2580
attgggaagt tggctctcct tggagccagt gctccgtgcg gtgcggccgg ggccagagaa 2640
gccggcaggt tcgctgtgtt gggaacaacg gtgatgaagt gagcgagcag gagtgtgcgt 2700
caggcccccc acagcccccc agcagagagg cctgtgacat ggggccctgt actactgcct 2760
ggttccacag cgactggagc tccaagtgct cagccgagtg tgggacggga atccagcggc 2820
gctctgtggt ctgccttggg agtggggcag ccactcgggc caggccaggg ggaagcagga 2880
gcaggaactg ggcagagctg tccaacagga agccggcccc ctgacatgcg cgcctgcagc 2940
ctggggccct gtgagagaac ttggcgctgg tacacagggc cctggggtga gtgctcctcc 3000
gaatgtggct ctggcacaca gcgtagagac atcatctgtg tatccaaact ggggacggag 3060
ttcaacgtga cttctccgag caactgttct cacctcccca ggccccctgc cctgcagccc 3120
tgtcaagggc aggcctgcca ggaccgatgg ttttccacgc cctggagccc atgttctcgc 3180
tcctgccaag ggggaacgca gacacgggag gtccagtgcc tgagcaccaa ccagaccctc 3240
agcacccgat gccctcctca actgcggccc tccaggaagc gcccctgtaa cagccaaccc 3300
tgcagccagc gccctgatga tcaatgcaag gacagctctc cacattgccc cctggtggta 3360
caggcccggc tctgcgtcta cccctactac acagccacct gttgccgctc ttgcgcacat 3420
gtcctggagc ggtctcccca ggatccctcc tgaaaggggt ccggggcacc ttcacggttt 3480
38145
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
tctgtgccac catcggtcac ccattgatcg gcccactctg aaccccctgg ctctccagcc 3540
tgtcccagtc tcagcaggga tgtcctccag gtgacagagg gtggcaaggt gactgacaca 3600
aagtgacttt cagggctgtg gtcaggccca tgtggtggtg tgatgggtgt gtgcacatat 3660
gcctcaggtg tgcttttggg actgcatgga tatgtgtgtg ctcaaacgtg tatcactttt 3720
caaaaagagg ttacacagac tgagaaggac aagacctgtt tccttgagac tttcctaggt 3780
ggaaaggaaa gcaagtctgc agttccttgc taatctgagc tacttagagt gtggtctccc 3840
caccaactcc agttttgtgc cctaagcctc atttctcatg ttcagacctc acatcttcta 3900
agccgccctg tgtctctgac cccttctcat ttgcctagta tctctgcccc tgcctcccta 3960
attagctagg gctggggtca gccactgcca atcctgcctt actcaggaag gcaggaggaa 4020
agagactgcc tctccagagc aaggcccagc tgggcagagg gtgaaaaaga gaaatgtgag 4080
catccgctcc cccaccaccc cgcccagccc ctagccccac tccctgcctc ctgaaatggt 4140
tcccacccag aactaattta ttttttatta aagatggtca tgacaaatga aaaaaaaaaa 4200
aaaaaaataa aaaaacaaaa aaaaaaaata 4230
<210> 34
<211> 3699
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3473847CB1
<400> 34
cgcagtgtgc tggcaaagct tgactttccc agcaggccta tgtcataggt actgtggtct 60
ctacaataca gcagaggtat ctgaggctcc gagaggttga gtgacttgct catggctgca 120
caaccagtaa atattggagc tggaattcag gtccacggtt tcctggctcc aaagcccatg 180
attttttccc tcaatttatt ctgactgggg catgggggag ggggtggcct ttgggcaggg 240
ccaccaggag cgaccaggcc cgtagagagc tgggtgcagg tacagaggaa aacctgttgt 300
cgagtgtggc ccgtagttcc catttttgcc tgaatggcac'atttgaaagt gttatataac 360
catgtgaata ataatagttg gcctatatga gttttttaat ttgctttttg gtccgcattt 420
ggtaacttct ttatcatcta ctatactctg ttgtgtctct tttgttgtaa tttgtaagta 480
ggggtgagat aaagtacacc tagggtttgc tgggtttctt ccatgtcatc atgttcctcc 540
ttgcatgggg ccaggatccg tggaggttgc ctggcaccta cgtggtggtg ctgaaggagg 600
agacccacct ctcgcagtca gagcgcactg cccgccgcct gcaggcccag gctgcccgcc 660.
ggggatacct caccaagatc ctgcatgtct tccatggcct tcttcctggc ttcctggtga 720
agatgagtgg cgacctgctg gagctggcct tgaagttgcc ccatgtcgac tacatcgagg 780
aggactcctc tgtctttgcc cagagcatcc cgtggaacct ggagcggatt acccctccac 840
ggtaccgggc ggatgaatac cagccccccg acggaggcag cctggtggag gtgtatctcc 900
tagacaccag catacagagt gaccaccggg aaatcgaggg cagggtcatg gtcaccgact 9-60
tcgagaatgt gcccgaggag gacgggaccc gcttccacag acaggccagc aagtgtgaca 1020
gtcatggcac ccacctggca ggggtggtca gcggccggga tgccggcgtg gccaagggtg 1080
ccagcatgcg cagcctgcgc gtgctcaact gccaagggaa gggcacggtt agcggcaccc 1140
tcataggcct ggagtttatt cggaaaagcc agctggtcca gcctgtgggg ccactggtgg 1200
tgctgctgcc cctggcgggt gggtacagcc gcgtcctcaa cgccgcctgc cagcgcctgg 1260
cgagggctgg ggtcgtgctg gtcaccgctg ccggcaactt ccgggacgat gcctgcetct 1320
actccccagc ctcagctccc gaggtcatca cagttggggc caccaatgcc caggaccagc 1380
cggtgaccct ggggactttg gggaccaact ttggccgctg tgtggacctc tttgccccag 1440
gggaggacat cattggtgcc tccagcgact gcagcacctg ctttgtgtca cagagtggga 1500
catcacaggc tgctgcccac gtggctggca ttgcagccat gatgctgtct gccgagccgg 1560
agctcaccct ggccgagttg aggcagagac tgatccactt ctctgccaaa gatgtcatca 1620
atgaggcctg gttccctgag gaccagcggg tactgacccc caacctggtg gccgccctgc 1680
cccccagcac ccatggggca ggttggcagc tgttttgcag gactgtgtgg tcagcacact 1740
cggggcctac acggatggcc acagccatcg cccgctgcgc cccagatgag gagctgctga 1800
gctgctccag tttctccagg agtgggaagc ggcggggcga gcgcatggag gcccaagggg 1860
gcaagctggt ctgccgggcc cacaacgctt ttgggggtga gggtgtctac gccattgcca 1920
ggtgctgcct gctaccccag gccaactgca gcgtccacac agctccacca gctgaggcca 1980
gcatggggac ccgtgtccac tgccaccaac agggccacgt cctcacaggc tgcagctccc 2040
actgggaggt ggaggacctt ggcacccaca agccgcctgt gctgaggcca cgaggtcagc 2100
ccaaccagtg cgtgggccac agggaggcca gcatccacgc ttcctgctgc catgccccag 2160
gtctggaatg caaagtcaag gagcatggaa tcccggcccc tcaggagcag gtgaccgtgg 2220
cctgcgagga gggctggacc ctgactggct gcagtgccct ccctgggacc tcccacgtcc 2280
tgggggccta cgccgtagac aacacgtgtg tagtcaggag ccgggacgtc agcactacag 2340
gcagcaccag cgaagaggcc gtgacagccg ttgccatctg ctgccggagc cggcacctgg 2400
cgcaggcctc ccaggagctc cagtgacagc cccatcccag gatgggtgtc tggggagggt 2460
caagggctgg ggctgagctt taaaatggtt ccgacttgtc cctctctcag ccctccatgg 2520
cctggcacga ggggatgggg atgcttccgc ctttccgggg ctgctggcct ggcccttgag 2580
39/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
tggggcagcc tccttgcctg gaactcactc actctgggtg cctcctcccc aggtggaggt 2640
gccaggaagc tccctccctc actgtggggc atttcaccat tcaaacaggt cgagctgtgc 2700
tcgggtgctg ccagctgctc ccaatgtgcc gatgtccgtg ggcagaatga cttttattga 2760
gctcttgttc cgtgccaggc attcaatcct caggtctcca ccaaggaggc aggattcttc 2820
ccatggatag gggagggggc ggtaggggct gcagggacaa acatcgttgg ggggtgagtg 2880
tgaaaggtgc tgatggccct catctccagc taactgtgga gaagcccctg ggggctccct 2940
gattaatgga ggcttagctt tctggatggc atctagccag aggctggaga caggtgtgcc 3000
cctggtggtc acaggctgtg ccttggtttc ctgagccacc tttactctgc tctatgccag 3060
gctgtgctag caacacccaa aggtggcctg cggggagcca tcacctagga ctgactcggc 3120
agtgtgcagt ggtgcatgca ctgtctcagc caacccgctc cactacccgg cagggtacac 3180
attcgcaccc ctacttcaca gaggaagaaa cctggaacca gagggggcgt gcctgccaag 3240
ctcacacagc aggaactgag ccagaaacgc agattgggct ggctctgaag ccaagcctct 3300
tcttacttca cccggctggg ctcctcattt ttacgggtaa cagtgaggct gggaagggga 3360
acacagacca ggaagctcgg tgagtgatgg cagaacgatg cctgcaggca tggaactttt 3420
tccgttatca cccaggcctg attcactggc ctggcggaga tgcttctaag gcatggtcgg 3480
gggagagggc caacaactgt ccctccttga gcaccagccc cacccaagc,a agcagacatt 3540
tatcttttgg gtctgtcctc tctgttgcct ttttacagcc aacttttcta gacctgtttt 3600
gcttttgtaa cttgaagata tttattctgg gttttgtagc atttttatta atatggtgac 3660
tttttaaaat aaaaacaaac aaacgttgtc ctaaaaaaa 3699
<210> 35
<211> 2410
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3750004CB1
<400> 35
cctcagcagt ggccccttcc ctccacgggc tgccccggag ctcagtccca ccccctccgc 60
cgatgaggcc atgaggcacc gaacggacct gggccagaac ctcctgctct tcctgtgggc 120
cctgctgaac tgtggtttgg gggtcagtgc tcagggtccg ggcgagtgga ccccgtgggt 180
gtcctggacc cgctgctcca gctcctgcgg gcgtggcgtc tctgtgcgca gccggcgctg 240
cctccggctt cctggggaag aaccgtgctg gggagactcc catgagtacc gcctctgcca 300
gttgccagac tgccccccag gggctgtgcc cttccgagac ctacagtgtg ccctgtacaa 360
tggccgccct gtcctgggca cccagaagac ctaccagtgg gtgcccttcc atggggcgcc 420
caaccagtgc gacctcaact gcctggctga ggggcacgcc ttctaccaca gcttcggccg 480
cgtcctggac ggcaccgcct gcagcccggg tgcccagggg gtctgcgtgg ctggccgctg 540
ccttagcgcc ggctgtgatg ggttgttggg ctcgggtgcc ctcgaggacc gctgtggccg 600
ctgcggaggc gccaacgact cgtgcctttt cgtgcagcgc gtgtttcgtg acgccggtgc 660
cttcgctggg tactggaacg tgaccctgat ccccgagggc gccagacaca tccgcgtgga 720
acacaggagc cgcaaccacc tgggtatcct aggatcactg atggggggcg atgggcgcta 780
cgtgcttaat gggcactggg tggtcagccc accagggacc tacgaggcgg ccggcacgca 840
tgtggtctac acccgagaca cagggcccca ggagacattg caagcagccg ggcccacctc 900
ccatgacctg ctcctacagg tcctcctgca ggagcccaac cctggcatcg agtttgagtt 960
ctggctccct cgggagcgct acagcccctt ccaggctcgt gtgcaggccc tgggctggcc 1020
cctgaggcag cctcagcccc ggggggtgga gcctcagccc cccgcagccc ctgctgtcac 1080
ccctgcacag accccaacgc tggccccaga cccctgccca ccctgccctg acacccgcgg 1140
ccgcgcccac cgactactcc actattgcgg cagtgacttt gtgttccagg cccgagtgct 1200
gggccaccac caccaggccc aggagacccg ctatgaggtg cgcatccagc tcgtctacaa 1260
gaaccgctcg ccactgcggg cacgcgagta cgtgtgggcg ccaggccact gcccctgccc 1320
gatgctggca ccccaccggg actacctgat ggctgtccag cgtcttgtca gccccgacgg 1380
cacacaggac cagctgctgc tgccccacgc cggctacgcc cggccctgga gccctgcgga 1440
ggacagccgc atacgcctga ctgcccggcg ctgtcctggc tgagcccctg caggagcccc 1500
ggccacacac agcaagaaag atacatctga ccagcctcaa cgtcaacgta tttcccctct 1560
caccctggct tccaggcagc tctgaaatac gtcccacctg tgcagctatg tgactccctc 1620
ccacacacgc ttaagacacc tctgcatgca gtcaaagcca ctgtcacaag ccggcaggca 1680
ctggtgagga ggcactaagg agactctgac ttttatttcg cctctctcct tggctgccag 1740
gaagctcata gctatttata ctcagaaagt ttaacgctgc tttctttctc tttgcgcgcg 1800
tcacacttgc ttggagacac tgtcatgaac gagcatgaca ccctgctgcc ctgggtaccc 1860
agaagatcat ctgtttactt cccagacact gtgctgtctc tgctctctgc tactcacaca 1920
caccctcatg tgtgaagggc agagacactg tcacaaacag gcatgcccct tagaagacat 1980
gcctaaccag gcactgtaac gtaccaacgt accaatttcc ccttttcccc tggctaccag 2040
gaaactcgga gacaatcttt tcagcctcag catttctggc tggatttcca cccatcaaca 2100
cgtgcttgct cctccttttt tttttttctg aggtagacct tgctctgtca cctaggctga 2160
agtgcggtgg tgcaatcatg gctcactgca gcctcaaatt cctgggctca agcgatcctc 2220
40/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
ccactcagct ccacagcagt ggaactcacg tgtgatcaca tgccggctaa tttaaatttt 2280
gtagagatgg gcttgtacgt gccaaatgtc tcactatggc tcaacatctc tgctgggtcc 2340
aagactgaat aggatgacat gatggtgtac cccttatcct tatttcagct ttaaaaattc 2400
taaaaaaaaa 2410
<210> 36
<211> 549
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4904126CB1
<400> 36
gggaggagag aaaagccatg gccgacaagg tcctgaagga gaagagaaag cagtttatcc 60
gttcagtggg cgaaggtaca ataaatggct tactgggtga attattggag acaagggtgc 120
tgagccagga agagatagag atagtaaaat gtgaaaatgc tacagttatg gataaggccc 180
gagctttgct tgactctgtt attcggaaag gggctccagc atgccaaatt tgcatcacat 240
acatttgtga agaagacagt cacctggcag ggacgctggg actctcagca ggtccaacat 300
ctggaaatca ccttactaca caagattctc aaatagtact tccttcctag gtaatgctgt 360
ttttaaagaa agagcattct ttgaaccgtg gcttcccgtg acattaatgt tgtaggatga 420
accacagtta aaggggctat gaagaattcc catagagtga tcatacaatt ttctttttgt 480
aatctattct gcttttgtag caactgtcaa aacagcttca ctatctatgt ctacattaaa 540
atttggaat 549
<210> 37
<211> 2755
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 71268415CB1
<400> 37
ttgctaggag ggtggagttc atccacttat gatataaatg tctcttttta ttttttgcag 60
gcaacttttt gctccttcct acacagaaac ccattatact tcaagtggta accctcaaac 120
caccacacgg aaattggagg atcactgctt ttaccacggc acggtgaggg agacagaact 180
gtccagcgtc acgctcagca cttgccgagg aattagagga ctgattacgg tgagcagcaa 240
cctcagctac gtcatcgagc ccctccctga cagcaagggc caacacctta tttacagatc 300
tgaacatctc aagccgcccc cgggaaactg tgggttcgag cactccaagc ccaccaccag 360
ggactgggct cttcagttta cacaacagac caagaagcga cctcgcagga tgaaaaggga 420
agatttaaac tccatgaagt atgtggagct ttacctcgtg gctgattatt tagagtttca 480
gaagaatcga cgagaccagg acgccaccaa acacaagctc atagagatcg ccaactatgt 540
tgataagttt taccgatcct tgaacatccg gattgctctc gtgggcttgg aagtgtggac 600
ccacgggaac atgtgtgaag tttcagagaa tccatattct accctctggt cctttctcag 660
ttggaggcgc aagctgcttg cccagaagta ccatgacaac gcccaattaa tcacgggcat 720
gtccttccac ggcaccacca tcggcctggc ccccctcatg gccatgtgct ctgtgtacca 780
gtctggagga gtcaacatgg accactccga gaatgccatt ggcgtggctg ccaccatggc 840
ccacgagatg ggccacaact ttggcatgac ccatgattct gcagattgct gctcggccag 900
tgcggctgat ggtgggtgca tcatggcagc tgccactggg cacccctttc ccaaagtgtt 960
caatggatgc aacaggaggg agctggacag gtatctgcag tcaggtggtg gaatgtgtct 1020
ctccaacatg ccagacacca ggatgttgta tggaggccgg aggtgtggga acgggtatct 1080
ggaagatggg gaagagtgtg actgtggaga agaagaggaa tgtaacaacc cctgctgcaa 1.140
tgcctctaat tgtaccctga ggccgggggc ggagtgtgct cacggctcct gctgccacca 1200
gtgtaagctg ttggctcctg ggaccctgtg ccgcgagcag gccaggcagt gtgacctccc 1260
ggagttctgt acgggcaagt ctccccactg ccctaccaac ttctaccaga tggatggtac 1320
cccctgtgag ggcggccagg cctactgcta caacggcatg tgcctcacct accaggagca 1380
gtgccagcag ctgtggggac ccggagcccg acctgcccct gacctctgct tcgagaaggt 1440
gaatgtggca ggagacacct ttggaaactg tggaaaggac atgaatggtg aacacaggaa 1500
gtgcaacatg agagatgcga agtgtgggaa gatccagtgt cagagctctg aggcccggcc 1560
cctggagtcc aacgcggtgc ccattgacac cactatcatc atgaatggga ggcagatcca 1620
gtgccggggc acccacgtct accgaggtcc tgaggaggag ggtgacatgc tggacccagg 1680
gctggtgatg actggaacca agtgtggcta caaccatatt tgctttgagg ggcagtgcag 1740
gaacacctcc ttctttgaaa ctgaaggctg tgggaagaag tgcaatggcc atggggtctg 1800
taacaacaac cagaactgcc actgcctgcc gggctgggcc ccgcccttct gcaacacacc 1860
41/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
gggccacggg ggcagtatcg acagtgggcc tatgccccct gagagtgtgg gtcctgtggt 1920
agctggagtg ttggtggcca tcttggtgct ggcggtcctc atgctgatgt actactgctg 1980
cagacagaac aacaaactag gccaactcaa gccctcagct ctcccttcca agctgaggca 2040
acagttcagt tgtcccttca gggtttctca gaacagcggg actggtcatg ccaacccaac 2100
tttcaagctg cagacgcccc agggcaagcg aaaggtgatc aacactccgg aaatcctgcg 2160
gaagccctcc cagcctcctc cccggccccc tccagattat ctgcgtggtg ggtccccacc 2220
tgcaccactg ccagctcacc tgagcagggc tgctaggaac tccccagggc ccgggtctca 2280
aatagagagg acggagtcgt ccaggaggcc tcctccaagc cggccaattc cccccgcacc 2340
aaattgcatc gtttcccagg acttctccag gcctcggccg ccccagaagg cactcccggc 2400
aaacccagtg ccaggccgca ggagcctccc caggccagga ggtgcatccc cactgcggcc 2460
ccctggtgct ggccctcagc agtcccggcc tctggcagca cttgccccaa aggtgagtcc 2520
acgggaagcc ctcaaggtga aagctggtac cagagggctc caggggggca ggtgtagagt 2580
tgagaaaaca aagcaattca tgcttcttgt ggtctggact gaacttccag aacaaaagcc 2640
aagggcaaaa cattcatgtt tcttggtgcc cgcttgactg tggagttttg gcttcatgtg 2700
aaaggtgatt cttagaatcc tgagctgtgg tggcttcagt cctgcccctg cacct 2755
<210> 38
<211> 2553
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 7473301CB1
<400> 38
atggacaaag aaaacagcga tgtttcagcc gcacctgctg acctgaaaat atccaatatc 60
tcagtccaag tggtcagtgc ccaaaagaag ctgccagtga gacgaccacc gttgccaggg 120
agacgactac cattgccagg aagacgacca ccacaaagac ccattggcaa agccaaaccc 180
aagaagcaat ccaagaaaaa agttcccttt tggaatgtac aaaataaaat cattctcttc 240
acagtatttt tattcatcct agcagtcata gcctggacac ttctgtggct gtatatcagt 300
aagacagaaa gcaaagatgc tttttacttt gctgggatgt ttcgcatcac caacatcgag 360
tttcttcccg aataccgaca aaaggagtcc agggaatttc tttcagtgtc acggactgtg 420
cagcaagtga taaacctggt ttatacaaca tctgccttct ccaaatttta tgagcagtct 480
gttgttgcag atgtcagcag caacaacaaa ggcggcctcc ttgtccactt ttggattgtt 540
tttgtcatgc cacgtgccaa aggccacatc ttctgtgaag actgtgttgc cgccatcttg 600
aaggactcca tccagacaag catcataaac cggacctctg tggggagctt gcagggactg 660
gctgtggaca tggactctgt ggtactaaat ggtgattgtt ggtcattcct aaaaaaaaag 720
aaaagaaagg aaaatggtgc tgtctccaca gacaaaggct gctctcagta cttctatgca 780
gagcatctgt ctctccacta cccgctggag atttctgcag cctcagggag gctgatgtgt 840
cacttcaagc tggtggccat agtgggctac ctgattcgtc tctcaatcaa gtccatccaa 900
atcgaagccg acaactgtgt cactgactcc ctgaccattt acgactccct tttgcccatc 960
cggagcagca tcttgtacag aatttgtgaa cccacaagaa cattaatgtc atttgtttct 1020
acaaataatc tcatgttggt gacatttaag tctcctcata tacggaggct ctcaggaatc 1080
cgggcatatt ttgaggtcat tccagaacaa aagtgtgaaa acacagtgtt ggtcaaagac 1140
atcactggct ttgaagggaa aatttcaagc ccatattacc cgagctacta tcctccaaaa 1200
tgcaagtgta cctggaaatt tcagacttct ctatcaactc ttggcatagc actgaaattc 1260
tataactatt caataaccaa gaagagtatg aaaggctgtg agcatggatg gtgggaaatt 1320
tatgagcaca tgtactgtgg ctcctacatg gatcatcaga caatttttcg agtgcccagc 1380
cctctggttc acattcagct ccagtgcagt tcaaggcttt caggcaagcc acttttggca 1440
gaatatggca gttacaacat cagtcaaccc tgccctgtgg gatcttttag atgctcctcc 1500
ggtttatgtg tccctcaggc ccagcgtggt gatggagtaa atgactgctt tgatgaaagt 1560
gatgaactgt tttgcgtgag ccctcaacct gcctgcaata ccagctcctt caggcagcat 1620
ggccctctca tctgtgatgg cttcagggac tgtgagaatg gccgggatga gcaaaactgc 1680
actcaaagta ttccatgcaa caacagaact tttaagtgtg gcaatgatat ttgctttagg 1740
aaacaaaatg caaaatgtga tgggacagtg gattgtccag atggaagtga tgaagaaggc 1800
tgcacctgca gcaggagttc ctccgccctt caccgcatca tcggaggcac agacaccctg 1860
gaggggggtt ggccgtggca ggtcagcctc cactttgttg gatctgccta ctgtggtgcc 1920
tcagtcatct ccagggagtg gcttctttct gcagcccact gttttcatgg aaacaggctg 1980
tcagatccca caccatggac tgcacacctc gggatgtatg ttcaggggaa tgccaagttt 2040
gtctccccgg tgagaagaat tgtggtccac gagtactata acagtcagac ttttgattat 2100
gatattgctt tgctacagct cagtattgcc tggcctgaga ccctgaaaca gctcattcag 2160
ccaatatgca ttcctcccac tggtcagaga gttcgcagtg gggagaagtg ctgggtaact 2220
ggctgggggc gaagacacga agcagataat aaaggctccc tcgttctgca gcaagcggag 2280
gtagagctca ttgatcaaac gctctgtgtt tccacctacg ggatcatcac ttctcggatg 2340
ctctgtgcag gcataatgtc aggcaagaga gatgcctgca aaggagattc gggtggacct 2400
ttatcttgtc gaagaaaaag tgatggaaaa tggattttga ctggcattgt tagctgggga 2460
42/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
catggatgtg gacgaccaaa ctttcctggt gtttacacaa gggtgtcaaa ctttgttccc 2520
tggattcata aatatgtccc ttctcttttg taa 2553
<210> 39
<211> 1041
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473308CB1
<400> 39
atgttcagcg gcaacacagg aaaaacccat attatcaatg ctcaaaaacc tggccacctc 60
aggcttagcc agttattcgt gagcagagag gtgtgtcatc tacatggcag tcatggcctg 120
gatgggtctg gaactgtggc aagaatcctt ccaggaaaca gccggtctcc ctctctgctc 180
tcagaaggca agtttcctta tcacctgtct gctctcagaa ggcaagtttc cttatcacct 240
gtgaatcaca aacccacaga gtggccaaac atactgatgc aagaccatag gaaggggaaa 300
gctgcagttg gtgtctcctt tgatgatgat gacaagattg ttgggggcta caactgtgag 360
gagaattctg tcccctacca ggtgtccctg aattctggct accacttctg tgttggctcc 420
ctcaacaggg aatactgcat ccaggtgaga ctgggagagc acaacatcga agtcctagag 480
gggaatgaac agttcatcta tgcggtcaag atcatccgcc accccaaata caacagctgg 540
actctggaca atgacatcct gctgatcaag ctctccacac ctgccatcat caatgcccat 600
gtgtccacca tctctctgcc caccacccct ccagctgctg gcactgagtg cctcatctct 660
ggctggggca acactctgag ttctggcgcc gactacccag acgagctgca gtgcctggat 720
gctcctgtgc tgagccaggc tgagtatgaa gcctcctacc ctggaaagat taccaacaac 780
gtgttttgtg tgggtttcct tgagggaggc aaggattcct gccagattat tcctatcaaa 840
gtgcagcagc tggttacctc aagccaagag acagacataa ggatccctat ggccttgcag 900
acagctgctt ccacctccta cctgggcccc ttagactctt tacacaggaa agtgagtcac 960
cccactgaga agcgttgcca gcagaaacag ggcatgaaaa tcacagataa ccatgggatt 1020
acttccaagt ggtcagtata a 1041
<210> 40
<211> 1707
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7478021CB1
<400> 40
atgctcgccg cctccatctt ccgtccgaca ctgctgctct gctggctggc tgctccctgg 60
cccacccagc ccgagagtct cttccacagc cgggaccgct cggacctgga gccgtcccca 120
ctgcgccagg ccaagcccat tgccgacctc cacgctgctc agcggttcct gtccagatac 180
ggctggtcag gggtgtgggc ggcctggggg cccagtcccg aggggccgcc ggagaccccc 240
aagggcgccg ccctggccga ggcggtgcgc aggttccagc gggcgaacgc gctgccggcc 300
agcggggagc tggacgcggc caccctagcg gccatgaacc ggccgcgctg cggggtcccg 360
gacatgcgcc caccgccccc ctccgccccg ccttcgcccc cgggcccgcc ccccagagcc 420
cgctccaggc gctccccgcg ggcgccgctg tccttgtccc ggcggggttg gcagccccgg 480
ggctaccccg acggcggagc tgcccaggcc ttctccaaga ggacgctgag ctggcggctg 540
ctgggcgagg ccctgagcag ccaactgtcc gtggccgacc agcggcgcat agaggcgctg 600
gccttcagga tgtggagcga ggtgacgccg ctggacttcc gcgaggacct ggccgccccc 660
ggggccgcgg tcgacatcaa gctgggcttt gggagacggc acctgggctg tccgcgggcc 720
ttcgatggga gcgggcagga gtttgcacac gcctggcgcc taggtgacat tcactttgac 780
gacgacgagc acttcacacc tcccaccagt gacacgggca tcagccttct caaggtggcc 840
gtccatgaaa ttggccatgt cctgggcttg cctcacacct acaggacggg atccataatg 900
caaccaaatt acattcccca ggagcctgcc tttgagttgg actggtcaga caggaaagca 960
attcaaaagc tgtatggttc ctgtgaggga tcatttgata ctgcgtttga ctggattcgc 1020
aaagagagaa accaatatgg agaggtgatg gtgagattta gcacatattt cttccgtaac 1080
agctggtact ggctttatga aaatcgaaac aataggacac gctatgggga ccctatccaa 1140
atcctcactg gctggcctgg aatcccaaca cacaacatag atgcctttgt tcacatctgg 1200
acatggaaaa gagatgaacg ttattttttt caaggaaatc aatactggag atatgacagt 1260
gacaaggatc aggccctcac agaagatgaa caaggaaaaa gctatcccaa attgatttca 1320
gaaggatttc ctggcatccc aagtccccta gacacggcgt tttatgaccg aagacagaag 1380
ttaatttact tcttcaagga gtcccttgta tttgcatttg atgtcaacag aaatcgagta 1440
cttaattctt atccaaagag gattactgaa gtttttccag cagtaatacc acaaaatcat 1500
43/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
cctttcagaa atatagattc cgcttattac tcctatgcat acaactccat tttctttttc 1560
aaaggcaatg catactggaa ggtagttaat gacaaggaca aacaacagaa ttcctggctt 1620
cctgctaatg gcttatttcc aaaaaagttt atttcagaga agtggtttga tgtttgtgac 1680
gtccatatct ccacactgaa catgtaa ' 1707
<210> 41
<211> 1262
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4333459CB1
<400> 41
aaaagatctt tgcgaaacac tacattcaga aacatcagat ggacatgctt gattcaccac 60
gtcttggtta atgaataaac ttgttttaaa ttggcttatt gctggtctct caaggcttcc 120
tatttttgtt tgctttagtc tctctaaaat ttcagggaaa aactatgagt ctcaaaatgc 180
ttataagcag gaacaagctg attttactac taggaatagt cttttttgaa cgaggtaaat 240
ctgcaactct ttcgctcccc aaagctccca gttgtgggca gagtctggtt aaggtacagc 300
cttggaatta ttttaacatt ttcagtcgca ttcttggagg aagccaagtg gagaagggtt 360
cctatccctg gcaggtatct ctgaaacaaa ggcagaagca tatttgtgga ggaagcatcg 420
tctcaccaca gtgggtgatc acggcggctc actgcattgc aaacagaaac attgtgtcta 480
ctttgaatgt tactgctgga gagtatgact taagccagac agacccagga gagcaaactc 540
tcactattga aactgtcatc atacatccac atttctccac caagaaacca atggactatg 600
atattgccct ,tttgaagatg gctggagcct tccaatttgg ccactttgtg gggcccatat 660
gtcttccaga gctgcgggag caatttgagg ctggttttat ttgtacaact gcaggctggg 720
gccgcttaac tgaaggtggc gtcctctcac aagtcttgca ggaagtgaat ctgcctattt 780
tgacctggga agagtgtgtg gcagctctgt taacactaaa gaggcccatc agtgggaaga 840
cctttctttg cacaggtttt cctgatggag ggagagacgc atgtcaggga gattcaggag 900
gttcactcat gtgccggaat aagaaagggg~cctggactct ggctggtgtg acttcctggg 960
gtttgggctg tggtcgaggc tggagaaaca atgtgaggaa aagtgatcaa ggatcccctg 1020
ggatcttcac agacattagt aaagtgcttt cctggatcca cgaacacatc caaactggta 1080
actaagccat cacacaaggt taagaagctg ccattctgct agggccagag acagcatcag 1140
cagagtcctg gcaaatcaga gcacctgaac caacaggctc tacctctgtt ctcagtgtag 1200
cacacaagga ttgtgaggtt taccaagtct aaataaaaca agagttaaat atggtaaaaa 1260
as 1262
<210> 42
<211> 3067
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6817347CB1
<400> 42
gcactgtgaa cgttggttgc atccaaatct gaattttgtc tgggaccagg gtcagggacc 60
agaatacacc agagctgagg gccagcccta cctgagaacc atcaacaaac ttaccccaca 120
tcccattata cctcctcact ccctgcagcc tgtcagcttc cccaatctcc cacactcact 180
gtcacctggg gctctggtgc accagatgac actacttgct ccctggtaca caggccccat 240
gatccccatg gatgttaatg agcccagctc cgtgaccacg gctcctaccc tcagctctag 300
cctgcagcat atctcctcat tcctggccac tggtaagaaa ctttccctcc attttggtca 360
tccacgtgag tgtgaagtca ccaggattga tgacaaaaat agaagaggat tggaagacag 420
tgagccaggt gccaaactct tcaataatga tggagtctgt tgttgcctgc aaaaacgggg 480
gccagtgaac attacatcag tgtgtgtgag tcccaggacc ttacaaatat cagtttttgt 540
gttatcagag aaatacgagg gtattgttaa atttgaatcg gatgaattac cttttggtgt 600
aattggttct aatattggtg atgcacattt tcaagaattc agggctggaa tctcctggaa 660
gcctgtggta gatcctgatg accccattcc tcagttccct gattgctgca gcagcagcag 720
cagcaggatt ccttcagtga gtgtgctagt tgcagttcct ctggttgcag gccacaaagg 780
gcaggcattt attgaaagga tgctggggtg cttcaaggaa ttgaagcaag agctgactca 840
ggaagggccg ggcgggggac accccaggtc tgcgtggccc ccgcgccgcc acgcccagtg 900
gccgcccgag ccctgcgagc agggggagga gccgccgcca gtggaggcgg aggaggtaga 960
ggaggcggag acggcggaga aggcggagag gaaggtggag gcggaggcga aggtggaggg 1020
gaaggcggag gcggcgggga aggcggaggc ggcggggaag gtggacgcca ccgagaaggt 1080
ggagacggcg gggaaggtgg acgccgctgg gaaggtggag acggcggagg gtccgggccg 1140
44/45
CA 02411971 2002-12-10
WO 01/98468 PCT/USO1/19178
ccgggctgag ctcaagctgg agcccgaacc cgagccggtc cgggaggcgg agcaggagcc 1200
gaagcaggag ctggaggatg agaacccagc gcggagcggc ggtggcggca acagcgacga 1260
ggttcctccc cccacccttc cctccgatcc accgcggccc cccgatccct ctccgcgtcg 1320
cagtcgtgcg ccgcgccgcc gaccccggcc ccggccccag acccggctcc gtaccccgcc 1380
gcagcctagg ccccggcccc cgccccggcc ccggccccgg cgcggccctg ggggcggatg 1440
cctggatgtg gattttgccg tggggccacc aggctgttct cacgtgaaca gctttaaggt 1500
gggagagaac tggaggcagg aactgcgggt tatctaccag tgcttcgtgt ggtgtggaac 1560
cccagagacc aggaaaagca aggcaaagtc ctgcatctgc catgtgtgtg gcacccatct 1620
gaacagactc cactcttgcc tttcctgtgt cttctttggc tgcttcacgg agaaacacat 1680
tcacgagcac gcagagacga aacaacacaa cttagcagta gacctgtatt acggaggtat 1740
atactgcttt atgtgtaagg actatgtata tgacaaagac attgagcaaa ttgccaaaga 1800
agagcaagga gaagctttga aattacaagc ctccacctca acagaggttt ctcaccagca 1860
gtgttcagtg ccaggccttg gtgagaaatt cccaacctgg gaaacaacca aaccagaatt 1920
agaactgctg gggcacaacc cgaggagaag aagaatcacc tccagcttta cgatcggttt 1980
aagaggactc atcaatcttg gcaacacgtg ctttatgaac tgcattgtcc aggccctcac 2040
ccacacgccg atactgagag atttctttct ctctgacagg caccgatgtg agatgccgag 2100
tcccgagttg tgtctggtct gtgagatgtc gtcgctgttt cgggagttgt attctggaaa 2160
cccgtctcct catgtgccct ataagttact gcacctggtg tggatacatg cccgccattt 2220
agcagggtac aggcaacagg atgcccacga gttcctcatt gcagcgttag atgtcctgca 2280
caggcactgc aaaggtgatg atgtcgggaa ggcggccaac aatcccaacc actgtaactg 2340
catcatagac caaatcttca caggtggcct gcagtctgat gtcacctgtc aagcctgcca 2400
tggcgtctcc accacgatag acccatgctg ggacattagt ttggacttgc ctggctcttg 2460
cacctccttc tggcccatga gcccagggag ggagagcagt gtgaacgggg aaagccacat 2520
accaggaatc accaccctca cggactgctt gcggaggttt acgaggccag agcacttagg 2580
aagcagtgcc aaaatcaaat gtggtagttg ccaaagctac caggaatcta ccaaacagct 2640
cacaatgaat aaattacctg tcgttgcctg ttttcatttc aaacggtttg aacattcagc 2700
gaaacagagg cgcaagatca ctacatacat ttcctttcct ctggagctgg atatgacgcc 2760
gtttatggcc tcaagtaaag agagcagaat gaatggacaa ttgcagctgc caaccaatag 2820
tggaaacaac gaaaataagt attccttgtt tgctgtggtt aatcaccaag gaaccttgga 2880
gagtggccac tataccagct tcatccggca ccacaaggac cagtggttca agtgtgatga 2940
tgccgtcatc actaaggcca gtattaagga cgtactggac agtgaagggt atttactgtt 3000
ctatcacaaa caggtgctag aacatgagtc agaaaaagtg aaagaaatga acacacaagc 3060
ctactga 3067
45/45
