Note: Descriptions are shown in the official language in which they were submitted.
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PROTEASES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of proteases
and to the use of
these sequences in the diagnosis, treatment, and prevention of
gastrointestinal, cardiovascular,
autoimmune/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) Proteol 'c 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. Barren (1994) Meth. Enzymol. 244:19-
61).
Most mammalian serine proteases are synthesized as zymogens, inactive
precursors that are
activated by proteolysis. For example, trypsinogen is converted to its active
form, trypsin, by
enteropeptidase. Enteropeptidase is an intestinal protease that removes an N-
terminal fragment from
. trypsinogen. The:remaining active fragment is trypsin, which in turn
activates the precursors of the
other pancreatic enzymes. Likewise proteolysis of prothrombin, the precursor
of thrombin generates
three separate polypeptide fragments. The N-terminal fragment is released
while the other two
fragments, .which comprise active thrombin, remain associatedthrough disulfide
bonds.
The two largest SP subfamilies are the chymotrypsin (S1) and subtilisin (S8)
families. Some
members of the chymotrypsin family contain two structural domains unique to
this family. Kringle
domains are triple-looped, disulfide cross-linked domains found in varying
copy number. Kringles are
thought to play a role in binding mediators such as membranes, other proteins
or phospholipids, and in
the regulation of proteolytic activity (PROSITE PDOC00020). Apple domains are
90 amino-acid
. repeated domains; each containing six conserved cysteines. Three disulfide
bonds link the first and
sixth, second and fifth, and third and fourth cysteines (PROSITE PDOC00376).
Apple domains are
involved in protein-protein interactions. S 1 family members include trypsin,
chymotrypsin, coagulation
factors IX-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 PC 1, PC2, PC3, PC6, and PACE4
(Rawlings and Barrett,
supra).
SPs have functions in many normal processes and some have been implicated in
the etiology or
treatment of disease. Enterokinase, the initiator of intestinal digestion, is
found in the intestinal brush
border, where it cleaves the acidic propeptide from trypsinogen to yield
active trypsin (Kitamoto, Y. et
al. (1994) Proc. Natl. Acad. Sci. USA 91:7588-7592). Prolylcarboxypeptidase, a
lysosomal serine
peptidase that cleaves peptides such as angiotensin II and III and [des-Arg9]
bradykinin, shares
sequence homology with members of both the serine carboxypeptidase and
prolylendopeptidase families
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(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). Tissueplasminogen 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 phosphorylation,
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
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
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types, including proteins that function to activate or repress cellular
processes such as transcription and
cell cycle progression (Ciechanover, A. (1994) Cell 79:13-21). In the UCS
pathway, proteins targeted
for degradation are conjugated to ubiquitin, a small heat stable protein. The
ubiquitinated protein is
then recognized and degraded by the proteasome. The resultant ubiquitin-
peptide complex is
hydrolyzed by a ubiquitin carboxyl terminal hydrolase, and free ubiquitin is
released for reutilization by
the UCS. Ubiquitin-proteasome systems are implicated in the degradation of
mitotic cyclic kinases,
oncoproteins, tumor suppressor genes (p53), cell surface receptors associated
with signal transduction,
transcriptional regulators, and mutated or damaged proteins (Ciechanover,
supra). This pathway has
been implicated in a number of diseases, including cystic fibrosis, Angelman's
syndrome, and Liddle
syndrome (reviewed in Schwartz, A.L. and A. Ciechanover (1999) Annu. Rev. Med.
50:57-74). A
marine proto-oncogene, Unp, encodes a nuclear ubiquitin protease whose
overexpression leads to
oncogenic transformation of NIH3T3 cells. The human homologue of this gene is
consistently elevated
in small cell tumors and adenocarcinomas of the lung (Gray, D.A. (1995)
Oncogene 10:2179-2183).
Ubiquitin carboxyl terminal hydrolase is involved in the differentiation of a
lymphoblastic leukemia cell
line to a non-dividing mature state (Maki, A. et al. (1996) Differentiation
60:59-66). In neurons,
ubiquitin carboxyl terminal hydrolase (PGP 9.5) expression is strong in the
abnormal structures that
occur in human neurodegenerative diseases (Lowe, J. et al. (1990) J. Pathol.
161:153-160). The
proteasome is a large (2000 kDa) multisubunit complex composed of a central
catalytic core
containing a variety of proteases arranged in four seven-membered rings with
the active sites facing
inwards into the central cavity, and terminal ATPase subunits covering the
outer port of the cavity and
regulating substrate entry (for review, see Schmidt, M. et al. (1999) Curr.
Opin. Chem. Biol. 3:584-
591).
Cysteine Proteases
Cysteine proteases (CPs) are involved in diverse cellular processes ranging
from the processing
of precursor proteins to intracellular degradation. Nearly half of the CPs
known are present only in
viruses. CPs have a cysteine as the major catalytic residue at the active site
where catalysis proceeds
via a thioester intermediate and is facilitated by nearby histidine and
asparagine residues. A glutamine
residue is also important, as it helps to form an oxyanion hole. Two important
CP families include the
papain-like enzymes (C 1) and the calpains (C2). Papain-like family members
are generally lysosomal
or secreted and therefore are synthesized with signal peptides as well as
propeptides. Most members
bear a conserved motif in the propeptide that may have structural significance
(Karrer, K.M. et al.
(1993) Proc. Natl. Acad. Sci. USA 90:3063-3067). Three-dimensional structures
of papain family
members show a bilobed molecule with the catalytic site located between the
two lobes. Papains
include cathepsins B, C, H, L, and S, certain plant allergens and dipeptidyl
peptidase (for a review, see
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Rawlings, N.D. and A.J. Barrett (1994) Meth. 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
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 specific aspartate residue (D297 in the caspase-
1 numbering convention)
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and removal of the spacer and prodomain, leaving a p 10/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, supra; Salveson, G.S. and V.M. Dixit
(1999) Proc. Natl.
Acad. Sci. USA 96:10964-10967).
Caspases have been implicated in a number of diseases. Mice lacking some
caspases have
severe nervous system defects due to failed apoptosis in the neuroepithelium
and suffer early lethality.
Others show severe defects in the inflammatory response, as caspases are
responsible for processing IL-
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, supra;
Thompson, C.B. (1995) Science 267:1456-1462).
Aspartyl proteases
Aspartyl proteases (APs) include the lysosomal proteases cathepsins D and E,
as well as
chymosin, renin, and the gastric pepsins. Most retroviruses encode an AP,
usually as part of the Col
polyprotein. APs, also called acid proteases, are monomeric enzymes consisting
of two domains, each
domain containing one half of the active site with its own catalytic aspartic
acid residue. APs are most
active in the range of pH 2-3, at which one of the aspartate residues is
ionized and the other neutral.
The pepsin family of APs contains many secreted enzymes, and all are likely to
be synthesized with
signal peptides and propeptides. Most family members have three disulfide
loops, the first ~5 residue
loop following the first aspartate, the second 5-6 residue loop preceding the
second aspartate, and the
third and largest loop occurring toward the C terminus. Retropepsins, on the
other hand, are analogous
to a single domain of pepsin, and become active as homodimers with each
retropepsin monomer
contributing one half of the active site. Retropepsins are required for
processing the viral polyproteins.
APs have roles in various tissues, and some have been associated with disease.
Renin mediates
the first step in processing the hormone angiotensin, which is responsible for
regulating electrolyte
balance and blood pressure (reviewed in Crews, D.E. and S.R. Williams (1999)
Hum. Biol. 71:475-
503). Abnormal regulation and expression of cathepsins are evident in various
inflammatory disease
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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
Most zinc-dependent metalloproteases share a common sequence in the zinc-
binding domain.
The active site is made up of two histidines which act as zinc ligands and a
catalytic glutamic acid C-
terminal to the first histidine. Proteins containing this signature sequence
are known as the metzincins
and include aminopeptidase N, angiotensin-converting enzyme, neurolysin, the
matrix metalloproteases
and the adamalysins (ADAMS). An alternate sequence is found in the zinc
carboxypeptidases, in which
all three conserved residues - two histidines and a glutamic acid - are
involved in zinc binding.
A number of the neutral metalloendopeptidases, including angiotensin
converting enzyme and
the aminopeptidases, are involved in the metabolism of peptide hormones. High
aminopeptidase B
activity, for example, is found in the adrenal glands and neurohypophyses of
hypertensive rats (Prieto,
I. et al. (1998) Horm. Metab. Res. 30:246-248). Oligopeptidase Mlneurolysin
can hydrolyze
bradykinin as well as neurotensin (Serizawa, A. et al. (1995) J. Biol. Chem
270:2092-2098).
Neurotensin is a vasoactive peptide that can act as a neurotransmitter in the
brain, where it has been
implicated in limiting food intake (Tritos, N.A. et al. (1999) Neuropeptides
33:339-349).
The matrix metalloproteases (MMPs) are a family of at least 23 enzymes that
can degrade
components of the extracellular matrix (ECM). They are Zn+2 endopeptidases
with an N-terminal
catalytic domain. Nearly all members of the family have a hinge peptide and C-
terminal domain which
can bind to substrate molecules in the ECM or to inhibitors produced by the
tissue (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).
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
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(Saarialho-Kere, U.K. et al. (1994) J. Clip. Invest. 94:79), bone resorption
(Blavier, L. and J.M.
Delaisse (1995) J. Cell Sci. 108:3649), age-related macular degeneration
(Steep, 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. Clip. 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.
ADAMs are implicated in such processes as sperm-egg binding and fusion,
myoblast fusion,
and protein-ectodomain processing or shedding of cytokines, cytokine
receptors, adhesion proteins and
other extracellular protein domains (Schlondorff, J. and C.P. Blobel (1999) J.
Cell. Sci. 112:3603-
3617). The Kuzbanian protein cleaves a substrate in the NOTCH pathway
(possibly NOTCH itself),
activating the program for lateral inhibition in 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.
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(1997) J. Biol. Chem. 272:556). To date eleven members are recognized by the
Human Genome
Organization (HUGO;
http://www.gene.ucl.ac.uk/users/pester/adamts.htsr~l.#Approved). Members of
this family have the ability to degrade aggrecan, a high molecular weight
proteoglycan which provides
cartilage with important. mechanical properties including compressibility, and
which is, lost during the
development of arthritis. Enzymes which degrade aggrecan are thus considered
attractive targets to
prevent and slow the degradation of articular cartilage (See, e.g.,
Tortorella, M.D. (1999) Science
284:1664; Abbaszade, I. (1999) J. Biol. Chem. 274:23443). Other members are
reported to have
antiangiogenic potential (Kuno et al., supra) and/or procollagen processing
(Colige, A. et al. (1997)
Proc. Natl. Acad. Sci. USA 94:2374).
Protease inhibitors
Protease inhibitors and other regulators of protease activity control the
activity and effects of
proteases. Protease inhibitors have been shown to control pathogenesis in
animal models of proteolytic
disorders (Murphy, G. (1991) Agents Actions Suppl. 35:69-76). Low levels of
the cystatins, low
molecular weight inhibitors of the cysteine proteases, correlate with
malignant progression of tumors
(Calkins, C. et al. (1995) Biol. Biochem. Hoppe Seyler 376:71-80). Serpins are
inhibitors of
mammalian plasma serine proteases. Many serpins serve to regulate the blood
clotting cascade and/or
the complement cascade in mammals. Sp32 is a positive regulator of the
mammalian acrosomal
protease, acrosin, that binds the proenzyme, proacrosin, and thereby aides in
packaging the enzyme into
the acrosomal matrix (Baba, T. et al. (1994) J. Biol. Chem. 269:10133-10140).
The Kunitz family of
serine protease inhibitors are characterized by one or more "Kunitz domains"
containing a series of
cysteine residues that are regularly spaced over approximately 50 amino acid
residues and form three
intrachain disulfide bonds. Members of this family include aprotinin, tissue
factor pathway inhibitor
(TFPI-1 and TFPI-2), inter-a-trypsin inhibitor, and bikunin. (Manor, C.W. et
al. (1997) J. Biol. Chem.
272:12202-12208.) Members of this family are potent inhibitors (in the
nanomolar range) against
serine proteases such as kallikrein and plasmin. Aprotinin has clinical
utility in reduction of
perioperative blood loss.
The discovery of new proteases and the polynucleotides encoding them satisfies
a need in the
art by providing new compositions which are useful in the diagnosis,
prevention, and treatment of
gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative,
developmental, epithelial,
neurological, and reproductive disorders, and in the assessment of the effects
of exogenous compounds
on the expression of nucleic acid and amino acid sequences of proteases.
SUMMARY OF THE INVENTION
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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,"
"PRTS-8," "PRTS-9," "PRTS-10," "PRTS-11," "PRTS-12," "PRTS-13," and "PRTS-14."
In one
aspect, the invention provides an isolated polypeptide comprising an amino
acid sequence selected from
the group consisting of a) an amino acid sequence selected from the group
consisting of SEQ ID N0:1-
14, b) a naturally occurring amino acid sequence having at least 90% sequence
identity to an amino
acid sequence selected from the group consisting of SEQ ID N0:1-14, c) a
biologically active fragment
of an amino acid sequence selected from the group consisting of SEQ ID N0:1-
14, and d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID N0:1-
14. In one alternative, the invention provides an isolated polypeptide
comprising the amino acid
sequence of SEQ ID N0:1-14.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising an
amino acid sequence selected from the group consisting of a) an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-14, b) a naturally occurring amino acid
sequence having at least
90% sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID N0:1-
14, c) a biologically active fragment of an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-14, and d) an immunogenic fragment of an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-14. In one alternative, the polynucleotide encodes a
polypeptide selected
from the group consisting of SEQ ID N0:1-14. In another alternative, the
polynucleotide is selected
from the group consisting of SEQ ID N0:15-28.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising
an amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-14, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID N0:1-14, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
N0:1-14, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID N0:1-14. 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 comprising an
amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-14, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID N0:1-14, c)
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a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
NO:1-14, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID N0:1-14. 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 comprising an amino acid sequence selected from the group
consisting of a) an amino acid
sequence selected from the group consisting of SEQ ID NO:l-14, b) a naturally
occurring amino acid
sequence having at least 90% sequence identity to an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-14, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-14, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID N0:1-14.
The invention further provides an isolated polynucleotide comprising a
polynucleotide sequence
selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID N0:15-28, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:15-28, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to 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 comprising a
polynucleotide sequence
selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID N0:15-28, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:15-28, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to 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
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target polynucleotide having a sequence of a polynucleotide comprising a
polynucleotide sequence
selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID N0:15-28, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID NO:15-28, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to 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
comprising an amino acid sequence selected from the group consisting of a) an
amino acid sequence
selected from the group consisting of SEQ ID N0:1-14, b) a naturally occurring
amino acid sequence
having at least 90% sequence identity to an amino acid sequence selected from
the group consisting of
SEQ ID N0:1-14, c) a biologically active fragment of an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-14, and d) an immunogenic fragment of an amino acid
sequence selected
from the group consisting of SEQ ID N0:1-14, and a pharmaceutically acceptable
excipieni" In one
embodiment, the composition comprises an amino acid sequence selected from the
group consisting of
SEQ ID N0:1-14. 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 comprising an amino acid sequence selected from the
group consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID N0:1-14, b) a
naturally occurring
amino acid sequence having at least 90% sequence identity to an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-14, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-14, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:l-14. 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 comprising an amino acid sequence selected from
the group consisting
of a) an amino acid sequence selected from the group consisting of SEQ ID N0:1-
14, b) a naturally
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occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID N0:1-14, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID N0:1-14, and d) an
immunogenic fragment
of an amino acid sequence selected from the group consisting of SEQ ID N0:1-
14. 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 comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID N0:1-14, b) a
naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-14, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-14, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID N0:1-14. 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 comprising an amino acid sequence selected from the
group consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID N0:1-14, b) a
naturally occurring
amino acid sequence having at least 90% sequence identity to an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-14, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID N0:1-14, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID N0:1-14. The method
comprises a)
combining the polypeptide with at least one test compound under conditions
permissive for the
activity of the polypeptide, b) assessing the activity of the polypeptide in
the presence of the test
compound, and c) comparing the activity of the polypeptide in the presence of
the test compound with
the activity of the polypeptide in the absence of the test compound, wherein a
change in the activity of
the polypeptide in the presence of the test compound is indicative of a
compound that modulates the
activity of the polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
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sequence selected from the group consisting of SEQ ID N0:15-28, 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 comprising a polynucleotide
sequence selected from the
group consisting of i) a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:15-28, ii) a naturally occurring polynucleotide sequence having at least
90°lo sequence identity to
a polynucleotide sequence selected from the group consisting of SEQ ID N0:15-
28, iii) a
polynucleotide sequence complementary to i), iv) a polynucleotide sequence
complementary to 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 comprising a polynucleotide sequence
selected from the group
consisting of i) a polynucleotide sequence selected from the group consisting
of SEQ ID N0:15-28,
ii) a naturally occurring polynucleotide sequence having at least 90% sequence
identity to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:15-28,
iii) a
polynucleotide sequence complementary to i), iv) a polynucleotide sequence
complementary to 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 and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for each polypeptide of the invention. The probability score for the
match between each
polypeptide and its GenBank homolog is also shown.
Table 3 shows structural features of each polypeptide sequence, including
predicted motifs and
domains, along with the methods, algorithms, and searchable databases used for
analysis of each
polypeptide.
Table 4 lists the cDNA and genomic DNA fragments which were used to assemble
each
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polynucleotide sequence, along with selected fragments of the polynucleotide
sequences.
Table 5 shows the representative cDNA library for each polynucleotide 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 lines,
protocols, reagents and vectors which are reported in the publications and
which might be used in
connection with the invention. Nothing herein is to be construed as an
admission that the invention is
not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"PRTS" refers to the amino acid sequences of substantially purified PRTS
obtained from any
species, particularly a mammalian species, including bovine, ovine, porcine,
marine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
PRTS. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of PRTS either by
directly interacting with
PRTS or by acting on components of the biological pathway in which PRTS
participates.
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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, andlor 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
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid sequence
to the complete native amino acid sequence associated with the recited protein
molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well known
in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity of
PRTS. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of PRTS either by
directly interacting with PRTS or by acting on components of the biological
pathway in which PRTS
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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. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic PRTS, or of
any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
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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 least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Tle 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 Tle, Leu, Thr
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Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of the
side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
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.
A "fragment" is a unique portion of PRTS or the polynucleotide encoding PRTS
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:15-28 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:15-28, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID NO:15-28 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:1S-28 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:15-28 and the region of SEQ ID N0:15-28 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 N0:1-14 is encoded by a fragment of SEQ ID N0:15-28. A
fragment
of SEQ ID N0:1-14 comprises a region of unique amino acid sequence that
specifically identifies
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SEQ ID N0:1-14. For example, a fragment of SEQ ID N0:1-14 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID N0:1-14.
The precise length of
a fragment of SEQ ID NO:l-14 and the region of SEQ ID N0:1-14 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full
length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer to
the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible'way, gaps in
the sequences being compared in order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence
alignment program. This program is part of the LASERGENE software package, a
suite of molecular
biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in
Higgins, D.G.
and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992)
CABIOS 8:189-191.
For pairwise 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
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
CA 02394789 2002-06-18
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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 foY match: 1
Penalty for mismatch: -2
Open Gap: 5 ai2d Extension Gap: 2 penalties
Gap x drop-off. 50
Expect: 1 D
Word Size: 1l
Filter: ora
Percent identity may be measured over the length of an entire defined
sequence, for example, as
defined by a particular SEQ ID number, or may be measured over a shorter
length, for example, over
the length of a fragment taken from a larger, defined sequence, for instance,
a fragment of at least 20, at
least 30, at least 40, at least 50, at least 70, at least 100, or at least 200
contiguous nucleotides. Such
lengths are exemplary only, and it is understood that any fragment length
supported by the sequences
shown herein, in the tables, figures, or Sequence Listing, may be used to
describe a length over which
percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes in
a nucleic acid sequence can be made using this degeneracy to produce multiple
nucleic acid sequences
that all encode substantially the same protein.
The phrases "percent identity" and "% 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=S, 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.
21
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Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12
(April-21-2000) with blastp set at default parameters. Such default parameters
may be, for example:
Matrix: BLOSUM62
Open Gap: II afad Extension Gap: 1 penalties
Gap x drop-off. 50
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence, for
example, as defined by a particular SEQ ID number, or may be measured over a
shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for instance,
a fragment of at least 15, at least 20, at least 30, at least 40, at least 50,
at least 70 or at least 150
contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment length
supported by the sequences shown herein, in the tables, figures or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the stringency
of the hybridization process, with more stringent conditions allowing less non-
specific binding, i.e.,
binding between pairs of nucleic acid strands that are not perfectly matched.
Permissive conditions for
annealing of nucleic acid sequences are routinely determinable by one of
ordinary skill in the art and
may be consistent among hybridization experiments, whereas wash conditions may
be varied among
experiments to achieve the desired stringency, and therefore hybridization
specificity. 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 pg/ml sheared, denatured salmon sperm DNA.
22
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WO 01/46443 PCT/US00/34811
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 Tm is the temperature (under defined ionic strength and
pH) at which 50% of the
target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and conditions
for nucleic acid hybridization are well known and can be found in Sambrook, J.
et al. (1989) Molecular
Cloning: A Laboratory Manual, 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
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 an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment of
23
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
PRTS which is useful in any of the antibody production methods disclosed
herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides, polypeptides,
or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of PRTS. For example,
modulation may
cause an increase or a decrease in protein activity, binding characteristics,
or any other biological,
functional, or immunological properties of PRTS.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an PRTS may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in the
art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary by
cell type depending on the enzymatic milieu of PRTS.
"Probe" refers to nucleic acid sequences encoding PRTS, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule. Typical
labels include radioactive isotopes, ligands, chemiluminescent agents, and
enzymes. "Primers" are
short nucleic acids, usually DNA oligonucleotides, which may be annealed to a
target polynucleotide by
complementary base-pairing. The primer may then be extended along the target
DNA strand by a DNA
polymerase enzyme. Primer pairs can be used for amplification (and
identification) of a nucleic acid
sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
24
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers may
be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas South
West Medical Center, Dallas TX) is capable of choosing specific primers from
megabase sequences
and is thus useful for designing primers on a genome-wide scope. The Primer3
primer selection
program (available to the public from the Whitehead InstitutelMIT Center for
Genome Research,
Cambridge MA) allows the user to input a "mispriming library," in which
sequences to avoid'as primer
binding sites are user-specified. Primer3 is useful, in particular, for the
selection of oligonucleotides for
microarrays. (The source code for the latter two primer selection programs may
also be obtained from
their respective sources and modified to meet the user's specific needs.) The
PrimeGen program
(available to the public from the UK Human Genome Mapping Project Resource
Centre, Cambridge
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
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for
example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions (UTRs).
Regulatory elements interact with host or viral proteins which control
transcription, translation, or RNA
stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or 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
26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75 % free, and most preferably at least 90% free from
other components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides by
different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" 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 eukaxyotic 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
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.
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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 95% or
at least 98% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an "allelic"
(as defined above), "splice," "species," or "polymorphic" variant. A splice
variant may have significant
identity to a reference molecule, but will generally have a greater or lesser
number of polynucleotides
due to alternative splicing of exons during mRNA processing. The corresponding
polypeptide may
possess additional functional domains or lack domains that are present in the
reference molecule.
Species variants are polynucleotide sequences that vary from one species to
another. The resulting
polypeptides will generally have significant amino acid identity relative to
each other. A polymorphic
variant is a variation in the polynucleotide sequence of a particular gene
between individuals of a given
species. Polymorphic variants also may encompass "single nucleotide
polymorphisms" (SNPs) in
which the polynucleotide sequence varies by one nucleotide base. The presence
of SNPs may be
indicative of, for example, a certain population, a disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having at
least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of the
polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
set at default parameters. Such a pair of polypeptides may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, at least 95 %, or at least 98 %
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, autoimmunelinflammatory, cell proliferative,
developmental, epitheliah
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.
2~
CA 02394789 2002-06-18
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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 each polypeptide 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
homolog. Column 5 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 each of the polypeptides of the
invention. Columns
1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and
the corresponding
Incyte polypeptide sequence number (Incyte Polypeptide ID) for each
polypeptide of the invention.
Column 3 shows the number of amino acid residues in each polypeptide. Column 4
shows potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the MOTIFS
program of the GCG sequence analysis software package (Genetics Computer
Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structurelfunction analysis and in some
cases, searchable
databases to which the analytical methods were applied.
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 N0:15-28 or that distinguish between SEQ ID
N0:15-28 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 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,
7032724H1 is the
identification number of an Incyte cDNA sequence, and BRAXTDRl2 is the cDNA
library from which
29
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
it is derived. Incyte cDNAs for which cDNA libraries are not indicated were
derived from pooled
cDNA libraries (e.g., 70152356V1). Alternatively, the identification numbers
in column 5 may refer to
GenBank cDNAs or ESTs (e.g., g5364348) 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.g6436155 002.edit is the
identification number of a Genscan-predicted coding sequence, with g6436155
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 assemblages of both cDNA and Genscan-
predicted exons brought
together by an "exon stitching" 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. (See Example V.) In some cases, Incyte cDNA
coverage redundant
with the sequence coverage shown in column 5 was obtained to conf'~rm the
final consensus
polynucleotide sequence, but the relevant Incyte cDNA identification numbers
are not shown.
Table S 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 N0:15-28, which encodes PRTS. The
polynucleotide sequences of
SEQ ID N0:15-28, 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
N0:15-28 which has at
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
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 N0:15-28.
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
by selecting combinations based on possible codon choices. These combinations
are made in
accordance with the standard triplet genetic code as applied to the
polynucleotide sequence of naturally
occurring PRTS, and all such variations are to be considered as being
specifically disclosed.
Although nucleotide sequences which encode PRTS and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally 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 codon usage, e.g., inclusion
of non-naturally occurring
codons. Codons may be selected to increase the rate at which expression of the
peptide occurs in a
particular prokaryotic or eukaryotic host in accordance with the frequency
with which particular codons
are utilized by the host. Other reasons for substantially altering the
nucleotide sequence encoding
PRTS and its derivatives without altering the encoded amino acid sequences
include the production of
RNA transcripts having more desirable properties, such as a greater half-life,
than transcripts produced .
from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode PRTS
and PRTS
derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the synthetic
sequence may be inserted into any of the many available expression vectors and
cell 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
N0:15-28 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
31
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Applied
Biosystems), thermostable T7 polymerise (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerises and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA sequencing
system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system
(Molecular Dynamics,
Sunnyvale CA), or other systems known in the art. The resulting sequences are
analyzed using a
variety of algorithms which are well known in the art. (See, e.g., Ausubel,
F.M. (1997) Short Protocols
in Molecular Biolo~y, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
(1995) Molecular
Biology 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.)
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
findeng intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 primer analysis software
(National Biosciences,
Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides
in length, to have a
GC content of about SO% 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
32
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5'regions of genes, are preferable for situations in
which an oligo d(T) library
does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence into 5'
non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze the
size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode PRTS may be cloned in recombinant DNA molecules that direct expression
of PRTS, or
fragments or functional equivalents thereof, in appropriate host cells. Due to
the inherent degeneracy of
the genetic code, other DNA sequences which encode substantially the same or a
functionally equivalent
amino acid sequence may be produced and used to express PRTS.
The nucleotide sequences of the present invention can be engineered using
methods generally
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"
33
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding PRTS may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively,
PRTS itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide
synthesis can be performed using various solution-phase or solid-phase
techniques. (See, e.g.,
Creighton, T. (1984) Proteins, Structures and Molecular 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'~rmed 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 cases where only coding sequence, or a fragment thereof,
is inserted, exogenous
translational control signals including an in-frame ATG initiation codon
should be provided by the
vector. Exogenous translational elements and initiation codons may be of
various origins, both natural
and synthetic. The efficiency of expression may be enhanced by the inclusion
of enhancers appropriate
34
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
for the particular host cell system used. (See, e.g., Schaxf, 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
franslational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. (1995)
Current Protocols in Molecular Biology, 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, supra; Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.I~. 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 Technology (1992) 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.
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
264:5503-5509.) When large quantities of PRTS are needed, e.g. for the
production of antibodies,
vectors which direct high level expression of PRTS may be used. For example,
vectors containing the
strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of PRTS. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, 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 Technoloay (1992) McGraw Hill,
New York NY, pp.
191-196.)
In mammalan cells, a number of viral-based expression systems may be utilized.
In cases
where an adenovirus, is used as an expression vector, sequenFes 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 mammalan host
cells. 5V40 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 mammalan systems, stable
expression of
PRTS in cell lines is preferred. For example, sequences encoding PRTS can be
transformed into cell
ones using expression vectors which may contain viral origins of replication
and/or endogenous
36
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include,
but are not limited to, the herpes simplex virus thymidine kinase and adenine
phosphoribosyltransferase
genes, for use in tk and app 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 axninoglycosides 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 IvisD, 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 J3-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 and/or quantification of nucleic acid or
protein sequences.
Immunological methods for detecting and measuring the expression of PRTS using
either
37
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques include
enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and
fluorescence
activated cell sorting (FACS). A two-site, monoclonal-based immunoassay
utilizing monoclonal
antibodies reactive to two non-interfering epitopes on PRTS is preferred, but
a competitive binding
assay may be employed. These and other assays are well known in the art. (See,
e.g., Hampton, R. et
al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN,
Sect. IV; Coligan, J.E.
et al. (1997) Current Protocols in 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 retained 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
38
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding PRTS may be ligated to a heterologous sequence resulting in
translation of a fusion
protein in any of the aforementioned host systems. For example, a chimeric
PRTS protein containing a ,
heterologous moiety that can be recognized by a commercially available
antibody may facilitate the
screening of peptide libraries for inhibitors of PRTS activity. Heterologous
protein and peptide
moieties may also facilitate purification of fusion proteins using
commercially available afFnity
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-nryc, 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-niyc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
proteins using commercially available monoclonal and polyclonal antibodies
that specifically recognize
these epitope tags. A fusion protein may also be engineered to contain a
proteolytic cleavage site
located between the PRTS encoding sequence and the heterologous protein
sequence, so that PRTS may
be cleaved away from the heterologous moiety following purification. Methods
for fusion protein
expression and purification are discussed in Ausubel (1995, sera, ch. 10). A
variety of commercially
available kits may also be used to facilitate expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled PRTS may
be achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These systems
couple transcription and translation of protein-coding sequences operably
associated with the T7, T3, or
SP6 promoters. Translation takes place in the presence of a radiolabeled amino
acid precursor, for
example, 35S-methionine.
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.
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coli. Cells expressing PRTS or cell membrane fractions which contain PRTS are
then contacted with
a test compound and binding, stimulation, or inhibition of activity of either
PRTS or the compound is
analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is.
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
PRTS, either in
solution or affixed to a solid support, and detecting the binding of PRTS to
the compound.
Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carried out using cell-free
preparations, chemical
libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to a
solid support.
PRTS of the present invention or fragments thereof may be used to screen for
compounds that
modulate the activity of PRTS. Such compounds may include agonists,
antagonists, or partial or'
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for PRTS
activity, wherein PRTS is combined with at least one test compound, and the
activity of PRTS in the
presence of a test compound is compared with the activity of PRTS in the
absence of the test
compound. A change in the activity of PRTS in the presence of the test
compound is indicative of a
compound that modulates the activity of PRTS. Alternatively, a test compound
is combined with an in
vitro or cell-free system comprising PRTS under conditions suitable for PRTS
activity, and the assay is
performed. In either of these assays, a test compound which modulates the
activity of PRTS may do so
indirectly and need not come in direct contact with the test compound. At
least one and up to a plurality
of test compounds may be screened.
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
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 gastrointestinal, epithelial, reproductive, cardiovascular, cancerous,
and inflamed tissues, and
with normal kidney and normal skin tissues. Therefore, PRTS appears to play a
role in
gastrointestinal, cardiovascular, autoimmunelinflammatory, 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-
Weiss syndrome, colonic carcinoma, colonic obstruction, irritable bowel
syndrome, short bowel
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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, verso-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, endocaxditis of systemic lupus
erythematosus, carcinoid heart
disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease,
congenital heart disease,
and complications of cardiac transplantation; an autoimmunelinflammatory
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 particular,
cancers of the adrenal
gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal tract,
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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, spiny bifida, anencephaly,
craniorachischisis, congenital
glaucoma, cataract, age-related macular degeneration, and sensorineural
hearing loss; an epithelial
disorder, such as dyshidrotic eczema, allergic contact dermatitis, keratosis
pilaris, melasma, vitiligo,
actinic keratosis, basal cell carcinoma, squamous cell carcinoma, seborrheic
keratosis, folliculitis,
herpes simplex, herpes zoster, varicella, candidiasis, dermatophytosis,
scabies, insect bites, cherry
angioma, keloid, dermatofibroma, acrochordons, urticaria, transient
acantholytic dermatosis, xerosis,
eczema, atopic dermatitis, contact dermatitis, hand eczema, nummular eczema,
lichen simplex
chronicus, asteatotic eczema, stasis dermatitis and stasis ulceration,
seborrheic dermatitis, psoriasis,
lichen planus, pityriasis rosea, impetigo, ecthyma, dermatophytosis, tinea
versicolor, warts, acne
vulgaris, acne rosacea, pemphigus vulgaris, pemphigus foliaceus,
paraneoplastic pemphigus, bullous
pemphigoid, herpes gestationis, dermatitis herpetiformis, linear IgA disease,
epidermolysis bullosa
acquisita, dermatomyositis, lupus erythematosus, scleroderma and morphea,
erythroderma, alopecia,
figurate skin lesions, telangiectasias, hypopigmentation, hyperpigmentation,
vesicles/bullae, exanthems,
cutaneous drug reactions, papulonodular skin lesions, chronic non-healing
wounds, photosensitivity
diseases, epidermolysis bullosa simplex, epidermolytic hyperkeratosis,
epidermolytic and
nonepidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens,
ichthyosis exfoliativa,
keratosis palmaris et plantaris, keratosis palmoplantaris, palmoplantar
keratoderma, keratosis punctata,
Meesmann's corneal dystrophy, pachyonychia congenita, white sponge nevus,
steatocystoma multiplex,
epidermal nevi/epidermolytic hyperkeratosis type, monilethrix,
trichothiodystrophy, chronic
hepatitis/cryptogenic cirrhosis, and colorectal hyperplasia; a neurological
disorder, such as epilepsy,
ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's
disease, Pick's disease,
Huntington's disease, dementia, Parkinson's disease and other extrapyramidal
disorders, amyotrophic
lateral sclerosis and other motor neuron disorders, progressive neural
muscular atrophy, retinitis
pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating
diseases, bacterial and viral
meningitis, brain abscess, subdural empyema, epidural abscess, suppurative
intracranial
thrombophlebitis, myelitis and radiculitis, viral central nervous system
disease, prion diseases including
kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome,
fatal familial
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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,
autoimmunelinflammatory, cell proliferative, developmental, epithelial,
neurological, and reproductive
disorders described above. In one aspect, an antibody which specifically binds
PRTS may be used
directly as an antagonist or indirectly as a targeting or delivery mechanism
for bringing a
pharmaceutical agent to cells or tissues which express PRTS.
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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
fiherapy may be made by
one of ordinary skill in the art, according to conventional pharmaceutical
principles. The combination
of therapeutic agents may act synergistically to effect the treatment or
prevention of the various
disorders described above. Using this approach, one may be able to achieve
therapeutic efficacy with
lower dosages of each agent, thus reducing the potential for adverse side
effects.
An antagonist of PRTS may be produced using methods which are generally known
in the art.
In particular, purified PRTS may be used to produce antibodies or to screen
libraries of pharmaceutical
agents to identify those which specifically bind PRTS. Antibodies to PRTS may
also be generated
using methods that are well known in the art. Such antibodies may include, but
are not limited to,
polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments,
and fragments produced
by a Fab expression library. Neutralizing antibodies (i.e., those which
inhibit dimer formation) are
generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with PRTS or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral gels
such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants
used in humans, BCG
(bacilli Calmette-Guerin) and Corynebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to PRTS
have an amino acid sequence consisting of at least about 5 amino acids, and
generally will consist of at
least about 10 amino acids. It is also preferable that these oligopeptides,
peptides, or fragments are
identical to a portion of the amino acid sequence of the natural protein.
Short stretches of PRTS amino
acids may be fused with those of another protein, such as KLH, and antibodies
to the chimeric molecule
may be produced.
Monoclonal antibodies to PRTS may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not limited
to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-
hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
CA 02394789 2002-06-18
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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 for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
PRTS-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g., Burton,
D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter,
G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for PRTS may also be
generated. For
example, such fragments include, but are not limited to, F(ab')2 fragments
produced by pepsin digestion
of the antibody molecule and Fab fragments generated by reducing the disulfide
bridges of the F(ab')2
fragments. Alternatively, Fab expression libraries may be constructed to allow
rapid and easy
identification of monoclonal Fab fragments with the desired specificity. (See,
e.g., Huse, W.D. et al.
(1989) Science 246:1275-1281.)
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 are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
PRTS and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two
non-interfering PRTS epitopes is generally used, but a competitive binding
assay may also be employed
(Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for PRTS. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of PRTS-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The I~ determined
for a preparation of polyclonal antibodies, which are heterogeneous in their
affinities for multiple PRTS
epitopes, represents the average affinity, or avidity, of the antibodies for
PRTS. The Ka determined for
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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 I~
ranging from about 106
to 10' L/mole are preferred for use in immunopurification and similar
procedures which ultimately
require dissociation of PRTS, preferably in active form, from the antibody
(Catty, D. (1988)
Antibodies, Volume I: A Practical 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 antibodylml, is generally employed in procedures requiring
precipitation of PRTS-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines' for
antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and
Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding PRTS, or
any fragment
or complement thereof, may be used for therapeutic purposes. In one aspect,
modifications of gene
expression can be achieved by designing complementary sequences or antisense
molecules (DNA, RNA,
PNA, or modified oligonucleotides) to the coding or regulatory regions of the
gene encoding PRTS.
Such technology is well known in the art, and antisense oligonucleotides or
larger fragments 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, I~.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding PRTS may be
used for
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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 falciparum and
Trypanosoma cruzi). In the
case where a genetic deficiency in PRTS expression or regulation causes
disease, the expression of
PRTS from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in PRTS
are treated by constructing mammalian expression vectors encoding PRTS and
introducing 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, PTK-HYG (Clontech, Palo Alto CA). PRTS may be
expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV),
Rous sarcoma virus
(RSV), SV40 virus, thymidine kinase (TK), or (1-actin genes), (ii) an
inducible promoter (e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998)
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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 KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), 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. Retrovirus 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
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
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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 al. (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
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.,
CA 02394789 2002-06-18
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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.
Fox example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding PRTS.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared by
any method known in the art for the synthesis of nucleic acid molecules. These
include techniques for
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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 treament 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
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
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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 Schizosaccharomvces pombe gene
expression system (Atkins,
D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic
Acids Res. 28:E15) or a
human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem.
Biophys. Res. Commun.
268:8-13). A particular embodiment of the present invention involves screening
a combinatorial
library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides,
peptide nucleic acids, and
modified oligonucleotides) for antisense activity against a specific
polynucleotide sequence (Bruice,
T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable for
use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells taken
from the patient and clonally propagated for autologous transplant back into
that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
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 . on's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of PRTS,
antibodies to PRTS, and mimetics, agonists, antagonists, or inhibitors of
PRTS.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the case
of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-acting
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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-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, monkeys,
or pigs. An animal model may also be used to determine the appropriate
concentration range and route .
of administration. Such information can then be used to determine useful doses
and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example PRTS or
fragments thereof, antibodies of PRTS, and agonists, antagonists or inhibitors
of PRTS, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDso (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDso ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the subject
requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the active
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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 ,ug to 100,000 fig, 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
extr acts 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 FACS,
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,
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.
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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
N0:15-28 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 avidinlbiotin 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,
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,
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atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms,
arterial dissections, varicose
veins, thrombophlebitis and phlebothrombosis, vascular tumors, and
complications of tbrombolysis,
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
autoimmunelinflammatory 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
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,
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hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral
palsy, spina bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, age-related
macular degeneration, and
sensorineural hearing loss; an epithelial disorder, such as dyshidrotic
eczema, allergic contact
dermatitis, keratosis pilaris, melasma, vitiligo, actinic keratosis, basal
cell carcinoma, squamous cell
carcinoma, seborrheic keratosis, folliculitis, herpes simplex, herpes zoster,
varicella, candidiasis,
dermatophytosis, scabies, insect bites, cherry angioma, keloid,
dermatofibroma, acrochordons,
urticaria, transient acantholytic dermatosis, xerosis, eczema, atopic
dermatitis, contact dermatitis, hand
eczema, nummular eczema, lichen simplex chronicus, asteatotic eczema, stasis
dermatitis and stasis
ulceration, seborrheic dermatitis, psoriasis, lichen planus, pityriasis rosea,
impetigo, ecthyma,
dermatophytosis, tinea versicolor, warts, acne vulgaris, acne rosacea,
pemphigus vulgaris, pemphigus
foliaceus, paraneoplastic pemphigus, bullous pemphigoid, herpes gestationis,
dermatitis herpetiformis,
linear IgA disease, epidermolysis bullosa acquisita,. dermatomyositis, lupus
erythematosus, scleroderma
and morphea, erythroderma, alopecia, figurate skin lesions, telangiectasias,
hypopigmentation,
hyperpigmentation, vesicles/bullae, exanthems, cutaneous drug reactions,
papulonodular skin lesions,
chronic non-healing wounds, photosensitivity diseases, epidermolysis bullosa
simplex, epidermolytic
hyperkeratosis, epidermolytic and nonepidermolytic palmoplantar keratoderma,
ichthyosis bullosa of
Siemens, ichthyosis exfoliativa, keratosis palmaris et plantaris, keratosis
palmoplantaris, palmoplantar
keratoderma, keratosis punctata, Meesmann's corneal dystrophy, pachyonychia
congenita, white sponge
nevus, steatocystoma multiplex, epidermal nevi/epidermolytic hyperkeratosis
type, monilethrix,
trichothiodystrophy, chronic hepatitis/cryptogenic cirrhosis, and colorectal
hyperplasia; a neurological
disorder, such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral
neoplasms, Alzheimer's
disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease
and other extrapyramidal
disorders, amyotrophic lateral sclerosis and other motor neuron disorders,
progressive neural muscular
atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and
other demyelinating diseases,
bacterial and viral meningitis, brain abscess, subdural empyema, epidural
abscess, suppurative
intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous
system disease, prion
diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-
Scheinker syndrome,
fatal familial insomnia, nutritional and metabolic diseases of the nervous
system, neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central nervous system
including Down syndrome,
cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders,
cranial nerve disorders,
spinal cord diseases, muscular dystrophy and other neuromuscular disorders,
peripheral nervous system
disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine,
and toxic myopathies,
myasthenia gravis, periodic paralysis, mental disorders including mood,
anxiety, and schizophrenic
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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
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.
Standaxd 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,
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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
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
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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
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,
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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:l/www.niehs.nih.gov/oc/news/toxchip.htm.)
Therefore, it is
important and desirable in toxicological screening using toxicant signatures
to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological sample
containing nucleic acids with the test compound. Nucleic acids that are
expressed in the treated
biological sample are hybridized with one or more probes specific to the
polynucleotides of the
present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a proteome
can be subjected individually to further analysis. Proteome expression
patterns, or profiles, are
analyzed by quantifying the number of expressed proteins and their relative
abundance under given
conditions and at a given time. A profile of a cell's proteome may thus be
generated by separating and
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analyzing the polypeptides of a particular tissue or cell type. In one
embodiment, the separation is
achieved using two-dimensional gel electrophoresis, in which proteins from a
sample are separated by
isoelectric focusing in the first dimension, and then according to molecular
weight by sodium dodecyl
sulfate slab gel electrophoresis in the second dimension (Steiner and
Anderson, supra). The proteins are
visualized in the gel as discrete and uniquely positioned spots, typically by
staining the gel with an agent
such as .Coomassie Blue or silver or fluorescent stains. The optical density
of each protein spot is
generally proportional to the level of the protein in the sample. The optical
densities of equivalently
positioned protein spots from different samples, for example, from biological
samples either treated or
untreated with a test compound or therapeutic agent, are compared to identify
any changes in protein
spot density related to the treatment. The proteins in the spots are partially
sequenced using, for
example, standard methods employing chemical or enzymatic cleavage followed by
mass spectrometry.
The identity of the protein in a spot may be determined by comparing its
partial sequence, preferably of
at least 5 contiguous amino acid residues, to the polypeptide sequences of the
present invention. In
some cases, further sequence data may be obtained for definitive protein
identification.
A proteomic profile may also be generated using antibodies specific for PRTS
to quantify the
levels of PRTS expression. In one embodiment, the antibodies are used as
elements on a microarray,
and protein expression levels are quantified by exposing the microarray to the
sample and detecting the
levels of protein bound to each array element (Lueking, A. et al. (1999) Anal.
Biochem. 270:103-111;
Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a variety of
methods known in the art, for example, by reacting the proteins in the sample
with a thiol- or amino-
reactive fluorescent compound and detecting the amount of fluorescence bound
at each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and should
be analyzed in parallel with toxicant signatures at the transcript level.
There is a poor correlation
between transcript and protein abundances for some proteins in some tissues
(Anderson, N.L. and J.
Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures
may be useful in the
analysis of compounds which do not significantly affect the transcript image,
but which alter the
proteomic profile. In addition, the analysis of transcripts in body fluids is
difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated biological
sample are separated so that the amount of each protein can be quantified. The
amount of each protein
is compared to the amount of the corresponding protein in an untreated
biological sample. A difference
in the amount of protein between the two samples is indicative of a toxic
response to the test compound
in the treated sample. Individual proteins are identified by sequencing the
amino acid residues of the
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individual proteins and comparing these partial sequences to the polypeptides
of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides. of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared with
the amount in an untreated biological sample. A difference in the amount of
protein between the two
samples is indicative of a toxic response to the test compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:20614-20619; Baldeschweiler et al. (1995) PCT application W0951251216;
Shalom D. et al.
(1995) PCT application W095135505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are well
known and thoroughly described in DNA Microarrays: A Practical Approach, M.
Schena, ed. (1999)
Oxford University Press, London, hereby expressly incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding PRTS
may be used to
generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
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. 7:127-234; and Trask, B.J.
(2991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop genetic
linkage maps, for example, which correlate the inheritance of a disease state
with the inheritance of a
particular chromosome region or restriction fragment length polymorphism
(RFLP). (See, for
example, 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
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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 11q22-23, any sequences
mapping to that area may represent associated or regulatory genes for further
investigation. (See, e.g.,
Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the
instant invention may
also be used to detect differences in the chromosomal location due to
translocation, inversion, etc.,
among normal, carrier, or affected individuals.
In another embodiment of the invention, PRTS, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between PRTS and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with PRTS, or
fragments thereof, and
washed. Bound PRTS is then detected by methods well known in the art. Purified
PRTS can also be
coated directly onto plates for use in the aforementioned drug screening
techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and immobilize
it on a solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding PRTS specifically compete with a test compound
for binding PRTS. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with PRTS.
In additional embodiments, the nucleotide sequences which encode PRTS may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are,
therefore, to be construed as merely illustrative, and not limitative of the
remainder of the disclosure
in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below, in
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particular U.S. Ser. No. 601172,055, U.S. Ser. No. 60/177,334, U.S. Ser. No.
60/178,884, and U.S.
Ser. No. 601179,903, are expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database
(Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. The Incyte
cDNAs shown for SEQ
ID N0:15 were derived from cDNA libraries constructed from small intestine,
ovary, lung, skin, breast,
prostate epilthelium, and mixed myometrial tissues; umbilical cord blood, and
teratocarcinoma cells
which contained neuronal precursors. The Incyte cDNAs shown for SEQ ID N0:17
were derived from
cDNA libraries constructed from a bronchial epithelium primary cell line,
dermal microvascular
endothelial cells, pancreas, ileum tissue associated with Crohn's disease, rib
bone tissue associated with
Patau's syndrome, kidney, thoracic dorsal root ganglion, and penis corpus
cavernosum tissue. The
Incyte cDNA shown for SEQ ID N0:18 was derived from a cDNA library constructed
from brain
tumor tissue. The Incyte cDNAs shown for SEQ ID NO:19 were derived from cDNA
libraries
constructed from adrenal gland, colon, and breast tissue. The Incyte cDNAs
shown for SEQ ID N0:20
were derived from cDNA libraries constructed from T-lymphocytes, lung, breast,
and penis corpus
cavernosum tissues. Some tissues were homogenized and lysed in guanidinium
isothiocyanate, while
others were homogenized and lysed in phenol or in a suitable mixture of
denaturants, such as TRIZOL
(Life Technologies), a monophasic solution of phenol and guanidine
isothiocyanate. The resulting
lysates were centrifuged over 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
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, sutra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
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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
ElectxoMAX
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 FLUOROSI~AN 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
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
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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 Consed, and
cDNA assemblages
were screened for open reading frames using programs based on GeneMark, BLAST,
and FASTA.
The full length polynucleotide sequences were translated to derive the
corresponding full length
polypeptide 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
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
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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
NO:15-28. 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. KarHn (1997) J. Mol. Biol. 268:78-94, and Burge, C. and S.
Karlin.(1998) Curr.
Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to
form an assembled
cDNA sequence extending from a methionine to a stop codon. The output of
Genscan is a FASTA
database of polynucleotide and polypeptide sequences. The maximum range of
sequence for Genscan
to analyze at once was set to 30 kb. To determine which of these Genscan
predicted cDNA sequences
encode proteases, the encoded polypeptides were analyzed by querying against
PFAM models for
proteases. Potential proteases were also identified by homology to Incyte cDNA
sequences that had
been annotated as proteases. These selected Genscan-predicted sequences were
then compared by
BLAST analysis to the genpept and gbpri public databases. Where necessary, the
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
infoxmation was used to
correct or confirm the Genscan predicted sequence. Full length polynucleotide
sequences were obtained
by assembling Genscan-predicted coding sequences with Incyte cDI~A 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 ir<
Example III were mapped
to genomic DNA and parsed into clusters containing related cDNAs and Genscan
exon predictions from
one or more genomic sequences. Each cluster was analyzed using an algorithm
based on graph theory
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and dynamic programming to integrate cDNA and genomic information, generating
possible splice
variants that were subsequently conf'~rmed, 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" Seauences
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 GenB ank protein homolog. The
GenB ank protein homolog,
the chimeric protein, or both were used as probes to search fox 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 N0:15-28 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:15-28 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
CA 02394789 2002-06-18
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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, or 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:l/www.ncbi.nlm.nih.gov/genemapn, can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a gene
and involves the hybridization of a labeled nucleotide sequence to a membrane
on which RNAs from a
particular cell type or tissue have been bound. (See, e.g., Sambrook, supra,
ch. 7; Ausubel (1995)
supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
search can be modified to determine whether any particular match is
categorized as exact or similar.
The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
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
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
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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
disease/condition categories: cancer,
cell line, developmental, inflammation, neurological, trauma, cardiovascular,
pooled, and other, and the
number of libraries in each category is counted and divided by the total
number of libraries across all
categories. The resulting percentages reflect the tissue- and disease-specific
expression of cDNA
encoding PRTS. cDNA sequences and cDNA library/tissue information are found in
the 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.
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)ZS 04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
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(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, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and 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 ~l of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~1 to 10 ~cl aliquot of the reaction mixture was
analyzed by electrophoresis
on a 1 % agaxose 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
using the same conditions as described above. Samples were diluted with 20%
dimethysulfoxide (1:2,
v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the
DYENAMIC
DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator
cycle
sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5' regulatory sequences using the above procedure along
with oligonucleotides
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designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:15-28 are employed to screen
cDNAs, genomic
DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about
20 base pairs, is
specifically described, essentially the same procedure is used with larger
nucleotide fragments.
Oligonucleotides are designed using state-of the-art software such as OLIGO
4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~cCi of
['y-32P] adenosine
triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase
(DuPont NEN, Boston
MA). The labeled oligonucleotides are substantially purified using a SEPHADEX
G-25 superfine size
exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot
containing 10' counts per
minute of the labeled probe is used in a typical membrane-based hybridization
analysis of human
genomic DNA digested with one of the following endonucleases: Ase I, Bgl II,
Eco 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°1o 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, su ra.), 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, W, chemical, or mechanical bonding procedures. A
typical array may be
produced using available methods and machines well known to those of ordinary
skill in the art and may
contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470;
Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson
(1998) Nat. Biotechnol.
16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The array
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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/~1 oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 unitsl~..il RNase inhibitor, 500 ~.iM dATP, 500 ~iM dGTP,
500 NM dTTP, 40 ~M
dCTP, 40 ~iM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium
hydroxide and
incubated for 20 minutes at 85° C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 ~.il 5X SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element is
amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
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 watex, and
CA 02394789 2002-06-18
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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 iil of the array
element DNA, at an average
concentration of 100 nglE.ff, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 ~l 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 ail 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.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
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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 S' sequence and
used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is
designed to prevent ribosomal binding to the PRTS-encoding transcript.
XII. Expression of PRTS
Expression and purification of PRTS is achieved using bacterial or virus-based
expression
systems. For expression of PRTS in bacteria, cDNA is subcloned into an
appropriate vector containing
an antibiotic resistance gene and an inducible promoter that directs high
levels of cDNA transcription.
Examples of such promoters include, but are not limited to, the trp-lac (tac)
hybrid promoter and the
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
77
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
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~iperda (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. Aced. 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 iaponicum, 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, suf 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. F~xnctional Assays
PRTS function is assessed by expressing the sequences encoding PRTS at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA),
both of which
contain the cytomegalovirus promoter. 5-10 /.cg of recombinant vector are
transiently transfected into a
human cell line, for example, an endothelial or hematopoietic cell line, using
either liposome
formulations or electroporation. 1-2 ~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-
78
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
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 polyacrylaxnide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the PRTS amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, su ra, ch. 11.)
Typically, oligopeptides of about 15 residues in length axe synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
I~LH (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.
79
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
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 lasl 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 al. (2000) U.S. Patent
No.6,057,101).
XVII. Demonstration of PRTS Activity
Protease activity is measured by the hydrolysis of appropriate synthetic
peptide substrates
conjugated with various chromogenic molecules in which the degree of
hydrolysis is quantified by
spectrophotometric (or fluorometric) absorption of the released chromophore
(Beynon, R.J. and J.S.
Bond (1994) Proteolytic Enzvmes; A Practical Approach, Oxford University
Press, New York NY,
pp.25-55). Peptide substrates are designed according to the category of
protease activity as
endopeptidase (serine, cysteine, aspartic proteases, or metalloproteases),
aminopeptidase (leucine
aminopeptidase), or carboxypeptidase (carboxypeptidases A and B, procollagen C-
proteinase).
Commonly used chromogens are 2-naphthylamine, 4-nitroaniline, and furylacrylic
acid. Assays are
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
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. Chemically synthesized human
ubiquitin-valine may be used
as substrate. Cleavage of the C-terminal valine residue from the substrate is
monitored by capillary
electrophoresis (Franklin, K. et al. (1997) Anal. Biochem. 247:305-309).
In the alternative, an assay for protease activity takes advantage of
fluorescence resonance
energy transfer (FRET) that occurs when one donor and one acceptor fluorophore
with an appropriate
spectral overlap are in close proximity. A flexible peptide linker containing
a cleavage site specific for
PRTS is fused between a red-shifted variant (RSGFP4) and a blue variant (BFPS)
of Green Fluorescent
Protein. This fusion protein has spectral properties that suggest energy
transfer is occurring from BFPS
1S 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.D. et al.
(1996) Gene 173:13-17). This assay can also be performed in living cells. In
this case the fluorescent
substrate protein is expressed constitutively in cells and PRTS is 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-S7).
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
XyII, 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
81
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
expression library displayed on the surface of phage particles (T7SELECTTM10-3
Phage display
vector, Novagen, Madison, WI) or yeast cells (pYD1 yeast display vector kit,
Invitrogen, Carlsbad,
CA). In this case, entire cDNAs are fused between Gene III and the appropriate
epitope.
XIX. Identification of PRTS Inhibitors
Compounds to be tested are arrayed in the wells of a mufti-well plate in
varying concentrations
along with an appropriate buffer and substrate, as described in the assays in
Example XVII. PRTS
activity is measured for each well and the ability of each compound to inhibit
PRTS activity can be
determined, as well as the dose-response kinetics. This assay could also be
used to identify molecules
which enhance PRTS activity.
In the alternative, phage display libraries can be used to screen for peptide
PRTS inhibitors.
Candidates are found among peptides which bind tightly to a protease. In this
case, mufti-well plate
wells are coated with PRTS and incubated with a random peptide phage display
library or a cyclic
peptide library (Koivunen, E. et al. (1999) Nat. Biotechnol. 17:768-774).
Unbound phage are washed
away and selected phage amplified and rescreened for several more rounds.
Candidates 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.
82
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
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98
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
<110> INCYTE GENOMICS, INC.
YANG, Junming
BAUGHN, Mariah R.
BURFORD, Neil
AU-YOUNG, Janice
LU, Dyung Aina M.
REDDY, Roopa
YUE, Henry
NGUYEN, Danniel B.
TANG, Y. Tom
YAO, Monique G.
LAL, Preeti
<120> PROTEASES
<130> PI-0003 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/172,055; 60/177,334; 60/178,884; 60/179,903
<151> 1999-12-23; 2000-01-21; 2000-01-28; 2000-02-02
<160> 28
<170> PERL Program
<210> 1
<211> 1055
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1714846CD1
<400> 1
Met Thr Val Glu Gln Asn Val Leu Gln Gln Ser Ala Ala Gln Lys
1 5 10 15
His Gln Gln Thr Phe Leu Asn Gln Leu Arg G1u Ile Thr Gly Ile
20 25 30
Asn Asp Thr Gln Ile Leu Gln Gln Ala Leu Lys Asp Ser Asn Gly
35 40 45
Asn Leu Glu Leu Ala Val Ala Phe Leu Thr Ala Lys Asn Ala Lys
50 55 . 60
Thr Pro Gln Gln Glu Glu Thr Thr Tyr Tyr Gln Thr Ala Leu Pro
65 70 75
Gly Asn Asp Arg Tyr Ile Ser Val Gly Ser Gln Ala Asp Thr Asn
80 85 90
Val Ile Asp Leu Thr Gly Asp Asp Lys Asp Asp Leu Gln Arg Ala
95 100 105
Ile Ala Leu Ser Leu Ala Glu Ser Asn Arg Ala Phe Arg Glu Thr
110 115 120
Gly Tle Thr Asp Glu Glu Gln Ala Ile Ser Arg Val Leu Glu Ala
125 130 135
Ser Ile Ala Glu Asn Lys Ala Cys Leu Lys Arg Thr Pro Thr Glu
140 145 150
Val Trp Arg Asp Ser Arg Asn Pro Tyr Asp Arg Lys Arg Gln Asp
155 160 165
Lys Ala Pro Val Gly Leu Lys Asn Val Gly Asn Thr Cys Trp Phe
170 175 180
Ser Ala Val Ile Gln Ser Leu Phe Asn Leu Leu Glu Phe Arg Arg
185 190 195
Leu Val Leu Asn Tyr Lys Pro Pro Ser Asn Ala Gln Asp Leu Pro
200 205 210
Arg Asn Gln Lys Glu His Arg Asn Leu Pro Phe Met Arg Glu Leu
215 220 225
Arg Tyr Leu Phe Ala Leu Leu Val Gly Thr Lys Arg Lys Tyr Val
1/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
230 235 240
Asp Pro Ser Arg Ala Val G1u Ile Leu Lys Asp Ala Phe Lys Ser
245 250 255
Asn Asp Ser Gln Gln Gln Asp Val Ser Glu Phe Thr His Lys Leu
260 265 270
Leu Asp Trp Leu Glu Asp Ala Phe Gln Met Lys Ala Glu Glu Glu
275 280 285
Thr Asp Glu Glu Lys Pro Lys Asn Pro Met Val Glu Leu Phe,Tyr
290 295 300
G1y Arg Phe Leu Ala Val Gly Val Leu Glu Gly Lys Lys Phe Glu
305 310 315
Asn Thr Glu Met Phe Gly Gln Tyr Pro Leu Gln Val Asn Gly Phe
320 325 330
Lys Asp Leu His Glu Cys Leu G1u Ala Ala Met Ile Glu Gly Glu
335 340 345
Ile Glu Ser Leu His Ser Glu Asn Ser Gly Lys Ser Gly Gln Glu
350 355 360
His Trp Phe Thr Glu Leu Pro Pro Val Leu Thr Phe Glu Leu Ser
365 370 375
Arg Phe G1u Phe Asn Gln Ala Leu Gly Arg Pro Glu Lys Ile His
380 385 390
Asn Lys Leu Glu Phe Pro Gln Val Leu Tyr Leu Asp Arg Tyr Met
395 400 405
His Arg Asn Arg Glu I1e Thr Arg Ile Lys Arg Glu Glu Ile Lys
410 4l5 420
Arg Leu Lys Asp Tyr Leu Thr Val Leu Gln Gln Arg Leu Glu Arg
425 430 435
Tyr Leu Ser Tyr Gly Ser Gly Pro Lys Arg Phe Pro Leu Val Asp
440 445 450
Val Leu Gln Tyr Ala Leu Glu Phe Ala Ser Ser Lys Pro Val Cys
455 460 465
Thr Ser Pro Val Asp Asp I1e Asp Ala Ser Ser Pro Pro Ser Gly
470 475 480
Ser Ile Pro Ser Gln Thr Leu Pro Ser Thr Thr Glu Gln Gln Gly
485 490 495
Ala Leu Ser Ser Glu Leu Pro Ser Thr Ser Pro Ser Ser Val Ala
500 505 510
Ala Ile Ser Ser Arg Ser Val Ile His Lys Pro Phe Thr Gln Ser
515 520 525
Arg Ile Pro Pro Asp Leu Pro Met His Pro Ala Pro Arg His Ile
530 535 540
Thr Glu Glu G1u Leu Ser Val Leu Glu Ser Cys Leu His Arg Trp
545 550 555
Arg Thr Glu I1e Glu Asn Asp Thr Arg Asp Leu Gln Glu Ser Ile
560 565 570
Ser Arg Ile His Arg Thr Tle Glu Leu Met Tyr Ser Asp Lys Ser
575 580 585
Met Ile Gln Val Pro Tyr Arg Leu His Ala Val Leu Val His Glu
590 595 600
Gly Gln Ala Asn A1a Gly His Tyr Trp Ala Tyr Ile Phe Asp His
605 610 615
Arg Glu Ser Arg Trp Met Lys Tyr Asn Asp Ile Ala Val Thr Lys
620 625 630
Ser Ser Trp Glu Glu Leu Val Arg Asp Ser Phe Gly Gly Tyr Arg
635 640 645
Asn Ala Ser Ala Tyr Cys Leu Met Tyr Ile Asn Asp Lys Ala Gln
650 655 660
Phe Leu Ile Gln Glu Glu Phe Asn Lys Glu Thr Gly Gln Pro Leu
665 670 675
Val Gly Ile Glu Thr Leu Pro Pro Asp Leu Arg Asp Phe Val Glu
680 685 690
Glu Asp Asn Gln Arg Phe Glu Lys Glu Leu Glu Glu Trp Asp Ala
695 700 705
Gln Leu Ala Gln Lys Ala Leu Gln Glu Lys Leu Leu Ala Ser Gln
710 715 720
Lys Leu Arg Glu Ser Glu Thr Ser Val Thr Thr Ala Gln Ala A1a
725 730 735
2/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
Gly Asp Pro Glu Tyr Leu Glu Gln Pro Ser Arg Ser Asp Phe Ser
740 745 750
Lys His Leu Lys GIu Glu Thr Ile Gln Ile Ile Thr Lys Ala Ser
755 760 765
His Glu His Glu Asp Lys Ser Pro Glu Thr Val Leu Gln Ser Ala
770 775 780
Ile Lys Leu Glu Tyr Ala Arg Leu Val Lys Leu Ala G1n Glu Asp
785 790 795
Thr Pro Pro Glu Thr Asp Tyr Arg Leu His His Val Val Val Tyr
800 805 810
Phe Ile Gln Asn Gln Ala Pro Lys Lys Ile Ile Glu Lys Thr Leu
815 820 825
Leu Glu Gln Phe Gly Asp Arg Asn Leu Ser Phe Asp Glu Arg Cys
830 835 840
His Asn Ile Met Lys Val Ala Gln Ala Lys Leu Glu Met Ile Lys
845 850 855
Pro Glu Glu Val Asn Leu Glu Glu Tyr Glu Glu Trp His Gln Asp
860 865 870
Tyr Arg Lys Phe Arg Glu Thr Thr Met Tyr Leu Ile Ile Gly Leu
875 880 885
Glu Asn Phe Gln Arg Glu Ser Tyr Ile Asp Ser Leu Leu Phe Leu
890 895 900
Ile Cys A1a Tyr Gln Asn Asn Lys Glu Leu Leu Ser Lys GIy Leu
905 910 915
Tyr Arg G1y His Asp Glu Glu Leu Ile Ser His Tyr Arg Arg Glu
920 925 930
Cys Leu Leu Lys Leu Asn Glu Gln Ala Ala Glu Leu Phe Glu Ser
935 940 945
Gly G1u Asp Arg Glu Val Asn Asn G1y Leu Ile I1e Met Asn Glu
950 955 960
Phe Ile Val Pro Phe Leu Pro Leu Leu Leu Va1 Asp Glu Met Glu
965 970 975
Glu Lys Asp Ile Leu Ala Val Glu Asp Met Arg Asn Arg Trp Cys
980 985 990
Ser Tyr Leu Gly Gln Glu Met Glu Pro His Leu Gln Glu Lys Leu
995 1000 1005
Thr,_Asp_Phe Leu Pro Lys Leu Leu Asp Cys Ser Met Glu Ile Lys
1010 1015 1020
Ser Phe His Glu Pro Pro Lys Leu Pro Ser Tyr Ser Thr His Glu
1025 1030 1035
Leu Cys Glu Arg Phe Ala Arg Ile Met Leu Ser Leu Ser Arg Thr
1040 1045 1050
Pro Ala Asp Gly Arg
1055
<210> 2
<211> 358
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1856589CD1
<400> 2
Met Gly Ala Ala Thr Cys Arg Gly Ser Arg Tle Pro Ser Gly Pro
1 5 10 ~.5
Pro Val Gln Gly Glu Arg Ser Ala Pro Arg Phe Gly Val Thr Ser
20 25 30
Leu Ser Leu Trp Pro Ala Asp Phe Lys Asp Asn Trp Arg Ile Ala
35 40 45
Gly Ser Arg Gln Glu Val Ala Leu Ala Gly Glu Pro Ala Asp Gln
50 55 60
Gln Gln Thr His Leu Arg Arg Leu Pro Tyr Arg Gln Thr Leu Gly
65 70 75
Tyr Lys Glu Asp Thr Thr Asn Pro Val Cys Gly Glu Pro Trp Trp
80 85 90
3/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
Ser Glu Asp Leu GIu Met Thr Arg His Trp Pro Trp GIu Val Ser
95 100 105
Leu Arg Met Glu Asn Glu His Val Cys Gly Gly Ala Leu Ile Asp
110 115 120
Pro Ser Trp Val Val Thr Ala Ala His Cys Ser Gln Gly Thr Lys
125 130 135
Glu Tyr Ser Val Val Leu Gly Thr Ser Lys Leu Gln Pro Met Asn
140 145 150
Phe Ser Arg Ala Leu Trp Val Pro Val Arg Asp Ile Ile Met His
155 160 165
Pro Lys Tyr Trp Gly Arg Ala Phe Ile Met Gly Asp Val Ala Leu
170 175 180
Val His Leu Gln Thr Pro Val Thr Phe Ser Glu Tyr Val Gln Pro
185 190 195
Ile Cys Leu Pro Glu Pro Asn Phe Asn Leu Lys Val Gly Thr Gln
200 205 210
Cys Trp Val Thr Gly Trp Ser Gln Val Lys Gln Arg Phe Ser Gly
215 220 225
Ser Thr Ala Asn Ser Met Leu Thr Pro Glu Leu Gln Glu Ala Glu
230 235 240
Val Phe Ile Met Asp Asn Lys Arg Cys Asp Arg His Tyr Lys Lys
245 250 255
Ser Phe Phe Pro Leu Val Val Pro Leu Val Leu Gly Asp Met Ile
260 265 270
Cys Ala Thr Asn Tyr Gly Glu Asn Leu Cys Tyr Gly Asp Ser Gly
275 280 285
Gly Pro Leu Ala Cys Glu Val Glu Gly Arg Trp Ile Leu Ala Gly
290 295 300
Val Leu Ser Trp Glu Lys Ala Cys Val Lys Ala Gln Asn Pro Gly
305 310 315
Val Tyr Thr Arg Val Thr Lys Tyr Thr Lys Trp Ile Lys Lys Gln
320 325 330
Met Ser Asn Gly Ala Phe Ser Gly Pro Cys Ala Ser Ala Cys Leu
335 340 345
Leu Phe Leu Cys Trp Pro Leu Gln Pro Gln Met Gly Ser
350 355
<210> 3
<211> 467
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2617672CD1
<400> 3
Met Trp Arg Cys Pro Leu Gly Leu Leu Leu Leu Leu Pro Leu Ala
1 5 10 15
Gly His Leu Ala Leu Gly Ala Gln Gln G1y Arg Gly Arg Arg Glu
20 25 30
Leu Ala Pro Gly Leu His Leu Arg Gly Ile Arg Asp Ala Gly Gly
35 40 45
Arg Tyr Cys Gln Glu Gln Asp Leu Cys Cys Arg Gly Arg Ala Asp
50 55 60
Asp Cys Ala Leu Pro Tyr Leu Gly Ala Ile Cys Tyr Cys Asp Leu
65 70 75
Phe Cys Asn Arg Thr Val Ser Asp Cys Cys Pro Asp Phe Trp Asp
80 85 90
Phe Cys Leu Gly Val Pro Pro Pro Phe Pro Pro Ile Gln Gly Cys
95 100 105
Met His Gly Gly Arg Ile Tyr Pro Val Leu Gly Thr Tyr Trp Asp
110 115 120
Asn Cys Asn Arg Cys Thr Cys Gln Glu Asn Arg Gln Trp Gln Cys
125 130 135
Asp Gln Glu Pro Cys Leu Val Asp Pro Asp Met Ile Lys Ala Ile
140 145 150
4/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
Asn Gln Gly Asn Tyr Gly Trp Gln Ala Gly Asn His Ser Ala Phe
155 160 165
Trp Gly Met Thr Leu Asp Glu Gly Ile Arg Tyr Arg Leu Gly Thr
170 175 180
Ile Arg Pro Ser Ser Ser Val Met Asn Met His Glu Ile Tyr Thr
185 190 195
Val Leu Asn Pro Gly Glu Val Leu Pro Thr Ala Phe Glu Ala Ser
200 205 210
Glu Lys Trp Pro Asn Leu Ile His Glu Pro Leu Asp Gln Gly Asn
215 220 225
Cys Ala Gly Ser Trp Ala Phe Ser Thr Ala Ala Val Ala Ser Asp
230 235 240
Arg Val Ser Ile His Ser Leu Gly His Met Thr Pro Val Leu Ser
245 250 255
Pro Gln Asn Leu Leu Ser Cys Asp Thr His Gln Gln Gln Gly Cys
260 265 270
Arg Gly Gly Arg Leu Asp Gly Ala Trp Trp Phe Leu Arg Arg Arg
275 280 285
Gly Val Val Ser Asp His Cys Tyr Pro Phe Ser Gly Arg Glu Arg
290 295 300
Asp Glu Ala Gly Pro Ala Pro Pro Cys Met Met His Ser Arg Ala
305 310 315
Met Gly Arg Gly Lys Arg Gln Ala Thr Ala His Cys Pro Asn Ser
320 325 330
Tyr Val Asn Asn Asn Asp Ile Tyr Gln Val Thr Pro Val Tyr Arg
335 340 345
Leu Gly Ser Asn Asp Lys Glu Ile Met Lys Glu Leu Met Glu Asn
350 355 360
Gly Pro Val Gln Ala Leu Met Glu Val His Glu Asp Phe Phe Leu
365 370 375
Tyr Lys Gly Gly Ile Tyr Ser His Thr Pro Val Ser Leu Gly Arg
380 385 390
Pro Glu Arg Tyr Arg Arg His Gly Thr His Ser Val Lys Ile Thr
395 400 405
Gly Trp Gly Glu Glu Thr Leu Pro Asp Gly Arg Thr Leu Lys Tyr
410 415 420
Trp Thr Ala Ala Asn Ser Trp Gly Pro Ala Trp Gly Glu Arg G1y
425 430 435
His Phe Arg Ile Val Arg Gly Val Asn Glu Cys Asp Ile Glu Ser
440 445 450
Phe Val Leu Gly Val Trp G1y Arg Val Gly Met Glu Asp Met Gly
455 4&0 465
His His
<210> 4
<211> 187
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2769104CD1
<400> 4
Met Pro Gly Pro Arg Val Trp Gly Lys Tyr Leu Trp Arg Ser Pro
1 5 10 15
His Ser Lys Gly Cys Pro Gly Ala Met Trp Trp Leu Leu Leu Trp
20 25 30
Gly Val Leu Gln Ala Cys Pro Thr Arg Gly Ser Val Leu Leu Ala
35 40 45
Gln Glu Leu Pro Gln Gln Leu Thr Ser Pro G1y Tyr Pro Glu Pro
50 55 60
Tyr Gly Lys Gly Gln Glu Ser Ser Thr Asp Ile Lys Ala Pro Glu
65 70 75
Gly Phe Ala Val Arg Leu Val Phe Gln Asp Phe Asp Leu Glu Pro
80 85 90
5/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
Ser Gln Asp Cys Ala Gly Asp Ser Val Thr Ile Ser Phe Val Gly
95 100 105
Ser Asp Pro Ser Gln Phe Cys Gly Gln Gln Gly Ser Pro Leu Gly
110 115 120
Arg Pro Pro Gly Gln Arg Glu Phe Val Ser Ser G1y Arg Ser Leu
125 130 135
Arg Leu Thr Phe Arg Thr Gln Pro Ser Ser Glu Asn Lys Thr Ala
140 145 150
His Leu His Lys Gly Phe Leu A1a Leu Tyr Gln Thr Val Gly Glu
155 160 165
Cys Pro Ser Trp Gly Cys Arg Glu Gly A1a Ser Val Pro Ser His
170 175 180
Asp Pro Gly Ile Phe Lys Pro
185
<210> 5
<211> 289
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4802789CD1
<400> 5
Met Arg Val Lys Asp Pro Thr Lys Ala Leu Pro Glu Lys Ala Lys
1 5 10 15
Arg Ser Lys Arg Pro Thr Val Pro His Asp Glu Asp Ser Ser Asp
20 25 30
Asp Ile Ala Val Gly Leu Thr Cys Gln His Val Ser His Ala Ile
35 40 45
Ser Val Asn His Val Lys Arg Ala Ile Ala Glu Asn Leu Trp Ser
50 55 60
Val Cys Ser Glu Cys Leu Lys Glu Arg Arg Phe Tyr Asp Gly Gln
65 70 75
Leu Val Leu Thr Ser Asp Ile Trp Leu Cys Leu Lys Cys Gly Phe
80 85 90
Gln Gly Cys Gly Lys Asn Ser Glu Ser G1n His Ser Leu Lys His
95 100 105
Phe Lys Ser Ser Arg Thr Glu Pro His Cys Ile Ile Ile Asn Leu
110 115 120
Ser Thr Trp Ile Ile Trp Cys Tyr GIu Cys Asp Glu Lys Leu Ser
125 13 0 13 5
Thr His Cys Asn Lys Lys Val Leu Ala Gln Ile Val Asp Phe Leu
140 145 150
Gln Lys His Ala Ser Lys Thr Gln Thr Ser A1a Phe Ser Arg Ile
155 260 165
Met Lys Leu Cys Glu Glu Lys Cys Glu Thr Asp Glu Ile Gln Lys
170 175 180
Gly Gly Lys Cys Arg Asn Leu Ser Val Arg Gly Ile Thr Asn Leu
185 190 195
Gly Asn Thr Cys Phe Phe Asn Ala Val Met Gln Asn Leu Ala Gln
200 205 210
Thr Tyr Thr Leu Thr Asp Leu Met Asn Glu Ile Lys Glu Ser Ser
215 220 225
Thr Lys Leu Lys Ile Phe Pro Ser Ser Asp Ser Gln Leu Asp Pro
230 235 240
Leu Val Val Glu Leu Ser Arg Pro Gly Pro Leu Thr Ser Ala Leu
245 250 255
Phe Leu Phe Leu His Ser Met Lys Glu Thr Glu Lys Gly Pro Leu
260 265 270
Ser Pro Lys Val Leu Phe Asn Gln Leu Cys Gln Lys Trp Val His
275 280 285
Leu His Leu Ile
<210> 6
6/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
<211> 960
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 60116897CD1
<400> 6
Met Phe His Ser Ser Ala Met Va1 Asn Ser His Arg Lys Pro Met
1 5 10 15
Phe Asn Ile His Arg Gly Phe Tyr Cys Leu Thr Ala Ile Leu Pro
20 25 30
Gln Ile Cys Ile Cys Ser Gln Phe Ser Val Pro Ser Ser Tyr His
35 40 45
Phe Thr Glu Asp Pro Gly Ala Phe Pro Val A1a Thr Asn Gly Glu
50 55 60
Arg Phe Pro Trp Gln Glu Leu Arg Leu Pro Ser Val Val Ile Pro
65 70 75
Leu His Tyr Asp Leu Phe Va1 His Pro Asn Leu Thr Ser Leu Asp
80 85 90
Phe Val Ala Ser Glu Lys Ile Glu Val Leu Val Ser Asn Ala Thr
95 100 105
G1n Phe Ile Ile Leu His Ser Lys Asp Leu Glu Ile Thr Asn Ala
110 115 120
Thr Leu Gln Ser Glu Glu Asp Ser Arg Tyr Met Lys Pro Gly Lys
125 130 135
Glu Leu Lys Val Leu Ser Tyr Pro Ala His Glu G1n Ile Ala Leu
140 145 150
Leu Val Pro Glu Lys Leu Thr Pro His Leu Lys Tyr Tyr Val Ala
155 160 165
Met Asp Phe Gln Ala Lys Leu GIy Asp Gly Phe Glu Gly Phe Tyr
170 175 180
Lys Ser Thr Tyr Arg Thr Leu Gly Gly Glu Thr Arg I1e Leu Ala
185 190 195
Val Thr Asp Phe Glu Pro Thr Gln Ala Arg Met Ala Phe Pro Cys
200 205 210
Phe Asp Glu Pro Leu Phe Lys Ala Asn Phe Ser Ile Lys Ile Arg
215 220 225
Arg Glu Ser Arg His Ile Ala Leu Ser Asn Met Pro Lys Val Lys
230 235 240
Thr I1e Glu Leu Glu Gly Gly Leu Leu Glu Asp His Phe Glu Thr
245 250 255
Thr Val Lys Met Ser Thr Tyr Leu Val Ala Tyr Ile Val Cys Asp
260 265 270
Phe His Ser Leu Ser Gly Phe Thr Ser Ser Gly Val Lys Val Ser
275 280 285
Ile Tyr Ala Ser Pro Asp Lys Arg Asn Gln Thr His Tyr Ala Leu
290 295 300
Gln Ala Ser Leu Lys Leu Leu Asp Phe Tyr Glu Lys Tyr Phe Asp
305 310 315
Ile Tyr Tyr Pro Leu Ser Lys Leu Asp Leu Ile Ala Ile Pro Asp
320 325 330
Phe Ala Pro Gly Ala Met Glu Asn Trp Gly Leu Ile Thr Tyr Arg
335 340 345
Glu Thr Ser Leu Leu Phe Asp Pro Lys Thr Ser Ser Ala Ser Asp
350 355 360
Lys Leu Trp Val Thr Arg Val I1e Ala His Glu Leu Ala His Gln
365 370 375
Trp Phe Gly Asn Leu Val Thr Met Glu Trp Trp Asn Asp Ile Trp
380 385 390
Leu Lys Glu Gly Phe Ala Lys Tyr Met Glu Leu Ile Ala Val Asn
395 400 405
Ala Thr Tyr Pro Glu Leu Gln Phe Asp Asp Tyr Phe Leu Asn Val
410 415 420
Cys Phe Glu Val Ile Thr Lys Asp Ser Leu Asn Ser Ser Arg Pro
425 430 435
7/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
Ile Ser Lys Pro Ala Glu Thr Pro Thr Gln Ile Gln Glu Met Phe
440 445 450
Asp Glu Val Ser Tyr Asn Lys Gly Ala Cys Ile Leu Asn Met Leu
455 460 465
Lys Asp Phe Leu Gly Glu Glu Lys Phe Gln Lys Gly Ile Ile Gln
470 475 480
Tyr Leu Lys Lys Phe Ser Tyr Arg Asn Ala Lys Asn Asp Asp Leu
485 490 495
Trp Ser Ser Leu Ser Asn Ser Cys Leu Glu Ser Asp Phe Thr Ser
500 505 510
G1y Gly Val Cys His Ser Asp Pro Lys Met Thr Ser Asn Met Leu
515 520 525
Ala Phe Leu Gly Glu Asn Ala Glu Val Lys Glu Met Met Thr Thr
530 535 540
Trp Thr Leu Gln Lys Gly Ile Pro Leu Leu Val Val Lys G1n Asp
545 550 555
Gly Cys Ser Leu Arg Leu Gln Gln Glu Arg Phe Leu Gln Gly Val
560 565 570
Phe Gln Glu Asp Pro Glu Trp Arg Ala Leu Gln Glu Arg Tyr Leu
575 580 585
Trp His Ile Pro Leu Thr Tyr Ser Thr Ser Ser Ser Asn Val Ile
590 595 600
His Arg His Ile Leu Lys Ser Lys Thr Asp Thr Leu Asp Leu Pro
605 610 615
Glu Lys Thr Ser Trp Val Lys Phe Asn Val Asp Ser Asn Gly Tyr
620 625 630
Tyr Ile Val His Tyr Glu Gly His Gly Trp Asp Gln Leu Ile Thr
635 640 645
Gln Leu Asn Gln Asn His Thr Leu Leu Arg Pro Lys Asp Arg Val
650 655 660
Gly Leu Ile His Asp Val Phe Gln Leu Val Gly Ala Gly Arg Leu
665 670 675
Thr Leu Asp Lys Ala Leu Asp Met Thr Tyr Tyr Leu Gln His Glu
680 685 690
Thr Ser Ser Pro Ala Leu Leu Glu Gly Leu Ser Tyr Leu Glu Ser
695 700 . 705
Phe Tyr His Met Met Asp Arg Arg Asn Ile Ser Asp Ile Ser Glu
710 715 720
Asn Leu Lys Arg Tyr Leu Leu Gln Tyr Phe Lys Pro Val Ile Asp
725 730 735
Arg Gln Ser Trp Ser Asp Lys Gly Ser Val Trp Asp Arg Met Leu
740 745 750
Arg Ser Ala Leu Leu Lys Leu Ala Cys Asp Leu Asn His Ala Pro
755 760 765
Cys Ile Gln Lys Ala Ala Glu Leu Phe Ser Gln Trp Met Glu Ser
770 775 780
Ser Gly Lys Leu Asn Ile Pro Thr Asp Val Leu Lys Ile Val Tyr
785 790 795
Ser Val Gly Ala Gln Thr Thr Ala Gly Trp Asn Tyr Leu Leu Glu
800 805 810
Gln Tyr Glu Leu Ser Met Ser Ser Ala G1u Gln Asn Lys Ile Leu
815 820 825
Tyr Ala Leu Ser Thr Ser Lys His Gln Glu Lys Leu Leu Lys Leu
830 835 840
I12 Glu Leu Gly Met Glu Gly Lys Val Ile Lys Thr Gln Asn Leu
845 850 855
Ala Ala Leu Leu His Ala Ile Ala Arg Arg Pro Lys Gly Gln Gln
860 865 870
Leu Ala Trp Asp Phe Val Arg Glu Asn Trp Thr His Leu Leu Lys
875 880 885
Lys Phe Asp Leu Gly Ser Tyr Asp Ile Arg Met Ile Ile Ser Gly
890 895 900
Thr Thr Ala His Phe Ser Ser Lys Asp Lys Leu Gln Glu Val Lys
905 910 915
Leu Phe Phe Glu Ser Leu Glu Ala Gln Gly Ser His Leu Asp Ile
920 925 930
Phe Gln Thr Val Leu Glu Thr Ile Thr Lys Asn Ile Lys Trp Leu
8/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
935 940 945
Glu Lys Asn Leu Pro Thr Leu Arg Thr Trp Leu Met Val Asn Thr
950 955 960
<210> 7
<211> 525
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1866356CD1
<400> 7
Met Ala Val Pro Gly Glu Ala Glu G1u Glu Ala Thr Val Tyr Leu
1 5 10 15
Val Val Ser Gly Ile Pro Ser Val Leu Arg Ser Ala His Leu Arg
20 25 30
Ser Tyr Phe Ser Gln Phe Arg Glu Glu Arg Gly Gly Gly Phe Leu
35 40 45
Cys Phe His Tyr Arg His Arg Pro Glu Arg Ala Pro Pro Gln Ala
50 55 60
Ala Pro Asn Ser Ala Leu Ile Pro Thr Asp Pro Ala Ala Glu Gly
65 70 75
Gln Leu Leu Ser Gln Thr Ser Ala Thr Asp Val Arg Pro Leu Ser
80 85 90
Thr Arg Asp Ser Thr Pro Ile Gln Thr Arg Thr Cys Cys Cys Val
95 100 105
Ile Ser Val Arg Gly Leu Ala Gln Ala Gln Arg Leu Ile Arg Met
110 115 120
Tyr Ser Gly Arg Arg Trp Leu Asp Ser His Gly Thr Trp Leu Pro
125 130 135
Gly Arg Cys Leu Ile Arg Arg Leu Arg Leu Pro Thr Glu Ala Ser
140 145 150
Gly Leu Gly Ser Phe Pro Phe Lys Thr Arg Lys Glu Leu Gln Ser
155 160 165
Trp Lys Ala Glu Asn Glu Ala Phe Thr Leu Ala Asp Leu Lys Gln
170 175 180
Leu Pro Glu Leu Asn Pro Pro Val Leu Met Pro Arg Gly Asn Val
185 190 195
Gly Thr Pro Leu Arg Va1 Phe Leu Glu Leu Ile Arg Ala Cys Arg
200 205 210
Leu Pro Pro Arg Ile Ile Thr Gln Leu Gln Leu Gln Phe Pro Lys
215 220 225
Thr Gly Ser Ser Arg Arg Tyr Gly Asn Val Pro Phe Glu Tyr Glu
230 235 240
Asp Ser Glu Thr Val Glu Gln Glu Glu Leu Val Tyr Thr Ala Glu
245 250 255
Gly Glu Glu Ile Pro Gln Gly Thr Tyr Leu Ala Asp Ile Pro Ala
260 265 270
Ser Pro Cys Gly Glu Pro Glu Glu Glu Val Gly Lys Glu Glu Glu
275 280 285
Glu Glu Ser His Ser Asp Glu Asp Asp Asp Arg Gly Glu Glu Trp
290 295 300
Glu Arg His Glu Ala Leu His Glu Asp Val Thr Gly Gln Glu Arg
305 310 315
Thr Thr Glu Gln Leu Phe Glu Glu Glu Ile Glu Leu Lys Trp Glu
320 325 330
Lys Gly Gly Ser Gly Leu Val Phe Tyr Thr Asp Ala Gln Phe Trp
335 340 345
Gln Glu Glu Glu G1y Asp Phe Asp Glu Gln Thr Ala Asp Asp Trp
350 355 360
Asp Val Asp Met Ser Val Tyr Tyr Asp Arg Asp Gly Gly Asp Lys
365 370 375
Asp Ala Arg Asp Ser Val Gln Met Arg Leu Glu Gln Arg Leu Arg
380 385 390
9/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
Asp Gly Gln Glu Asp Gly Ser Val Ile Glu Arg Gln Val Gly Thr
395 ~ 400 405
Phe Glu Arg His Thr Lys Gly Ile Gly Arg Lys Val Met Glu Arg
410 415 420
Gln Gly Trp AIa Glu Gly Gln Gly Leu Gly Cys Arg Cys Ser Gly
425 430 435
Val Pro Glu Ala Leu Asp Ser Asp Gly Gln His Pro Arg Cys Lys
440 445 450
Arg Gly Leu Gly Tyr His Gly Glu Lys Leu Gln Pro Phe Gly Gln
455 460 465
Leu Lys Arg Pro Arg Arg Asn Gly Leu GIy Leu Ile Ser Thr Ile
470 475 480
Tyr Asp Glu Pro Leu Pro Gln Asp Gln Thr Glu Ser Leu Leu Arg
485 490 495
Arg G1n Pro Pro Thr Ser Met Lys Phe Arg Thr Asp Met Ala Phe
500 505 510
Val Arg Gly Ser Ser Cys Ala Ser Asp Ser Pro Ser Leu Pro Asp
515 520 525
<210> 8
<211> 795
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1872095CD1
<400> 8
Met Ile Thr Val Leu Ile Arg Ser Leu Thr Thr Asp Pro Asn Val
1 ~ 5 10 15
Lys Asp Ala Ser Met Thr Gln Ala Leu Cys Arg Met Ile Asp Trp
20 25 30
Leu Ser Trp Pro Leu Ala Gln His Val Asp Thr Trp Val I1e Ala
35 40 45
Leu Leu Lys Gly Leu Ala Ala Val Gln Lys Phe Thr Ile Leu Ile
50 55 60
Asp Val Thr Leu Leu Lys Ile Glu Leu Val Phe Asn Arg Leu Trp
65 70 75
Phe Pro Leu Val Arg Pro Gly Ala Leu Ala Val Leu Ser His Met
80 85 90
Leu Leu Ser Phe GIn His Ser Pro Glu Ala Phe His Leu I1e Val
95 100 105
Pro His Val Val Asn Leu Val His Ser Phe Lys Asn Asp Gly Leu
110 115 120
Pro Ser Ser Thr Ala Phe Leu Val Gln Leu Thr Glu Leu Ile His
125 13 0 13 5
Cys Met Met Tyr His Tyr Ser Gly Phe Pro Asp Leu Tyr Glu Pro
140 145 150
Ile Leu Glu Ala I1e Lys Asp Phe Pro Lys Pro Ser Glu Glu Lys
155 160 165
Ile Lys Leu Ile Leu Asn Gln Ser Ala Trp Thr Ser Gln Ser Asn
170 175 180
Ser Leu Ala Ser Cys Leu Ser Arg Leu Ser Gly Lys Ser Glu Thr
185 190 195
Gly Lys Thr Gly Leu Ile Asn Leu Gly Asn Thr Cys Tyr Met Asn
200 205 210
Ser VaI Ile Gln Ala Leu Phe Met Ala Thr Asp Phe Arg Arg Gln
215 220 225
VaI Leu Ser Leu Asn Leu Asn Gly Cys Asn Ser Leu Met Lys Lys
230 235 240
Leu Gln His Leu Phe Ala Phe Leu Ala His Thr Gln Arg Glu Ala
245 250 255
Tyr Ala Pro Arg Ile Phe Phe Glu Ala Ser Arg Pro Pro Trp Phe
260 265 270
Thr Pro Arg Ser Gln Gln Asp Cys Ser Glu Tyr Leu Arg Phe Leu
10/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
275 280 285
Leu Asp Arg Leu His Glu Glu Glu Lys Ile Leu Lys Val G1n Ala
290 295 300
Ser His Lys Pro Ser Glu Ile Leu G1u Cys Ser G1u Thr Ser Leu
305 310 315
Gln Glu Val Ala Ser Lys Ala Ala Val Leu Thr Glu Thr Pro Arg
320 325 330
Thr Ser Asp Gly Glu Lys Thr Leu Ile Glu Lys Met Phe Gly Gly
335 340 345
Lys Leu Arg Thr His Ile Arg Cys Leu Asn Cy_s Arg Ser Thr Ser
350 355 360
Gln Lys Val Glu Ala Phe Thr Asp Leu Ser Leu Ala Phe Cys Pro
365 370 375
Ser Ser Ser Leu Glu Asn Met Ser Val Gln Asp Pro Ala Ser Ser
380 385 390
Pro Ser Ile Gln Asp Gly Gly Leu Met Gln Ala Ser Val Pro Gly
395 400 405
Pro Ser Glu Glu Pro Val Val Tyr Asn Pro Thr Thr Ala Ala Phe
410 415 420
Ile Cys Asp Ser Leu Va1 Asn Glu Lys Thr Ile Gly Ser Pro Pro
425 430 435
Asn Glu Phe Tyr Cys Ser Glu Asn Thr Ser Val Pro Asn Glu Ser
440 445 450
Asn Lys Ile Leu Val Asn Lys Asp Val Pro Gln Lys Pro Gly Gly
455 460 465
Glu Thr Thr Pro Ser Va1 Thr Asp Leu Leu Asn Tyr Phe Leu A1a
470 475 480
Pro Glu Ile Leu Thr Gly Asp Asn Gln Tyr Tyr Cys Glu Asn Cys
485 490 495
Ala Ser Leu Gln Asn Ala Glu Lys Thr Met Gln Ile Thr Glu Glu
500 505 510
Pro Glu Tyr Leu Ile Leu Thr Leu Leu Arg Phe Ser Tyr Asp Gln
515 520 525
Lys Tyr His Val Arg Arg Lys Ile Leu Asp Asn Val Ser Leu Pro
530 535 540
Leu Val Leu Glu Leu Pro Val Lys Arg Ile Thr Ser Phe Ser Ser
545 550 555
Leu Ser Glu Ser Trp Ser Val Asp Val Asp Phe Thr Asp Leu Ser
560 565 570
Glu Asn Leu Ala Lys Lys Leu Lys Pro Ser Gly Thr Asp Glu Ala
575 580 585
Ser Cys Thr Lys Leu Val Pro Tyr Leu Leu Ser Ser Val Val Val
590 595 600
His Ser Gly Ile Ser Ser Glu Ser Gly His Tyr Tyr Ser Tyr Ala
605 610 615
Arg Asn Ile Thr Ser Thr Asp Ser Ser Tyr Gln Met Tyr His Gln
620 625 630
Ser Glu Ala Leu Ala Leu A1a Ser Ser Gln Sex His Leu Leu Gly
635 640 645
Arg Asp Ser Pro Ser Ala Val Phe Glu Gln Asp Leu Glu Asn Lys
650 655 660
Glu Met Ser Lys Glu Trp Phe Leu Phe Asn Asp Ser Arg Val Thr
665 670 675
Phe Thr Ser Phe Gln Ser Val Gln Lys Ile Thr Ser Arg Phe Pro
680 685 690
Lys Asp Thr Ala Tyr Val Leu Leu Tyr Lys Lys G1n His Ser Thr
695 700 705
Asn Gly Leu Ser Gly Asn Asn Pro Thr Ser Gly Leu Trp Ile Asn
710 715 720
Gly Asp Pro Pro Leu Gln Lys Glu Leu Met Asp Ala Ile Thr Lys
725 730 735
Asp Asn Lys Leu Tyr Leu Gln Glu Gln Glu Leu Asn Ala Arg Ala
740 745 750
Arg Ala Leu Gln Ala Ala Ser Ala Ser Cys Ser Phe Arg Pro Asn
755 760 765
Gly Phe Asp Asp Asn Asp Pro Pro Gly Ser Cys Gly Pro Thr Gly
770 775 780
11/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
Gly Gly Gly Gly Gly Gly Phe Asn Thr Val Gly Arg Leu Val Phe
785 790 795
<210> 9
<211> 919
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2278688CD1
<400> 9
Met Trp Leu Ala Ala Ala Ala Pro Ser Leu Ala Arg Arg Leu Leu
1 5 10 15
Phe Leu Gly Pro Pro Pro Pro Pro Leu Leu Leu Leu Val Phe Ser
20 25 30
Arg Ser Ser Arg Arg Arg Leu His Ser Leu Gly Leu Ala Ala Met
35 40 45
Pro Glu Lys Arg Pro Phe G1u Arg Leu Pro Ala Asp Val Ser Pro
50 55 60
Ile Asn Cys Ser Leu Cys Leu Lys Pro Asp Leu Leu Asp Phe Thr
65 70 75
Phe Glu Gly Lys Leu Glu Ala Ala Ala Gln Val Arg Gln Ala Thr
80 85 90
Asn Gln Ile Val Met Asn Cys Ala Asp Ile Asp Ile Ile Thr Ala
95 100 105
Ser Tyr Ala Pro Glu Gly Asp Glu Glu Ile His Ala Thr Gly Phe
110 115 120
Asn Tyr Gln Asn Glu Asp Glu Lys Val Thr Leu Ser Phe Pro Ser
125 130 135
Thr Leu Gln Thr Gly Thr Gly Thr Leu Lys Ile Asp Phe Val Gly
140 145 150
Glu Leu Asn Asp Lys Met Lys Gly Phe Tyr Arg Ser Lys Tyr Thr
155 160 165
Thr Pro Ser Gly Glu Val Arg Tyr Ala Ala Val Thr Gln Phe Glu
170 175 180
A1a Thr Asp Ala Arg Arg Ala Phe Pro Cys Trp Asp Glu Pro Ala
185 190 195
Ile Lys Ala Thr Phe Asp Ile Ser Leu Val Val Pro Lys Asp Arg
200 205 210
Val Ala Leu Ser Asn Met Asn Val Ile Asp Arg Lys Pro Tyr Pro
215 220 225
Asp Asp Glu Asn Leu Val Glu Val Lys Phe Ala Arg Thr Pro Val
230 235 240
Met Ser Thr Tyr Leu Val Ala Phe Val Val Gly Glu Tyr Asp Phe
245 250 255
Val Glu Thr Arg Ser Lys Asp Gly Val Cys Val Arg Val Tyr Thr
260 265 270
Pro Val Gly Lys Ala Glu Gln G1y Lys Phe Ala Leu Glu Val Ala
275 280 285
Ala Lys Thr Leu Pro Phe Tyr Lys Asp Tyr Phe Asn Val Pro Tyr
290 295 300
Pro Leu Pro Lys Ile Asp Leu Ile Ala Tle Ala Asp Phe Ala Ala
305 310 315
Gly Ala Met Glu Asn Trp Gly Leu Val Thr Tyr Arg Glu Thr Ala
320 325 330
Leu Leu Ile Asp Pro Lys Asn Ser Cys Ser Ser Ser Arg Gln Trp
335 340 345
Val Ala Leu Val Val Gly His Glu Leu Ala His Gln Trp Phe Gly
350 355 360
Asn Leu Val Thr Met Glu Trp Trp Thr His Leu Trp Leu Asn Glu
365 370 375
Gly Phe Ala Ser Trp Ile Glu Tyr Leu Cys Val Asp His Cys Phe
380 385 390
Pro Glu Tyr Asp Ile Trp Thr Gln Phe Val Ser Ala Asp Tyr Thr
12/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
395 400 405
Arg Ala Gln G1u Leu Asp Ala Leu Asp Asn Ser His Pro Ile Glu
410 415 420
Val Ser Val Gly His Pro Ser Glu Val Asp Glu Ile Phe Asp Ala
425 430 435
I1e Ser Tyr Ser Lys Gly Ala Ser Val Ile Arg Met Leu His Asp
440 445 450
Tyr Ile Gly Asp Lys Asp Phe Lys Lys Gly Met Asn Met Tyr Leu
455 460 465
Thr Lys Phe Gln Gln Lys Asn Ala Ala Thr Glu Asp Leu Trp Glu
470 475 480
Ser Leu Glu Asn Ala Ser Gly Lys Pro Tle Ala Ala Val Met Asn
485 490 495
Thr Trp Thr Lys Gln Met Gly Phe Pro Leu Ile Tyr Val Glu Ala
500 505 510
Glu Gln Val Glu Asp Asp Arg Leu Leu Arg Leu Ser Gln Lys Lys
515 520 525
Phe Cys Ala Gly Gly Ser Tyr Val Gly Glu Asp Cys Pro Gln Trp
530 535 540
Met Val Pro Tle Thr Ile Ser Thr Ser G1u Asp Pro Asn Gln Ala
545 550 555
Lys Leu Lys Ile Leu Met Asp Lys Pro Glu Met Asn Val Val Leu
560 565 570
Lys Asn Val Lys Pro Asp Gln Trp Val Lys Leu Asn Leu Gly Thr
575 580 585
Val Gly Phe Tyr Arg Thr Gln Tyr Ser Ser Ala Met Leu Glu Ser
590 595 600
Leu Leu Pro Gly Ile Arg Asp Leu Ser Leu Pro Pro Val Asp Arg
605 610 615
Leu G1y Leu Gln Asn Asp Leu Phe Ser Leu Ala Arg Ala Gly Ile
620 625 630
Ile Ser Thr Val Glu Val Leu Lys Val Met Glu Ala Phe Va1 Asn
635 640 645
Glu Pro Asn Tyr Thr Val Trp Ser Asp Leu Ser Cys Asn Leu Gly
650 655 660
Ile Leu Ser Thr Leu Leu Ser His Thr Asp Phe Tyr G1u Glu Ile
665 670 675
G1n Glu Phe Val Lys Asp Val Phe Ser Pro Ile Gly Glu Arg Leu
680 685 690
Gly Trp Asp Pro Lys Pro Gly Glu Gly His Leu Asp Ala Leu Leu
695 700 705
Arg Gly Leu Val Leu Gly Lys Leu Gly Lys Ala Gly His Lys Ala
710 715 720
Thr Leu Glu Glu Ala Arg Arg Arg Phe Lys Asp His Val Glu Gly
725 730 735
Lys Gln Ile Leu Ser Ala Asp Leu Arg Ser Pro Val Tyr Leu Thr
740 745 750
Val Leu Lys His Gly Asp Gly Thr Thr Leu Asp Ile Met Leu Lys
755 760 765
Leu His Lys Gln Ala Asp Met Gln Glu Glu Lys Asn Arg Ile Glu
770 775 780
Arg Val Leu Gly Ala Thr Leu Leu Pro Asp Leu Ile Gln Lys Val
785 790 795
Leu Thr Phe Ala Leu Ser Glu Glu Val Arg Pro Gln Asp Thr Val
800 805 810
Ser Val Ile Gly Gly Val Ala Gly Gly Ser Lys His Gly Arg Lys
815 820 825
Ala Ala Trp Lys Phe Ile Lys Asp Asn Trp Glu Glu Leu Tyr Asn
830 835 840
Arg Tyr Gln Gly Gly Phe Leu Ile Ser Arg Leu Ile Lys Leu Ser
845 850 855
Val Glu Gly Phe Ala Va1 Asp Lys Met Ala Gly Glu Val Lys Ala
860 865 870
Phe Phe Glu Ser His Pro Ala Pro Ser Ala Glu Arg Thr Ile Gln
875 880 885
Gln Cys Cys Glu Asn Ile Leu Leu Asn Ala Ala Trp Leu Lys Arg
890 895 900
13126
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
Asp Ala Glu Ser Ile His Gln Tyr Leu Leu Gln Arg Lys Ala Ser
905 910 915
Pro Pro Thr Va1
<210> 10
<211> 209
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4043361CD1
<400> 10
Met Glu Gln Pro Arg Lys Ala Val Val Val Thr Gly Phe Gly Pro
1 5 10 15
Phe Gly Glu His Thr Val Asn Ala Ser Trp Ile Ala Val Gln Glu
20 25 30
Leu Glu Lys Leu G1y Leu Gly Asp Ser Val Asp Leu His Val Tyr
35 40 45
Glu Ile Pro Val Glu Tyr Gln Thr Val Gln Arg Leu Ile Pro Ala
50 55 60
Leu Trp Glu Lys His Ser Pro G1n Leu Val Val His Val Gly Val
65 70 75
Ser Gly Met Ala Thr Thr Val Thr Leu Glu Lys Cys Gly His Asn
80 85 90
Lys Gly Tyr Lys G1y Leu Asp Asn Cys Arg Phe Cys Pro Gly Ser
95 100 105
Gln Cys Cys Val Glu Asp Gly Pro Glu Ser Ile Asp Ser Ile Ile
110 115 120
Asp Met Asp Ala Val Cys Lys Arg Val Thr Thr Leu Gly Leu Asp
125 130 135
Val Ser Val Thr Ile Ser Gln Asp Ala Gly Arg Lys Lys Pro Phe
140 145 150
Pro Ala Lys Gly Asp Cys Val Phe Cys Arg Arg Arg Arg Ala Arg
155 160 165
Ser Leu Gln A1a Gln Cys Gly Phe Ser Leu Thr Pro Ala Leu Glu
170 175 180
Leu Leu Pro Val Pro Phe Leu Lys Leu Leu Cys Pro Gly Pro Pro
185 190 195
Arg Arg Arg Arg Ile Cys Arg Ile Leu Pro Gly Ala Gly Leu
200 205
<210> 11
<211> 77
<212> pRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3937958CD1
<400> 11
Met Gly Lys G1u Lys Ala Leu Ser Leu Gln Met Met Lys Tyr Trp
1 5 10 15
Ala Asn Phe Ala Arg Thr Gly Asn Pro Asn Asp Gly Asn Leu Pro
20 25 30
Cys Trp Pro Arg Tyr Asn Lys Asp Glu Lys Tyr Leu Gln Leu Asp
35 40 45
Phe Thr Thr Arg Val Gly Met Lys Leu Lys Glu Lys Lys Met Ala
50 55 60
Phe Trp Met Ser Leu Tyr Gln Ser Gln Arg Pro Glu Lys Gln Arg
65 70 75
Gln Phe
14!26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
<210> 12
<211> 414
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7257324CD1
<400> 12
Met Asn Pro Thr Leu Gly Leu Ala Ile Phe Leu Ala Val Leu Leu
1 5 10 15
Thr Val Lys Gly Leu Leu Lys Pro Ser Phe Ser Pro Arg Asn Tyr
20 25 30
Lys Ala Leu Ser Glu Val Gln G1y Trp Lys Gln Arg Met Ala Ala
35 40 45
Lys Glu Leu Ala Arg Gln Asn Met Asp Leu G1y Phe Lys Leu Leu
50 55 60
Lys Lys Leu Ala Phe Tyr Asn Pro Gly Arg Asn Ile Phe Leu Ser
65 70 75
Pro Leu Ser Ile Ser Thr Ala Phe Ser Met Leu Cys Leu Gly Ala
80 85 90
Gln Asp Ser Thr Leu Asp Glu Ile Lys Gln Gly Phe Asn Phe Arg
95 100 105
Lys Met Pro G1u Lys Asp Leu His Glu Gly Phe His Tyr Ile Ile
110 115 120
His Glu Leu Thr Gln Lys Thr Gln Asp Leu Lys Leu Ser Ile Gly
125 130 135
Asn Thr Leu Phe Ile Asp Gln Arg Leu G1n Pro Gln Arg Lys Phe
140 145 150
Leu Glu Asp Ala Lys Asn Phe Tyr Ser Ala Glu Thr Ile Leu Thr
155 160 165
Asn Phe Gln Asn Leu Glu Met Ala Gln Lys Gln Tle Asn Asp Phe
170 175 180
Ile Ser Gln Lys Thr His Gly Lys Ile Asn Asn Leu Ile Glu Asn
185 190 195
Ile Asp Pro Gly Thr Val Met Leu Leu A1a Asn Tyr Ile Phe Phe
200 205 210
Arg Ala Arg Trp Lys His Glu Phe Asp Pro Asn Val Thr Lys Glu
215 220 225
Glu Asp Phe Phe Leu Glu Lys Asn Ser Ser Val Lys Val Pro Met
230 235 240
Met Phe Arg Ser Gly Ile Tyr Gln Val Gly Tyr Asp Asp Lys Leu
245 250 255
Ser Cys Thr Ile Leu Glu Ile Pro Tyr Gln Lys Asn I1e Thr Ala
260 265 270
Ile Phe Ile Leu Pro Asp Glu Gly Lys Leu Lys His Leu Glu Lys
275 280 285
Gly Leu Glri Val Asp Thr Phe Ser Arg Trp Lys Thr Leu Leu Ser
290 295 300
Arg Arg Val Val Asp Val Ser Val Pro Arg Leu His Met Thr Gly
305 310 315
Thr Phe Asp Leu Lys Lys Thr Leu Ser Tyr Ile Gly Val Ser Lys
320 325 330
Ile Phe Glu Glu His Gly Asp Leu Thr Lys Ile Ala Pro His Arg
335 340 345
Ser Leu Lys Val Gly G1u Ala Val His Lys Ala Glu Leu Lys Met
350 355 360
Asp Glu Arg Gly Thr Glu Gly Ala Ala Gly Thr Gly Ala Gln Thr
365 370 375
Leu Pro Met Glu Thr Pro Leu Val Val Lys Ile Asp Lys Pro Tyr
380 385 390
Leu Leu Leu Ile Tyr Ser Glu Lys Ile Pro Ser Val Leu Phe Leu
395 400 405
Gly Lys Ile Val Asn Pro Ile Gly Lys
410
15/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
<210> 13
<211> 397
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472038CD1
<400> 13
Met Pro Arg Ala Ile Ser Pro Leu Met Arg Phe Gln His Pro Val
1 5 10 15
Ser Cys Lys Leu Gln Leu Tyr Arg Val Pro Leu Arg Arg Phe Pro
20 25 30
Ser Ala Arg His Arg Phe Glu Lys Leu Gly Ile Arg Met Asp Arg
35 40 45
Leu Arg Leu Lys Tyr Ala Glu Glu Val Ser His Phe Arg Gly Glu
50 55 60
Trp Asn Ser Ala Val Lys Ser Thr Pro Leu Ser Asn Tyr Leu Asp
65 70 75
Ala Gln Tyr Phe Gly Pro Ile Thr Ile Gly Thr Pro Pro Gln Thr
80 85 90
Phe Lys Val Ile Phe Asp Thr Gly Ser Ser Asn Leu Trp Val Pro
95 100 105
Ser Ala Thr Cys Ala Ser Thr Met Val Ala Cys Arg Va1 His Asn
110 115 120
Arg Tyr Phe Ala Lys Arg Ser Thr Ser His Gln Val Arg Gly Asp
125 13 0 13 5
His Phe Ala Ile His Tyr Gly Ser Gly Ser Leu Ser G1y Phe Leu
140 145 150
Ser Thr Asp Thr Val Arg Val Ala Gly Leu Glu Ile Arg Asp Gln
155 160 165
Thr Phe Ala Glu Ala Thr Glu Met Pro Gly Pro Ile Phe Leu Ala
170 175 180
Ala Lys Phe Asp Gly Ile Phe Gly Leu Ala Tyr Arg Ser Ile Ser
185 190 195
Met Gln Arg Ile Lys Pro Pro Phe Tyr Ala Met Met Glu Gln Gly
200 205 210
Leu Leu Thr Lys Pro Ile Phe Ser Val Tyr Leu Ser Arg Asn Gly
215 220 225
Glu Lys Asp Gly Gly A1a Ile Phe Phe Gly Gly Ser Asn Pro His
230 235 240
Tyr Tyr Thr Gly Asn Phe Thr Tyr Val Gln Val Ser His Arg Ala
245 250 255
Tyr Trp Gln Val Lys Met Asp Ser Ala Val Ile Arg Asn Leu Glu
260 265 270
Leu Cys Gln Gln Gly Cys Glu Val Ile Ile Asp Thr Gly Thr Ser
275 280 285
Phe Leu Ala Leu Pro Tyr Asp Gln Ala Ile Leu Ile Asn Glu Ser
290 295 300
Ile Gly Gly Thr Pro Ser Ser Phe Gly Gln Phe Leu Val Pro Cys
305 310 315
Asp Ser Val Pro Asp Leu Pro Lys Ile Thr Phe Thr Leu Gly Gly
320 325 330
Arg Arg Phe Phe Leu Glu Ser His Glu Tyr Val Phe Arg Asp Ile
335 340 345
Tyr Gln Asp Arg Arg Ile Cys Ser Ser Ala Phe Ile Ala Val Asp
350 355 360
Leu Pro Ser Pro Ser Gly Pro Leu Trp Ile Leu Gly Asp Val Phe
365 370 375
Leu Gly Lys Tyr Tyr Thr Glu Phe Asp Met Glu Arg His Arg Ile
380 385 390
Gly Phe Ala Asp Ala Arg Ser
395
<210> 14
<211> 145
16/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472041CD1
<400> 14
Met Gly Ile Gly Cys Trp Arg Asn Pro Leu Leu Leu Leu Ile Ala
1 5 10 15
Leu Val Leu Ser Ala Lys Leu Gly His Phe Gln Arg Trp Glu Gly
20 25 30
Phe Gln Gln Lys Leu Met Ser Lys Lys Asn Met Asn Ser Thr Leu
35 40 45
Asn Phe Phe Tle Gln Ser Tyr Asn Asn A1a Ser Asn Asp Thr Tyr
50 55 60
Leu Tyr Arg Val Gln Arg Leu Ile Arg Ser Gln Met Gln Leu Thr
65 70 75
Thr Gly Val G1u Tyr I1e Val Thr Val Lys Ile Gly Trp Thr Lys
80 85 90
Cys Lys Arg Asn Asp Thr Ser Asn Ser Ser Cys Pro Leu G1n Ser
95 100 105
Lys Lys Leu Arg Lys Ser Leu Ile Cys Glu Ser Leu Ile Tyr ~Thr
110 115 120
Met Pro Trp Ile Asn Tyr Phe Gln Leu Trp Asn Asn Ser Cys Leu
125 13 0 135
Glu Ala Glu His Val Gly Arg Asn Leu Arg
140 145
<210> 15
<211> 4028
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1714846CB1
<400> 15
gccattccgg gcggccgctc cctccggtcc cctctctccc ttccccaaag cagcccgcgg 60
accggcagca aaggaacgtg cgaacgcgtg acgccgcccg actggctcgc gctctcccgt 120
gccccggcgt cctccgcccg ctcatggccc gggccgccgc ggacgagcgg cgctgaggcg 180
ggccgcgtgg agacgtgagg cggccgccgt ggccctcaca gtcggcgttt cgccgcctgc 240
ccgcggtgcc cgcgcacgcc ggccgccatc gccttcgcgc ctggctggcg ggggcgctgt 300
cctcccaggc cgtccgcgcc gctccctgga gctcggcgga gcgcggcagc cagggccggc 360
ggaggcgcga ggagccgggc gccaccgccg ccgccgccgc cgccgccgcg ggggccatga 420
ccgtggagca gaacgtgctg cagcagagcg cggcgcagaa gcaccagcag acgtttttga 480
atcaactgag agaaattacg gggattaatg acacccagat actacagcaa gccttgaagg 540
atagtaatgg aaacttggaa ttagcagtgg ctttccttac tgcgaagaat gctaagaccc 600
ctcagcagga ggagacaact tactaccaaa cagcacttcc tggcaatgat agatacatca 660
gtgtgggaag ccaagcagat acaaatgtga ttgatctcac tggagatgat aaagatgatc 720
ttcagagagc aattgccttg agtttggccg aatcaaacag ggcattcagg gagactggaa 780
taactgatga ggaacaagcc attagcagag ttcttgaagc cagcatagca gagaataaag 840
catgtttgaa gaggacacct acagaagttt ggagggattc tcgaaaccct tatgatagaa 900
aaagacagga caaagctccc gttgggctaa agaatgttgg caatacttgt tggtttagtg 960
ctgttattca gtcattattt aatcttttgg aatttagaag attagttctg aattacaagc 1020
ctccatcaaa tgctcaagat ttaccccgaa accaaaagga acatcggaat ttgcctttta 1080
tgcgtgagct gaggtatcta tttgcacttc ttgttggtac caaaaggaag tatgttgatc 1140
catcaagagc agttgaaatt cttaaggatg ctttcaaatc aaatgactca cagcagcaag 1200
atgtgagtga gtttacacac aaattattag attggttaga agatgccttc caaatgaaag 1260
ctgaagagga gacggatgaa gagaagccaa agaaccccat ggtagagttg ttctatggca 1320
gattcctggc tgtgggagta cttgaaggta aaaaatttga aaacactgaa atgtttggtc 1380
agtacccact tcaggtcaat gggttcaaag atctgcatga gtgcctagaa gctgcaatga 1440
ttgaaggaga aattgagtct ttacattcag agaattcagg aaaatcaggc caagagcatt 1500
ggtttactga attaccacct gtgttaacat ttgaattgtc aagatttgaa tttaatcagg 1560
cattgggaag accagaaaaa attcacaaca aattagaatt tccccaagtt ttatatttgg 1620
acagatacat gcacagaaac agagaaataa caagaattaa gagggaagag atcaagagac 1680
17/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
tgaaagatta cctcacggta ttacaacaaa ggctagaaag atatttaagc tatggttccg 1740
gtcccaaacg attccccttg gtagatgttc ttcagtatgc attggaattt gcctcaagta 1800
aacctgtttg cacttctcct gttgacgata ttgacgctag ttccccacct agtggttcca 1860
taccatcaca gacattacca agcacaacag aacaacaggg agccctatct tcagaactgc 1920
caagcacatc accttcatca gttgctgcca tttcatcgag atcagtaata cacaaaccat 1980
ttactcagtc ccggatacct ccagatttgc ccatgcatcc ggcaccaagg cacataacgg 2040
aggaagaact ttctgtgctg gaaagttgtt tacatcgctg gaggacagaa atagaaaatg 2100
acaccagaga tttgcaggaa agcatatcca gaatccatcg aacaattgaa ttaatgtact 2160
ctgacaaatc tatgatacaa gttccttatc gattacatgc cgttttagtt cacgaaggcc 2220
aagctaatgc tgggcactac tgggcatata tttttgatca tcgtgaaagc agatggatga 2280
agtacaatga tattgctgtg acaaaatcat catgggaaga gctagtgagg gactcttttg 2340
gtggttatag aaatgccagt gcatactgtt taatgtacat aaatgataag gcacagttcc 2400
taatacaaga ggagtttaat aaagaaactg ggcagcccct tgttggtata gaaacattac 2460
caccggattt gagagatttt gttgaggaag acaaccaacg atttgaaaaa gaactagaag 2520
aatgggatgc acaacttgcc cagaaagctt tgcaggaaaa gcttttagcg tctcagaaat 2580
tgagagagtc agagacttct gtgacaacag cacaagcagc aggagaccca gaatatctag 2640
agcagccatc aagaagtgat ttctcaaagc acttgaaaga agaaactatt caaataatta 2700
ccaaggcatc acatgagcat gaagataaaa gtcctgaaac agttttgcag tcggcaatta 2760
agttggaata tgcaaggttg gttaagttgg cccaagaaga caccccacca gaaaccgatt 2820
atcgtttaca tcatgtagtg gtctacttta tccagaacca ggcaccaaag aaaattattg 2880
agaaaacatt actagaacaa tttggagata gaaatttgag ttttgatgaa aggtgtcaca 2940
acataatgaa agttgctcaa gccaaactgg aaatgataaa acctgaagaa gtaaacttgg 3000
aggaatatga ggagtggcat caggattata ggaaattcag ggaaacaact atgtatctca 30&0
taattgggct agaaaatttt caaagagaaa gttatataga ttccttgctg ttcctcatct 3120
gtgcttatca gaataacaaa gaactcttgt ctaaaggctt atacagagga catgatgaag 3180
aattgatatc acattataga agagaatgtt tgctaaaatt aaatgagcaa gccgcagaac 3240
tcttcgaatc tggagaggat cgagaagtaa acaatggttt gattatcatg aatgagttta 3300
ttgtcccatt tttgccatta ttactggtgg atgaaatgga agaaaaggat atactagctg 3360
tagaagatat gagaaatcga tggtgttcct accttggtca agaaatggaa ccacacctcc 3420
aagaaaagct gacagatttt ttgccaaaac tgcttgattg ttctatggag attaaaagtt 3480
tccatgagcc accgaagtta ccttcatatt ccacgcatga actctgtgag cgatttgccc 3540
gaatcatgtt gtccctcagt cgaactcctg ctgatggaag ataaactgca cactttccct 3600
gaacacactg tataaactct ttttagttct taacccttgc cttcctgtca cagggtttgc 3660
ttgttgctgc tatagttttt aacttttttt tattttaata actgcaaaag acaaaatgac 3720
tatacagact ttagtcagac tgcagacaat aaagctgaaa atcgcatggc gctcagacat 3780
tttaaccgga actgatgtat aatcacaaat ctaattgatt ttattatggc aaaactatgc 3840
ttttgccacc ttcctgttgc agtattactt tgcttttatc ttttctttct caacagcttt 3900
ccattcagtc tggatccttc catgactaca gccatttaag tgttcagcac tgtgtacgat 3960.
acataatatt tggtagcttg taaatgaaat aaagaataaa gttttattta tggctaccta 4020
aaaaaaaa 4028
<210> 16
<211> 1422
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1856589CB1
<400> 16
ggcccgggca ggcagggtgg gtgcgcaggg aggcgtacac tgctcttccc ctccgcgctc 60
ccctcagggc caggcggcca ggaccccgga gcgagcggat gggagccgcc acctgtaggg 120
gctccaggat ccccagcggc cccccagtcc agggggaacg cagtgcgccc cgcttcggtg 180
ttacttccct cagcctgtgg ccagcggact tcaaggataa ctggaggatt gccggctcca 240
gacaggaagt ggccctggca ggtgagcctg cagaccagca acagacacat ctgcggaggc 300
tcccttatcg ccagacactg ggttataaag aggacacaac caatccagtt tgtggtgagc 360
cctggtggtc ggaggatttg gaaatgaccc gccattggcc ctgggaggtg agcctccgga 420
tggaaaatga gcacgtgtgt ggaggggccc tcattgaccc cagctgggtg gtgactgcgg 480
cccactgcag ccaaggcacc aaagagtact cagtggtgct tggcacctcc aagctgcagc 540
ccatgaactt cagcagggcc ctctgggtcc ctgtgaggga catcattatg caccccaagt 600
actggggccg ggccttcatc atgggtgacg ttgcccttgt ccaccttcaa acacctgtca 660
ccttcagtga gtacgtgcag cccatctgcc tcccggagcc caatttcaac ctgaaggttg 720
ggacgcagtg ttgggtgact ggctggagcc aggttaagca gcgcttttca ggctccacag 780
ccaactccat gctgacccca gagctgcagg aggctgaggt gtttatcatg gacaacaaga 840
ggtgtgaccg gcattacaag aagtecttct tccccetagt tgtcccectt gtcctggggg 900
acatgatctg tgccaccaat tatggggaaa acttgtgcta tggggattct ggagggccat 960
18/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
tggcttgtga agttgagggc agatggattc tggctggggt gttgtcctgg gaaaaggcct 1020
gcgtgaaggc acagaatcca ggtgtgtaca cccgcgtcac caaatacacc aaatggatca 1080
agaagcaaat gagcaatgga gccttctcag gtccctgtgc ctctgcctgc ctcctgttcc 1140
tgtgctggcc gctgcagccc cagatgggct cctgacctcc ctaccttttc ctcctcctgc 1200
cttgcctctg ctgaatgggg ccagatggtt tgaccaaggt catgtgtcca tcttcaaaaa 1260
gagtcagggt ggggaagagt aacccctggg agaatgggtc tggctttggc atcccggtga 1320
ggagaagtgt ggtggatgac taggccttgg gtgagcagga gaagggaagt gtggcctaga 1380
aggattctgg aatctgggac caggagagca gggattaaac at 1422
<210> 17
<211> 1911
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2617672CB1
<400> 17
cccacgcgtc cgccggcggt cgcagagcca ggaggcggag gcgcgcgggc cagcctgggc 60
cccagcccac accttcacca gggcccagga gccaccatgt ggcgatgtcc actggggcta 120
ctgctgttgc tgccgctggc tggccacttg gctctgggtg cccagcaggg tcgtgggcgc 180
cgggagctag caccgggtct gcacctgcgg ggcatccggg acgcgggagg ccggtactgc 240
caggagcagg acctgtgctg ccgcggccgt gccgacgact gtgccctgcc ctacctgggc 300
gccatctgtt actgtgacct cttctgcaac cgcacggtct ccgactgctg ccctgacttc 360
tgggacttct gcctcggcgt gccaccccct tttcccccga tccaaggatg tatgcatgga 420
ggtcgtatct atccagtctt gggaacgtac tgggacaact gtaaccgttg cacctgccag 480
gagaacaggc agtggcagtg tgaccaagaa ccatgcctgg tggatccaga catgatcaaa 540
gccatcaacc agggcaacta tggctggcag gctgggaacc acagcgcctt ctggggcatg 600
accctggatg agggcattcg ctaccgcctg ggcaccatcc gcccatcttc ctcggtcatg 660
aacatgcatg aaatttatac agtgctgaac ccaggggagg tgcttcccac agccttcgag 720
gcctctgaga agtggcccaa cctgattcat gagcctcttg accaaggcaa ctgtgcaggc 780
tcctgggcct tctccacagc agctgtggca tccgatcgtg tctcaatcca ttctctggga 840
cacatgacgc ctgtcctgtc gccccagaac ctgctgtctt gtgacaccca ccagcagcag 900
ggctgccgcg gtgggcgtct cgatggtgcc tggtggttcc tgcgtcgccg aggggtggtg 960
tctgaccact gctacccctt ctcgggccgt gaacgagacg aggctggccc tgcgcccccc 1020
tgtatgatgc acagccgagc catgggtcgg ggcaagcgcc aggccactgc ccactgcccc 1080
aacagctatg ttaataacaa tgacatctac caggtcactc ctgtctaccg cctcggctcc 1140
aacgacaagg agatcatgaa ggagctgatg gagaatggcc ctgtccaagc cctcatggag 1200
gtgcatgagg acttcttcct atacaaggga ggcatctaca gccacacgcc agtgagcctt 1260
gggaggccag agagataccg ccggcatggg acccactcag tcaagatcac aggatgggga 1320
gaggagacgc tgccagatgg aaggacgctc aaatactgga ctgcggccaa ctcctggggc 2380
ccagcctggg gcgagagggg ccacttccgc atcgtgcgcg gcgtcaatga gtgcgacatc 1440
gagagcttcg tgctgggcgt ctggggccgc gtgggcatgg aggacatggg tcatcactga 1500
ggctgcgggc accacgcggg gtccggcctg ggatccaggc taagggccgg cggaagaggc 1560
cccaatgggg cggtgacccc agcctcgccc gacagagccc ggggcgcagg cgggcgccag 1620
ggcgctaatc ccggcgcggg ttccgctgac gcagcgcccc gcctgggagc cgcgggcagg 1680
cgagactggc ggagccccag acctcccagt ggggacgggg cagggcctgg cctgggaaga 1740
gcacagctgc agatcccagg cctctggcgc ccccactcaa gactaccaaa gccaggacac 1800
ctcaagtctc cagccccact accccacccc acccctgtat tcttattctt cagatattta 1860
tttttctttt cactgtttta aaataaaacc aaagtattga taaaaaaaaa a 1911
<210> 18
<211> 854
<212> DNA
<213> Homo Sapiens
<220>
<222> misc_feature
<223> Incyte ID No: 2769104CB1
<400> 18
caccttttgt tccctatcct gggccagttc tctcgcaggt cccagatgtc cagttccaga 60
tgcctggacc cagagtgtgg gggaaatatc tctggagaag ccctcactcc aaaggctgtc 120
caggcgcaat gtggtggctg cttctctggg gagtcctcca ggcttgccca acccggggct 180
ccgtcctctt ggcccaagag ctaccccagc agctgacatc ccccgggtac ccagagccgt 240
atggcaaagg ccaagagagc agcacggaca tcaaggctcc agagggcttt gctgtgaggc 300
19/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
tcgtcttcca ggacttcgac ctggagccgt cccaggactg tgcaggggac tctgtcacaa 360
tctcattcgt cgggtcggat ccaagccagt tctgtggtca gcaaggctcc cctctgggca 420
ggccccctgg tcagagggag tttgtatcct cagggaggag tttgcggctg accttccgca 480
cacagccttc ctcggagaac aagactgccc acctccacaa gggcttcctg gccctctacc 540
aaaccgtggg tgagtgtccc tcctgggggt gcagggaggg agcctctgtt cccagccatg 600
accctggtat cttcaagcct taagtggaag cttgagtgac agctgaggct ggggactcag 660
ggacacctgg gctggatccc agccctgccc ctgctggcaa gcaaccctat taagagacag 720
ccgtagctga gcccccagcg gttgtttcca tgcagattta caggcccagt gtttgcagat 780
catctcattc ttaaagagat gccaaaaatc cagattttta agtaaaatta taaattttca 840
aaaaaaaaaa aaaa 854
<210> 19
<211> 1386
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4802789CB1
<400> 19
gacgctgcgg cccggcccgg cgggtaaata acagatgcgg gtgaaagatc caactaaagc 60
tttacctgag aaagccaaaa gaagtaaaag gcctactgta cctcatgatg aagactcttc 120
agatgatatt gctgtaggtt taacttgcca acatgtaagt catgctatca gcgtgaatca 180
tgtaaagaga gcaatagctg agaatctgtg gtcagtttgc tcagaatgtt taaaagaaag 240
aagattctat gatgggcagc tagtacttac ttctgatatt tggttgtgcc tcaagtgtgg 300
cttccaggga tgtggtaaaa actcagaaag ccaacattca ttgaagcact ttaagagttc 360
cagaacagag ccccattgta ttataattaa tctgagcaca tggattatat ggtgttatga 420
atgtgatgaa aaattatcaa cgcattgtaa taagaaggtt ttggctcaga tagttgattt 480
tctccagaaa catgcttcta aaacacaaac aagtgcattt tctagaatca tgaaactttg 540
tgaagaaaaa tgtgaaacag atgaaataca gaagggagga aaatgcagaa atttatctgt 600
aagaggaatt acaaatttag gaaatacttg cttttttaat gcagtcatgc agaacttggc 660
acagacttat actcttactg atctgatgaa tgagatcaaa gaaagtagta caaaactcaa 720
gatttttcct tcctcagact ctcagctgga cccattggtg gtggaacttt caaggcctgg 780
accactgacc tcagccttgt tcctgtttct tcacagcatg aaggagactg aaaaaggacc 840
actttctcct aaagttcttt ttaatcagct ttgtcagaag tgggtgcatc tacatttaat 900
ataaataatt atgagttaca aaatactaat gtattcatca tttaacatga atagtcgttt 960
ttactgtaac tttgctctta ttgccctgac tatgaagaga actaaaattt gttacagctc 1020
tatgctttat gaaaattata tctcagtcct cagaagaagc agcttatcct catatataag 1080
gaaatggaga cacagaaatt aaatggctca cctagtctga gtgaaaagct gagaatcaaa 1140
tggagatctg tcctgacttg gatgcctatg ttgtaatacc ataaagtgag aaaaccatag 1200
agttgtaaaa tctagaaagt accgtaagat aacatctaat ctagctttct tattttaaaa 1260
gatgagctgt gaggcaaata gagtttaagt gaatttctca aggtattaca gtatgtttaa 1320
aaaccaaatc cttatgtgcc tggaaataaa cacataaagg atctgacttg aaaaaaaaaa 1380
aaaaaa 2386
<210> 20
<211> 3323
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 60116897CB1
<400> 20
caaatctgca gcagcatgat ttaagattaa attcatgtat tgaaaatatt gttcagaccc 60
catgtgacat aactggagcc agtgcagtgc catgaagaac tacgagatta gcctggatat 120
taacttgtct tctagagaat agatttcatg ttccattctt ctgcaatggt taattcacac 180
agaaaaccaa tgtttaacat tcacagagga ttttactgct taacagccat cttgccccaa 240
atatgcattt gttctcagtt ctcagtgcca tctagttatc acttcactga ggatcctggg 300
gctttcccag tagccactaa tggggaacga tttccttggc aggagctaag gctccccagt 360
gtggtcattc ctctccatta tgacctcttt gtccacccca atctcacctc tctggacttt 420
gttgcatctg agaagattga agtcttggtc agcaatgcta cccagtttat catcttgcac 480
agcaaagatc ttgaaatcac gaatgccacc cttcagtcag aggaagattc aagatacatg 540
aaaccaggaa aagaactgaa agttttgagt taccctgctc atgaacaaat tgcactgctg 600
gttccagaga aacttacgcc tcacctgaaa tactatgtgg ctatggactt ccaagccaag 660
20/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
ttaggtgatg gctttgaagg gttttataaa agcacataca gaactcttgg tggtgaaaca 720
agaattcttg cagtaacaga ttttgagcca acccaggcac gcatggcttt cccttgcttt 780
gatgaaccgt tgttcaaagc caacttttca atcaagatac gaagagagag caggcatatt 840
gcactatcca acatgccaaa ggttaagaca attgaacttg aaggaggtct tttggaagat 900
cactttgaaa ctactgtaaa aatgagtaca taccttgtag cctacatagt ttgtgatttc 960
cactctctga gtggcttcac ttcatcaggg gtcaaggtgt ccatctatgc atccccagac 1020
aaacggaatc aaacacatta tgctttgcag gcatcactga agctacttga tttttatgaa 1080
aagtactttg atatctacta tccactctcc aaactggatt taattgctat tcctgacttt 1140
gcacctggag ccatggaaaa ttggggcctc attacatata gggagacgtc actgcttttt 1200
gaccccaaga cctcttctgc ttccgataaa ctgtgggtca ccagagtcat agcccatgaa 1260
ctggcgcacc agtggtttgg caacctggtc acaatggaat ggtggaatga tatttggctt 1320
aaggagggtt ttgcaaaata catggaactt atcgctgtta atgctacata tccagagctg 1380
caatttgatg actatttttt gaatgtgtgt tttgaagtaa ttacaaaaga ttcattgaat 1440
tcatcccgcc ctatctccaa accagcggaa accccgactc aaatacagga aatgtttgat 1500
gaagtttcct ataacaaggg agcttgtatt ttgaatatgc tcaaggattt tctgggtgag 1560
gagaaattcc agaaaggaat aattcagtac ttaaagaagt tcagctatag aaatgctaag 1620
aatgatgact tgtggagcag tctgtcaaat agttgtttag aaagtgattt tacatctggt 1680
ggagtttgtc attcggatcc caagatgaca agtaacatgc tcgcctttct gggggaaaat 1740
gcagaggtca aagagatgat gactacatgg actctccaga aaggaatccc cctgctggtg 1800
gttaaacaag acgggtgttc actccgactg caacaggagc gcttcctcca gggggttttc 1860
caggaagacc ctgaatggag ggccctgcag gagaggtacc tgtggcatat cccattgacc 1920
tactccacga gttcttctaa tgtgatccac agacacattc taaaatcaaa gacagatact 1980
ctggatctac ctgaaaagac cagttgggtg aaatttaatg tggactcaaa tggttactac 2040
atcgttcact,atgagggtca tggatgggac caactcatta cacagctgaa tcagaaccac 2100
acacttctca gacctaagga cagagtaggt ctgattcatg atgtgtttca gctagttggt 2160
gcagggagac tgaccctaga caaagctctt gacatgactt actacctcca acatgaaaca 2220
agcagccccg cacttctcga aggtctgagt tacttggaat cgttttacca catgatggac 2280
agaaggaata tttcagatat ctctgaaaac ctcaagcgtt accttcttca gtattttaag 2340
ccagtgattg acaggcaaag ctggagtgac aagggctcag tctgggacag gatgctccgc 2400
tcggctctct tgaagctggc ctgtgacctg aaccatgctc cttgcatcca gaaagctgct 2460
gaactcttct cccagtggat ggaatccagt ggaaaattaa atataccaac agatgtttta 2520
aagattgtgt attctgtggg tgctcagaca acagcaggat ggaattacct tttagagcaa 2580
tatgaactgt caatgtcaag tgctgaacaa aacaaaattc tgtatgcttt gtcaacgagc 2640
aagcatcagg aaaagttact gaagttaatt gaactaggaa tggaaggaaa ggttatcaag 2700
acacagaact tggcagctct ccttcatgcg attgccagac gtccaaaggg gcagcaacta 2760
gcatgggatt ttgtaagaga aaattggacc catcttctga aaaaatttga cttgggctca 2820
tatgacataa ggatgatcat ctctggcaca acagctcact tttcttccaa ggataagttg 2880
caagaggtga aactattttt tgaatctctt gaggctcaag gatcacatct ggatattttt 2940
caaactgttc tggaaacgat aaccaaaaat ataaaatggc tggagaagaa tcttccgact 3000
ctgaggactt ggctaatggt taatacttaa atggtcaata gaaaaagtag gctgggcgcg 3060
gtggctcacg cctgtaatcc cagcactttg ggaggctgag aagggcggat cacgaggtca 3120
ggagatggag accatcctgg ctaacacggt gagaccccgt ctccgctaaa aatacaaaaa 3180
attagccggg catggtggca ggtgcctgta gtcccagcta ctcggcaggc tgcagcagga 3240
aaatggcata aacccgggag gtggagcttg cagtgagccg agattgcacc actgcattcc 3300
agcctgggtg actgagcgag act 3323
<210> 21
<211> 2123
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1866356CB1
<400> 21
tgacaatcca agatggcggt gcccggcgag gcggaggagg aggcgacagt ttacctggta 60
gtgagcggta tcccctccgt gttgcgctcg gcccatttac ggagctattt tagccagttc 120
cgagaagagc gcggcggtgg cttcctctgt ttccactacc ggcatcggcc tgagcgggcc 180
cctccgcagg ccgctcctaa ctctgcccta attcctaccg acccagccgc tgagggccag 240
cttctctctc agacttcggc caccgatgtc cggcctctct ccactcgaga ctctactcca 300
atccagaccc gcacctgctg ctgcgtcatc tcggtaaggg ggttggctca agctcagagg 360
cttattcgca tgtactcggg ccgccggtgg ctggattctc acgggacttg gctaccgggt 420
cgctgtctca tccgcagact tcggctacct acggaggcat caggtctggg ctcctttccc 480
ttcaagaccc ggaaggaact gcagagttgg aaggcagaga atgaagcctt caccctggct 540
gacctgaagc aactgccgga gctgaaccca ccagtgctga tgcccagagg gaatgtgggg 600
actcccctgc gggtcttttt ggagttgatc cgggcctgcc gcctaccccc tcggatcatc 660
21/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
acccagctgc agctccagtt ccccaagaca ggttcctccc ggcgctacgg caatgtgcct 720
tttgagtatg aggactcaga gactgtggag caggaagagc ttgtgtatac agcagagggt 780
gaagaaatac cccaaggaac ctacctggca gatataccag ccagcccctg tggagagcct 840
gaggaagaag tggggaagga agaggaagaa gagtctcact cagatgagga cgatgaccgg 900
ggtgaggaat gggaacggca tgaagcgctg catgaggacg tgaccgggca ggagcggacc 960
actgagcagc tctttgagga ggagattgag ctcaagtggg agaagggtgg ctctggcctg 1020
gtgttttata ctgatgccca gttctggcag gaggaagaag gagattttga tgaacagaca 1080
gccgatgact gggatgtgga catgagtgtg tactatgaca gagatggtgg agacaaggat 1140
gcccgagact ctgtccaaat gcgtctggaa cagagactcc gagatggaca ggaagatggc 1200
tctgtgatcg aacgccaggt gggcaccttt gagcgccaca ccaagggcat tgggcggaag 1260
gtgatggagc ggcagggctg ggctgagggc cagggcctgg gctgcaggtg ctcaggggtg 1320
cctgaggccc tggatagtga tggccaacac cccagatgca agcgtggatt ggggtaccat 1380
ggagagaagc tacagccatt tgggcaactg aagaggcccc gtagaaatgg cttggggctc 1440
atctccacca tctatgatga gcctctaccc caagaccaga cggagtcact gctccgccgc 1500
cagccaccca ccagcatgaa gtttcggaca gacatggcct ttgtgagggg ttccagttgt 1560
gcttcagaca gcccctcatt gcctgactga ccgggttggg ggcttccttt catagctaca 1620
tgatgaaaac cctctgccct ggcctcatct accactgaag cagaaaggag tctgggagca 1680
gcagtcttcg tggctggttc agggtgtttt gttccgagcc tgcctgcctg ccggttctat 1740
acctcagggg catttttaca aaaagccccc tcccgtcccc tccccttgga tattaggggt 1800
aacgaccgct tgtctttggt ctctaaccct aatctctggg cttgcccttt gcctcctgca 1860
gaactttgaa aagctgggtt gagtgaggct atcagcacag ccttccttgg ggactctgaa 1920
ggtgtcccca cgaaggccag aaagggggaa agggacctgg gcgaggagag gatttgtggt 1980
gcttggaaga gccggccttg ggtgggccct ccaccgcctc taccctcact gggtgggact 2040
gccagcggag agtccgcggg aggtggcttg ggtgtgcgac gtcacggaag aataaagacg 2100
tttactactg gaaaaaaaaa aaa 2123
<210> 22
<211> 2893
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1872095CB1
<400> 22
atgcatcatt tgaaccttct gtagcattgg caagccttgt gcagcatatt cctcttcaga 60
tgattacagt tctcatcagg agccttacta cggatccaaa tgtaaaagat gcaagtatga 120
cccaagccct ttgcagaatg attgactggc tatcctggcc attggctcag catgtggata 180
catgggtaat tgcactcctg aaaggactgg cagctgtcca gaagtttact attttgatag 240
atgttacttt gctgaaaata gaactggttt ttaatcgact ttggtttcct cttgtgagac 300
ctggtgctct tgcagttctt tctcacatgc tgcttagctt tcagcattct ccagaggcgt 360
tccatttgat tgttcctcat gtggttaatt tggttcattc tttcaaaaat gatggtctgc 420
cttcaagtac agccttctta gtacaattaa cagaattgat acactgtatg atgtatcatt 480
attctggatt tccagatctc tatgaaccta ttctggaggc aataaaggat tttcctaagc 540
ccagtgaaga gaagattaag ttaattctca atcaaagtgc ctggacttct caatccaatt 600
ctttggcgtc ttgcttgtct agactttctg gaaaatctga aactgggaaa actggtctta 660
ttaacctagg aaatacatgt tatatgaaca gtgttataca agccttgttt atggccacag 720
atttcaggag acaagtatta tctttaaatc taaatgggtg caattcatta atgaaaaaat 780
tacagcatct ttttgccttt ctggcccata cacagaggga agcatacgca cctcggatat 840
tctttgaggc ttccagacct ccatggttta ctcccagatc acagcaagac tgttctgaat 900
acctcagatt tctccttgac aggctccatg aagaagaaaa gatcttgaaa gttcaggcct 960
cacacaagcc ttctgaaatt ctggaatgca gtgaaacttc tttacaggaa gtagctagta 1020
aagcagcagt actaacagag acccctcgta caagtgacgg tgagaagact ttaatagaaa 1080
aaatgtttgg aggaaaacta cgaactcaca tacgttgttt gaactgcagg agtacctcac 1140
aaaaagtgga agcctttaca gatctttcgc ttgccttttg tccttcctct tctttggaaa 1200
acatgtctgt ccaagatcca gcatcatcac ccagtataca agatggtggt ctaatgcaag 1260
cctctgtacc cggtccttca gaagaaccag tagtttataa tccaacaaca gctgccttca 1320
tctgtgactc acttgtgaat gaaaaaacca taggcagtcc tcctaatgag ttttactgtt 1380
ctgaaaacac ttctgtccct aacgaatcta acaagattct tgttaataaa gatgtacctc 1440
agaaaccagg aggtgaaacc acaccttcag taactgactt actaaattat tttttggctc 1500
cagagattct tactggtgat aaccaatatt attgtgaaaa ctgtgcctct ctgcaaaatg 1560
ctgagaaaac tatgcaaatc acggaggaac ctgaatacct tattcttact ctcctgagat 1620
tttcatatga tcagaagtat catgtgagaa ggaaaatttt agacaatgta tcactgccac 1680
tggttttgga gttgccagtt aaaagaatta cttctttctc ttcattgtca gaaagttggt 1740
ctgtagatgt tgacttcact gatcttagtg agaaccttgc taaaaaatta aagccttcag 1800
ggactgatga agcttcctgc acaaaattgg tgccctatct attaagttcc gttgtggttc 1860
22/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
actctggtat atcctctgaa agtgggcatt actattctta tgccagaaat atcacaagta 1920
cagactcttc atatcagatg taccaccagt ctgaggctct ggcattagca tcctcccaga 1980
gtcatttact agggagagat agtcccagtg cagtttttga acaggatttg gaaaataagg 2040
aaatgtcaaa agaatggttt ttatttaatg acagtagagt gacatttact tcatttcagt 2100
cagtccagaa aattacgagc aggtttccaa aggacacagc ttatgtgctt ttgtataaaa 2160
aacagcatag tactaatggt ttaagtggta ataacccaac cagtggactc tggataaatg 2220
gagacccacc tctacagaaa gaacttatgg atgctataac aaaagacaat aaactatatt 2280
tacaggaaca agagttgaat gctcgagccc gggccctcca agctgcatct gcttcatgtt 2340
catttcggcc caatggattt gatgacaacg acccaccagg aagctgtgga ccaactggtg 2400
gagggggtgg aggaggattt aatacagttg gcagactcgt attttgatcc tgagagagtc 2460
caaaatgcac tggtcacgaa acgtctaata ctatgactgt taaaatgtca gactataaca 2520
aatatctatc ttttattttt cattagaccc ttatacttca agagaacaca ctcagtgctt 2580
gtttttattt tcttgacaca tttattaaca aaatgcatca tggaaaaaaa aatctacctc 2640
ttaaaattcc atttgctttt atggttagac atgcttgacc aaaaatgttc agaagaaaat 2700
atgtacctgg tccctaatta agctgcgtta aatttggtag aagcatttaa atggtctatc 2760
ttcagtttta ctgaacaaaa aatgtaattt atttagcatt ctttataaaa gaattgatgc 2820
tagaggtaaa aaaaaatact tgtttttaaa aaatccttta cgtcttgtgt aattaccccg 2880
attattaaat tca 2893
<210> 23
<211> 4170
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2278688CB1
<400> 23
gctcccccgg tcgctctcct ccggcggtcg cccgcgctcg gtggatgtgg cttgcagctg 60
ccgccccctc cctcgctcgc cgcctgctct tcctcggccc tccgcctcct cccctcctcc 120
ttctcgtctt cagccgctcc tctcgccgcc gcctccacag cctgggcctc gccgcgatgc 180
cggagaagag gcccttcgag cggctgcctg ccgatgtctc ccccatcaac tgcagccttt 240
gcctcaagcc cgacttgctg gacttcacct tcgagggcaa gctggaggcc gccgcccagg 300
tgaggcaggc gactaatcag attgtgatga attgtgctga tattgatatt attacagctt 360
catatgcacc agaaggagat gaagaaatac atgctacagg atttaactat cagaatgaag 420
atgaaaaagt caccttgtct ttccctagta ctctgcaaac aggtacggga accttaaaga 480.
tagattttgt tggagagctg aatgacaaaa tgaaaggttt ctatagaagt aaatatacta 540
ccccttctgg agaggtgcgc tatgctgctg taacacagtt tgaggctact gatgcccgaa 600
gggcttttcc ttgctgggat gagcctgcta tcaaagcaac ttttgatatc tcattggttg 660
ttcctaaaga cagagtagct ttatcaaaca tgaatgtaat tgaccggaaa ccataccctg 720
atgatgaaaa tttagtggaa gtgaagtttg cccgcacacc tgttatgtct acatatctgg 780
tggcatttgt tgtgggtgaa tatgactttg tagaaacaag gtcaaaagat ggtgtgtgtg 840
tccgtgttta cactcctgtt ggcaaagcag aacaaggaaa atttgcgtta gaggttgctg 900
ctaaaacctt gcctttttat aaggactact tcaatgttcc ttatcctcta cctaaaattg 960
atctcattgc tattgcagac tttgcagctg gtgccatgga gaactggggc cttgttactt 1020
atagggagac tgcattgctt attgatccaa aaaattcctg ttcttcatcc cgccagtggg 1080
ttgctctggt tgtgggacat gaactcgccc atcaatggtt tggaaatctt gttactatgg 1140
aatggtggac tcatctttgg ttaaatgaag gttttgcatc ctggattgaa tatctgtgtg 1200
tagaccactg cttcccagag tatgatattt ggactcagtt tgtttctgct gattacaccc 1260
gtgcccagga gcttgacgcc ttagataaca gccatcctat tgaagtcagt gtgggccatc 1320
catctgaggt tgatgagata tttgatgcta tatcatatag caaaggtgca tctgtcatcc 1380
gaatgctgca tgactacatt ggggataagg actttaagaa aggaatgaac atgtatttaa 1440
ccaagttcca acaaaagaat gctgccacag aggatctctg ggaaagttta gaaaatgcta 1500
gtggtaaacc tatagcagct gtgatgaata cctggaccaa acaaatggga tttcccctca 1560
tttatgtgga agctgaacag gtagaagatg acagattatt gaggttgtcc caaaagaagt 1620
tctgtgctgg tgggtcatat gttggtgaag attgtcccca gtggatggtc cctatcacaa 1680
tctctactag tgaagacccc aaccaggcca aactaaaaat tctaatggac aagccagaga 1740
tgaatgtggt tttgaaaaat gtcaaaccag accaatgggt gaagttaaac ttaggaacag 1800
ttgggtttta tcggacccag tacagctctg ccatgctgga aagtttatta ccaggcattc 1860
gtgacctttc tctgccccct gtggatcgac ttggattaca gaatgacctc ttctccttgg 1920
ctcgagctgg aatcattagc actgtagagg ttctaaaagt catggaggct tttgtgaatg 1980
agcccaatta tactgtatgg agcgacctga gctgtaacct ggggattctc tcaactctct 2040
tgtcccacac agacttctat gaggaaatcc aggagtttgt gaaagatgtc ttttcaccta 2100
taggggagag actgggctgg gaccccaaac ctggagaagg tcatctcgat gcactcctga 2160
ggggcttggt tctgggaaaa ctaggaaaag caggacataa ggcaacgtta gaagaagccc 2220
gtcgtcggtt taaggaccac gtggaaggaa aacagattct ctccgctgat ctgaggagtc 2280
23/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
ctgtctatct gactgttttg aagcatggtg atggcactac tttagatatt atgttaaaac 2340
ttcataaaca agcagatatg caagaagaga aaaaccgaat cgaaagagtc cttggcgcta 2400
ctcttttgcc tgacctgatt caaaaagtcc tcacgtttgc actttcagaa gaggtacgtc 2460
cacaggacac tgtatcggta attggtggag tagctggagg cagcaagcat ggtaggaaag 2520
ctgcttggaa attcataaag gacaactggg aagaacttta taaccgatac cagggaggat 2580
tcttaatatc cagactaata aagctatcag ttgagggatt tgeagttgat aaaatggctg 2640
gagaggttaa ggctttcttc gagagtcacc cagctccttc agctgagcgt accatccagc 2700
agtgttgtga aaatattctg ctgaatgctg cctggctaaa gcgagatgct gagagcatcc 2760
accagtacct ccttcagcgg aaggcctcac cacccacagt gtgaatcctg aggtgccgcc 2820
attggcggtt ctgctcgttc gctgcaggga taaggtggag ctaccgaaca gctgattcat 2880
atgccaagaa tttggagtct tctttcaaac cagtgggggt tggacaatga atgtagttaa 2940
ctggttcctg ctcacactcc agaattaaat tctattgaaa aaggaaaatc agcaattcag 3000
caaaaaataa ataaaaaata aaaatgtaaa tatgatagta ataaaataga gcataacgaa 3060
actgtgaaac tttctgaagc cttgtcagtg gttaaaagta tttaacactc tactgttaat 3120
gacagatgtt ctgtttttat aacctaccaa aaggaaacta gaggcttctt ggtgaagagc 3180
atttttgtga agtgggttct gcaaggagcc tataaagcca agggtggtgt ccatttctgg 3240
gaatggttaa acacaaaagg ctgatagctg gtatcacata gttggagtca gtgcataatt 3300
ccaagtggct tttttttttt ttggcacggg gactgatcag gaagatatat tcctgcataa 3360
ctcaatctga accaaggatt gtagtttagt tttcctcctt gccttccctt ctgtgtgacc 3420
gaccccttgg ccaaaaaaaa aaacaaaaag caaaaaacaa aaacctaccc tgttctggtt 3480
tttttcctcc ctttagttcc acccccaacc cccattccct ggtgtccttc ttagagatga 3540
agaaataata aggaaacatc tttcatagcc acattaaata agagaaactg atatacatta 3600
tttttttctt tttaaagatg acttataaga accctgaaat ttatataggt gagacaatag 3660
aaataaaaag atcttcagcc aggcctttct gaaggagtta ttctgctaaa aatggtctta 3720
gttgtctgaa aagccagctc ttgaacctct tcacaacagt atcaacactg gcttctcccg 3780
gttcatttta tgcgtgcgag aagtcagtgg taactgctgc agggcttaat acattagtgg 3840
taactggttt aaaaaacaaa gactgtaagc ctgtgtgtgc cactgtttgc ttcaacagta 3900
tatcctacta ataagcctca cctatttaat ccaatgagtt ttaaatctaa atctcattcc 3960
cttcttcttt ccctaccttt tttttctttt tttcttaaaa aaatattttg tgttattaac 4020
agaaattcat atttggtgtg gcttaacggt atttcagaag gtcatcagat tgtgagactg 4080
cttccttgaa acatttttgt gctattgttt taaaaaaata attaaaaaac agttggcgtt 4140
aataaaaatg tcaatgtgaa aaaaaaaaaa 4170
<210> 24
<211> 767
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4043361CB1
<400> 24
ccgagaggct gcagcggcac agctgtcgcg ccagtcgcaa cagaagcagg tccgaggcac 60
agcccgatcc cgccatggag cagccgagga aggcggtggt agtgacggga tttggccctt 120
ttggggaaca caccgtgaac gccagttgga ttgcagttca ggagctagaa aagctaggcc 180
ttggcgacag cgtggacctg catgtgtacg agattccggt tgagtaccaa acagtccaga 240
gactcatccc cgccctgtgg gagaagcaca gtccacagct ggtggtgcat gtgggggtgt 300
caggcatggc gaccacagtc acactggaga aatgtggaca caacaagggc tacaaggggc 360
tggacaactg ccgcttttgc cccggctccc agtgctgcgt ggaggacggg cctgaaagca 420
ttgactccat catcgacatg gatgctgtgt gcaagcgagt caccacgttg ggcctggatg 480
tgtcggtgac catctcgcag gatgccggca ggaaaaaacc ettccctgcc aaaggtgact 540
gtgttttctg ccgccgaagg agggcccggt ccctccaggc tcagtgtggc ttctccctga 600
cccccgccct agaacttttg ccagtgcctt ttctgaaact cctgtgtccc gggcccccca 660
ggcggagaag gatatgccgg attctgcctg gggctgggct ctaggagacc ccaaatttga 720
caccacagaa agcaaataaa acacttgaaa tacgcaaaaa aaaaaaa 767
<210> 25
<211> 1538
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3937958CB1
<400> 25
24/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
ggtgagtggg aggcatgggg tggatgagaa gcctaggcag aggcttttcc tgcatccctc 60
ctcagtttcc ctattcacag atgccggcct ccctgtctac ctgtatgaat ttgagcacca 120
cgctcgtgga ataatcgtca aaccccgcac tgatggggca gaccatgggg atgagatgta 180
cttcctcttt gggggcccct tcgccacagg tgcaaaggtc ccacctgata ccccaactgg 240
gtgtccagtc tcccacctct ggatgcagac ccacccctcc attggctggc cacagggagc 300
tcaccagttc ctaatctgtt atgctctccc aaatgaaagt cttctgctcc ggaagcagca 360
gaagcagcag gagtagggtg ggaggtcagt gtcccctgct ctgtccgaaa tcccacatcc 420
cattctgccc ccaggccttt ccatgggtaa ggagaaggca cttagcctcc agatgatgaa 480
atactgggcc aactttgccc gcacaggaaa ccccaatgat gggaatctgc cctgctggcc 540
acgctacaac aaggatgaaa agtacctgca gctggatttt accacaagag tgggcatgaa 600
gctcaaggag aagaagatgg ctttttggat gagtctgtac cagtctcaaa gacctgagaa 660
gcagaggcaa ttctaagggt ggctatgcag gaaggagcca aagaggggtt tgcccccacc 720
atccaggccc tggggagact agccatggac atacctgggg acaagagttc tacccacccc 780
agtttagaac tgcaggagct ccctgctgcc tccaggccaa agctagagct tttgcctgtt 840
gtgtgggacc tgcactgccc tttccagcct gacatcccat gatgcccctc tacttcactg 900
ttgacatcca gttaggccag gccctgtcaa caccacactg tgctcagctc tccagcctca 960
ggacaacctc tttttttccc ttcttcaaat cctcccaccc ttcaatgtct ccttgtgact 1020
ccttcttatg ggaggtcgac ccagactgcc actgcccctg tcactgcacc cagcttggca 1080
tttaccatcc atcctgctca accttgtgcc tgtctgttca cattggcctg gaggcctagg 1140
gcaggttgtg acatggagca aacttttggt agtttgggat cttctctccc acccacactt 1200
atctccccca gggccactcc aaagtctata cacaggggtg gtctcttcaa taaagaagtg 1260
ttgattagac ctgaatttct ccacctataa aatgggtgtg tgaagtgaat gatgtctcaa 1320
tttgagccct gagagaaagg aagtattgct gcctgttcct tagtgggctg tgcctggatg 1380
ctacactcag tcaaagggtg ctactgcaaa gttgcctggg gtacaaaaca cttgcctttg 1440
gcccttcatg gtctcaagtg cacccctcag gacagccaca ccccacgctc acttgtccat 1500
cagtttaggt cttagtgcca catctagatt cctctggc 1538
<210> 26
<211> 1497
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7257324CB1
<400> 26
ggccttactc ttccaagagg ccatggaagt ataaataata aagcaagaaa ggcagatgca 60
tttggctggc tcagtggact tctgaatgta ctgtgagtat gagaccttcc cttccaaaag 120
atccggtgct tcttgtctat tccacacgaa gcttgcttca gatcgaggga ggatgtagca 180
ctgtccacag gtctactact caacaggata ttcttcaagg aaaatgaacc ccacactagg 240
cctggccatt tttctggctg ttctcctcac ggtgaaaggt cttctaaagc cgagcttctc 300
accaaggaat tataaagctt tgagcgaggt ccaaggatgg aagcaaagga tggcagccaa 360
ggagcttgca aggcagaaca tggacttagg ctttaagctg ctcaagaagc tggcctttta 420
caaccctggc aggaacatct tcctatcccc cttgagcatc tctacagctt tctccatgct 480
gtgcctgggt gcccaggaca gcaccctgga cgagatcaag caggggttca acttcagaaa 540
gatgccagaa aaagatcttc atgagggctt ccattacatc atccacgagc tgacccagaa 600
gacccaggac ctcaaactga gcattgggaa cacgctgttc attgaccaga ggctgcagcc 660
acagcgtaag tttttggaag atgccaagaa cttttacagt gccgaaacca tccttaccaa 720
ctttcagaat ttggaaatgg ctcagaagca gatcaatgac tttatcagtc aaaaaaccca 780
tgggaaaatt aacaacctga tcgagaatat agaccccggc actgtgatgc ttcttgcaaa 840
ttatattttc tttcgagcca ggtggaaaca tgagtttgat ccaaatgtaa ctaaagagga 900
agatttcttt ctggagaaaa acagttcagt caaggtgccc atgatgttcc gtagtggcat 960
ataccaagtt ggctatgacg ataagctctc ttgcaccatc ctggaaatac cctaccagaa 1020
aaatatcaca gccatcttca tccttcctga tgagggcaag ctgaagcact tggagaaggg 1080
attgcaggtg gacactttct ccagatggaa aacattactg tcacgcaggg tcgtagacgt 1140
gtctgtaccc agactccaca tgacgggcac cttcgacctg aagaagactc tctcctacat 1200
aggtgtctcc aaaatctttg aggaacatgg tgatctcacc aagatcgccc ctcatcgcag 1260
cctgaaagtg ggcgaggctg tgcacaaggc tgagctgaag atggatgaga ggggtacgga 1320
aggggccgct ggcaccggag cacagactet gcccatggag acaccacteg tcgtcaagat 2380
agacaaaccc tatctgctgc tgatttacag cgagaaaata ccttccgtgc tcttcctggg 1440
aaagattgtt aaccctattg gaaaataaag gagaattcct gcttgccaca aaaaaaa 1497
<210> 27
<211> 1194
<212> DNA
<213> Homo Sapiens
25/26
CA 02394789 2002-06-18
WO 01/46443 PCT/US00/34811
<220>
<221> misc_feature
<223> ~Incyte ID No: 7472038CB1
<400> 27
atgccccggg ccattagtcc cctgatgagg tttcaacatc cggtcagttg caagctgcag 60
ctgtaccgcg ttcccctgcg ccgcttcccc tccgcccgtc atcgcttcga gaagttgggc 120
atccggatgg accggctgcg tttaaagtac gccgaggagg tcagccattt ccgtggcgag 180
tggaactcgg cggtgaagag cacaccactg agcaattacc tagacgccca gtactttggc 240
cccatcacca ttggtacgcc gccgcagaca ttcaaggtga tattcgatac gggttcctcg 300
aatctctggg tgccatccgc cacgtgtgcg tccacaatgg tggcctgtcg tgtgcacaat 360
cgctactttg ccaagcggtc gaccagtcac caggtgaggg gagaccactt tgccatccac 420
tatggcagcg gcagtctgtc cggcttcctt tccaccgaca ccgttcgggt ggctggccta 480
gagattcggg atcagacctt cgcggaggcc accgaaatgc cgggtcccat cttcctggca 540
gcaaaattcg acggcatctt tggattggcc tatcgcagca tctctatgca gcgcatcaag 600
ccaccattct atgcgatgat ggagcaagga cttctaacga aacccatatt cagtgtttac 660
cttagcagaa atggcgaaaa ggatggtgga gccatcttct ttggcggatc caatccgcat 720
tactacaccg gcaactttac ttatgtccag gtgagccatc gtgcctattg gcaggtgaaa 780
atggattcag cagttatccg gaatctcgag ctatgtcagc agggatgtga agtgattatc 840
gacacgggca cctctttcct ggcattgccc tacgaccagg ctatacttat caatgaatcc 900
attgggggaa ctccctcctc ctttggacag tttctagttc cgtgcgacag cgtaccagac 960
ctgcccaaaa tcacctttac cttgggtggg cgtagatttt tcctggagtc tcacgagtat 1020
gtctttcggg atatctacca ggatcgaagg atctgctcct cggcgttcat tgccgtggac 1080
ctgccatcgc ccagtggacc gctctggatt ctgggggatg tgtttttggg caaatactat 1140
actgagttcg acatggagag gcatcgcatt ggattcgccg atgccaggag ttga 1194
<210> 28
<212> 438
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472041CB1
<400> 28
atggggatcg gatgctggag aaaccccctg ctgctgctga ttgccctggt cctgtcagcc 60
aagctgggtc acttccaaag gtgggagggc ttccagcaga agctcatgag caagaagaac 120
atgaattcaa cactcaactt cttcattcaa tcctacaaca atgccagcaa cgacacctac 180
ttatatcgag tccagaggct aattcgaagt cagatgcagc tgacgacggg agtggagtat 240
atagtcactg tgaagattgg ctggaccaaa tgcaagagga atgacacgag caattcttcc 300
tgccccctgc aaagcaagaa gctgagaaag agtttaattt gcgagtcttt gatatacacc 360
atgccctgga taaactattt ccagctctgg aacaattcct gtctggaggc cgagcatgtg 420
ggcagaaacc tcagatga 438
26/26
