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PROTEIN MODIFICATION AND MAINTENANCE MOLECULES
TECHNICAL FIELD
The invention relates to novel nucleic acids, protein modification and
maintenance molecules
encoded by these nucleic acids, and to the use of these nucleic acids and
proteins in the diagnosis,
treatment, and prevention of gastrointestinal, cardiovascular,
autoimmunelinflammatory, cell
proliferative, developmental, epithelial, neurological, reproductive,
endocrine, pancreatic, adrenal, and
metabolic disorders; as well as lipid, copper, and carbohydrate metabolism
disorders, disorders
associated with gonadal steroid hormones, the immune system, and infections.
The invention also
relates to the assessment of the effects of exogenous compounds on the
expression of nucleic acid
and amino acid sequences of protein modification and maintenance molecules.
BACKGROUND OF THE INVENTION
'The cellular processes regulating modification and maintenance of protein
molecules
coordinate their function, conformation, stabilization, and degradation. Each
of these processes is
mediated by key enzymes or proteins such as kinases, phosphatases, proteases,
protease inhibitors,
isomerases, transferases, and molecular chaperones.
Kinases
Kinases catalyze the transfer of high-energy phosphate groups from adenosine
triphosphate
(ATP) to target proteins on the hydroxyamino acid residues serine, threonine,
or tyrosine. Addition of
a phosphate group alters the local charge on the acceptor molecule, causing
internal conformational
changes and potentially influencing intermolecular contacts. Reversible
protein phosphorylation is the
ubiquitous strategy used to control many of the intracellular events in
eukaryotic cells. It is estimated
that more than ten percent of proteins active in a typical mammalian cell are
phosphorylated.
Extracellular signals including hormones, neurotransmitters, and growth and
differentiation factors can
activate kinases, which can occur as cell surface receptors or as the
activators of the final effector
protein, as well as elsewhere along the signal transduction pathway. Kinases
are involved in all
aspects of a cell's function, from basic metabolic processes, such as
glycolysis, to cell-cycle
regulation, differentiation, and communication with the extracellular
environment through signal
transduction cascades. Inappropriate phosphorylation of proteins in cells has
been linked to changes in
cell cycle progression and cell differentiation. Changes in the cell cycle
have been linked to induction
of apoptosis or cancer. Changes in cell differentiation have been licked to
diseases and disorders of
the reproductive system, immune system, and skeletal muscle.
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There are two classes of protein kinases. One class, protein tyrosine kinases
(PTKs),
phosphorylates tyrosine residues, and the other class, protein
serine/threonine kinases (STKs),
phosphorylates serine and threonine residues. Some PTKs and STKs possess
structural
characteristics of both families and have dual specificity for both tyrosine
and serine/threonine
residues. Almost all kinases contain a conserved 250-300 amino acid catalytic
domain containing
specific residues and sequence motifs characteristic of the kinase family.
(Reviewed in Hardie, G.
and S. Hanks (1995) The Protein Kinase Facts Book, Vol I, Academic Press, San
Diego, CA, pp. 17-
20).
Phosphatases
Phosphatases hydrolytically remove phosphate groups from proteins.
Phosphatases are
essential in determining the extent of phosphorylation in the cell and,
together with kinases, regulate
key cellular processes such as metabolic enzyme activity, proliferation, cell
growth and differentiation,
cell adhesion, and cell cycle progression. Protein phosphatases are
characterized as either
serine/threonine- or tyrosine-specific based on their preferred phospho-amino
acid substrate. Some
phosphatases (DSPs, for dual specificity phosphatases) can act on
phosphorylated tyrosine, serine, or
threonine residues. The protein serine/threonine phosphatases (PSPs) are
important regulators of
many cAMP-mediated hormone responses in cells. Protein tyrosine phosphatases
(PTPs) play a
significant role in cell cycle and cell signaling processes.
Proteases
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,
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,
CA 02460476 2004-03-15
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cleave at residues within the peptide. Four principal categories of mammalian
proteases have been
identified based on active site structure, mechanism of action, and overall
three-dimensional structure.
(See Beynon, R.J. and J.S. Bond (1994) Proteolytic Enzymes: A Practical
Approach, Oxford
University Press, New York NY, pp. 1-5.)
Serine Proteases
The serine proteases (SPs) are a large, widespread family of proteolytic
enzymes that include
the digestive enzymes trypsin and chymotrypsin, components of the complement
and blood-clotting
cascades, and enzymes that control the degradation and turnover of
macromolecules within the cell
and in the extracellular matrix. Most of the more than 20 subfamilies can be
grouped into six clans,
each with a common ancestor. These six clans are hypothesized to have
descended from at least four
evolutionarily distinct ancestors. SPs are named for the presence of a serine
residue found in the
active catalytic site of most families. The active site is defined by the
catalytic triad, a set of
conserved asparagine, histidine, and serine residues critical for catalysis.
These residues form a
charge relay network that facilitates substrate binding. Other residues
outside the active site form an
oxyanion hole that stabilizes the tetrahedral transition intermediate formed
during catalysis. SPs have
a wide range of substrates and can be subdivided into subfamilies on the basis
of their substrate
specificity. The main subfamilies are named for the residues) after which they
cleave: trypases
(after arginine or lysine), aspases (after aspartate), chymases (after
phenylalanine or leucine),
metases (methionine), and serases (after serine) (Rawlings, N.D. and A.J.
Barrett (1994) Methods
2o Enzymo1.244:19-61).
Most mammalian serine proteases are synthesized as zymogens, inactive
precursors that are
activated by proteolysis. For example, trypsinogen is converted to its active
form, trypsin, by
enteropeptidase. Enteropeptidase is an intestinal protease that removes an N-
terminal fragment from
trypsinogen. The remaining active fragment is trypsin, which in turn activates
the precursors of the
other pancreatic enzymes. Likewise, proteolysis of prothrombin, the precursor
of thrombin, generates
three separate polypeptide fragments. The N-terminal fragment is released
while the other two
fragments, which comprise active thrombin, remain associated through disulfide
bonds.
The two largest SP subfamilies are the chymotrypsin (S1) and subtilisin (S8)
families. Some
members of the chymotrypsin family contain two structural domains unique to
this family. I~ringle
domains are triple-looped, disulfide cross-linked domains found in varying
copy number. Kringle
domains are thought to play a role in binding mediators such as membranes,
other proteins or
phospholipids, and in the regulation of proteolytic activity (PROSTTE
PDOC00020). Apple domains
are 90 amino-acid repeated domains, each containing six conserved cysteines.
Three disulfide bonds
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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 urokiuase-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 PCl,
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 (Tan, F. et al. (1993) J. Biol. Chem. 268:16631-16638). The protease
neuropsin may
influence synapse formation and neuronal connectivity in the hippocampus in
response to neural
signaling (Chen, Z.-L. et al. (1995) J. Neurosci. 15:5088-5097). Tissue
plasminogen activator is useful
for acute management of stroke (Zivin, J.A. (1999) Neurology 53:14-19) and
myocardial infarction
(Ross, A.M. (1999) Clip. Cardiol. 22:165-171). Some receptors (PAR, for
proteinase-activated
receptor), highly expressed throughout the digestive tract, are activated by
proteolytic cleavage of an
2o 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).
Signal peptidases
The mechanism for the trauslocation process into the endoplasmic reticulum
(ER) involves the
recognition of an N-terminal signal peptide on the elongating protein. The
signal peptide directs the
protein and attached ribosome to a receptor on the ER membrane. The
polypeptide chain passes
through a pore in the ER membrane into the lumen while the N-terminal signal
peptide remains
attached at the membrane surface. The process is completed when signal
peptidase located inside the
4
CA 02460476 2004-03-15
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ER cleaves the signal peptide from the protein and releases the protein into
the lumen.
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,
l0 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.
Thrombin is a serine protease with an essential role in the process of blood
coagulation.
Prothrombin, synthesized in the liver, is converted to active thrombin by
Factor Xa. Activated
thrombin then cleaves soluble fibrinogen to polymer-forming fibrin, a primary
component of blood
clots. In addition, thrombin activates Factor XBIa, which plays a role in
cross-linking fibrin.
Thrombin also stimulates platelet aggregation through proteolytic processing
of a 41-residue
amino-terminal peptide from protease-activated receptor 1 (PAR-1), formerly
known as the thrombin
receptor. The cleavage of the amino-terminal peptide exposes n new amino
terminus and may also be
associated with PAR-1 internalization (Stubbs, M.T. and W. Bode (1994) Curr.
Opin. Struct. Biol.
4:823-832; and Ofoso, F.A. et al. (1998.) Biochem. J. 336:283-285). In
addition to stimulating platelet
activation through cleavage of the PAR-1 receptor, thrombin also induces
platelet aggregation
following cleavage of glycoprotein V, also on the surface of platelets.
Glycoprotein V appears to be
the major thrombin substrate on intact platelets. Platelets deficient for
glycoprotein V are
hypersensitive to thrombin, which is still required to cleave PAR-1. While
platelet aggregation is
required for normal hemostasis in mammals, excessive platelet aggregation can
result in arterial
thrombosis, atherosclerotic arteries, acute myocardial infarction, and stroke
(Ramakrishnan, V. et al.
(1999) Proc. Natl. Acad. Sci. U.S.A. 96:13336-13341 and references within).
Proteases in another family have a serine in their active site and 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
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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. 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).
Proteasomes are
associated with the ubiquitin conjugation system (UCS), a major pathway for
the degradation of
cellular proteins of all types, including proteins that function to activate
or repress cellular processes
such as transcription and cell cycle progression (Ciechanover, A. (1994) Cell
79:13-21). In the UCS
pathway, proteins targeted for degradation are conjugated to ubiquitin, a
small heat stable protein. The
ubiquitinated protein is then recognized and degraded by the proteasome. The
resultant ubiquitin-
peptide complex is hydrolyzed by a ubiquitin carboxyl terminal hydrolase, and
free ubiquitin is released
for reutilizationby 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,
Angelinan's syndrome, and Liddle syndrome (reviewed in Schwartz, A.L. and A.
Ciechanover (1999) ,
Annu. Rev. Med. 50:57-74). A murine proto-oncogene, Unp, encodes a nuclear
ubiquitin protease
whose overexpression leads to oncogenic transformation of NIH3T3 cells. The
human homolog 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).
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
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CP families include the papain-like enzymes (C1) and the calpains (C2). Papain-
like family members
are generally lysosomal or secreted and therefore are synthesized with signal
peptides as well as
propeptides. Most members bear a conserved motif in the propeptide that may
have structural
significance (Karrer, K.M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3063-
3067). Three-
dimensional structures of papain family members show a bilobed molecule with
the catalytic site
located between the two lobes. Papains include cathepsins B, C, H, L, and S,
certain plant allergens
and dipeptidyl peptidase (for a review, see Rawlings, N.D. and A.J. Barrett
(1994) Methods Enzymol.
244:461-486).
Some CPs are expressed ubiquitously, while others are produced only by cells
of the immune
system. Of particular note, CPs are produced by monocytes, macrophages and
other cells which
migrate to sites of inflammation and secrete molecules involved in tissue
repair. Overabundance of
these repair molecules plays a role in certain disorders. In autoimmune
diseases such as rheumatoid
arthritis, secretion of the cysteine peptidase cathepsin C degrades collagen,
laminin, elastin and other
structural proteins found in the extracellular matrix of bones. Bone weakened
by such degradation is
also more susceptible to tumor invasion and metastasis. Cathepsin L expression
may also contribute
to the influx of mononuclear cells which exacerbates the destruction of the
rheumatoid synovium
(Keyszer, G.M. (1995) Arthritis Rheum. 38:976-984).
Human cathepsin O is a novel cysteine protease with 94% identity to rabbit OC2
and 50%
identity to both human cathepsin S and cathepsin L. It displays potent
endoprotease activity against .
fibrinogen at acid pH. The mature protein contains 215 amino acids with one
potential N-glycosylation
site. The gene for cathepsin O is expressed in osteoclastoma and ovary. Its
extremely high
expression levels in osteoclastoma suggests a major role in bone remodelling
and bone related diseases
(Shi, G. P. et al. (1995) FEBS Lett. 357:129-134; Bromine, D. and Okamoto, K.
(1995) Biol. Chem.
Hoppe Seyler 376:379-384).
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 (Char, 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
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neurodegenerative disorders, including ALS, Parkinson's disease and
Alzheimer's disease are
associated with increased calpain expression (Char and Mattson, supra).
Calpain-mediated
breakdown of the cytoskeleton has been proposed to contribute to brain damage
resulting from head
injury (McCracken, E. et al. (1999) J. Neurotrauma 16:749-761). Calpain-3 is
predominantly
expressed in skeletal muscle, and is responsible for limb-girdle muscular
dystrophy type 2A (Minami,
N. et al. (1999) J. Neurol. Sci. 171:31-37).
Another family of thiol proteases is the caspases, which are involved in the
initiation and
execution phases of apoptosis. A pro-apoptotic signal can activate initiator
caspases that trigger a
proteolytic caspase cascade, leading to the hydrolysis of target proteins and
the classic apoptotic death
of the cell. Two active site residues, a cysteine and a histidine, have been
implicated in the catalytic
mechanism. Caspases 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)
and removal of the spacer and prodomain, leaving a p101p20 heterodimer. Two of
these heterodimers
interact via their small subunits to form the catalytically active tetramer.
The long prodomains of some
caspase family members have been shown to promote dimerization and auto-
processing of
procaspases. Some caspases contain a "death effector domain" in their
prodomain by which they can
be recruited into self activating complexes with other caspases and FADD
protein associated death
receptors or the TNF receptor complex. In addition, two dimers from different
caspase family
members can associate, changing the substrate specificity of the resultant
tetramer. Endogenous
caspase inhibitors (inhibitor of apoptosis proteins, or IAPs) also exist. All
these interactions have clear
effects on the control of apoptosis (reviewed in Chan and Mattson, supt-a;
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 (Char 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,
supf'a; Thompson, C.B. (1995) Science 267:1456-1462).
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As~artyl proteases
Aspartyl proteases (APs) include the lysosomal proteases cathepsins D and E,
as well as
chymosin, resin, and the gastric pepsins. Most retroviruses encode au AP,
usually as part of the pol
polyprotein. APs, also called acid proteases, are monomeric enzymes consisting
of two domains, each
domain containing one half of the active site with its own catalytic aspartic
acid residue. APs are
most active in the range of pH 2-3, at which one of the aspartate residues is
ionized and the other
neutral. The pepsin family of 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.
Resin
mediates the first step in processing the hormone angiotensin, which is
responsible for regulating
electrolyte balance and 'blood pressure (reviewed in Crews, D.E. and S.R.
Williams (1999) Hum. Biol.
71:475-503). Abnormal regulation and expression of cathepsins are evident in
various inflammatory
disease states. Expression of cathepsin D is elevated in synovial tissues from
patients with rheumatoid
arthritis and osteoarthritis. The increased expression and differential
regulation of the cathepsins are
linked to the metastatic potential of a variety of cancers (Chambers; A.F. et
al. (1993) Crit. Rev.
Oncol. 4:95-114).
Metalloproteases
Metalloproteases require a metal ion for activity, usually manganese or zinc.
Examples of
manganese metalloenzymes include aminopeptidase P and human proline
dipeptidase (PEPD).
Aminopeptidase P can degrade bradykinin, a nonapeptide activated in a variety
of inflammatory
responses. Aminopeptidase P has been implicated in coronary
ischemia/reperfusion injury.
Administration of aminopeptidase P inhibitors has been shown to have a
cardioprotective effect in rats
(Ersahin, C. et al (1999) J. Cardiovasc. Pharmacol. 34:604-611). Most zinc-
dependent
metalloproteases share a common sequence in the zinc-binding domain. The
active site is made up of
two histidines which act as zinc ligands and a catalytic glutamic acid C-
terminal to the first histidine.
Proteins containing this signature sequence are known as the metzincins and
include aminopeptidase
N, angiotensin-converting enzyme, neurolysin, the matrix metalloproteases and
the adamalysins
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(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.
Porcine pancreatic preprocarboxypeptidase A1 (prePCPAI) is the proenzyme of
carboxypeptidase A (CPA), a metalloprotease secreted from panceratic juice.
The open reading
frame of the prePCPAI nucleotide sequence is 1260 nucleotides in length and
encodes a protein of
419 amino acids. The molecular mass of the mature proenzyme is 45561 Da. The
amino acid
sequence of the enzyme shows 85% identity and 55% identity to that of
procarboxypeptidases A1 and
A2, respectively (Darnis S. (1999) Eur. J. Biochem. 259:719-725).
A number of the neutral metalloendopeptidases, including angiotensin
converting enzyme and
the aminopeptidases, are involved in the metabolism of peptide hormones. High
aminopeptidase B
activity, for example, is found in the adrenal glands and neurohypophyses of
hypertensive rats (Prieto,
I. et al. (1998) Horm. Metab. Res. 30:246-248). Oligopeptidase M/neurolysin
can hydrolyze
bradykinin as well as neurotensin (Serizawa, A. et al. (1995) J. Biol. Chem
270:2092-2098).
Neurotensin is a vasoactive peptide that can act as a neurotransmitter in the
brain, where it has been
implicated in limiting food intake (Tritos, N.A. et al. (1999) Neuropeptides
33:339-349).
The matrix metalloproteases (MMPs) are a family of at least 23 enzymes that
can degrade
components of the extracellular matrix (ECM). They are Zn2+ endopeptidases
with an N-terminal
catalytic domain. Nearly all members of the family have a hinge peptide and a
C-terminal domain
which can bind to substrate molecules in the ECM or to inhibitors produced by
the tissue-(TllVIPs, for '
tissue inhibitor of metalloprotease; Campbell, LL. and A. Pagenstecher (1999)
Trends Neurosci.
22:285-287). The presence of fibronectin-like repeats, transmembrane domains,
or C-terminal
hemopexinase-like domains can be used to separate MMPs into collagenase,
gelatinise, stromelysin
and membrane-type MMP subfamilies. In the inactive form, the Znz+ ion in the
active site interacts
with a cysteine in the pro-sequence. Activating factors disrupt the Zn2+-
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. 21:75-80).
MMPs are implicated in a number of diseases including osteoarthritis
(Mitchell, P. et al.
(1996) J. Clip. Invest. 97:761-768), atherosclerotic plaque rupture (Sukhova,
G.K. et al. (1999)
Circulation 99:2503-2509), aortic aneurysm (Schneiderman, J. et al. (1998) Am.
J. Path. 152:703-710),
non-healing wounds (Saarialho-Kere, U.K. et al. (1994) J. Clip. Invest. 94:79-
88), bone resorption
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(Blavier, L. and J.M. Delaisse (1995) J. Cell Sci. 108:3649-3659), age-related
macular degeneration
(Steep, B. et al. (1998) Invest. Ophthalmol. Vis. Sci. 39:2194-2200),
emphysema (Finlay, G.A. et al.
(1997) Thorax 52:502-506), myocardial infarction (Rohde, L.E. et al. (1999)
Circulation 99:3063-3070)
and dilated cardiomyopathy (Thomas, C.V. et al. (1998) Circulation 97:1708-
1715). 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. 56:2815-2822; Anderson et al. (1996) Cancer Res. 56:715-718; Volpert,
O.V. et al. (1996) J.
Clip. Invest. 98:671-679; Taraboletti, G. et al. (1995) J. Natl. Cancer Inst.
87:293-298; Davies, B. et
al. (1993) Cancer Res. 53:2087-2091). 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 et al., 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 TIIuVIP-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 itselt~,
activating the program for lateral inhibition in I~r-osophila 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 385:729-733). 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.
Proteins of the ADAMTS sub-family have all of the features of ADAM family
metalloproteases and contain an additional thrombospondin domain (TS). The
prototypic ADAMTS
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was identified in mouse, and found to be expressed in heart and kidney and
upregulated by
proinflammatory stimuli (Kuno, K. et al. (1997) J. Biol. Chem. 272:556-562).
To date eleven members
are recognized by the Human Genome Organization (HUGO;
http://www.gene.ucl.ac.uk/users/hester/adamts.html#Approved). Members of this
family have the
ability to degrade aggrecan, a high molecular weight proteoglycan which
provides cartilage with
important mechanical properties including compressibility, and which is lost
during the development of
arthritis. Enzymes which degrade aggrecan are thus considered attractive
targets to prevent and slow
the degradation of articular cartilage (See, e.g., Tortorella, M.D. (1999)
Science 284:1664-1666;
Abbaszade, I. (1999) J. Biol. Chem. 274:23443-23450). 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 94:2374-2379).
Proteaseinhibitors
Protease inhibitors and other regulators of protease activity control the
activity and effects of
proteases. Protease inhibitors have been shown to control pathogenesis in
animal models of
proteolytic disorders (Murphy, G. (1991) Agents Actions Suppl. 35:69-76). Low
levels of the
cystatins, low molecular weight inhibitors of the cysteine proteases,
correlate with malignant
progression of tumors (Catkins, C. et al. (1995) Biol. Biochem. Hoppe Seyler
376:71-80). The cystatin
superfamily of protease inhibitors is characterized by a particular pattern of
linearly arranged and
tandemly repeated disulfide loops (Kellermann, J. et al. (1989) J. Biol. Chem.
264:14121-14128).
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
(Marlor, 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.
A major portion of all proteins synthesized in eukaryotic cells are
synthesized on the cytosolic
surface of the endoplasmic reticulum (ER). Before these immature proteins are
distributed to other
organelles in the cell or are secreted, they must be transported into the
interior lumen of the ER where
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post-translational modifications are performed. These modifications include
protein folding and the
formation of disulfide bonds, and N-linked glycosylations.
Protein Isomerases
Protein folding in the ER is aided by two principal types of protein
isomerases, protein disulfide
isomerase (PDI), and peptidyl-prolyl isomerase (PPI). PDI catalyzes the
oxidation of free sulfhydryl
groups in cysteine residues to form intramolecular disulfide bonds in
proteins. PPI, au enzyme that
catalyzes the isomerization of certain proline imidic bonds in oligopeptides
and proteins, is considered
to govern one of the rate limiting steps in the folding of many proteins to
their final functional
conformation. The cyclophilins represent a major class of PPI that was
originally identified as the
to major receptor for the immunosuppressive drug cyclosporin A
(Handschumacher, R.E. et al. (1984)
Science 226: 544-547).
Protein Glycosylation
The glycosylation of most soluble secreted and membrane bound proteins by
oligosaccharides
linked to asparagine residues in proteins is also performed in the ER. This
reaction is catalyzed by a
membrane-bound enzyme, oligosaccharyl transferase. Although the exact purpose
of this "N-linked"
glycosylation is unknown, the presence of oligosaccharides tends to make a
glycoprotein resistant to
protease digestion. In addition, oligosaccharides attached to cell-surface
proteins called selectins are
known to function in cell-cell adhesion processes (Alberts, B. et al. (1994)
Molecular Biology of the
Cell Garland Publishing Co., New York, NY, p. 608). "O-linked" glycosylation
of proteins also occurs
in the ER by the addition of N-acetylgalactosamine to the hydroxyl group of a
serine or threonine
residue followed by the sequential addition of other sugar residues to the
first. This process is
catalyzed by a series of glycosyltrausferases, each specific for a particular
donor sugar nucleotide and
acceptor molecule (Lodish, H. et al. (1995) Molecular Cell Biolo~y, W. H.
Freeman and Co., New
York, NY, pp. 700-708). In many cases, both N- and O-linked oligosaccharides
appear to be required
for the secretion of proteins or the movement of plasma membrane glycoproteins
to the cell surface.
An additional glycosylation mechanism operates in the ER specifically to
target lysosomal
enzymes to lysosomes and prevent their secretion. Lysosomal enzymes in the ER
receive an N-linked
oligosaccharide, like plasma membrane and secreted proteins, but are then
phosphorylated on one or
two mannose residues. The phosphorylation of mannose residues occurs in two
steps, the first step
being the addition of an N-acetylglucosamine phosphate residue by N-
acetylglucosamine
phosphotransferase, and the second the removal of the N-acetylglucosamine
group by
phosphodiesterase. The phosphorylated mannose residue then targets the
lysosomal enzyme to a
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mannose 6-phosphate receptor which transports it to a lysosome vesicle (Lodish
et al. supra, pp. 708-
711).
Chaperones
Molecular chaperones are proteins that aid in the proper folding of immature
proteins and
refolding of improperly folded ones, the assembly of protein subunits, and in
the transport of unfolded
proteins across membranes. Chaperones are also called heat-shock proteins
(hsp) because of their
tendency to be expressed in dramatically increased amounts following brief
exposure of cells to
elevated temperatures. This latter property most likely reflects their need in
the refolding of proteins
that have become denatured by the high temperatures. Chaperones may be divided
into several
classes according to their location, function, and molecular weight, and
include hsp60, TCP1, hsp70,
hsp40 (also called DnaJ), and hsp90. For example, hsp90 binds to steroid
hormone receptors,
represses transcription in the absence of the ligand, and provides proper
folding of the ligand binding
domain of the receptor in the presence of the hormone (Burston, S.G. and A.R.
Clarke (1995) Essays
Biochem. 29:125-136). Hsp60 and hsp70 chaperones aid in the transport and
folding of newly .
synthesized proteins. Hsp70 acts early in protein folding, binding a newly
synthesized protein before it
leaves the ribosome and transporting the protein to the mitochondria or ER
before releasing the folded
protein. Hsp60, along with hspl0, binds misfolded proteins and gives them the
opportunity to refold
correctly. All chaperones share an affinity for hydrophobic patches on
incompletely folded proteins
and the ability to hydrolyze ATP. The energy of ATP hydrolysis is used to
release the lisp bound
2o protein in its properly folded state (Alberts et al., supra;: pp. 214, 571-
572).
The putative human lysyl oxidase-like 3 (humanLOXL3) contains 754 amino acids.
The
protein contains a copper-binding site with four histidyl residues, the lysyl
and tyrosyl residues known
to be involved in LOX enzyme in the formation of the quinone cofactor and
surrounding sequences,
and a cytokine receptor-like domain. It also contains four scavenger receptor
cysteine-rich (SRCR)
domains in its N-terminal region with the second and fourth of these SRCR
domains truncated.
Further the potential BMP-1 cleavage site is not present. The gene encoding
the cDNA has been
mapped to chromosome 2p13.3, overlapping at its 3' end the HtrA2 serine
protease gene. The central
nervous system, neurons, and leukocytes express humanLOXL3. The gene contains
14 exons and
there are at least two alternative splice variants of LOXL3, that lack exon 5
and exon 8 (Jourdan-Le
Saux, C. et al. (2001) Genomics 74:211-218).
E~ression profiling
Microarrays are analytical tools used in bioanalysis. A microarray has a
plurality of molecules
spatially distributed over, and stably associated with, the surface of a solid
support. Microarrays of
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polypeptides, polynucleotides, and/or antibodies have been developed and find
use in a variety of
applications, such as gene sequencing, monitoring gene expression, gene
mapping, bacterial
identification, drug discovery, and combinatorial chemistry.
One area in particular in which microarrays end use is in gene expression
analysis. Array
technology can provide a simple way to explore the expression of a single
polymorphic gene or the
expression profile of a large number of related or unrelated genes. When the
expression of a single
gene is examined, arrays are employed to detect the expression of a specific
gene or its variants.
When an expression profile is examined, arrays provide a platform for
identifying genes that are tissue
specific, are affected by a substance being tested in a toxicology assay, are
part of a signaling
cascade, carry out housekeeping functions, or are specifically related to a
particular genetic
predisposition, condition, disease, or disorder. The potential application of
gene expression profiling is
particularly relevant to improving diagnosis, prognosis, and treatment of
disease. For example, both
the levels and sequences expressed in tissues from subjects with diabetes may
be compared with the
levels and sequences expressed in normal tissue.
Lung cancer
The potential application of gene. expression profiling is particularly
relevant to improving
diagnosis, prognosis, and treatment of cancers such as lung cancer. Lung
cancer is the leading cause
of cancer death for men and the second leading cause of cancer death for women
in the U.S. The
vast majority of lung cancer cases are attributed to smoking tobacco, and
increased use of tobacco
products in third world countries is projected to lead to an epidemic of lung
cancer in these countries.
Exposure of the bronchial epithelium to tobacco smoke appears to result in
changes in tissue
morphology, which are thought to be precursors of cancer. Lung cancers are
divided into four
histopathologically distinct groups. Three groups (squamous cell carcinoma,
adenocarcinoma, and
large cell carcinoma) are classified as non-small cell lung cancers (NSCLCs).
The fourth group of
cancers is referred to as small cell lung cancer (SCLC). Collectively, NSCLCs
account for ~70% of
cases while SCLCs account for ~18% of cases. The molecular and cellular
biology underlying the
development and progression of lung cancer are incompletely understood.
Deletions on chromosome 3 are common in this disease and are thought to
indicate the
presence of a tumor suppressor gene in this region. Activating mutations in K-
ras are commonly
found in lung cancer and are the basis of one of the mouse models for the
disease.
Preadipoc~te Cells
The potential application of gene expression profiling is particularly
relevant to improving
diagnosis, prognosis, and treatment of obesity. The most important function of
adipose tissue is its
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ability to store and release fat during periods of feeding and fasting. White
adipose tissue is the major
energy reserve in periods of excess energy use, and its primary purpose is
mobilization during energy
deprivation. Understanding how the various molecules regulate adiposity and
energy balance in
physiological and pathophysiological situations may lead to the development of
novel therapeutics for
human obesity. Adipose tissue is also one of the important target tissues for
insulin. Adipogenesis and
insulin resistance in type II diabetes are linked and present intriguing
relations. Most patients with type
II diabetes are obese and obesity in turn causes insulin resistance.
The majority of research in adipocyte biology to date has been done using
transformed mouse
preadi-pocyte cell lines. The culture condition, which stimulates mouse
preadipocyte differentiation is
different from that for inducing human primary preadipocyte differentiation.
In addition, primary cells
are diploid and may therefore reflect the in vivo context better than
aneuploid cell lines.
Understanding the gene expression profile during adipogenesis in human will
lead to understanding the
fundamental mechanism of adiposity regulation. Furthermore, through comparing
the gene expression
profiles of adipogenesis between donor with normal weight and donor with
obesity, identification of
crucial genes, potential drug targets for obesity and type II diabetes, will
be possible.
Peroxisome Proliferator-activated Receptor Gamma A~onist
Thiazolidinediones (TZDs) act as agonists for the peroxisome-proliferator-
activated receptor
gamma (PPARy), a member of the nuclear hormone receptor superfamily. TZDs
reduce
hyperglycemia, hyperinsulinemia, and hypertension, in part by promoting
glucose metabolism and
inhibiting gluconeogenesis. Roles for PPARy and its agonists have been
demonstrated in a wide range
of pathological conditions including diabetes, obesity, hypertension,
atherosclerosis, polycystic ovarian
syndrome, and cancers such as breast, prostate, liposarcoma, and colon cancer.
The mechanism by which TZDs and other PPAR~y agonists enhance insulin
sensitivity is not
fully understood, but may involve the ability of PPARy to promote
adipogenesis. When ectopically
expressed in cultured preadipocytes, PPARy is a potent inducer of adipocyte
differentiation. TZDs, in
combination with insulin and other factors, can also enhance differentiation
of human preadipocytes in
culture (Adams et al. (1997) J. Clip. Invest. 100:3149-3153). The relative
potency of different TZDs
in promoting adipogenesis in vitro is proportional to both their insulin
sensitizing effects in vivo, and
their ability to bind and activate PPARy in vitro. Interestingly, adipocytes
derived from omental
adipose depots are refractory to the effects of TZDs. It has therefore been
suggested that the insulin
sensitizing effects of TZDs may result from their ability to promote
adipogenesis in subcutaneous
adipose depots (Adams et al., ibid). Further, dominant negative mutations in
the PPARY gene have
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been identified in two non-obese subjects with severe insulin resistance,
hypertension, and overt non-
insulin dependent diabetes mellitus (NIDDM) (Barroso et al. (1998) Nature
402:880-883).
NIDDM is the most common form of diabetes mellitus, a chronic metabolic
disease that
affects 143 million people worldwide. NIDDM is characterized by abnormal
glucose and lipid
metabolism that result from a combination of peripheral insulin resistance and
defective insulin
secretion. NIDDM has a complex, progressive etiology and a high degree of
heritability. Numerous
complications of diabetes including heart disease, stroke, renal failure,
retinopathy, and peripheral
neuropathy contribute to the high rate of morbidity and mortality.
At the molecular level, PPARy functions as a ligand activated transcription
factor. In the
presence of ligand, PPARy forms a heterodimer with the retinoid X receptor
(RXR) which then
activates transcription of target genes containing one or more copies of a
PPARy response element
(PPRE). Many genes important in lipid storage and metabolism contain PPREs and
have been
identified as PPARy targets, including PEPCK, aP2, LPL, ACS, and FAT-P
(Auwerx, J. (1999) .
Diabetologia 42:1033-1049. Multiple ligands for PPARy have been identified.
These include a .
variety of fatty acid metabolites; synthetic drugs belonging to the TZD class,
such as Pioglitazone and
Rosiglitazone (BRL49653); and certain non-glitazone tyrosine analogs such as
GI262570 and
GW1929. The prostaglandin derivative 15-dPGJ2 is a potent endogenous ligand
for PPARY.
Expression of PPARy is very high in adipose but barely detectable in skeletal
muscle, the
primary site for insulin stimulated glucose disposal in the body. PPARy is
also moderately expressed.
2o in large intestine, kidney, liver, vascular smooth muscle, hematopoietic
cells, and macrophages. The
high expression of PPARy in adipose suggests that the insulin sensitizing
effects of TZDs may result
from alterations in the expression of one or more PPARy regulated genes in
adipose tissue.
Identification of PPAR~y target genes will contribute to better drug design
and the development of
novel therapeutic strategies for diabetes, obesity, and other conditions.
Systematic attempts to identify PPARy target genes have been made in several
rodent models
of obesity and diabetes (Suzuki et al. (2000) Jpn. J. Pharmacol. 84:113-123;
Way et al. (2001)
Endocrinology 142:1269-1277). However, a serious drawback of the rodent gene
expression studies is
that significant differences exist between human and rodent models of
adipogenesis, diabetes, and
obesity (Taylor (1999) Cell 97:9-12; Gregoire et al. (1998) Physiol. Reviews
78:783-809). Therefore,
an unbiased approach to identifying TZD regulated genes in primary cultures of
human tissues is
necessary to fully elucidate the molecular basis for diseases associated with
PPARY activity.
Colon Cancer
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'The potential application of gene expression profiling is particularly
relevant to improving
diagnosis, prognosis, and treatment of cancer, such as colon cancer.
Colorectal cancer is the second
leading cause of cancer deaths in the United States. Colon cancer is
associated with aging, since 90%
of the total cases occur in individuals over the age of 55. A widely accepted
hypothesis is that several
contributing genetic mutations must accumulate over time in an individual who
develops the disease.
To understand the nature of genetic alterations in colorectal cancer, a number
of studies have focused
on the inherited syndromes. The first known inherited syndrome, Familial
Adenomatous Polyposis
(FAP), is caused by mutations in the Adenomatous Polyposis Coli gene (APC),
resulting in truncated
or inactive forms of the protein. This tumor suppressor gene has been mapped
to chromosome 5q.
The second known inherited syndrome is hereditary nonpolyposis colorectal
cancer (HNPCC), which
is caused by mutations in mismatch repair genes.
Although hereditary colon cancer syndromes occur in a small percentage of the
population
and most colorectal cancers are considered sporadic, knowledge from studies of
the hereditary
syndromes can be generally applied. For instance, somatic mutations in APC
occur in at least 80% of
indiscriminate colon tumors. APC mutations are thought to be the initiating
event in the disease:
Other mutations occur subsequently. Approximately 50% of colorectal cancers
contain activating
mutations in ras, while 85 % contain inactivating mutations in p53. Changes in
these genes lead to
gene expression changes in colon cancer. Less is understood about downstream
targets of these
mutations and the role they may play in cancer.development and progression.
Ovarian cancer
The potential application of gene expression profiling is particularly
relevant to improving
diagnosis, prognosis, and treatment of cancer, such as ovarian cancer. Ovarian
cancer is the leading
cause of death from a gynecologic cancer. The majority of ovarian cancers are
derived from
epithelial cells, and 70% of patients with epithelial ovarian cancers present
with late-stage disease. As
a result the long term survival rates for this disease are very low.
Identification of early stage markers
for ovarian cancer would significantly increase the survival rate. The
molecular events that lead to
ovarian cancer are poorly understood. Some of the known aberrations include
mutation of p53 and
microsatellite instability.
Breast cancer
The potential application of gene expression profiling is particularly
relevant to improving
diagnosis, prognosis, and treatment of cancer, such as breast cancer. More
than 180,000 new cases
of breast cancer are diagnosed each year, and the mortality rate for breast
cancer approaches 10% of
all deaths in females between the ages of 45-54 (Gish, K. (1999) ASS Magazine
28:7-10). However
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the survival rate based on early diagnosis of localized breast cancer is
extremely high (97 %),
compared with the advanced stage of the disease in which the tumor has spread
beyond the breast
(22%). Current procedures for clinical breast examination are lacking in
sensitivity and specificity,
and efforts are underway to develop comprehensive gene expression profiles for
breast cancer that
may be used in conjunction with conventional screening methods to improve
diagnosis and prognosis
of this disease (Perou, C.M. et al. (2000) Nature 406:747-752).
Mutations in two genes, BRCA1 and BRCA2, are known to greatly predispose a
woman to
breast cancer and may be passed on from parents to children (Gish, supra).
However, this type of
hereditary breast cancer accounts for only about 5% to 9% of breast cancers,
while the vast majority
of breast cancer is due to non-inherited mutations that occur in breast
epithelial cells.
The relationship between expression of epidermal growth factor (EGF) and its
receptor,
EGFR, to human mammary carcinoma has been particularly well studied. (See
Khazaie, K. et al.
(1993) Cancer and Metastasis Rev. 12:255-274, and references cited therein for
a review of this
area.) Overexpression of EGFR, particularly coupled with down-regulation of
the estrogen receptor,
is a marker of poor prognosis in breast cancer patients. In addition, EGFR
expression in breast tumor
metastases is frequently elevated relative to the primary tumor, suggesting
that EGFR is involved in
tumor progression and metastasis. This is supported by accumulating evidence
that EGF has effects
on cell functions related to metastatic potential, such as cell motility,
chemotaxis, secretion and
differentiation. Changes in expression of other members of the erbB receptor
family, of which EGFR
is one, have also been implicated in breast cancer. The abundance of erbB
receptors, such as HER-
2/neu, HER-3, and HER-4, and their ligands in breast cancer points to their
functional importance in
the pathogenesis of the disease, and may therefore provide targets for therapy
of the disease (Bacus,
S.S. et al. (1994) Am. J. Clin. Pathol. 102:513-S24). Other known markers of
breast cancer include a
human secreted frizzled protein mRNA that is downregulated in breast tumors;
the matrix G1a protein
which is overexpressed in human breast carcinoma cells; Drg1 or RTP, a gene
whose expression is
diminished in colon, breast, and prostate tumors; maspin, a tumor suppressor
gene downregulated in
invasive breast carcinomas; and CaNl9, a member of the S 100 protein family,
all of which are down-
regulated in mammary carcinoma cells relative to normal mammary epithelial
cells (Zhou, Z. et al.
(1998) Int. J. Cancer 78:95-99; Chen, L. et al. (1990) Oncogene 5:1391-1395;
Ulrix, W. et al (1999)
FEBS Lett 455:23-26; Sager, R. et al. (1996) Curr. Top. Microbiol. Tmmunol.
213:51-64; and Lee,
S.W. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2504-2508).
Cell lines derived from human mammary epithelial cells at various stages of
breast cancer
provide a useful model to study the process of malignant transformation and
tumor progression as it
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has been shown that these cell lines retain many of the properties of their
parental tumors for lengthy
culture periods (Wistuba, LI. et al. (1998) Clip. Cancer Res. 4:2931-2938).
Such a model is
particularly useful for comparing phenotypic and molecular characteristics of
human mammary
epithelial cells at various stages of malignant transformation.
There is a need in the art for new compositions, including nucleic acids and
proteins, for the
diagnosis, prevention, and treatment of gastrointestinal, cardiovascular,
autoimmune/inflammatory, cell
proliferative, developmental, epithelial, neurological, reproductive,
endocrine, pancreatic, adrenal, and
metabolic disorders; as well as lipid, copper, and carbohydrate metabolism
disorders, disorders
associated with gonadal steroid hormones, the immune system, and infections.
SUMMARY OF THE INVENTION
Various embodiments of the invention provide purified polypeptides, protein
modification and
maintenance molecules, referred to collectively as 'PNIIvIM' and individually
as 'PNIIVIM-1,'
<P~_2~> <P_3~> <P_4~> <P~_5~> <P_6~> <P~_7~> <P~_8~> 'P_
9,' <P~~llVIM_10,> 'PNIIVIM-11,' 'PMMM-12,' <P~~VIM_13,> <P~_14,> <PM~~I_15,>
'PNIMM-
16,' 'PNNIIVVIM-17,' <P1~~VIM_18,> 'PMMM-19,' 'PMMM-20,' 'PMMM-21,' 'PIVInMM-
22,' 'Pn~VIM_
23,' 'PMIvVIIVVI-24,' <P_25,> <P~~VIM_26,' 'PMMM-27,' <PMIVIT~I_28,> 'PNllVIM-
29,' <PN1~4M_
30,> <P~_31~> <P_32,> <P~_33~> <P~_34,> <P_35~> <P~_36,> <P_
37,' 'PNIMM-38,' 'P-39,' and 'Pl~wIM-40' and methods for using these proteins
and their
encoding polynucleotides for the detection, diagnosis, and treatment of
diseases and medical
conditions. Embodiments also provide methods for utilizing the purified
protein modification and
maintenance molecules and/or their encoding polynucleotides for facilitating
the drug discovery
process, including determination of efficacy, dosage, toxicity, and
pharmacology. Related
embodiments provide methods for utilizing the purified protein modification
and maintenance molecules
and/or their encoding polynucleotides for investigating the pathogenesis of
diseases and medical
conditions.
An embodiment provides an isolated polypeptide selected from the group
consisting of a) a
polypeptide comprising an amino acid sequence selected from the group
consisting of SEQ ID N0:1-
40, b) a polypeptide comprising a naturally occurring amino acid sequence at
least 90% identical or at
least about 90% identical to an amino acid sequence selected from the group
consisting of SEQ ~
NO:1-40, c) a biologically active fragment of a polypeptide having an amino
acid sequence selected
from the group consisting of SEQ 117 N0:1-40, and d) an immunogenic fragment
of a polypeptide
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having an amino acid sequence selected from the group consisting of SEQ m N0:1-
40. Another
embodiment provides an isolated polypeptide comprising an amino acid sequence
of SEQ m N0:1-40.
Still another embodiment provides an isolated polynucleotide encoding a
polypeptide selected
from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ m N0:1-40, b) a polypeptide comprising a naturally
occurring amino acid
sequence at least 90% identical or at least about 90% identical to an amino
acid sequence selected
from the group consisting of SEQ DJ N0:1-40, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ m N0:1-
40, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m N0:1-40. In another embodiment, the polynucleotide encodes
a polypeptide
selected from the group consisting of SEQ m N0:1-40. In an alternative
embodiment, the
polynucleotide is selected from the group consisting of SEQ m N0:41-80.
Still another embodiment provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ m N0:1-40, b) a polypeptide comprising a naturally occurring amino acid
sequence at least
90% identical or at least about 90% identical to an amino acid sequence
selected from the group
consisting of SEQ m N0:1-40, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ m N0:1-40, and d) an
immunogenic fragment of .
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ~ N0:1-40.
Another embodiment provides a cell transformed with the recombinant
polynucleotide. Yet another
embodiment provides a transgenic organism comprising the recombinant
polynucleotide.
Another embodiment provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ m N0:1-40, b) a polypeptide comprising a naturally occurring amino acid
sequence at least
90% identical or at least about 90% identical to an amino acid sequence
selected from the group
consisting of SEQ m NO:1-40, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ m NO:1-40, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ~ NO:1-40.
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.
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Yet another embodiment provides an isolated antibody which specifically binds
to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid sequence
selected from the group consisting of SEQ )D N0:1-40, b) a polypeptide
comprising a naturally
occurring amino acid sequence at least 90% identical or at least about 90%
identical to an amino acid
sequence selected from the group consisting of SEQ )D N0:1-40, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ )D NO:1-40,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ m N0:1-40.
Still yet another embodiment provides an isolated polynucleotide selected from
the group
consisting of a) a polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of SEQ ~ N0:41-80, b) a polynucleotide comprising a naturally
occurring polynucleotide
sequence at least 90% identical or at least about 90% identical to a
polynucleotide sequence selected
from the group consisting of SEQ m N0:41-80, c) a polynucleotide complementary
to the
polynucleotide of a), d) a polynucleotide complementary to the polynucleotide
of b), and e) an RNA
equivalent of a)-d). In other embodiments, the polynucleotide can comprise at
least about 20, 30, 40,
60, 80, or 100 contiguous nucleotides.
Yet another embodiment provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide being selected from,the group consisting of a) a
polynucleotide comprising a
polynucleotide sequence selected from the group consisting of SEQ ID N0:41-80,
b) a polynucleotide
comprising a naturally occurring polynucleotide sequence at least 90%
identical or at least about 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ )D N0:41-80, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). 'The method comprises
a) hybridizing the
sample with a probe comprising at least 20 contiguous nucleotides comprising a
sequence
complementary to said target polynucleotide in the sample, and which probe
specifically hybridizes to
said target polynucleotide, under conditions whereby a hybridization complex
is formed between said
probe and said target polynucleotide or fragments thereof, and b) detecting
the presence or absence of
said hybridization complex. In a related embodiment, the method can include
detecting the amount of
the hybridization complex. In still other embodiments, the probe can comprise
at least about 20, 30,
40, 60, 80, or 100 contiguous nucleotides.
Still yet another embodiment provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide being selected from the group consisting of
a) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
)D N0:41-80, b) a
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polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical or at
least about 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ D7
NO:41-80, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method
comprises a) amplifying said target polynucleotide or fragment thereof using
polymerase chain
reaction amplification, and b) detecting the presence or absence of said
amplified target polynucleotide
or fragment thereof. In a related embodiment, the method can include detecting
the amount of the
amplified target polynucleotide or fragment thereof.
Another embodiment provides a composition comprising an effective amount of a
polypeptide
selected from the group consisting of a) a polypeptide comprising an amino
acid sequence selected
from the group consisting of SEQ ~ N0:1-40, b) a polypeptide comprising a
naturally occurring
amino acid sequence at least 90% identical or at least about 90% identical to
an amino acid sequence
selected from the group consisting of SEQ D7 N0:1-40, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ~ N0:1-40,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ m N0:1-40, and a pharmaceutically acceptable
excipient. In one
embodiment, the composition can comprise an amino acid sequence selected from
the group consisting
of SEQ m N0:1-40. Other embodiments provide a method of treating a disease or
condition
associated with decreased or abnormal expression of functional PNNnVVIM,
comprising administering to a
patient in need of such treatment the composition.
Yet another embodiment provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ m N0:1-40, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
amino acid sequence selected from the group consisting of SEQ D7 N0:1-40, c) a
biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
B7 N0:1-40, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ m NO:1-40. The method comprises a)
exposing a sample
comprising the polypeptide to a compound, and b) detecting agonist activity in
the sample. Another
embodiment provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. Yet another embodiment provides a
method of treating a
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disease or condition associated with decreased expression of functional
PNJIVIM, comprising
administering to a patient in need of such treatment the composition.
Still yet another embodiment provides a method for screening a compound for
effectiveness
as an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ >D N0:1-40, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical or
at least about 90%
identical to an amino acid sequence selected from the group consisting of SEQ
)D N0:1-40, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m N0:1-40, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-40. The method
comprises a)
exposing a sample comprising the polypeptide to a compound, and b) detecting
antagonist activity in
the sample. Another embodiment provides a composition comprising an antagonist
compound
identified by the method and a pharmaceutically acceptable excipient. Yet
another embodiment
provides a method of treating a disease or condition associated with
overexpression of functional
Pte, comprising administering to a patient in need of such treatment the
composition.
Another embodiment provides a method of screening for a compound that
specifically binds to
a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID N0:1-40,: b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
amino acid sequence selected from the group consisting of SEQ ID N0:1-40, c) a
biologically active ,
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
m N0:1-40, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ 117 N0:1-40. 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.
Yet another embodiment provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ m N0:1-40, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
a_rrLno acid sequence selected from the group consisting of SEQ ID N0:1-40, c)
a biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
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>D N0:1-40, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ ID N0:1-40. 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.
Still yet another embodiment provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of SEQ )D N0:41-80,
the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, b) detecting
altered expression of the target polynucleotide; and c) comparing the
expression of the target .
polynucleotide in the presence of varying amounts of the compound and in the
absence of the
compound.
Another embodiment provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound; b)
hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
177 N0:41-80, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical or at
least about 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:41-80, iii) a polynucleotide having a sequence complementary to i), iv) a
polynucleotide
complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-
iv). Hybridization occurs
under conditions whereby a specific hybridization complex is formed between
said probe and a target
polynucleotide in the biological sample, said target polynucleotide selected
from the group consisting of
i) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
>D N0:41-80, ii) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical or at least about 90% identical to a polynucleotide sequence
selected from the group
consisting of SEQ >D N0:41-80, iii) a polynucleotide complementary to the
polynucleotide of i), iv) a
polynucleotide complementary to the polynucleotide of ii), and v) an RNA
equivalent of i)-iv).
Alternatively, the target polynucleotide can comprise a fragment of a
polynucleotide selected from the
CA 02460476 2004-03-15
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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 full length polynucleotide and
polypeptide
embodiments of the invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog, and the PROTEOME database identification numbers and annotations of
PROTBOME
database homologs, for polypeptide embodiments of the invention. 'The
probability scores.for the
matches between each polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide embodiments, including
predicted motifs and
domains, along with the methods, algorithms, and searchable databases used for
analysis of the
polypeptides.
Table 4 lists the cDNA andlor genomic DNA fragments which were used to
assemble
polynucleotide embodiments, along with selected fragments of the
polynucleotides.
Table 5 shows representative cDNA libraries for polynucleotide embodiments.
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
polynucleotides and
polypeptides, along with applicable descriptions, references, and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleic acids, and methods are described, it is
understood that
embodiments of the invention are not limited to the particular machines,
instruments, 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
invention.
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
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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 various embodiments of 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
"PNPVIM" refers to the amino acid sequences of substantially purified Pl~~VIM
obtained from
any species, particularly a mammalian species, including bovine, ovine,
porcine, murine, equine, and .
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
PNEVIM. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of PMNINI either by
directly interacting with
PIV11VIM or by acting on components of the biological pathway in which PM1VINI
participates.
An "allelic variant" is an alternative form of the gene encoding P1VBVIM.
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 PNNIIVVIM include those sequences
with deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as PM1VIT~I or a
polypeptide with at least one functional characteristic of PNIMM. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding Pn~llVIM, and improper or unexpected hybridization
to allelic variants, with
a locus other than the normal chromosomal locus for the polynucleotide
encoding P1~~VIM. The
encoded protein may also be "altered," and may contain deletions, insertions,
or substitutions of amino
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acid residues which produce a silent change and result in a functionally
equivalent PIVllVIM.
Deliberate amino acid substitutions may be made on the basis of one or more
similarities in polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic
nature of the residues, as long
as the biological or immunological activity of Pis 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" can refer to an oligopeptide,
a peptide, a
polypeptide, or a 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. Amplification
may be carried out using polymerase chain reaction (PCR) technologies or other
nucleic acid
amplification technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of PM1VIM. Antagonists may include proteins such as antibodies, anticalins,
nucleic acids,
carbohydrates, small molecules, or any other compound or composition which
modulates the activity of
pN~~M either by directly interacting with PNhVIM or by acting on components of
the biological
pathway in which PMIVIM participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')Z, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind P~ 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 Garners that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLI~. 'The coupled peptide is
then used to immunize
the animal.
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The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen used
to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an in vitro evolutionary
process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large
combinatorial libraries.
Aptamer compositions maybe double-stranded or single-stranded, and may include
deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other
nucleotide-like molecules. The
nucleotide components of an aptamer may have rriodified sugar groups (e.g.,
the 2'-OH group of a
ribonucleotide may be replaced by 2'-F or 2'-NHZ), wluch may improve a desired
property, e.g.,
resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
e.g., a high molecular weight carrier to slow clearance of the aptamer from
the circulatory system.
Aptamers may be specifically cross-licked to their cognate ligands, e.g., by
photo-activation of a
cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
The term "intramer" refers to an aptamer which is expressed in vivo. For
example, a
vaccinia virus-based RNA expression system has been used to express specific
RNA aptamers at
high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc.
Natl. Acad. Sci. USA
96:3606-3610).
The term "spiegeliner" refers to an aptamer which includes L-DNA, L-RNA, or
other left-
handed nucleotide derivatives or nucleotide-like molecules. Aptamers
containing left-handed
nucleotides are resistant to degradation by naturally occurring enzymes, which
normally act on
substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a polynucleotide having 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'-
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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 PMMM, or of
any oligopeptide
thereof, to induce a specific immune response in appropriate animals or cells
and to bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide" and a "composition
comprising a given
polypeptide" can refer to any composition containing the given polynucleotide
or polypeptide. The
composition may comprise a dry formulation or an aqueous solution.
Compositions comprising
polynucleotides encoding PMMM or fragments of PMMM 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 genornic DNA fragments
using a computer
program for fragment assembly, such as the GELV1EW fragment assembly system
(Accelrys,
Burlington MA) 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
CA 02460476 2004-03-15
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acids which may be substituted for an original amino acid in a protein and
wluch 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
l0 Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr IIis, Phe, Trp '
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, andlor (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
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comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of PMMM or a polynucleotide encoding Pwhich
can
be identical i_n 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 about 5 to about 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 B7 N0:41-80 can comprise a region of unique polynucleotide
sequence
that specifically identifies SEQ m NO:41-80, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ m N0:41-80 can
be employed
in one or more embodiments of methods of the invention, for example, in
hybridization and
amplification technologies and in analogous methods that distinguish SEQ m
N0:41-80 from related
polynucleotides. The precise length of a fragment of SEQ m NO:41-80 and the
region of SEQ m
NO:41-80 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 m N0:1-40 is encoded by a fragment of SEQ m NO:41-80. A
fragment of SEQ m N0:1-40 can comprise a region of unique amino acid sequence
that specifically
identifies SEQ D7 N0:1-40. For example, a fragment of SEQ m N0:1-40 can be
used as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ m N0:1-40.
The precise length of a fragment of SEQ m N0:1-40 and the region of SEQ m N0:1-
40 to which
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the fragment corresponds can be determined based on the intended purpose for
the fragment using
one or more analytical methods described herein or otherwise known in the art.
A "full length" polynucleotide 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, alternatively, 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 identical 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 one
or more
computer algorithms or programs known in the art or described herein. For
example, percent identity
can 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 iwHiggins, 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.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
which can be used 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
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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 for match: 1
Penalty for' rnisrnatch: -2
Open Gap: 5 arid Extension Gap: 2 penalties
Gap x drop-off. 50
Expect: 10
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
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 identical 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. The
phrases "percent similarity' and "% similarity," as applied to polypeptide
sequences, refer to the
percentage of residue matches, including identical residue matches and
conservative substitutions,
between at least two polypeptide sequences aligned using a standardized
algorithm. In contrast,
conservative substitutions are not included in the calculation of percent
identity between polypeptide
sequences.
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Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table.
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:
Matt~ix: BLOSUM62
Open Gap: 11 and Exterzsio~ Gap: 1 penalties
Gap x dt-op-off. ~'0
Expect: 10
Word Size: 3
Fi~tet-: 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
2o 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 shownherein, 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.
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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 p,g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (T"~ for the
specific sequence at a defined ionic
strength and pH. The 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. and D.W.
Russell (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold
Spring Harbor Press,
Cold Spring Harbor NY, ch. 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 maybe 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 acids 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 present in
solution and another nucleic acid immobilized on a solid support (e.g., paper,
membranes, filters, chips,
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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
polynucleotide
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 P~
which is
to 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 PM1VIM 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,
15 polypeptides, antibodies, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, antibody, or
other chemical compound having a unique and defined position on a microarray.
' The term "modulate" refers to a change in the activity of PMIVINI. For
example, modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
20 functional, or immunological properties of PMMM.
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 genoxnic 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.
25 "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.
30 "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
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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 PMMM 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 P1VIIVIM.
"Probe" refers to nucleic acids encoding Pl~~VIM, their complements, or
fragments thereof,
which are used to detect identical, allelic or related nucleic acids. 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, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in, for
example, Sambrook,
J. and D.W. Russell (2001; Molecular Cloninw A Laboratory Manual, 3rd ed.,
vol. 1-3, Cold Spring
Harbor Press, Cold Spring Harbor NY),. Ausubel, F.M. et al. (1999; Short
Protocols in Molecular
Biolo , 4t'' ed., John Wiley & Sons, New York NY), and 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
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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 Institute/1VRT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UI~) designs primers. based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for example,
as PCR or sequencing primers, microarray elements, or specific probes to
identify fully or partially
complementary polynucleotides in a sample of nucleic acids. Methods of
oligonucleotide selection are
not limited to those described above.
A "recombinant nucleic acid" is a nucleic acid 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. Tlus 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 and Russell (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
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(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 molecule, is composed of the same
linear
sequence of nucleotides as the reference DNA molecule 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 PI~~VIM,
nucleic acids encoding Pte, or fragments thereof may comprise a bodily fluid;
an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or cDNA,
in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recognized by the
binding molecule. For
example, if an antibody is specific for epitope "A," the presence of a
polypeptide comprising the
epitope A, or the presence of free unlabeled A, in a reaction containing free
labeled A and the
antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least about 60°7o
free, preferably at least about 75% free, and most preferably at least about
90% free from other
components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
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A "transcript image" or "expression profile" refers to the collective pattern
of gene expression
by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host 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. In another embodiment, the nucleic acid can be introduced
by infection with a
recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002)
Science 295:868-872). 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 and Russell (supra).
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant maybe described as,
for example, an
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"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotides 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
l0 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 or sequence similarity 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 85%,
at least 90%, at least
91%, at least 92%, at least 93%, at least 94%,. at least 95%, at least 96%, at
least 97%, at least 98%,
or at least 99% or greater sequence identity or sequence similarity over a
certain defined length of one
of the polypeptides.
THE INVENTION
Various embodiments of the invention include new human protein modification
and
maintenance molecules (PMMM), the polynucleotides encoding PMMM, and the use
of these
compositions for the diagnosis, treatment, or prevention of gastrointestinal,
cardiovascular,
autoimmune/inflammatory, cell proliferative, developmental, epithelial,
neurological, reproductive,
endocrine, pancreatic, adrenal, and metabolic disorders; as well as lipid,
copper, and carbohydrate
metabolism disorders, disorders associated with gonadal steroid hormones, the
immune system, and
infections.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
embodiments 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
)D NO:) and an
42
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Incyte polypeptide sequence number (Incyte Polypeptide 117) 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 m) as shown.
Column 6 shows the Incyte ID numbers of physical, full length clones
corresponding to the polypeptide
and polynucleotide sequences of the invention. The full length clones encode
polypeptides which have
at least 95oIo sequence identity to the polypeptide sequences shown in column
3.
Table 2 shows sequences with homology to polypeptide embodiments of the
invention as
identified by BLAST analysis against the GenBank protein (genpept) database
and the PROTEOME
database. Columns 1 and 2 show the polypeptide sequence identification number
(Polypeptide SEQ
ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte
Polypeptide ID) for
polypeptides of the invention. Column 3 shows the GenBank identification
number (GenBank ID NO:)
of the nearest GenBank homolog and the PROTEOME database identification
numbers
(PROTEOME ID NO:) of the nearest PROTEOME database homologs. Column 4 shows
the
probability scores for the matches between each polypeptide and its
homolog(s). Column 5 shows the
~ annotation of the GenBank and PROTEOME database homolog(s) along with
relevant citations where ,
applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and -
2 show the polypeptide sequence identification number (SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention. Column
3 shows the number of amino acid residues in each polypeptide. Column 4 shows
potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the MOTIFS
program of the GCG sequence analysis software package (Accekys, Burlington
MA). Column 6
shows amino acid residues comprising signature sequences, domains, and motifs.
Column 7 shows
analytical methods for protein structure/function analysis and in some cases,
searchable databases to
which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are protein modification
and maintenance molecules.
For example, SEQ ID N0:2 is 100% identical from residue T41 to residue M288,
and 95% identical
from residue M1 to residue E43, to cathepsin 02 (GenBank m 81195556) as
determined by the Basic
Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability
scores are both
1.8e-158, which indicates the probability of obtaining the observed
polypeptide sequence alignment by
chance. SEQ ID N0:2 is localized to the cytoplasmic lysosome/vacuole, has a
protease function, and
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is a human cathepsin O (cathepsin K), as determined by BLAST analysis using
the PROTEOME
database. SEQ ID N0:2 also contains a papain family cysteine protease domain
as determined by
searching for statistically significant matches in the hidden Markov model
(IEVVIM)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from
BLllVll'S, MOTIFS, and
PROFILESCAN, and other BLAST analyses provide further corroborative evidence
that SEQ ID
N0:2 is a cathepsin O. In an alternative example, SEQ ID N0:4 is 100%
identical, from residue M1
to residue R68, and 99% identical, from residue K69 to residue P218, to human
prostate-specific
antigen (GenBank ID g618464) as determined by the Basic Local Alignment Search
Tool (BLAST).
(See Table 2.) The BLAST probability score is 2.0e-118, which indicates the
probability of obtaining
1o the observed polypeptide sequence alignment by chance. SEQ ID NO:4 has
serine protease activity,
and is a kallikrein serine protease, as determined by BLAST analysis using the
PROTEOME .
database. SEQ ID N0:4 also contains a trypsin domain as determined by
searching for statistically
significant matches in the hidden Markov model (I~VIM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from BLIIVVIPS, MOTIFS, BLAST and
PROFILESCAN
analyses provide further corroborative evidence that SEQ ~ N0:4 is a serine
protease in the trypsin
family. In an alternative example, SEQ ID N0:10 is 99% identical, from residue
F123 to residue I824,
to human dipeptidyl peptidse (GenBank ID g11095188) as determined by the Basic
Local Alignment,
Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which
indicates the
probability of obtaining the observed polypeptide sequence alignment by
chance. SEQ ID N0:10 is
localized to the subcellular region, has peptidase function, and is a
dipeptidyl aminopeptidase 8, as
determined by BLAST analysis using the PROTEOME database. SEQ ID N0:10 also
contains a
prolyl oligopeptidase domain as determined by searching for statistically
significant matches in the
hidden Markov model (I~~IM)-based PFAM database of conserved protein family
domains. (See
Table 3.) Data from BLllVIPS, MOTIFS, and PROFILESCAN analyses provide further
corroborative evidence that SEQ ~ NO:10 is a peptidase. In an alternative
example, SEQ ID N0:14
is 96% identical, from residue M1 to residue I429, and 100% identical, from
residue V412 to residue
N945, to human UnpEL, a ubiquitin specific protease (GenBank ~ g2656141) as
determined by the
Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 0.0,
which indicates the probability of obtaining the observed polypeptide sequence
alignment by chance.
SEQ ID N0:14 also has homology to proteins that are ubiquitin specific
proteases and have altered
expression in small cell lung carcinoma cell lines, as determined by BLAST
analysis using the
PROTEOME database. SEQ D7 N0:14 also contains a ubiquitin carboxyl-terminal
hydrolases family
44.
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domain as determined by searching for statistically significant matches in the
ludden Markov model
(I~VIM) based PFAM database of conserved protein family domains. (See Table
3.) Data from
BLIIVVIPS and MOTIFS analyses provide further corroborative evidence that SEQ
ID N0:14 is a
ubiquitin specific protease. In an alternative example, SEQ LD NO:27 is 99%
identical, from residue
I49 to residue H521 and 93% identical, from residue M1 to residue V58, to
human chaperonin
containing t-complex polypeptide 1, eta subunit (GenBank ID g2559010) as
determined by the Basic
Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability
scores are both
1.4e-272, which indicates the probability of obtaining the observed
polypeptide sequence alignment by
chance. SEQ ID N0:27 also has homology to proteins that are localized to the
cytoskeleton, function
as chaperones, and are the eta subunit of the cytosolic chaperonin containing
TCP-1 (CCT), as
determined by BLAST analysis using the PROTEOME database. SEQ ID N0:27 also
contains a
TCP-1/cpn60 chaperonin family domain as determined by searching for
statistically significant
matches in the hidden Markov model (I~~IM)-based PFAM database of conserved.
protein family
domains. (See Table 3.) Data from BLILVVIPS, MOT1FS, and additional BLAST
analyses provide
further corroborative evidence that SEQ ID N0:27 is a chaperonin containing t-
complex polypeptide
1, eta subunit. In an alternative example, SEQ D7 NO:34 is 97% identical, from
residue M1 to residue
N83, and 100% identical, from residue A82 to residue M146, to human
cyclophilin (GenBank ID
g3647230, from M1 to D83 and from A113 to M177, respectively) as determined by
the Basic Local:
Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is
1.8e-75, which
indicates the probability of obtaining the observed polypeptide sequence
alignment by chance. SEQ
ID N0:34 also has homology to proteins that are localized to the nucleus, have
peptidyl-prolyl
isomerase activity and are cyclophilins, as determined by BLAST analysis using
the PROTEOME
database. SEQ LD NO:34 also contains a cyclophilin-type peptidyl-prolyl cis-
traps isomerase domain
as determined by searching for statistically significant matches in the hidden
Markov model (I~VVIM)-
based PFAM database of conserved protein family domains. (See Table 3.) Data
from BLM'S,
BLAST, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence
that SEQ
DJ N0:34 is a member of the class of cyclophilin peptidyl-prolyl isomerases.
SEQ LD N0:1, SEQ ID
NO:3, SEQ ~ N0:5-9, SEQ )D N0:11-13, SEQ ID N0:15-26, SEQ ID NO:28-33, and SEQ
~
N0:35-40 were analyzed and annotated in a similar manner. The algorithms and
parameters for the
analysis of SEQ ID N0:1-40 are described in Table 7.
As shown in Table 4, the full length polynucleotide embodiments were assembled
using cDNA
sequences or coding (exon) sequences derived from genomic DNA, or any
combination of these two
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types of sequences. Column 1 lists the polynucleotide sequence identification
number (Polynucleotide
SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number
(Incyte ID) for
each polynucleotide of the invention, and the length of each polynucleotide
sequence in basepairs.
Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA
and/or genomic
sequences used to assemble the full length polynucleotide embodiments, and of
fragments of the
polynucleotides which are useful, for example, in hybridization or
amplification technologies that
identify SEQ ID N0:41-80 or that distinguish between SEQ ID N0:41-80 and
related polynucleotides.
'The polynucleotide fragments described in Column 2 of Table 4 may refer
specifically, for
example, to Iucyte cDNAs derived from tissue-specific cDNA libraries or from
pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2
may refer to GenBank
cDNAs or ESTs which contributed to the assembly of the full length
polynucleotides. In addition, the
polynucleotide fragments described in column 2 may identify sequences derived
from the ENSEMBL
(The Sanger Centre, Cambridge, UK) database (i.e., those sequences including
the designation
"ENST"). Alternatively, the polynucleotide fragments described in column 2 may
be derived from the
NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences
including the
designation "NM" or "NT") or the NCBI RefSeq Protein Sequence Records (i.e.,
those sequences
including the designation "NP"). Alternatively, the polynucleotide fragments
described in column 2
may refer to assemblages of both cDNA and Genscan-predicted exons brought
together by an "exon
stitching" algorithm. For example, a polynucleotide sequence identified as
FL XXXXXX-Nj NZ YYYYY_N3 Na represents a "stitched" sequence in which XXXXXX
is the
identification number of the cluster of sequences to which the algorithm was
applied, and YYYYY is the
number of the prediction generated by the algorithm, and NI,z~3...~ if
present, represent specific exons
that may have been manually edited during analysis (See Example V).
Alternatively, the
polynucleotide fragments in column 2 may refer to assemblages of exons brought
together by an
"exon-stretching" algorithm. For example, a polynucleotide sequence identified
as
FZ,~'~~YX'XI~ gAAAAA~BBBBB_1 N is a "stretched" sequence, with XXXXXX being
the Incyte
project identification number, gAAAAA being the GenBank identification number
of the human
genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB
being the GenBank
identification number or NCBI RefSeq identification number of the nearest
GenBank protein homolog,
and N referring to specific exons (See Example V). In instances where a RefSeq
sequence was used
as a protein homolog for the "exon-stretching" algorithm, a RefSeq identifier
(denoted by "NM,"
"NP," or "NT") may be used in place of the GenBank identifier (i. e., gBBBBB).
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Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs
GNN, GFG, Exon prediction from genomic sequences using,
for example,
ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK)
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example V).
INCY Full length transcript and exon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the exons and
resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in
Table 4 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant Incyte
cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotides 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 polynucleotides. The tissues and vectors
which were used to
construct the cDNA libraries shown in Table 5 are described in Table 6.
2o The invention also encompasses PNIIV1M variants. Various embodiments of
PI~~VIM variants
can have at least about 80%, at least about 90%, or at least about 95% amino
acid sequence identity
to the PNnVIM amino acid sequence, and can contain at least one functional or
structural characteristic
of P1~~VIM.
Various embodiments also encompass polynucleotides which encode Pte. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:41-80, which encodes Pn~VIM. The
polynucleotide
sequences of SEQ D7 N0:41-80, 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.
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The invention also encompasses variants of a polynucleotide encoding PMMM. In
particular,
such a variant polynucleotide will have at least about 70%, or alternatively
at least about 85%, or even
at least about 95% polynucleotide sequence identity to a polynucleotide
encoding PNIIVIM. A
particular aspect of the invention encompasses a variant of a polynucleotide
comprising a sequence
selected from the group consisting of SEQ ID N0:41-80 which has at least about
70%, or alternatively
at least about 85%, or even at least about 95% polynucleotide sequence
identity to a nucleic acid
sequence selected from the group consisting of SEQ ID NO:41-80. Any one of the
polynucleotide
variants described above can encode a polypeptide which contains at least one
functional or structural
characteristic of PD~VIM.
In addition, or in the alternative, a polynucleotide variant of the invention
is a splice variant of a
polynucleotide encoding P1~~VIM. A splice variant may have portions which have
significant sequence
identity to a polynucleotide encoding PI~~VIM, but will generally have a
greater or lesser number of
polynucleotides due to additions or deletions of blocks of sequence arising
from alternate splicing of
exons during mRNA processing. A splice variant may have less than about 70%,
or alternatively less
than about 60%, or alternatively less than about 50% polynucleotide sequence
identity to a
polynucleotide encoding P1~~V1M over its entire length; however, portions of
the splice variant will
have at least about 70%, or alternatively at least about 85%, or alternatively
at least about 95%, or
alternatively 100% polynucleotide sequence identity to portions of the
polynucleotide encoding
PMn~VI. For example, a polynucleotide comprising a sequence of SEQ ID NO:43
and a
polynucleotide comprising a sequence of SEQ ID N0:52 are splice variants of
each other; a
polynucleotide comprising a sequence of SEQ,ID NO:44 and a polynucleotide
comprising a sequence
of SEQ ll~ NO:69 are splice variants of each other; and a polynucleotide
comprising a sequence of
SEQ ID NO:46, a polynucleotide comprising a sequence of SEQ ID N0:49, and a
polynucleotide
comprising a sequence of SEQ ID N0:50 are splice variants of each other. Any
one of the splice
variants described above can encode a polypeptide which contains at least one
functional or structural
characteristic of PNNI1VVIM.
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 P1~~VIM, 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
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polynucleotide sequence of naturally occurring PMMM, and all such variations
are to be considered as
being specifically disclosed.
Although polynucleotides which encode PMMM and its variants are generally
capable of
hybridizing to polynucleotides encoding naturally occurring PNN11VVIM under
appropriately selected
conditions of stringency, it may be advantageous to produce polynucleotides
encoding PMMM 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 PMMM 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 polynucleotides which encode PMMM
and
PMMM derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the
synthetic polynucleotide 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 polynucleotide encoding PMMM or any fragment
thereof.
Embodiments of the invention can also include polynucleotides that are capable
of hybridizing
to the claimed polynucleotides, and, in particular, to those having the
sequences shown in SEQ ID
N0:41-80 and fragments thereof, under various conditions of stringency (Wahl,
G.M. and S.L. Berger
(1987) Methods Enzymol. 152:399-407; IKimmel, 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
I~lenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ),
or combinations
of polymerases and proofreading exonucleases such as those found in the
ELONGASE amplification
system (Invitrogen, Carlsbad CA). 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 (Amersham Biosciences),
or other
49
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systems known in the art. The resulting sequences are analyzed using a variety
of algorithms which
are well known in the art (Ausubel et al., supf~a, ch. 7; Meyers, R.A. (1995)
Molecular Biology and
Biotechnology, Wiley VCH, New York NY, pp. 856-853).
The nucleic acids encoding PMIVINI 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 (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 (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 (Lagerstrom, M. et al. (1991) PCR Methods
Applic. 1:111-119). In
this method, multiple restriction enzyme digestions and ligations may be used
to insert an engineered
double-stranded sequence into a region of unknown sequence before performing
PCR. Other
methods which may be used to retrieve unknown sequences are known in the art
(Parker, J.D. et al. .
(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested
primers, and
PROMOTERF1NDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This
procedure
avoids the need to screen libraries and is useful in finding intron/exon
junctions. For all PCR based
methods, primers may be designed using commercially available software, such
as OLIGO 4.06
primer analysis software (National Biosciences, Plymouth MN) or another
appropriate program, to be
about 22 to 30 nucleotides in length, to have a GC content of about 50% or
more, and to anneal to the
template at temperatures of about 68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 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
CA 02460476 2004-03-15
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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, polynucleotides or fragments thereof
which encode
PMMM may be cloned in recombinant DNA molecules that direct expression of
PMMM, or
fragments or functional equivalents thereof, in appropriate host cells. Due to
the inherent degeneracy
of the genetic code, other polynucleotides which encode substantially the same
or a functionally
equivalent polypeptides may be produced and 'used to express PMMM.
The polynucleotides of the invention can be engineered using methods generally
known in the
art in order. to alter PMMM-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 examples 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 MOLECULARBREED1NG (Maxygen Inc., Santa Clara CA; described in U.S. Patent
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or improve
the biological properties of PMMM, such as its biological or enzymatic
activity or its ability to bind to
other molecules or compounds. DNA shuffling is a process by which a library of
gene variants is
produced using PCR-mediated recombination of gene fragments. The library is
then subjected to
selection or screening procedures that identify those gene variants with the
desired properties. These
preferred variants may then be pooled and further subjected to recursive
rounds of DNA shuffling and
selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid
molecular evolution. For example, fragments of a single gene containing random
point mutations may
be recombined, screened, and then reshuffled until the desired properties are
optimized. Alternatively,
fragments of a given gene may be recombined with fragments of homologous genes
in the same gene
family, either from the same or different species, thereby maximizing the
genetic diversity of multiple
naturally occurring genes in a directed and controllable manner.
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In another embodiment, polynucleotides encoding PI~~VIM may be synthesized, in
whole or in
part, using one or more chemical methods well known in the art (Caruthers,
M.H. et al. (1980)
Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp.
Ser. 7:225-232).
Alternatively, PMMM itself or a fragment thereof may be synthesized using
chemical methods known
in the art. For example, peptide synthesis can be performed using various
solution-phase or
solid-phase techniques (Creighton, T. (1984) Proteins Structures and Molecular
Properties, WH
Freeman, New York NY, pp. 55-60; 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 PMMM, 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 (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-
421). .The
composition of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing
(Creighton, supra, pp. 28-53).
In order to express a biologically active PMMM, the polynucleotides encoding
P1~~VIM 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
polynucleotides encoding
PI~~VIM. Such elements may vary in their strength and specificity. Specific
initiation signals may also
be used to achieve more efficient translation of polynucleotides encoding
PMMM. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where a
polynucleotide sequence encoding PMMM and its initiation codon and upstream
regulatory sequences
are inserted into the appropriate expression vector, no additional
transcriptional or translational control
signals may be needed. However, in cases where only coding sequence, or a
fragment thereof, is
inserted, exogenous translational control signals including an in-frame ATG
initiation codon should be
provided by the vector. Exogenous translational elements and initiation codons
may be of various
origins, both natural and synthetic. The efficiency of expression may be
enhanced by the inclusion of
enhancers appropriate for the particular host cell system used (Scharf, D. et
al. (1994) Results Probl.
Cell Differ. 20:125-162).
52
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Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing polynucleotides encoding PMMM and appropriate
transcriptional and translational
control elements. These methods include itv vitt~o recombinant DNA techniques,
synthetic techniques,
and ira vivo genetic recombination (Sambrook and Russell, supra, ch. 1-4, and
8; Ausubel et al.,
supra, ch. 1, 3, and 15).
A variety of expression vector/host systems may be utilized to contain and
express
polynucleotides encoding PMMM. 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 (Sambrook and Russell,
supt~a; Ausubel et al.,
supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509;
Engelhard, E.K. et al.
(1994) Proc..Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.
Gene Ther. 7:1937-
1945; Takamatsu, N. (1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of
Science and
Technola~y (1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T.
Shenk (1984) Proc.:
Natl. Acad. Sci. USA 81:3655-3659; 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 polynucleotides to the
targeted organ, tissue, or
cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu,
M. et al. (1993) Proc:
Natl. Acad. Sci. USA 90:6340-6344; Buller, R.M. et al. (1985) Nature 317:813-
815; McGregor, D.P.
et al. (1994) Mol. Trmmunol. 31:219-226; 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 polynucleotides encoding PMMM. For example, routine
cloning, subcloning,
and propagation of polynucleotides encoding PMMM can be achieved using a
multifunctional E. eoli
vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid
(Invitrogen).
Ligation of polynucleotides encoding PMMM 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 ifi vitro
transcription, dideoxy
sequencing, single strand rescue with helper phage, and creation of nested
deletions in the cloned
sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-
5509). When large
53
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quantities of P1~~VIM are needed, e.g. for the production of antibodies,
vectors which direct high level
expression of Pl~~VIM may be used. For example, vectors containing the strong,
iuducible SP6 or T7
bacteriophage promoter may be used.
Yeast expression systems may be used for production of PMMM. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Sacchar'omyces cer~evisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign polynucleotide sequences into the host genome for
stable propagation (Ausubel
et al., supf'a; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544;
Scorer, C.A. et al. (1994)
to Bio/Technology 12:181-184).
Plant systems may also be used for expression of PNNIIVVIM. Transcription of
polynucleotides
encoding PMNIM 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
RUBISC~ or heat shock
15 promoters may be used (Coruzzi, G. et al. (1984) EMB~ J. 3:1671-1680;
Brogue, R. et al. (1984)
Science 224:838-843; 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
(The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New
York NY, pp.
191-196).
20 In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, polynucleotides encoding
PMMM may be
ligated into an adenovirus transcription/trauslation 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 PMMM in host cells (Logan, J. and T.
Shenk (1984) Proc.
25 Natl. Acad. Sci. USA 81:3655-3659). In addition, transcription enhancers,
such as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
30 constructed and delivered via conventional delivery methods (liposomes,
polycationic amino polymers,
or vesicles) for therapeutic purposes (Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355).
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For long term production of recombinant proteins in mammalian systems, stable
expression of
PMMM in cell lines is preferred. For example, polynucleotides encoding PMMM
can be transformed
into cell lines using expression vectors which may contain viral origins of
replication and/or
endogenous expression elements and a selectable marker gene on the same or on
a separate vector.
Following the introduction of the vector, cells may be allowed to grow for
about 1 to 2 days in enriched
media before being switched to selective media. The purpose of the selectable
marker is to confer
resistance to a selective agent, and its presence allows growth and recovery
of cells which
successfully express the introduced sequences. Resistant clones of stably
transformed cells may be
propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and apf~ cells, respectively
(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, dh, frv
confers resistance to
methotrexate; ~ceo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively (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., tfpB and
hisD, which alter cellular
requirements for metabolites (Hartxnan, 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), ~3-
glucuronidase and its substrate (3-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 (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 PMNIM is inserted within a marker gene sequence,
transformed cells
containing polynucleotides encoding PMMM can be identified by the absence of
marker gene
function. Alternatively, a marker gene can be placed in tandem with a sequence
encoding PMMM
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.
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In general, host cells that contain the polynucleotide encoding PMMM and that
express
PMMM 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.
T_mmunological methods for detecting and measuring the expression of PMMM
using either
specific polyclonal or monoclonal autibodies 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 PMMM is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art
(Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN, Sect.
IV; Coligan, J.E. et al. (1997) Current Protocols in Immunology, ~Greene Pub.
Associates and Wiley-
Interscience, New York NY; Pound, J.D. (1998) Tmmunochemical 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 polynticleotides encoding
PMMM include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, polynucleotides encoding PMMM, 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 i~c 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 Biosciences,
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 polynucleotides encoding PM1VBVI 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
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polynucleotides which encode PM1V>T~I may be designed to contain signal
sequences which direct
secretion of PMMM 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 polynucleotides or to process the expressed protein in the desired
fashion. Such modifications
of the polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
polynucleotides
encoding PMMM 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 PMMM
protein containing a
heterologous moiety that can be recognized by a commercially available
antibody may facilitate the
screening of peptide libraries for inhibitors of PMMM activity. Heterologous
protein and peptide
moieties may also facilitate purification of fusion proteins using
commercially available affinity
matrices. Such moieties include, but are not limited to,.glutathione S-
trausferase (GST), maltose
binding protein (MBP), thioredoxin (Trx), caltnodulin binding peptide (CBP), 6-
His, FLAG, c-myc, and
hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their
cognate fusion
proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
and metal-chelate resins,
respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
proteins using commercially available monoclonal and polyclonal antibodies
that specifically recognize
these epitope tags. A fusion protein may also be engineered to contain a
proteolytic cleavage site
located between the P11/IIVBVI encoding sequence and the heterologous protein
sequence, so that
PMMM may be cleaved away from the heterologous moiety following purification.
Methods for
fusion protein expression and purification are discussed in Ausubel et al.
(supf-a, ch. 10 and 16). A
variety of commercially available kits may also be used to facilitate
expression and purification of
fusion proteins.
In another embodiment, synthesis of radiolabeled PMIVITiI may be achieved in
vitt-o using the
TN'T 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
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promoters. Translation takes place in the presence of a radiolabeled amino
acid precursor, for
example, 35S-methionine.
pNnVIM, fragments of PNBVIM, or variants of PMMM may be used to screen for
compounds
that specifically bind to PI~~VIM. One or more test compounds may be screened
for specific binding
to PMMM. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test
compounds can be
screened for specific binding to PNMVI. Examples of test compounds can include
antibodies,
anticalins, oligonucleotides, proteins (e.g., ligands or receptors), or small
molecules.
In related embodiments, variants of PI~~VIM can be used to screen for binding
of test
compounds, such as antibodies, to PI~~VIM, a variant of PMMM, or a combination
of PMNIM andlor
one or more variants PM1VINI. In an embodiment, a variant of PMMM can be used
to screen for
compounds that bind to a variant of PMMM, but not to PMMM having the exact
sequence of a
sequence of SEQ ID N0:1-40. PI~~VIM variants used to perform such screening
can have a range of
about SO% to about 99% sequence identity to PMIVIIVI, with various embodiments
having 60%, 70%,
75%, 80%, 85%, 90%, and 95% sequence identity.
In an embodiment, a compound identified in a screen for specific binding to
P1~~VIM can be
closely related to the natural ligand of PNEVIM, e.g., a ligand or fragment
thereof, a natural substrate, a
structural or functional mimetic, or a natural binding partner (Coligan, J.E.
et al. (1991) Current
Protocols in hnmunolo~y 1(2):Chapter 5). In another embodiment, the compound
thus identified can
be a natural ligand of a receptor PNNIIVVIM (Howard, A.D. et al. (2001) Trends
Pharmacol. Sci.22:132-
140; Wise, A. et al: (2002) Drug Discovery Today 7:235-246). '
In other embodiments, a compound identified in a screen for specific binding
to PNIIVIM can
be closely related to the natural receptor to which PNINIM binds, at least a
fragment of the receptor,
or a fragment of the receptor including all or a portion of the ligand binding
site or binding pocket. For
example, the compound may be a receptor for PMMM which is capable of
propagating a signal, or a
decoy receptor for P1~~VIM which is not capable of propagating a signal
(Ashkenazi, A. and V.M.
Divit (1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001)
Trends Tm_m__unol. 22:328-
336). The compound can be rationally designed using known techniques. Examples
of such
techniques include those used to construct the compound etanercept (ENBREL;
Amgen Inc.,
'Thousand Oaks CA), which is efficacious for treating rheumatoid arthritis in
humans. Etanercept is
an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc
portion of human IgGl
(Taylor, P.C. et al. (2001) Curr. Opin. T_mmunol. 13:611-616).
5s
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In one embodiment, two or more antibodies having similar or, alternatively,
different
specificities can be screened for specific binding to PM1VW, fragments of
PMMM, or variants of
per. The binding specificity of the antibodies thus screened can thereby be
selected to identify
particular fragments or variants of P1~~VIM. In one embodiment, an antibody
can be selected such
that its binding specificity allows for preferential identification of
specific fragments or variants of
pT~VIM. In another embodiment, an antibody can be selected such that its
binding specificity allows
for preferential diagnosis of a specific disease or condition having
increased, decreased, or otherwise
abnormal production of PNNFvvIM.
In an embodiment, anticalins can be screened for specific binding to PMMM,
fragments of
PMMM, or variants of PT~VIM. Anticalins are ligand-binding proteins that have
been constructed
based on a lipocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol.
7:8177-8184;
Skerra, A. (2001) J. Biotechnol. 74:257-275). The protein architecture of
lipocalins can include a
beta-barrel having eight antiparallel beta-strands, which supports four loops
at its open end. These
loops form the natural ligand-binding site of the lipocalins, a site which can
be re-engineered ih vitro
by amino acid substitutions to impart novel binding specificities. The amino
acid substitutions can be .
made using methods known in the art or described herein, and can include
conservative substitutions
(e.g., substitutions that do not alter binding specificity) or substitutions
that modestly, moderately, or
significantly alter binding specificity.
In one embodiment, screening for compounds which specifically bind to,
stimulate; or inhibit
PMMMM involves producing appropriate cells which express PMMMM, either as a
secreted protein or on
the cell membrane. Preferred cells can include cells from mammals, yeast, Dt-
osopltila, or E. coli.
Cells expressing PNFVIM or cell membrane fractions which contain Pl~ are then
contacted with a
test compound and binding, stimulation, or inhibition of activity of either
PM1VPVI 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
PI~~VIM, either in solution
or affixed to a solid support, and detecting the binding of PMMM 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.
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An assay can be used to assess the ability of a compound to bind to its
natural ligand and/or to
inhibit the binding of its natural ligand to its natural receptors. Examples
of such assays include radio-
labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S.
Patent No. 6,372,724.
In a related embodiment, one or more amino acid substitutions can be
introduced into a polypeptide
compound (such as a receptor) to improve or alter its ability to bind to its
natural ligands (Matthews,
D.J. and J.A. Wells. (1994) Chem. Biol. 1:25-30). In another related
embodiment, one or more amino
acid substitutions can be introduced into a polypeptide compound (such as a
ligand) to improve or alter
its ability to bind to its natural receptors (Cumingham, B.C. and J.A. Wells
(1991) Proc. Natl. Acad.
Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-
10988).
Pte, fragments of PMMM, or variants of PMMM may be used to screen for
compounds
that modulate the activity of PMMM. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for PMMM
activity, wherein PMMM is combined with at least one test compound, and the
activity of PT~VIM in
the presence of a test compound is compared with the activity of PMMM in the
absence of the test
compound. A change in the activity of PMMM in the presence of the test
compound is indicative of a
compound that modulates the activity of PMMM. Alternatively, a test compound
is combined with an
i>1 vitro or cell-free system comprising PNNEVVIM under conditions suitable
for PMMM activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of
PM1VIM 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 PMMM or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem (ES)
cells. Such techniques are well known in the art and are useful for the
generation of animal models of
human disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No.
5,767,337). For example,
mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the
early mouse embryo and
grown in culture. The ES cells are transformed with a vector containing the
gene of interest disrupted
by a marker gene, e.g., the neomycin phosphotransferase gene (tteo; 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) Clip. 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
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the C57BL/6 mouse strain. The blastocysts are surgically transferred to
pseudopregnant dams, and
the resulting chimeric progeny are genotyped and bred to produce heterozygous
or homozygous
strains. Transgenic animals thus generated may be tested with potential
therapeutic or toxic agents.
Polynucleotides encoding PMMM may also be manipulated it2 vitf~o 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 PMMM 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 P~ 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 PNIMM, e.g., by secreting Pin 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 PM1VI1~~I and protein modification and maintenance molecules. In
addition, examples of
tissues expressing PNNIIVVIM can be found in Table 6 and can also be found in
Example XI. Therefore,
pI~~VIM appears to play a role in gastrointestinal, cardiovascular,
autoimmune/inflammatory, cell
proliferative, developmental, epithelial, neurological, reproductive,
endocrine, pancreatic, adrenal, and
metabolic disorders; as well as lipid, copper, and carbohydrate metabolism
disorders, disorders
associated with gonadal steroid hormones, the immune system, and infections.
In. the treatment of
disorders associated with increased PMNEVI expression or activity, it is
desirable to decrease the
expression or activity of PI~~VIM. In the treatment of disorders associated
with decreased P1~~VIM
expression or activity, it is desirable to increase the expression or activity
of PMIV>TZ.
Therefore, in one embodiment, PNll~~IM 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 Pte. 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,
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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, alphai 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; an endocrine disorder such as a
disorder of the
hypothalamus andlor pituitary resulting from lesions such as a primary brain
tumor, adenoma,
infarction associated with pregnancy, hypophysectomy, aneurysm, vascular
malformation, thrombosis,
infection, immunological disorder, and complication due to head trauma; a
disorder associated with
hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus,
Kallman's disease,
Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty
sella syndrome, and
dwarfism; a disorder associated with hyperpituitarism including acromegaly,
giantism, and syndrome of
inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by
benign adenoma; a
disorder associated with hypothyroidism including goiter, myxedema, acute
thyroiditis associated with
bacterial infection, subacute thyroiditis associated with viral infection,
autoimmune thyroiditis
(Hashimoto's disease), and cretinism; a disorder associated with
hyperthyroidism including
thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema,
toxic multinodular goiter,
thyroid carcinoma, and Plummer's disease; a disorder associated with
hyperparathyroidism including
Coon disease (chronic hypercalemia); a pancreatic disorder such as Type I or
Type lI diabetes
mellitus and associated complications; a disorder associated with the adrenals
such as hyperplasia,
carcinoma, or adenoma of the adrenal cortex, hypertension associated with
alkalosis, amyloidosis,
hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon
syndrome,
pheochromocytoma tumors, and Addison's disease; a disorder associated with
gonadal steroid
3o hormones such as: in women, abnormal prolactin production, infertility,
endometriosis, perturbation of
the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated
gonadotropin deficiency,
amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast
cancer, and, in post-
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menopausal women, osteoporosis; and, in men, Leydig cell deficiency, male
climacteric phase, and
germinal cell aplasia, a hypergonadal disorder associated with Leydig cell
tumors, androgen resistance
associated with absence of androgen receptors, syndrome of 5 a-reductase, and
gynecomastia; a
disorder of the immune system such as inflammation, actinic keratosis,
acquired immunodeficiency
syndrome (AmS), Addison's disease, adult respiratory distress syndrome,
allergies, ankylosing
spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis,
autoimmune hemolytic
anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis,
cirrhosis, contact dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria,
hepatitis, hypereosinophilia,
irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed
connective tissue
disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation,
myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythenua vera,
polymyositis, psoriasis,
Reiter's syndrome, rheumatoid arthritis, scleroderma,.Sjogren's syndrome,
systemic anaphylaxis,
systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia,
thrombocytopenic
purpura, ulcerative colitis, uveitis, Werner, syndrome, complications of
cancer, hemodialysis; and
extracorporeal circulation, trauma, and hematopoietic cancer including
lymphoma, leukemia, and
myeloma; an infection caused by a viral agent classified as adenovirus,
arenavirus, bunyavirus,
calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus,
orthomyxovirus, parvovirus,
papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus,
rhabdovirus, or togavirus; an
infection caused by a bacterial agent classified as pneumococcus,
staphylococcus, streptococcus,
bacillus, corynebacterium, clostridium, meningococcus, gonococcus, listeria,
moraxella, kingella,
haemophilus, legionella, bordetella, gram-negative enterobacterium including
shigella, salmonella, or
campylobacter, pseudomonas, vibrio, brucella, francisella, yersinia,
bartonella, norcardium,
actinomyces, mycobacterium, spirochaetale, rickettsia, chlamydia, or
mycoplasma; an infection caused
by a fungal agent classified as aspergillus, blastomyces, dermatophytes,
cryptococcus, coccidioides,
malasezzia, histoplasma, or other mycosis-causing fungal agent; and an
infection caused by a parasite
classified as plasmodium or malaria-causing, parasitic entamoeba, leishmania,
trypanosoma,
toxoplasma, pneumocystis carinii, intestinal protozoa such as giardia,
trichomonas, tissue nematode
such as trichinella, intestinal nematode such as ascaris, lymphatic filarial
nematode, trematode such as
schistosoma, and cestrode such as tapeworm; a metabolic disorder such as
Addison's disease,
cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin
resistance, cystic fibrosis,
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diabetes, fatty hepatocirrhosis, fructose-1,6-diphosphatase deficiency,
galactosemia, goiter,
glucagonoma, glycogen storage diseases, hereditary fructose intolerance,
hyperadrenalism,
hypoadrenalism, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia,
hyperthyroidism,
hypoglycemia, hypothyroidism, hyperlipidemia, hyperlipemia, lipid myopathies,
lipodystrophies,
lysosomal storage diseases, mannosidosis, neuraminidase deficiency, obesity,
pentosuria
phenylketonuria, pseudovitamin_ D-deficiency rickets; a disorder of
carbohydrate metabolism such as
congenital type II dyserythropoietic anemia, diabetes, insulin-dependent
diabetes mellitus,
non-insulin-dependent diabetes mellitus, fructose-1,6-diphosphatase
deficiency, galactosemia,
glucagonoma, hereditary fructose intolerance, hypoglycemia, mannosidosis,
neuraminidase deficiency,
obesity, galactose epimerase deficiency, glycogen storage diseases, lysosomal
storage diseases,
fructosuria, pentosuria, and inherited abnormalities of pyruvate metabolism; a
disorder of lipid
metabolism such as fatty liver, cholestasis, primary biliary cirrhosis,
carnitine deficiency, carnitine .
palmitoyltransferase deficiency, myoadenylate deaminase deficiency,
hypertriglyceridemia, lipid
storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's
disease, metachromatic
leukodystrophy, adrenoleukodystrophy, GMZ gangliosidosis, and ceroid
lipofuscinosis,
abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetes
mellitus, lipodystrophy,
lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa,
lipoid adrenal hyperplasia,
minimal change disease, lipomas, atherosclerosis, hypercholesterolemia,
hypercholesterolemia with
hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal
disease, liver disease,
lecithin:cholesterol acyltransferase deficiency, cerebrotendinous
xanthomatosis, sitosterolemia,
hypocholesterolemia, Tay-Sachs disease, Sandhoff's disease, hyperlipidemia,
hyperlipemia, lipid
myopathies, and obesity; a disorder of copper metabolism such as Menke's
disease, Wilson's disease,
and Ehlers-Danlos syndrome type IX.; a cardiovascular disorder, such as
arteriovenous fistula,
atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms,
arterial dissections, varicose
veins, thrombophlebitis and phlebothrombosis, vascular tumors, and
complications of thrombolysis,
balloon angioplasty, vascular replacement, and coronary artery bypass graft
surgery, congestive heart
failure, ischemic heart disease, angina pectoris, myocardial infarction,
hypertensive heart disease,
degenerative valvular heart disease, calcific aortic valve stenosis,
congenitally bicuspid aortic valve,
mitral annular calcification, mural valve prolapse, rheumatic fever and
rheumatic heart disease,
infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of
systemic lupus
erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis,
pericarditis, neoplastic heart
disease, congenital heart disease, and complications of cardiac
transplantation; an
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autoimmune/inflammatory disease, 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 hehninthic 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, C~xshing's syndrome, achondroplastic
dwarfism, Duchenne and
Becker muscular dystrophy, bone resorption, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wilms'
tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-
Magenis syndrome,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas, hereditary
neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,
hypothyroidism,
hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral
palsy, spina bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, age-related
macular degeneration, and
sensorineural hearing loss; an epithelial disorder, such as dyshidrotic
eczema, allergic contact
dermatitis, keratosis pilaris~ melasma, vitiligo, actinic keratosis, basal
cell carcinoma, squamous cell
carcinoma, seborrheic keratosis, folliculitis, herpes simplex, herpes zoster,
varicella, candidiasis,
dermatophytosis, scabies, insect bites, cherry angioma, keloid,
dermatofibroma, acrochordons,
urticaria, transient acantholytic dermatosis, xerosis, eczema, atopic
dermatitis, contact dermatitis, hand
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eczema, nurnmular 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
palinoplantaris, 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
neoplasrns, 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, priori
diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-
Scheiuker syndrome,
fatal familial insomnia, nutritional and metabolic diseases of the nervous
system, neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central nervous system
including Down
syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system
disorders, cranial nerve
disorders, spinal cord diseases, muscular dystrophy and other neuromuscular
disorders, peripheral
nervous system disorders, dermatomyositis and polymyositis, inherited,
metabolic, endocrine, and toxic
myopathies, myasthenia gravis, periodic paralysis, mental disorders including
mood, anxiety, and
schizophrenic disorders, seasonal affective disorder (SAD), akathesia,
amnesia, catatonia, diabetic
neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic
neuralgia, Tourette's
disorder, progressive supranuclear palsy, corticobasal degeneration, and
familial frontotemporal
dementia; and a reproductive disorder, such as infertility, including tubal
disease, ovulatory defects,
and endometriosis, a disorder of prolactin production, a disruption of the
estrous cycle, a disruption of
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the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation
syndrome, an endometrial or
ovarian tumor, a uterine fibroid, autoimmune disorders, au 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 PN)ZVJ:M 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 PNBVIM including, but not limited to, those
described above.
In a further embodiment, a composition comprising a substantially purified P~
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 P1~~V1M
including, but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of
PI\~VIM may be
administered to a,subject to treat or prevent a disorder associated with
decreased expression or
activity of PMMM including, but not limited to, those listed above.
In a further embodiment, an antagonist of PMMM maybe administered to a subject
to treat or
prevent a disorder associated with increased expression or activity of
Pl~~VIM. Examples of such
disorders include, but are not limited to, those gastrointestinal,
cardiovascular,
autoimmune/inflammatory, cell proliferative, developmental, epithelial,
neurological, reproductive,
endocrine, pancreatic, adrenal, and metabolic disorders; as well as lipid,
copper, and carbohydrate
metabolism disorders, disorders associated with gonadal steroid hormones, the
immune system, and
infections described above. In one aspect, an antibody which specifically
binds PMMM 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 PMMM.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding PI~~VIM may be administered to a subject to treat or prevent a
disorder associated with
increased expression or activity of Pn~VIM including, but not limited to,
those described above.
In other embodiments, any protein, agonist, antagonist, antibody,
complementary sequence, or
vector embodiments may be administered in combination with other appropriate
therapeutic agents.
Selection of the appropriate agents for use in combination therapy may be made
by one of ordinary
skill in the art, according to conventional pharmaceutical principles. The
combination of therapeutic
agents may act synergistically to effect the treatment or prevention of the
various disorders described
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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 PMMM may be produced using methods which are generally known
in the
art. In particular, purified PMMM may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind PMMM.
Antibodies to PMMM 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. In an embodiment, neutralizing
antibodies (i.e., those
which inhibit dimer formation) can be used therapeutically. Single chain
antibodies (e.g., from camels
or llamas) may be potent enzyme inhibitors and may have application in the
design of peptide mim__etics,
and in the development of immuno-adsorbents and biosensors (Muyldermans, S.
(2001) J. Biotechnol.
74:277-302).
For the production of autibodies, various hosts including goats, rabbits,
rats, mice, camels
dromedaries, llamas, humans, and others may be immunized by injection with
PMMM or with any
fragment or oligopeptide thereof which has immunogenic properties. Depending
on the host species,
various adjuvauts 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
PNnVlM 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 substantially identical to a portion of the amino acid sequence
of the natural protein.
Short stretches of PMMM amino acids may be fused with those of another
protein, such as I~LH, and
antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to PMMM 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 (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al.
(1985) J. Tm_m__unol. Methods
81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030;
Cole, S.P. et al. (1984)
Mol. Cell Biol. 62:109-120).
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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 (Morrison, S.L. et
al. (1984) Proc. Natl. Acad.
Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608;
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 PMMM-specific
single chain antibodies.
Antibodies with related specificity, but of distinct idiotypic composition,
may be generated by chain
shuffling from random combinatorial immunoglobulin libraries (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 (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 P~ 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
(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
PMMM and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering PMMM 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 Ph~VIM. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of PMIVINI-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The Ka
determined for a preparation of polyclonal antibodies, which are heterogeneous
in their affinities for
multiple PMMM epitopes, represents the average affinity, or avidity, of the
antibodies for Pte.
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CA 02460476 2004-03-15
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The Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a particular
PMMM 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 PMIVIIVI-
antibody complex must withstand rigorous manipulations. Low-affinity antibody
preparations with Ka
ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of PNNEVVIM, 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).
1o The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
the quality and suitability of such preparations for certain downstream
applications. For example, a
polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of Pl~~VIM-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available
(Catty, supt-a; Coligan et al.,
supra)
In another embodiment of the invention, polynucleotides encoding PI~~VIM, 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
PMMM. 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
Pl~~VIM (Agrawal, S., ed. (1996) Antisense Therapeutics, Humaua Press, 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 (Slater, J.E. et
al. (1998) J. Allergy Clin. Tmmunol. 102:469-475; Scanlon, K.J. et al. (1995)
9:1288-1296). Antisense
sequences can also be introduced intracellularly through the use of viral
vectors, such as retrovirus and
adeno-associated virus vectors (Miller, A.D. (1990) Blood 76:271; Ausubel et
al., sup~~a; Uckert, W.
and W. Walther (1994) Pharmacol. Ther. 63:323-347). Other gene delivery
mechanisms include
liposome-derived systems, artificial viral envelopes, and other systems known
in the art (Rossi, J.J.
7o
CA 02460476 2004-03-15
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(1995) Br. Med. Bull. 51:217-225; Boado, R.J. et al. (1998) J. Pharm. Sci.
87:1308-1315; Morris,
M.C. et al. (1997) Nucleic Acids Res. 25:2730-2736).
In another embodiment of the invention, polynucleotides encoding PMMM may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D. (1988)
Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA
93:11395-11399), hepatitis
B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Par-
acoccidioides
br-asiliensis; and protozoan parasites such as Plasmodium falcipaf~um and
Tfypanosoma cruzi). In
the case where a genetic deficiency in PMMM expression or regulation causes
disease, the
expression of PM1VII~~I 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
PMMM are treated by constructing mammalian expression vectors encoding PMMM
and introducing
these vectors by mechanical means into PMMM-deficient cells. Mechanical
transfer technologies for
use with cells iyi 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 PMMM include,
but are not
limited to, the PCDNA 3.1, EPTTAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
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and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
PMMM
may be expressed using (i) a constitutively active promoter, (e.g., from
cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40 virus, thymidine kiuase (TK), or (3-actin. genes),
(ii) an inducible promoter
(e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992)
Proc. Natl. Acad. Sci.
USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi,
F.M.V. and H.M. Blau
(1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding PMMM 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 PMMM expression are treated by constructing a retrovirus vector
consisting of (i) the
2o polynucleotide encoding PMIVIn~I 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 No. 5,910,434 to Rigg ("Method
for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retxoviral supernatant") discloses
a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
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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 an embodiment, an adenovirus-based gene therapy delivery system is used to
deliver
polynucleotides encoding PMMM to cells which have one or more genetic
abnormalities with respect
to the expression of PMMM. 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
to be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for
gene therapy"),
hereby incorporated by reference. Fox 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).
In another embodiment, a herpes-based; gene therapy delivery system is used to
deliver
polynucleotides encoding PMMM to target cells which have one or more genetic
abnormalities with
respect to the expression of PMMM. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing PNIn~IM to cells of the central nervous
system, for which HSV has
a tropism. The construction and packaging of herpes-based vectors are well
known to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of
recombinant HSV d92
which consists of a genome containing at least one exogenous gene to be
transferred to a cell under
the control of the appropriate promoter for purposes including human gene
therapy. Also taught by
tlus 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). 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.
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In another embodiment, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding PMMM to target cells. The biology of the
prototypic alphavil-us,
Semlik_i Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and I~.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
PMMM into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
PNIIVIM-coding RNAs and the synthesis of high levels of PMMM 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 PMMM 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 (Gee, J.E. et al. (1994) in Huber, B.E. and
B.I. Carr, Molecular and
T_mmunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177). A
complementary
sequence or antisense molecule may also be designed to block translation of
mRNA by preventing the
transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
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engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of RNA molecules encoding P1~~VIM.
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 may be prepared by any
method
known in the art for the synthesis of nucleic acid molecules. These include
techniques for chemically
synthesizing oligonucleotides such as solid phase phosphoramidite chemical
synthesis. Alternatively,
RNA molecules may be generated by in vitf-o and ire vivo transcription of DNA
molecules encoding
Pn~VIM. Such DNA sequences may be incorporated into a wide variety of vectors
with suitable
RNA polynierase 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.
In other embodiments of the invention, the expression of one or more selected
polynucleotides
of the present invention can be altered, inhibited, decreased, or silenced
using RNA interference
(RNAi) or post-transcriptional gene silencing (PTGS) methods known in the art.
RNAi is a post-
transcriptional mode of gene silencing in which double-stranded RNA (dsRNA)
introduced into a
targeted cell specifically suppresses the expression of the homologous gene
(i.e., the gene bearing the
sequence complementary to the dsRNA). This effectively knocks out or
substantially reduces the
expression of the targeted gene. PTGS can also be accomplished by use of DNA
or DNA fragments
as well. RNAi methods are described by Fire, A. et al. (1998; Nature 391:806-
811) and Gura, T.
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(2000; Nature 404:804-808). PTGS can also be initiated by introduction of a
complementary segment
of DNA into the selected tissue using gene delivery and/or viral vector
delivery methods described
herein or known in the art.
RNAi can be induced in mammalian cells by the use of small interfering RNA
also known as
siRNA. SiRNA are shorter segments of dsRNA (typically about 21 to 23
nucleotides in length) that
result i~. vivo from cleavage of introduced dsRNA by the action of an
endogenous ribonuclease.
SiRNA appear to be the mediators of the RNAi effect in mammals. The most
effective siRNAs
appear to be 21 nucleotide dsRNAs with 2 nucleotide 3' overhangs. The use of
siRNA for inducing
RNAi in mammalian cells is described by Elbashir, S.M. et al. (2001; Nature
411:494-498).
SiRNA can either be generated indirectly by introduction of dsRNA into the
targeted cell, or
directly by mammalian transfection methods and agents described herein or
known in the art (such as
liposome-mediated transfection, viral vector methods, or other polynucleotide
delivery/introductory
methods). Suitable Si_RNAs can be selected by examining a transcript of the
target polynucleotide
(e.g., mRNA) for nucleotide sequences downstream from the AUG start codon and
recording the
occurrence of each nucleotide and the 3' adjacent 19 to 23 nucleotides as
potential siRNA target sites,
with sequences having a 21 nucleotide length being preferred. Regions to be
avoided for target
siRNA sites include the 5' and 3' untranslated regions (U'TRs) and regions
near the start codon (within
75 bases), as these may be richer in regulatory protein binding sites. UTR-
binding proteins andlor
translation initiation complexes may interfere with binding of the siRNP
endonuclease complex. The
selected target sites for siRNA can then be compared to the appropriate genome
database (e.g.,
human, etc.) using BLAST or other sequence comparison algorithms known in the
art. Target
sequences with significant homology to other coding sequences can be
eliminated from consideration.
The selected SiRNAs can be produced by chemical synthesis methods known in the
art or by iii vitro
transcription using commercially available methods and kits such as the
SILENCER siRNA
construction kit (Ambion, Austin TX).
In alternative embodiments, long-term gene silencing and/or RNAi effects can
be induced in
selected tissue using expression vectors that continuously express siRNA. This
can be accomplished
using expression vectors that are engineered to express hairpin RNAs (shRNAs)
using methods
known in the art (see, e.g., Brummelkamp, T.R. et al. (2002) Science 296:550-
553; and Paddison, P.J.
et al. (2002) Genes Dev. 16:948-958). In these and related embodiments, shRNAs
can be delivered to
target cells using expression vectors known in the art. An example of a
suitable expression vector for
delivery of siRNA is the PSILENCER1.0-U6 (circular) plasmid (Ambion). Once
delivered to the
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target tissue, shRNAs are processed in vivo into siRNA-like molecules capable
of carrying out gene-
specific silencing.
In various embodiments, the expression levels of genes targeted by RNAi or
PTGS methods
can be determined by assays for mRNA and/or protein analysis. Expression
levels of the mRNA of a
targeted gene, can be determined by northern analysis methods using, for
example, the
NORTHERNMAX-GLY kit (Ambion); by microarray methods; by PCR methods; by real
time PCR
methods; and by other RNA/polynucleotide assays known in the art or described
herein. Expression
levels of the protein encoded by the targeted gene can be determined by
Western analysis using
standard techniques known in the art.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding PNIIVIM. 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 PMIVI1~~I
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding PMIvITiI may be therapeutically useful, and in the treatment of
disorders associated with
decreased PM1VINI expression or activity, a compound which specifically
promotes expression of the
polynucleotide encoding PMIV~~I may be therapeutically useful.
In various embodiments, one or more 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 PMIVW is exposed to at least one test compound thus
obtained. The sample
3o 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
Pl~~VIM are assayed
by any method commonly known in the art. Typically, the expression of a
specific nucleotide is
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detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
of the polynucleotide encoding PMMM. '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 Schizosacchar-omyces pornbe 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 (Goldman, C.K. et al. (1997)
Nat. Biotechnol..15:462-
466).
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition which
generally comprises an active ingredient formulated with a pharmaceutically
acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and
proteins. Various
formulations are commonly known and are thoroughly discussed in the latest
edition of Remin won's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of PMMM,
antibodies to PMMM, and mimetics, agonists, antagonists, or inhibitors of
PMMM.
In various embodiments, the compositions described herein, such as
pharmaceutical
compositions, may be administered by any number of routes including, but not
limited to, oral,
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intravenous, intramuscular, intra-arterial, intramedullary, intrathecal,
intraventricular, pulmonary,
transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-
acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and
proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the lung
have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton, J.S.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery allows 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 PMT~VI or fragments thereof. For example, liposome
preparations
containi_ug a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, PMMMM 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
2o 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
PI~~VIM or fragments thereof, antibodies of Pn~VIM, and agonists, antagonists
or inhibitors of PM1V~~I,
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
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therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDso
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and response
to therapy. Long-acting compositions may be administered every 3 to 4 days,
every week, or
biweekly depending on the half life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1,ug to 100,000 ,ug, 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 PI~~VIM may be used
for the
diagnosis of disorders characterized by expression of P1~~VIM, or in assays to
monitor patients being
treated with PNI1VIM or agonists, antagonists, or inhibitors of Pn~VIM.
Antibodies useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic
assays for Pinclude methods which utilize the antibody and a label to detect
PM1VIM in human
body fluids or in extracts of cells or tissues. The antibodies may be used
with or without modification,
and may be labeled by covalent or non-covalent attachment of a reporter
molecule. A wide variety of
reporter molecules, several of which are described above, are known in the art
and may be used.
A variety of protocols for measuring PM1VIM, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
PMIVIn~I expression. Normal
or standard values for PNIIVIM expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibodies to PI~MMM
under conditions suitable for complex formation. The amount of standard
complex formation may be
so
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quantitated by various methods, such as photometric means. Quantities of PMMM
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, polynucleotides encoding PMMM may be
used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotides, 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 Pmay be correlated with
disease. The
diagnostic assay may be used to determine absence, presence, and excess
expression of PMMM, and
to monitor regulation of PMMM levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotides,
including genomic sequences, encoding PNnVIM or closely related molecules may
be used to identify
nucleic acid sequences which encode PMMM. The specificity of the probe,
whether it is made from
a highly specific region, e.g., the '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 PNNnVVIM, 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 PMMM encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ m
N0:41-80 or from
genomic sequences including promoters, enhancers, and introns of the PMLVEVI
gene.
Means for producing specific hybridization probes for polynucleotides encoding
P
include the cloning of polynucleotides encoding PMMIVI or PNEVIM 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 vitt~o by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 355,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotides encoding PMMMM may be used for the diagnosis of disorders
associated with
expression of PNEVIM. 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
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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, alphai 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; an endocrine disorder such as a
disorder of the
hypothalamus and/or pituitary resulting from lesions such as a primary brain
tumor, adenoma,
infarction associated with pregnancy, hypophysectomy, aneurysm, vascular
malformation, thrombosis,
infection, imtnunological disorder, and complication due to head trauma; a
disorder associated with
hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus,
Kallman's disease,
Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty
sella syndrome,' and
dwarfism; a disorder associated with hyperpituitarism includilig acromegaly,
giantism, and syndrome of
inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by
benign adenoma; a.
disorder associated with hypothyroidism including goiter, myxedema, acute
thyroiditis associated with
bacterial infection, subacute thyroiditis associated with viral infection,
autoimmune thyroiditis
(Hashimoto's disease), and cretinism; a disorder associated with
hyperthyroidism including
thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema,
toxic multinodular goiter,
thyroid carcinoma, and Plummer's disease; a disorder associated with
hyperparathyroidism including
Conn disease (chronic hypercalemia); a pancreatic disorder such as Type I or
Type II diabetes
mellitus and associated complications; a disorder associated with the adrenals
such as hyperplasia,
carcinoma, or adenoma of the adrenal cortex, hypertension associated with
alkalosis, amyloidosis,
hypokalemia, C~xshing's disease, Liddle's syndrome, and Arnold-Healy-Gordon
syndrome,
pheochromocytoma tumors, and Addison's disease; a disorder associated with
gonadal steroid
hormones such as: in women, abnormal prolactin production, infertility,
endometriosis, perturbation of
the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated
gonadotropin deficiency,
amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast
cancer, and, in post-
menopausal women, osteoporosis; and, in men, Leydig cell deficiency, male
climacteric phase, and
germinal cell aplasia, a hypergonadal disorder associated with Leydig cell
tumors, androgen resistance
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associated with absence of androgen receptors, syndrome of 5 a-reductase, and
gynecomastia; a
disorder of the immune system such as inflammation, actinic keratosis,
acquired immunodeficiency
syndrome (AmS), Addison's disease, adult respiratory distress syndrome,
allergies, ankylosing
spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis,
autoimmune hemolytic
anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis,
cirrhosis, contact dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria,
hepatitis, hypereosinophilia,
irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed
connective tissue
disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation,
myelofibrosis, osteoartluitis, osteoporosis, pancreatitis, polycythemia vera,
polymyositis, psoriasis,
Reiter's syndrome, rheumatoid arrlu7.tis, scleroderma, Sjogren's syndrome,
systemic anaphylaxis,
systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia,
thrombocytopenic
purpura~ ulcerative colitis, uveitis, Werner syndrome, complications of
cancer, hemodialysis, and
extracorporeal circulation, trauma, and hematopoietic cancer including
lymphoma, leukemia, and
myeloma; an infection caused by a viral agent classified as adenovirus,
arenavirus, bunyavirus,
calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus,
orthomyxovirus; parvovirus, .
papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus,
rhabdovirus, or togavirus; an..
infection caused by a bacterial agent classified as pneumococcus,
staphylococcus, streptococcus
bacillus, corynebacterium, clostridium, meningococcus, gonococcus, listeria,
moraxella, kingella,
haemophilus, legionella, bordetella, gram-negative enterobacterium including
shigella, salmonella, or
campylobacter, pseudomonas, vibrio, brucella, francisella, yersinia,
bartonella, norcardium,
actinomyces, mycobacterium, spirochaetale, rickettsia, chlamydia, or
mycoplasma; an infection caused
by a fungal agent classified as aspergillus, blastomyces, dermatophytes,
cryptococcus, coccidioides,
malasezzia, histoplasma, or other mycosis-causing fungal agent; an infection
caused by a parasite
classified as plasmodium or malaria-causing, parasitic entamoeba, leishmania,
trypanosome,
toxoplasma, pneumocystis carinii, intestinal protozoa such as giardia,
trichomonas, tissue nematode
such as trichinella, intestinal nematode such as ascaris, lymphatic filarial
nematode, trematode such as
schistosoma, and cestrode such as tapeworm; a metabolic disorder such as
Addison's disease,
cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin
resistance, cystic fibrosis,
diabetes, fatty hepatocirrhosis, fructose-1,6-diphosphatase deficiency,
galactosemia, goiter,
glucagonoma, glycogen storage diseases, hereditary fructose intolerance,
hyperadrenalism,
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hypoadrenalism, hyperparathyroidisrri, hypoparathyroidism,
hypercholesterolemia, hyperthyroidism,
hypoglycemia, hypothyroidism, hyperlipidemia, hyperlipemia, lipid myopathies,
lipodystrophies,
lysosomal storage diseases, mannosidosis, neuraminidase deficiency, obesity,
pentosuria
phenylketonuria, pseudovitamin D-deficiency rickets; a disorder of
carbohydrate metabolism such as
congenital type II dyserythropoietic anemia, diabetes, insulin-dependent
diabetes mellitus,
non-insulin-dependent diabetes mellitus, fructose-1,6-diphosphatase
deficiency, galactosemia,
glucagonoma, hereditary fructose intolerance, hypoglycemia, mannosidosis,
neuraminidase deficiency,
obesity, galactose epimerase deficiency, glycogen storage diseases, lysosomal
storage diseases,
fructosuria, pentosuria, and inherited abnormalities of pyruvate metabolism; a
disorder of lipid
metabolism such as fatty liver, cholestasis, primary biliary cirrhosis,
carnitine deficiency, carnitine
palinitoyltransferase deficiency, myoadenylate deaminase deficiency,
hypertriglyceridemia,.lipid
storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's
disease, metachromatic
leukodystrophy, adrenoleukodystrophy, GMa gangliosidosis, and ceroid
lipofuscinosis,
abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetes
mellitus, lipodystrophy,
lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa,
lipoid adrenal hyperplasia,
minimal change disease, lipomas, atherosclerosis, hypercholesterolemia,
hypercholesterolemia with
hypertriglyceridemia~ primary hypoalphalipoproteinemia, hypothyroidism, renal
disease, liver disease, ~ .
lecithin:cholesterol acyltransferase deficiency, cerebrotendinous
xanthomatosis, sitosterolemia,
hypocholesterolemia, Tay-Sachs disease, Sandhoff's disease, hyperlipidemia,
hyperlipemia, lipid
myopathies, and obesity; a disorder of copper metabolism such as Menke's
disease, Wilson's disease,
and Ehlers-Danlos syndrome type IX.; 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,
mural annular calcification, mitral valve prolapse, rheumatic fever and
rheumatic heart disease,
infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of
systemic lupus
erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis,
pericarditis, neoplastic heart
disease, congenital heart disease, and complications of cardiac
transplantation; an
autoimmune/inflammatory disease, such as acquired immunodeficiency syndrome
(AIDS), Addison's
disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia,
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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 heltninthic 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
adenocarcindma, 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 (Wilins'
tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-
Magenis syndrome,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas, hereditary
neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,
hypothyroidism,
hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral
palsy, spina bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, age-related
macular degeneration, and
sensorineural hearing loss; an epithelial disorder, such as dyshidrotic
eczema, allergic contact
dermatitis, keratosis pilaris, melasma, vitiligo, actinic keratosis, basal
cell carcinoma, squamous cell
carcinoma, seborrheic keratosis, folliculitis, herpes simplex, herpes zoster,
varicella, candidiasis,
dermatophytosis, scabies, insect bites, cherry angioma, keloid,
dermatofibroma, acrochordons,
urticaria, transient acantholytic dermatosis, xerosis, eczema, atopic
dermatitis, contact dermatitis, hand
eczema, nummular eczema, lichen simplex chronicus, asteatotic eczema, stasis
dermatitis and stasis
ulceration, seborrheic dermatitis, psoriasis, lichen planus, pityriasis rosea,
impetigo, ecthyma,
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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, hgurate 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, priors
diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-
Scheinker syndrome,
fatal familial insomnia, nutritional and metabolic diseases of the nervous
system, neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central nervous system
including Down
syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system
disorders, cranial nerve
disorders, spinal cord diseases, muscular dystrophy and other neuromuscular
disorders, peripheral
nervous system disorders, dermatomyositis and polymyositis, inherited,
metabolic, endocrine, and toxic
myopathies, myasthenia gravis, periodic paralysis, mental disorders including
mood, anxiety, and
schizophrenic disorders, seasonal affective disorder (SAD), akathesia,
amnesia, catatonia, diabetic
neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic
neuralgia, Tourette's
disorder, progressive supranuclear palsy, corticobasal degeneration, and
familial frontotemporal
dementia; and a reproductive disorder, such as infertility, including tubal
disease, ovulatory defects,
and endometriosis, a disorder of prolactin production, a disruption of the
estrous cycle, a disruption of
the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation
syndrome, an endometrial or
ovarian tumor, a uterine fibroid, autoimmune disorders, an ectopic pregnancy,
and teratogenesis;
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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.
Polynucleotides encoding P1~~VIM 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 P1~~VIM expression.
Such qualitative or quantitative methods are well known in the art.
In a particular embodiment, polynucleotides encoding PNIIVIM may be used in
assays that
detect the presence of associated disorders, particularly those mentioned
above. Polynucleotides
complementary to sequences encoding PMIVI1~Z 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
polynucleotides encoding
Pl~~VIM 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
PT~VIM, 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 PNIIV1M, under conditions suitable
for hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from normal
subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
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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 PMMM
may involve the use of PCR. These oligomers may be chemically synthesized,
generated
enzymatically, or produced iti vitro. Oligomers will preferably contain a
fragment of a polynucleotide
encoding PMMM, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
PMMM, 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 polynucleotides
encoding PNRVIM
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 polynucleotides
encoding PMMM are used to amplify DNA using the polymerase chain reaction
(PCR). The DNA
may be derived, for example, from diseased or normal tissue, biopsy samples,
bodily fluids, and the
like. SNPs in the DNA cause differences in the secondary and tertiary
structures of PCR products in
single-stranded form, and these differences are detectable using gel
electrophoresis in non-denaturing
gels. In fSCCP, the oligonucleotide primers are fluorescently labeled, which
allows detection of the
amplimers in high-throughput equipment such as DNA sequencing machines.
Additionally, sequence
database analysis methods, termed in silico SNP (isSNP), are capable of
identifying polymorphisms by
comparing the sequence of individual overlapping DNA fragments which assemble
into a common
consensus sequence. These computer-based methods filter out sequence
variations due to laboratory
preparation of DNA and sequencing errors using statistical models and
automated analyses of DNA
sequence chromatograms. In the alternative, SNPs may be detected and
characterized by mass
spectrometry using, for example, the high throughput MASSARRAY system
(Sequenom, Inc., San
Diego CA).
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SNPs may be used to study the genetic basis of human disease. For example, at
least 16
common SNPs have been associated with non-insulin-dependent diabetes mellitus.
SNPs are also
useful for examining differences in disease outcomes in monogenic disorders,
such as cystic fibrosis,
sickle cell anemia, or chronic granulomatous disease. For example, variants in
the mannose-binding
lectin, MBL2, have been shown to be correlated with deleterious pulmonary
outcomes in cystic
fibrosis. SNPs also have utility in pharmacogenomics, the identification of
genetic variants that
influence a patient's response to a drug, such as life-threatening toxicity.
For example, a variation in
N-acetyl transferase is associated with a high incidence of peripheral
neuropathy in response to the
anti-tuberculosis drug isoniazid, while a variation in the core promoter of
the ALOXS gene results in
diminished clinical response to treatment with an anti-asthma drug that
targets the 5-lipoxygenase
pathway. Analysis of the distribution of SNPs in different populations is
useful for investigating
genetic drift, mutation, recombination, and selection, as well as for tracing
the origins of populations
and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y. and Z. Gu
(1999) Mol. Med. Today 5:53-543; Nowotny, P. et al. (2001) Curr. Opin.
Neurobiol. 11:637-641).
Methods which may also be used to quantify the expression of PMMM include
radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves (Melby, P.C. et al. (1993) J. Tmmunol. 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
polynucleotides described herein may be used as elements on a microarray. The
microarray can be
used in transcript imaging techniques which monitor the relative expression
levels of large numbers of
genes simultaneously as described below. The microarray may also be used to
identify genetic
variants, mutations, and polymorphisms. This information may be used to
determine gene function, to
understand the genetic basis of a disorder, to diagnose a disorder, to monitor
progression/regression of
disease as a function of gene expression, and to develop and monitor the
activities of therapeutic
agents in the treatment of disease. In particular, this information may be
used to develop a
pharmacogenomic profile of a patient in order to select the most appropriate
and effective treatment
regimen for that patient. For example, therapeutic agents which are highly
effective and display the
fewest side effects may be selected for a patient based on his/her
pharmacogenomic profile.
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In another embodiment, PMMM, fragments of PMMMM, or antibodies specific for
PMMM
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 (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S.
Patent No. 5,840,484;
hereby expressly incorporated by reference herein). Thus a transcript image
may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies, ;
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or iii vitro, as in the case of a cell
line.
Transcript images which proftle 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). 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
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matching of signatures which leads to prediction of toxicity (see, for
example, Press Release 00-02
from the National Institute of Environmental Health Sciences, released
February 29, 2000, available at
http://www.niehs.nih.gov/oc/news/toxchip.htm). Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In an embodiment, the toxicity of a test compound can be 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 embodiment relates to the use of the polypeptides disclosed herein to
analyze the
proteome of a tissue or cell type. The term proteome refers to the global
pattern of protein expression
in a particular tissue or cell type. Each protein component of a proteome can
be subjected individually
to further analysis. Proteome expression patterns, or profiles, are analyzed
by quantifying the number
of expressed proteins and their relative abundance under given conditions and
at a given time. A
profile of a cell's proteome may thus be generated by separating and analyzing
the polypeptides of a
particular tissue or cell type. In one embodiment, the separation is achieved
using two-dimensional gel
electrophoresis, in which proteins from a sample are separated by isoelectric
focusing in the first
dimension, and then according to molecular weight by sodium dodecyl sulfate
slab gel electrophoresis
in the second dimension (Steiner and Anderson, supra). The proteins are
visualized in the gel as
discrete and uniquely positioned spots, typically by staining the gel with an
agent such as Coomassie
Blue or silver or fluorescent stains. The optical density of each protein spot
is generally proportional to
the level of the protein in the sample. The optical densities of equivalently
positioned protein spots
from different samples, for example, from biological samples either treated or
untreated with a test
compound or therapeutic agent, are compared to identify any changes in protein
spot density related to
the treatment. The proteins in the spots are partially sequenced using, for
example, standard methods
employing chemical or enzymatic cleavage followed by mass spectrometry. The
identity of the protein
in a spot may be determined by comparing its partial sequence, preferably of
at least 5 contiguous
amino acid residues, to the polypeptide sequences of interest. In some cases,
further sequence data
may be obtained for definitive protein identification.
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A proteomic profile may also be generated using antibodies specific for PMMM
to quantify
the levels of P~ 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 maybe
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological sample:
A difference in the amount of protein between the two samples is indicative of
a toxic response to the
test compound in the treated sample. Individual proteins are identified by
sequencing the amino acid
residues of the individual proteins and comparing these partial sequences to
the polypeptides of the
present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared
with the amount in an untreated biological sample. A difference in the amount
of protein between the
two samples is indicative of a toxic response to the test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the art
(Brennan,
T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc.
Natl. Acad. Sci. USA
93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al. (1995)
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PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl. Aced. Sci.
USA 94:2150-2155;
Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662). Various types of
microarrays are well known
and thoroughly described in Schena, M., Bd. (1999; DNA Microarrays: A
Practical Approach, Oxford
University Press, London).
In another embodiment of the invention, nucleic acid sequences encoding PMMM
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 Boding sequences. For example, conservation of a coding
sequence among
members of a multi-gene family may potentially cause undesired cross
hybridization during
1o 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 (PACs), bacterial artificial chromosomes
(BACs), bacterial P1
constructions; or single chromosome cDNA libraries (Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-
355; Price, C.M. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet.
7:149-154). Once
mapped, the nucleic acid sequences 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) (Lender, E.S. and D. Botstein
(1986) Proc. Natl.
Aced. Sci. USA 83:7353-7357).
Fluorescent i~ situ hybridization (FISH) may be correlated with other physical
and genetic
map data (Heinz-Ulrich, et al. (1995) in Meyers, supr-~c, 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
PMMM on a physical
map and a specific disorder, or a predisposition to a specific disorder, may
help define the region of
DNA associated with that disorder and thus may further positional cloning
efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to 11q22-23, any
sequences mapping to that area may represent associated or regulatory genes
for further investigation
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(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, P1~MMM, 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 Pn~VIM 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 (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 PA~VIM, or fragments
thereof, and washed.
Bound PMMMM is then detected by methods well known in the art. Purified
PNNnVVIM 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 PM1VIT~I specifically compete with a test
compound for binding PNnVIM. -'
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more .
antigenic determinants with PNnVIM.
In additional embodiments, the nucleotide sequences which encode PM1VIM 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 preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below,
including U.S. Ser. No. 60/329,689, U.S. Ser. No. 60/335,703, U.S. Ser. No.
60/348,887, U.S. Ser.
No. 60/334,145, U.S. Ser. No. 60/337,451, and U.S. Ser. No. 60/340,584 are
hereby expressly
incorporated by reference.
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EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA). 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 (Invitrogen), 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 (Invitrogen),
using the
recommended procedures or similar methods known in the art (Ausubel et al.,
supra, ch. 5). Reverse
transcription was initiated using oligo d(T) or random primers. Synthetic
oligonucleotide adapters were
ligated to double stranded cDNA, and the cDNA was digested with the
appropriate restriction enzyme
or enzymes. For most libraries, the cDNA was.size-selected (300-1000 bp) using
SEPHACRYL
S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham
Biosciences) 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 (Invitrogen, Carlsbad CA), PCDNA2.1 plasmid
(Invitrogen), PBI~-
CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid
(Stratagene),
pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pllVCY
(Incyte Genomics),
or derivatives thereof. Recombinant plasmids were transformed into competent
E. coli cells including
XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DH5a, DH10B, or ElectroMAX
DH10B
from Invitrogen.
II. Isolation of cDNA Clones
CA 02460476 2004-03-15
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Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
IIL 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 Biosciences 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 (Amersham
Biosciences);
the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard
ABI protocols and base calling software; or other sequence analysis systems
known in the art.
Reading frames within the cDNA sequences were identified using standard
methods (Ausubel et al.,
supra, ch. 7). Some of the cDNA sequences were selected for extension using
the techniques
disclosed in Example VlIT.
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 prograrxnning, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
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databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Hotno
Sapiens, Rattus norvegicus, Mus rnusculus, Caenor~habditis elegans,
Sacclaarornyces cer~evisiae,
Schizosacchar-ornyces pombe, and Candida albicans (Incyte Genomics, Palo Alto
CA); hidden
Markov model (HMM)-based protein family databases such as PFAM, llVCY, and
TIGRFAM (Haft,
D.H. et al. (2001) Nucleic Acids Res. 29:41-43); and HMM-based protein domain
databases such as
SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864;
Letunic, I. et al. (2002)
Nucleic Acids Res. 30:242-244). (I~VIM 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,
BLIIVVIPS, and
~R. 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 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
2o databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS,
DOMO,
PRODOM, Prosite, hidden Markov model (I~VIM)-based protein family databases
such as PFAM,
INCY, and TIGRFAM; and I3T~IM-based protein domain databases such as SMART.
Full length
polynucleotide sequences are also analyzed using MACDNASIS PRO software
(MiraiBio, Alameda
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,
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where applicable, the scores, probability values, and other parameters used to
evaluate the strength of
a match between two sequences (the higher the score or the lower the
probability value, the greater
the identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide and
polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:41-80. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative protein modification and maintenance molecules 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 (Burge, C. and S. Karlin (1997) J. Mol.
Biol. 268:78-94; 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 protein modification and maintenance
molecules, the
encoded polypeptides were analyzed by querying against PFAM models for protein
modification and,
maintenance molecules. Potential protein modification and maintenance
molecules were also
identified by homology to Incyte cDNA sequences that had been annotated as
protein modification
and maintenance molecules. These selected Genscan-predicted sequences were
then compared by
BLAST analysis to the genpept and gbpri public databases. Where necessary, the
Genscan-predicted
sequences were then edited by comparison to the top BLAST hit from genpept to
correct errors in the
sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis
was also used to
find any Incyte cDNA or public cDNA coverage of the Genscan-predicted
sequences, thus providing
evidence for transcription. When Incyte cDNA coverage was available, this
information was used to
correct or confirm the Genscan predicted sequence. Full length polynucleotide
sequences were
obtained by assembling Genscan-predicted coding sequences with Incyte cDNA
sequences and/or
public cDNA sequences using the assembly process described in Example III.
Alternatively, full
length polynucleotide sequences were derived entirely from edited or unedited
Genscau-predicted
coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
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"Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length'sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for homologous
genomic sequences from the public human genome databases. Partial DNA
sequences were
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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 PMMM Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:41-80 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 N0:41-80 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 Iustitute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances
are based on genetic markers mapped by Genethon which provide boundaries for
radiation hybrid
markers whose sequences were includedin each of the clusters. Human genome
maps and other
resources available to the public, such as the NCBI "GeneMap'99" World Wide
Web site
(http://www.ncbi.nlm.nih.gov/genemapn, can be employed to determine if
previously identified disease
genes map within or in proximity to the intervals indicated above.
Association of PMMM polynucleotides with Parkinson's Disease
Several genes have been identified as showing linkage to autosomal dominant
forms of
Parkinson's Disease (PD). PD is a common neurodegenerative disorder causing
bradykinesia, resting
tremor, muscular rigidity, and postural instability. Cytoplasmic eosinophilic
inclusions called Lewy
bodies, and neuronal loss especially in the substantia nigra pats compacta,
are pathological hallmarks
of PD (Valente, E.M. et al (2001) Am. J. Hum. Genet. 68:895-900). Lewy body
Parkinson disease
has been thought to be a specific autosomal dominant disorder (Wakabayashi, K.
et al. (1998) Acta
Neuropath. 96:207-210). Juvenile parkinsonism may be a specific autosomal
recessive disorder
(Matsumine, H. et al. (1997) Am. J. Hum. Genet. 60: 588-596, 1997). (Online
Mendelian Inheritance
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in Man, OMIM. Johns Hopkins University, Baltimore, MD. MIM Number: 168600:
Sept. 9, 2002: .
World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/)
Association of a disease with a chromosomal locus can be determined by lod
score. Lod
score is a statistical method used to test the linkage of two or more loci
within families having a
genetic disease. The lod score is the logarithm to base 10 of the odds in
favor of linkage. Linkage is
defined as the tendency of two genes located on the same chromosome to be
inherited together
through meiosis (Gefaetics in Medicine, Fifth Edition, (1991) Thompson, M.W.
et al., W.B. Saunders
Co. Philadelphia). A lod score of +3 or greater (1000:1 odds in favor of
linkage) indicates a
probability of 1 in 1000 that a particular marker was found solely by chance
in affected individuals,
which is strong evidence that two genetic loci are linked.
One such gene implicated in PD is PARK3, which maps to 2p13 (Gasser, T. et al.
(1998)
Nature Genet. 18:262-265). A marker at chromosomal position D2S441 was found
to have a lod
score of 3.2 in the region of PARK3. This marker supported the disease
association of PARK3 in
the chromosomal interval from D2S 134 to D2S286 (Gasser et al., supra).
Markers located within
chromosomal intervals D2S134 and D2S286, which map.between 83.88 to 94.05
centiMorgans on the,
short arm of chromosome 2, were used to identify genes that map in the region
between D2S 134 and
D2S286.
PMMM polynucleotides were found to map within the chromosomal region in which
markers'
associated with disease or other physiological processes of interest were
located. Genomic contigs
available from NCBI were used to identidy PMMM polynucleotides which map to a
disease locus.
Contigs longer than 1Mb were broken into subcontigs of 1Mb in length with
overlapping sections of
100 kb. A preliminary step used an algorithm, similar to MEGABLAST (NCBI), to
identify mRNA
sequence/masked genomic DNA contig pairings. SIM4 (Florea, L. et al. (1998)
Genome Res. 8:967-
74, version May 2000 was optimized for high throughput and strand assignment
confidence, and used
to further select cDNA/genomic pairings. The SIM4-selected mRNA
sequence/genomic contig pairs
were further processed to determine the correct location of the P1~~VIM
polynucleotides on the
genomic contig and their strand identity.
SEQ >D NO:43 mapped to GBI:NT_005428_001.7 from the February 2002 Genbank
release
covering a 9.65 Mb region of the genome that also contains PD-associated
genetic markers D2S134
and D2S286. The maximum distance between SEQ ID NO:43 and markers D2S134 and
D2S286,
therefore, is 9.65 Mb. Thus, SEQ ID NO:43 is in proximity with genetic markers
shown to
consistently associate with PD. In various embodiments, SEQ )D N0:43 can be
used for one or more
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of the following: i) linkage analysis of persons andlor families to the PD
disease region at 2q12-q22, ii)
diagnostic assays for osteoarthritis and interleukin expression abnormalities,
and iii) developing
therapeutics andlor other treatments for PD.
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 (Sambrook and Russell,
supra, ch. 7; Ausubel et
al., supra, ch. 4).
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in databases such as GenBank or LIFESBQ (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 f length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences 'and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is .
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotides encoding PMMM are analyzed with respect to the
tissue
sources from which they were derived. For example, some full length sequences
are assembled, at
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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; hernic 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 PMMM. cDNA sequences and cDNA library/tissue
information are
found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of PMMM Encoding Polynucleotides
Full length polynucleotides are 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 dimeri2ations 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+, (NH~)ZSO4,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE
enzyme
(Invitrogen), 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:
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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 ~,l
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~,1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 /.d to 10 ~1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Biosciences). 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
Biosciences), 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 Biosciences) 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, vlv), and sequenced using DYENAMIC energy transfer
sequencing primers
and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotides are verified using the above
procedure or are used
to obtain 5' regulatory sequences using the above procedure along with
oligonucleotides designed for
such extension, and an appropriate genomic library.
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IX. Identification of Single Nucleotide Polymorphisms in PMMM Encoding
Polynucleotides
Common DNA sequence variants known as single nucleotide polymorphisms (SNPs)
were
identified in SEQ ID N0:41-80 using the L1FESEQ database (Iucyte Genomics).
Sequences from the
same gene were clustered together and assembled as described in Example III,
allowing the
identification of all sequence variants in the gene. An algorithm consisting
of a series of filters was
used to distinguish SNPs from other sequence variants. Preliminary filters
removed the majority of
basecall errors by requiring a minimum Phred quality score of 15, and removed
sequence alignment
errors and errors resulting from improper trimming of vector sequences,
chimeras, and splice variants.
An automated procedure of advanced chromosome analysis analysed the original
chromatogram files
in the vicinity of the putative SNP. Clone error filters used statistically
generated algorithms to identify
errors introduced during laboratory processing, such as those caused by
reverse transcriptase,
polymerase, or somatic mutation. Clustering error filters used statistically
generated algorithms to
identify errors resulting from clustering of close homologs or pseudogenes, or
due to contamination by
non-human sequences. A final set of filters removed duplicates and SNPs found
in immunoglobulins
or T-cell receptors.
Certain SNPs were selected for further characterization by mass spectrometry
using the high
throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at
the SNP sites in
four different human populations. The Caucasian population comprised 92
individuals (46 male, 46
female), including 83 from Utah, four French, three Venezualan, and two Amish
individuals. The
African population comprised 194 individuals (97 male, 97 female), all African
Americans. The
Hispanic population comprised 324 individuals (162 male, 162 female), all
Mexican Hispanic. The
Asian population comprised 126 individuals (64 male, 62 female) with a
reported parental breakdown
of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
Allele
frequencies were first analyzed in the Caucasian population; in some cases
those SNPs which showed
no allelic variance in this population were not further tested in the other
three populations.
X. Labeling and LTse of Individual Hybridization Probes
Hybridization probes derived from SEQ ID NO:41-80 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ,uCi of
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~~ 32P, adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide
kinase (DuPont NEN,
BostoJn MA). The labeled oligonucleotides are substantially purified using a
SEPHADEX G-25
superfine size exclusion dextran bead column (Amersham Biosciences). An
aliquot containing 10'
counts per minute of the labeled probe is used in a typical membrane-based
hybridization analysis of
human genomic DNA digested with one of the following endonucleases: Ase I, Bgl
II, Eco RI, Pst I,
Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40 °C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
XI. 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 et al., supra),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena, M., ed.
(1999) DNA Microarrays: A Practical Apt~roach, Oxford University Press,
London). Suggested
substrates include silicon, silica, glass sfides, glass chips, and silicon
wafers. Alternatively, a procedure
analogous to a dot or slot blot may also be used to arrange and link elements
to the surface of a
substrate using thermal, UV, chemical, or mechanical bonding procedures. A
typical array may be
produced using available methods and machines well known to those of ordinary
skill in the art and
may contain any appropriate number of elements (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. Biotechuol.
16:27-31 ).
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
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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 units/~.1 RNase inhibitor, 500 ACM dATP, 500 ~.M dGTP, 500
~,M dTTP, 40 ixM
dCTP, 40 ~,M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse
transcription .
reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with
GEMBRIGHT kits
(Incyte Genomics). Specific control poly(A)+ RNAs are synthesized by in vitf-o
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 CHRQMA SPIN 30 gel filtration spin columns (Clontech, Palo Alto CA)
and after
combining, both reaction samples are ethanol precipitated using 1 inl 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 ~,l 5X
SSC/0.2% SDS.
2o 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 ampliftcation uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5 ~,g.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Biosciences).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma). in 95% ethanol. Coated slides
are cured in a 110°C
oven.
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Array elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~.1 of the array
element DNA, at an average
concentration of 100 ng/~,1, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2%
SDS and distilled water as before.
1o Hybridization
Hybridization reactions contain 9 ~Cl of sample mixture consisting of 0.2 p.g
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cma 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
.. ~1 of 5X SSC in a corner of the chamber. The chamber containing the arrays
is incubated for about
6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in
a first wash buffer (1X SSC, 0.1 %
SDS), three times for 10 minutes each at 45° C in a second wash buffer
(0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Iunova 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 mn 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
fiuorophores 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 fiuorophores used are 565 nn1 for Cy3 and 650 nm for
CyS. Eaeh array is
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typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that location
to be correlated with a weight ratio of hybridizing species of 1:100,000. When
two samples from
different sources (e.g., representing test and control cells), each labeled
with a different fluorophore,
are hybridized to a single array for the purpose of identifying genes that are
differentially expressed,
the calibration is done by labeling samples of the calibrating cDNA with the
two fluorophores and
1o 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
Genomics). Array
elements that exhibit at least about a two-fold change in expression, a signal-
to-background ratio of at
least about 2.5, and an element spot size of at least about 40%, are
considered to be differentially
expressed.
Expression
For example, SEQ ll~ N0:41, SEQ D? NO:55, and SEQ ID NO:57 showed differential
expression in association with breast cancer, as determined by microarray
analysis. The expression of
SEQ LD N0:41 was decreased by at least 2.8-fold in breast tumor when matched
with normal tissue
from the same donor. The tumorous breast tissue was obtained from tumor from
the right breast of a
43-year-old female with invasive lobular carcinoma in situ. The tumor was well
differentiated and
metastatic to two out of 13 lymph nodes. Normal tissue was obtained from
grossly uninvolved breast
tissue from the same donor. Matched normal and tumorigenic breast tissue
samples were provided by
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the Huntsman Cancer Institute (Salt Lake City, ITT). In addition, the
expression of SEQ ID N0:55 in
several tumor cell lines representing various stages of breast tumor
progression was compared with
that in the non-malignant mammary epithelial cell line, MCF-10A. The
expression of SEQ )D N0:55
from six tumor cell lines (MCF7, T-47D, Sk-BR-3, BT20, MDA-mb-231, and MDA-mb-
435S) was
compared with that in MCF-10A cells grown in the supplier's recommended medium
or grown in
defined serum-free H14 medium to 70-80°~o confluence prior to
comparison. MCF-10A is a breast
mammary gland (luminal ductal characteristics) cell line that was isolated
from a 36-year-old woman
with fibrocystic breast disease. MCF-10A expresses cytoplasmic keratins,
epithelial sialomucins, and
milkfat globule antigens. This cell line exhibits three-dimensional growth in
collagen and forms domes
in confluent culture. MCF7 is a nonmalignant breast adenocarcinoma cell line
isolated from the pleural
effusion of a 69-year-old female. MCF7 has retained characteristics of the
mammary epithelium such
as the ability to process estradiol via cytoplasmic estrogen receptors and the
capacity to form domes in
culture. T-47D is a breast carcinoma cell line isolated from a pleural
effusion obtained from a 54-
year-old female with an infiltrating ductal carcinoma of the breast. Sk-BR-3
is a breast
adenocarcinoma cell line isolated from a malignant pleural effusion. of a 43-
year-old female. It forms
poorly differentiated adenocarcinoma when injected into nude mice. BT-20 is a
breast carcinoma cell
line derived in vitro from cells emigrating out of thin slices of the tumor
mass isolated from a 74-
year-old female. MDA-mb-231 is a breast tumor cell line isolated from the
pleural effusion of a 51-
year old female. It forms poorly differentiated adenocarcinoma in nude mice
and ALS treated
BALB/c mice. It also expresses the Wnt3 oncogene, EGF, and TGF-a. MDA-mb-435S
is a spindle
shaped strain that evolved from the parent line (435) as isolated in 1976 by
R. Cailleau from the
pleural effusion of a 31-year-old female with metastatic, ductal
adenocarcinoma of the breast. The
expression of SEQ ID N0:55 was increased by at least two-fold in T-47D and BT-
20 cells as
compared to the nonmalignant MCF-10A cells. In addition, the expression of SEQ
ID NO:57 in the
six breast cell tumor lines described above was compared to that in a primary
breast epithelial cell line
derived from a normal donor, HMEC. The expression of SEQ ll~ N0:57 was
increased by at least
two-fold in MCF7, T-47D, Sk-BR-3, BT-20, and MDA-mb-435S cells as compared to
HMEC cells.
Therefore, in various embodiments, SEQ ID N0:41, SEQ ID NO:55, and SEQ ID
N0:57 can be used
for one or more of the following: i) monitoring treatment of breast cancer,
ii) diagnostic assays for
breast cancer, and iii) developing therapeutics and/or other treatments for
breast cancer.
In another example, SEQ D7 N0:41-42, SEQ ID N0:46, SEQ ID N0:49 and SEQ ID
NO:78
showed differential expression in association with lung cancer, as determined
by microarray analysis.
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The expression of SEQ m N0:41 was decreased at least 2.4-fold in one of five
lung squamous cell
carcinoma when matched with normal tissue from the same donor. The tumorous
lung tissue was
obtained from lung squamous cells of a 66-year-old male. Normal tissue was
obtained from grossly
uninvolved lung tissue from the same donor. Matched normal and tumorigenic
lung tissue samples
were provided by the Roy Castle Lung Cancer Foundation (Liverpool, UK). In
addition, the
expression of SEQ ~ N0:42 was increased at least 2.9-fold in one of four lung
adenocarcinoma
when matched with normal tissue from the same donor. The expression of SEQ D7
N0:42 also was
increased at least two-fold in one of five lung squamous cell carcinoma when
matched with normal
tissue from the same donor. The tumorous lung tissue was obtained from lung
squamous cells of a 73-
year-old male. Normal tissue was obtained from grossly uninvolved lung tissue
from the same donor.
In a separate matched tissue experiment, the expression of SEQ ID N0:46 was
decreased by at least
two-fold in lung squamous cell carcinoma tissue, comprising 60% overt tumor
cells. In addition, the
expression of SEQ m N0:49 was decreased by at least two-fold in the lung tumor
tissue as
compared to normal lung tissue from the same donor. -In a separate matched
tissue experiment, the
expression of SEQ ID N0:78 was increased' more than two-fold in lung squamous
cell carcinoma
tissue, with 70% overt tumor cells, from a 75-year-old female, as compared to
grossly uninvolved
tissue from the same donor. Therefore, in various embodiments, SEQ ID N0:41-
42, SEQ D7 NO:46,
SEQ ID N0:49 and SEQ D7 N0:78 can be used for one or more of the following: i)
monitoring
treatment of lung cancer, ii) diagnostic assays for lung cancer, and iii)
developing therapeutics and/or
other treatments for lung cancer.
In a further example, SEQ ID NO: 41 showed differential expression in
association with
obesity, as determined by microarray analysis. The expression of SEQ m N0:41
was decreased at
least 2.3-fold, to as much as 11-fold, in treated human adipocytes from obese
and normal donors when
compared to non-treated adipocytes from the same donors. The normal human
primary subcutaneous
preadipocytes were isolated from adipose tissue of a 28-year-old healthy
female with a body mass
index (BMI) of 23.59. The obese human primary subcutaneous preadipocytes were
isolated from
adipose tissue of a 40-year-old healthy female with a body mass index (BMI) of
32.47. The
preadipocytes were cultured and induced to differentiate into adipocytes by
culturing them in the
differentiation medium containing the active components, PPAR-'y agonist and
human insulin. Human
preadipocytes were treated with human insulin and PPAR-'y agonist for three
days and subsequently
were switched to medium containing insulin for 24 hours, 48 hours, four days,
1.1, 2.1, or 2.6 weeks
before the cells were collected for analysis. Differentiated adipocytes were
compared to untreated
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preadipocytes maintained in culture in the absence of inducing agents. Between
80% and 90% of the
preadipocytes finally differentiated to adipocytes as observed under phase
contrast microscope.
In the alternative, obese human primary subcutaneous preadipocytes were
isolated from
adipose tissue of a 36-year-old healthy female with a body mass index (BMI) of
27.7. The
preadipocytes were cultured and induced to differentiate into adipocytes by
culturing them in a
proprietary differentiation medium containing active components such as PPAR-y
agonist and human
insulin. Human preadipocytes were treated with human insulin and PPAP-'y
agonist for 3 days and
subsequently switched to medium containing insulin only for 5, 9, and 12 more
days. Differentiated
adipocytes were compared to untreated preadipocytes maintained in culture in
the absence of inducing
agents. An overall differentiation rate of more than 60% was observed after 15
days in culture.
Therefore, in various embodiments, SEQ ID N0:41 can be used for one or more of
the following: i)
monitoring treatment of diabetes mellitus and other disorders, such as
obesity, hypertension, and
atherosclerosis, ii) diagnostic assays for diabetes mellitus and other
disorders, such as obesity,
hypertension, and atherosclerosis, and iii) developing therapeutics and/or
other treatments for diabetes
mellitus and other disorders, such as obesity, hypertension, and
atherosclerosis.
Matched normal and obese preadipocyte samples were provided by the (Zen-Bio,
Research
Triangle Park NC).
In an alternative example, SEQ m NO:63 showed differential expression in
association with
colon cancer, as determined by microarray analysis. The expression of SEQ ID
N0:63 was
decreased by at least two-fold in sigmoid colon tumor tissue when matched with
normal tissue from
the same donor. Tumorous tissue was obtained from a 48-year-old female with
sigmoid colon tumor
originating from a metastatic gastric sarcoma (stromal tumor). Normal tissue
was obtained from
grossly uninvolved sigmoid colon tissue from the same donor. Matched normal
and tumorigenic
sigmoid colon tissue samples are provided by the Huntsman Cancer Institute,
(Salt Lake City, UT).
Therefore, in various embodiments, SEQ ID N0:63 can be used for one or more of
the following: i)
monitoring treatment of colon cancer, ii) diagnostic assays for colon cancer,
and iii) developing
therapeutics and/or other treatments for colon cancer.
In another example, SEQ ID NO:78 showed differential expression in association
with ovarian
cancer, as determined by microarray analysis. The expression of SEQ ID N0:78
was increased by at
least 2-fold in ovarian tumor tissue as compared to normal tissue from the
same donor, a 79-year-old
female donor with ovarian adenocarcinoma. Matched normal and tumorigenic
ovarian tissue samples
were provided by the Huntsman Cancer Institute, (Salt Lake City, UT).
Therefore, in various
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embodiments, SEQ ID N0:78 can be used for one or more of the following: i)
monitoring treatment of
ovarian cancer, ii) diagnostic assays for ovarian cancer, and iii) developing
therapeutics and/or other
treatments for ovarian cancer.
XII. Complementary Polynucleotides
Sequences complementary to the PNNIIVVIM-encoding sequences, or any parts
thereof, are used
to detect, decrease, or inhibit expression of naturally occurring PN>ZVIM.
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 PIV>ZVIM.
To inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5' sequence
and used to prevent promoter binding to the coding sequence. To inhibit
translation, a complementary
oligonucleotide is designed to prevent ribosomal binding to the PM1V>IVI-
encoding transcript.
XIII. Expression of PMMM
Expression and purification of PNBVIM is achieved using bacterial or virus-
based expression
systems. For expression of PMIVIA~I 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-hac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express PM1V>NI upon induction with isopropyl
beta-D-
thiogalactopyranoside (IPTG). Expression of PA~VIM in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autogr-aphica cal iforwica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding Pl~~VIM 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 Spoeloptera frugiper-da (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus (Engelhard, E.K. et al.
(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.
Gene Ther. 7:1937-
1945).
In most expression systems, PI~~llVIM 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,
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affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Schistosorna japonicum, enables the purification of fusion
proteins on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Biosciences).
Following purification, the GST moiety can be proteolytically cleaved from
PMIVhVI at specifically
engineered sites. FLAG, an 8-amino acid peptide, enables immunoaf~nity
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 et
al. (supra, ch. 10 and 16).
Purified PMMM obtained by these methods can be used directly in the assays
shown in Examples
XVII, XVIJI, XIX, and XX, where applicable.
XIV. Functional Assays
PMMM function is assessed by expressing the sequences encoding PMMM 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 plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid
(Invitrogen), 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 ~g of an additional plasmid
containing sequences
encoding a marker protein are co-transfected. Expression. of a marker protein
provides a means to
distinguish transfected cells from nontransfected cells and, is a reliable
predictor of cDNA expression
from the recombinant vector. Marker proteins of choice include, e.g., Green
Fluorescent Protein
(GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser
optics based technique, is used to identify transfected cells expressing GFP
or CD64-GFP and to
evaluate the apoptotic state of the cells and other cellular properties. FCM
detects and quantifies the
uptake of fluorescent molecules that diagnose events preceding or coincident
with cell death. These
events include changes in nuclear DNA content as measured by staining of DNA
with propidium
iodide; changes in cell size and granularity as measured by forward light
scatter and 90 degree side
light scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine
uptake; alterations in expression of cell surface and intracellular proteins
as measured by reactivity
with specific antibodies; and alterations in plasma membrane composition as
measured by the binding
of fluorescein-conjugated Annexin V protein to the cell surface. Methods in
flow cytometry are
discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York NY).
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The influence of PNIIVIM on gene expression can be assessed using highly
purified populations
of cells transfected with sequences encoding PMMM 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 PMMM and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XV. Production of PMMM Specific Antibodies
PNNIIVVIM substantially purified using polyacrylamide gel electrophoresis
(PAGE; see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using
standard protocols.
Alternatively, the PMMM 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 (Ausubel et al., supra, ch. 11).
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with
the oligopeptide-KLH
complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-PNIIVIM
activity by, for example, binding the peptide or PMMM to a substrate, blocking
with 1 % BSA, reacting
with rabbit antisera, washing, and reacting with radio-iodinated goat anti-
rabbit IgG.
XVI. Purification of Naturally Occurring PMMM Using Specific Antibodies
Naturally occurring or recombinant PMMM is substantially purified by
immunoaffinity
chromatography using antibodies specific for PNIMM. An immunoaffinity column
is constructed by
covalently coupling anti-PI\~VIM antibody to an activated chromatographic
resin, such as
CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin
is blocked and
washed according to the manufacturer's instructions.
Media containing PMMMM are passed over the imrnunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of PMMM (e.g.,
high ionic strength
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buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/PMMM 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 PMMM is collected.
XVII. Identification of Molecules Which Interact with PMMM
PMMM, or biologically active fragments thereof, are labeled with 1'~I Bolton-
Hunter reagent
(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
PMMM, washed, and any
wells with labeled PMMM complex are assayed. Data obtained using different
concentrations of
PMMM are used to calculate values for the number, affinity, and association of
PMMM with the
candidate molecules.
Alternatively, molecules interacting with PMMM 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).
PMMM 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).
XVIII. Demonstration of PMMM Activity
PMMM activity can be demonstrated using a generic immunoblotting strategy or
through a
variety of specific activity assays, some of which are outlined below. As a
general approach, cell lines
or tissues transformed with a vector containing PMMM coding sequences can be
assayed for PNNnVVIM
activity by immunoblotting. Transformed cells are denatured in SDS in the
presence of b-
mercaptoethanol, nucleic acids are removed by ethanol precipitation, and
proteins are purified by
acetone precipitation. Pellets are resuspended in 20 mM Tris buffer at pH 7.5
and incubated with
Protein G-Sepharose pre-coated with an antibody specific for PM1VINI. After
washing, the Sepharose
beads are boiled in electrophoresis sample buffer, and the eluted proteins
subjected to SDS-PAGE.
The SDS-PAGE is transferred to a membrane for immunoblotting, and the PMMM
activity is
assessed by visualizing and quantifying bands on the blot using the antibody
specific for P1~RVIM as the
primary antibody and 1~I-labeled IgG specific for the primary antibody as the
secondary antibody.
PMMM kinase activity is measured by quantifying the phosphorylation of a
protein substrate
by PMMM in the presence of gamma-labeled 3aP-ATP. PMIVINI is incubated with
the protein
substrate, 32P-ATP, and an appropriate kinase buffer. The 32P incorporated
into the substrate is
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separated from free 32P-ATP by electrophoresis and the incorporated 32P is
counted using a
radioisotope counter. The amount of incorporated 3aP is proportional to the
activity of PMMM. A
determination of the specific amino acid residue phosphorylated is made by
phosphoamino acid
analysis of the hydrolyzed protein.
PMMM phosphatase activity is measured by the hydrolysis of p-nitrophenyl
phosphate
(PNPP). PMMM is incubated together with PNPP in HEPES buffer, pH 7.5, in the
presence of
0.1 % (i-mercaptoethanol at 37 °C for 60 min. The reaction is stopped
by the addition of 6 ml of 10 N
NaOH and the increase in light absorbance at 410 nm resulting from the
hydrolysis of PNPP is
measured using a spectrophotometer. The increase in light absorbance is
proportional to the activity of
PMMM in the assay (Diamond, R.H. et al. (1994) Mol. Cell. Biol. 14:3752-3762).
In the alternative, PMMM phosphatase activity is determined by measuring the
amount of
phosphate removed from a phosphorylated protein substrate. Reactions are
performed. with 2 or 4 nM
enzyme in a final volume of 30 ~.1 containing 60 mM Tris, pH 7.6, 1 mM EDTA, 1
mM EGTA, 0.1 %
2-mercaptoethanol and 10 ~,M substrate, 32P-labeled on serine/threonine or
tyrosine, as appropriate.
Reactions are initiated with substrate and incubated at 30° C for 10-15
min. Reactions are quenched
with 450 p,1 of 4% (w/v) activated charcoal in 0.6 M HCl, 90 mM Na4P20~, and 2
mM NaH2P04, then
centrifuged at 12,000 x g for 5 min. Acid-soluble 32Pi is quantified by liquid
scintillation counting
(Sinclair, C. et al. (1999) J. Biol. Chem. 274:23666-23672).
PMMM protease activity is measured by the hydrolysis of appropriate synthetic
peptide
substrates conjugated with various chromogenic molecules in which the degree
of hydrolysis is
quantified by spectrophotometric (or fluorometric) absorption of the released
chromophore (Beynon,
R.J. and J.S. Bond (1994) Proteol 'c Enzymes: A Practical Approach, Oxford
University Press, New
York, NY, pp. 25-55). Peptide substrates are designed according to the
category of protease activity
as endopeptidase (serine, cysteine, aspartic proteases, or metalloproteases),
aminopeptidase (leucine
aminopeptidase), or carboxypeptidase (carboxypeptidases A and B, procollagen C-
proteinase).
Commonly used chromogens are 2-naphthylamine, 4-nitroaniline, and furylacrylic
acid. Assays are
performed at ambient temperature and contain an aliquot of the enzyme and the
appropriate substrate
in a suitable buffer. Reactions are carried out in an optical cuvette, and the
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.
In the alternative, an assay for PMMM protease activity takes advantage of
fluorescence
resonance energy transfer (FRET) that occurs when one donor and one acceptor
fluorophore with an
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appropriate spectral overlap are in close proximity. A flexible peptide linker
containing a cleavage site
specific for PMMM 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 BFP5 to RSGFP4. When the fusion protein is incubated with PMMM,
the substrate is
cleaved, and the two fluorescent proteins dissociate. This is accompanied by a
marked decrease in
energy transfer which is quantified by compering the emission spectra before
and after the addition of
PI~~VIM (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 PMMM is
introduced on an inducible vector so that FRET can be monitored in the
presence and absence of
l0 PM)VIM (Sagot, I. et al (1999) FEBS Letters 447:53-57).
An assay for ubiquitin hydrolase activity measures the hydrolysis of a
ubiquitin precursor. The
assay is performed at ambient temperature and contains an aliquot of Pn~VIM
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, I~. et al. (1997) Anal. Biochem. 247:305-309).
PMMM protease inhibitor activity for alpha 2-HS-glycoprotein (AHSG) can be
measured as a
decrease in osteogenic activity in dexamethasone-treated rat bone marrow cell
cultures (dex-RBMC). .
Assays are carried out in 96-well culture plates containing minimal essential
medium supplemented
with 15°7o fetal bovine serum, ascorbic acid (50 mg/ml), antibiotics
(100 mg/ml penicillin G, 50 mg/ml
gentamicin, 0.3 mg/ml fungjzone), 10 mM B-glycerophosphate, dexamethasone (10-
8 M) and various
concentrations of PMMM for 12-14 days. Mineralized tissue formation in the
cultures is quantified by
measuring the absorbance at 525 rim using a 96-well plate reader (Binkert, C.
et al. (1999) J. Biol.
Chem. 274:28514-28520}.
PMMM protease inhibitor activity for inter-alpha-trypsin inhibitor (ITI) can
be measured by a
continuous spectrophotometric rate determination of trypsin activity. The
assay is performed at
ambient temperature in a quartz cuvette in pH 7.6 assay buffer containing 63
mM sodium phosphate,
0.23 mM N a-benzoyle-L-arginine ethyl ester, 0.06 mM hydrochloric acid, 100
units trypsin, and
various concentrations of Pte. Tmmediately after mixing by inversion, the
increase in Aa53 "", is
recorded for approximately 5 minutes and the enzyme activity is calculated
(Bergmeyer, H.U. et al.
(1974) Meth. Enzym. Anal. 1:515-516).
Pl~~VIM isomerase activity such as peptidyl prolyl cisltr-atvs isomerase
activity can be assayed
by au enzyme assay described by Rahfeld, J.U., et al. (1994; FEBS Lett.
352:180-184). The assay is
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performed at 10°C in 35 mM HEPES buffer, pH 7.8, containing
chymotrypsin (0.5 mg/ml) and
PMMM at a variety of concentrations. Under these assay conditions, the
substrate, Suc-Ala-Xaa-
Pro-Phe-4-NA, is in equilibrium with respect to the prolyl bond, with 80-95%
in tr~ans and 5-20% in
cis conformation. An aliquot (2 ml) of the substrate dissolved in dimethyl
sulfoxide (10 mg/ml) is
added to the reaction mixture described above. Only the cis isomer of the
substrate is a substrate for
cleavage by chymotrypsin. Thus, as the substrate is isomerized by PMMM, the
product is cleaved by
chymotrypsin to produce 4-nitroanilide, which is detected by it's absorbance
at 390 rim. 4-nitroanilide
appears in a time-dependent and a PMMM concentration-dependent manner.
PMMM galactosyltransferase activity can be determined by measuring the
trausfer of
to radiolabeled galactose from UDP-galactose to a GlcNAc-terminated
oligosaccharide chain
(Kolbinger, F. et al. (1998) J. Biol. Chem. 273:58-65). The sample is
incubated with 14 ~.l of assay
stock solution (180 mM sodium cacodylate, pH 6.5, 1 mg/ml bovine serum
albumin, 0.26 mM UDP-
galactose, 2 ~.l of UDP-[3H]galactose), 1 ~,1 of MnCl2 (500 mM), and 2.5 p1 of
GlcNAc(3O-(CHz)$
COZMe (37 mg/ml in dimethyl sulfoxide) for 60 minutes at 37 °C. The
reaction is quenched by the
addition of 1 ml of water and loaded on a C18 Sep-Pak cartridge (Waters), and
the column is washed
twice with 5 ml of water to remove unreacted UDP-['H]galactose. The
[3H]galactosylated
GlcNAc(30-(CHZ)8 COzMe remains bound to the column during the water washes and
is eluted with 5
ml of methanol. Radioactivity in the eluted material is measured by liquid
scintillation counting and is
proportional to galactosyltxansferase activity in the starting sample.
PMLVBVI induction by heat or toxins may be demonstrated using primary cultures
of human
fibroblasts or human cell lines such as CCL-13, HEK293, or HEP G2 (ATCC). To
heat induce
PMMM expression, aliquots of cells are incubated at 42°C for 15, 30, or
60 minutes. Control aliquots
are incubated at 37°C for the same time periods. To induce PMMM
expression by toxins, aliquots of
cells are treated with 100 ~.M arsenite or 20 mM azetidine-2-carboxylic acid
for 0, 3, 6, or 12 hours.
After exposure to heat, arsenite, or the amino acid analogue, samples of the
treated cells are
harvested and cell lysates prepared for analysis by western blot. Cells are
lysed in lysis buffer
containing 1% Nonidet P-40, 0.15 M NaCl, 50 mM Tris-HCl, 5 mM EDTA, 2 mM N-
ethylmaleimide,
2 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, and 1 mg/ml pepstatin.
Twenty micrograms of
the cell lysate is separated on an 8% SDS-PAGE gel and transferred to a
membrane. After blocking
with 5% nonfat dry milk/phosphate buffered saline for 1 h, the membrane is
incubated overnight at
4°C or at room temperature for 2-4 hours with an appropriate dilution
of anti-PMMM serum in 2%
nonfat dry milk/phosphate-buffered saline. The membrane is then washed and
incubated with a
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1:1000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG in
2~/o dry
milk/phosphate-buffered saline. After washing with 0.1% Tween 20 in phosphate-
buffered saline, the
PMMM protein is detected and compared to controls using chemiluminescence.
PNIIVIM lysyl hydroxylase activity is determined by measuring the production
of
hydroxy[14C]lysine from [14C]lysine. Radiolabeled protocollagen is incubated
with PMMM in buffer
containing ascorbic acid, iron sulfate, dithiothreitol, bovine serum albumin,
and catalase. Following a
30 minute incubation, the reaction is stopped by the addition of acetone, and
centrifuged. The
sedimented material is dried, and the hydroxy[14C]lysine is converted to
[14C]formaldehyde by
oxidation with periodate, and then extracted into toluene. The amount of 14C
extracted into toluene is
quantified by scintillation counting, and is proportional to the activity of
PMMM in the sample
(Kivirikko, K., and R. Myllyla (1982) Methods Enzymol. 82:245-304).
XIX. Identification of PMMM Substrates
Phage display libraries can be used to identify optimal substrate sequences
for PMIVIIbI. 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 PMMM under
proteolytic conditions so that the epitope will be removed if the hexamer
codes for a PMMM cleavage
site. An antibody that recognizes the epitope is added along with immobilized
protein A. TJncleaved
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 X~, and an optimal cleavage sequence can be derived (Ke, S.H. et al.
(1997) J. Biol.
Chem. 272:16603-16609).
To screen for itt vivo PMMM substrates, this method can be expanded to screen
a cDNA
expression library displayed on the surface of phage particles (T7SELECT10-3
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 DI and the appropriate epitope.
XX. Identification of PMMM Inhibitors
Compounds to be tested are arrayed in the wells of a multi-well plate in
varying
concentrations along with an appropriate buffer and substrate, as described in
the assays in Example
XVIII. PMMM activity is measured for each well and the ability of each
compound to inhibit PMMM
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activity can be determined, as well as the dose-response kinetics. This assay
could also be used to
identify molecules which enhance PMIVEVI activity.
In the alternative, phage display libraries can be used to screen for peptide
PMMM inhibitors.
Candidates are found among peptides which bind tightly to a protease. In this
case, multi-well plate
wells are coated with PMIVI~~I and incubated with a random peptide phage
display library or a cyclic
peptide library (Koivunen, E. et al. (1999) Nature Biotech 17:768-774).
Unbound phage are washed
away and selected phage amplified and rescreened for several more rounds.
Candidates are tested
for PMMM inhibitory activity using an assay described in Example XVIII.
Various modifications and variations of the described compositions, 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. It will be appreciated that the invention provides novel and
useful proteins, and their
encoding polynucleotides, which can be used in the drug discovery process, as
well as methods for
using these compositions for the detection, diagnosis, and treatment of
diseases and conditions.
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.
Nor should the description of such embodiments be considered exhaustive or
limit the invention to the
precise forms disclosed. Furthermore, elements from one embodiment can be
readily recombined with
elements from one or more other embodiments. Such combinations can form a
number of
embodiments within the scope of the invention. It is intended that the scope
of the invention be
defined by the following claims and their equivalents.
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CA 02460476 2004-03-15
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Table 5
PolynucleotideIncyte ProjectRepresentative Library
SEQ ID:
ID NO:
41 7500287CB BRAITUT21
1
42 7500511CB BONEUNROl
1
43 7500273CB PGANNON02
1
44 7500183CB1 PROSTUT16
45 7499957CB1 LUNGNOT14
46 7500001CB1 NEUTFMTO1
47 7503245CB SECP00006
1
48 7503237CB THYMNON04
1
49 7503230CB BRAFNOT02
1
50 7503231CB1 BRAFNOT02
51 71592234CB HEAPNOTO1
1
53 55141453CB SPLNNOT04
1
54 7503135CB GBLATUTO1
1
55 7503242CB SKINDIA01
1
56 7504774CB LIVRNOTOl
1
57 7503166CB BRACNOI~02
1
58 7503171CB1 LUNGNON07
59 7503220CB ADRENOT07
1
60 6145631CB1 FIBRUNT02
61 7504980CB ISLTNOTO1
1
62 3118830CB1 LUNGTUT13
63 7505187CB PROSTUS23
1
64 7506567CB I~IDNNOT 19
1
65 7503673CB PLACFERO1
1
67 7505765CB OVARTUTOl
1
68 7505775CB THYMFET03
1
69 7500181CB1 PROSTUT16
70 7503799CB1 BRSTTUT16
71 7504602CB DENDNOTO1
1
72 5873533CB1 COLTDIT04
73 71033239CB ISLTNOT01
1
74 7506001CB DUODNOT02
1
75 7506026CB U937NOT01
1
76 8167924CB BRSTNOT03
1
77 2365313CB PROSTUS23
1
78 7503156CB PROSTUT09
1
79 7505980CB BMARTXE01
1
80 7646577CB ESOGTUE01
1
209
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<110> INCYTE GENOMICS, INC.
RAMKUMAR, Jayalaxmi
GORVAD, Ann E.
BAUGHN, Mariah R.
EMERLING, Brooke M.
YANG, Junming
LEE, Soo Yeun
TRAN, Uyen K.
BECHA, Shanya D.
DUGGAN, Brendan M.
LEE, Ernestine A.
GRIFFIN, Jennifer A.
LI, Joana X.
SPRAGUE, William W.
HAFALIA, April J.A.
CHAWLA, Narinder K.
LEHR-MASON, Patricia M.
KABLE, Amy E.
YUE, Henry
MARQUIS Joseph P.
YAO, Monique G.
RICHARDSON, Thomas W.
TANG, Y. Tom
JIN, Pei
CHIEN, David
BHATIA, Umesh G.
BURRILL, John D.
LEE, Sally
BLAKE, Julie J.
HO, Anne
ZHENG, Wenjin
<120> PROTEIN MODIFICATION AND MAINTENANCE PROTEINS
<130> PF-1237 PCT
<140> To Be Assigned
<141> Herewith
<150> US 60/329,689
<151> 2001-10-12
<150> US 60/335,703
<151> 2001-10-25
<150> US 60/348,887
<151> 2001-11-09
<150> US 60/334,145
<151> 2001-11-28
<150> US 60/337,451
<151> 2001-12-06
<150> US 60/340,584
<151> 2001-12-14
1/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
<160> 80
<170> PERL Program
<210> 1
<211> 270
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500287CD1
<400> 1
Met His Gly Ser Cys Ser Phe Leu Met Leu Leu Leu Pro Leu Leu
1 5 10 15
Leu Leu Leu Val Ala Thr Thr Gly Pro Val Gly Ala Leu Thr Asp
20 25 30
Glu Glu Lys Arg Leu Met Val Glu Leu His Asn Leu Tyr Arg Ala
35 40 45
Gln Val Ser Pro Pro Ala Ser Asp Met Leu His Met Arg Trp Asp
50 55 60
Glu Glu Leu Ala Ala Phe Ala Lys Ala Tyr Ala Arg Gln Cys Val
65 70 75
Trp Gly His Asn Lys Glu Arg Gly Arg Arg Gly Glu Asn Leu Phe
80 85 90
Ala Ile Thr Asp Glu Gly Met Asp Val Pro Leu Ala Met Glu Glu
95 100 105
Trp His His Glu Arg Glu His Tyr Asn Leu Ser Ala Ala Thr Cys
110 115 120
Ser Pro Gly Gln Met Cys Gly His Tyr Thr Gln Val Val Trp Ala
125 130 135
Lys Thr Glu Arg Ile Gly Cys Gly Ser His Phe Cys Glu Lys Leu
140 145 150
Gln Gly Val Glu Glu Thr Asn Ile Glu Leu Leu Val Cys Asn Tyr
155 160 165
Glu Pro Pro Gly Asn Val Lys Gly Lys Arg Pro Tyr Gln Glu Gly
170 175 180
Thr Pro Cys Ser Gln Cys Pro Ser Gly Tyr His Cys Lys Asn Ser
185 190 195
Leu Cys Glu Pro Ile Gly Ser Pro Glu Asp Ala Gln Asp Leu Pro
200 205 210
Tyr Leu Val Thr Glu Ala Pro Ser Phe Arg Ala Thr Glu Ala Ser
215 220 225
Asp Ser Arg Lys Met Gly Ala Glu Gly Pro Asp Lys Pro Ser Val
230 235 240
Val Ser Gly Leu Asn Ser Gly Pro Gly His Val Trp Gly Pro Leu
245 250 255
Leu Gly Leu Leu Leu Leu Pro Pro Leu Val Leu Ala Gly Ile Phe
260 265 270
<210> 2
<211> 288
<212> PRT
<213> Homo Sapiens
2/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
<220>
<221> misc_feature
<223> Incyte ID No: 7500511CD1
<400> 2
Met Trp Gly Leu Lys Val Leu Leu Leu Pro Val Val Ser Phe Ala
1 5 10 15
Leu Tyr Pro Glu Glu Ile Leu Asp Thr His Trp Glu Leu Trp Lys
20 25 30
Lys Thr His Arg Lys Gln Tyr Asn Asn Lys Thr Ser Glu Glu Val
35 40 45
Val Gln Lys Met Thr Gly Leu Lys Val Pro Leu Ser His Ser Arg
50 55 60
Ser Asn Asp Thr Leu Tyr Ile Pro Glu Trp Glu Gly Arg Ala Pro
65 70 75
Asp Ser Val Asp Tyr Arg Lys Lys Gly Tyr Val Thr Pro Val Lys
80 85 90
Asn Gln Gly Gln Cys Gly Ser Cys Trp Ala Phe Ser Ser Val Gly
95 100 105
Ala Leu Glu Gly Gln Leu Lys Lys Lys Thr Gly Lys Leu Leu Asn
110 115 120
Leu Ser Pro Gln Asn Leu Val Asp Cys Val Ser Glu Asn Asp Gly
125 130 135
Cys Gly Gly Gly Tyr Met Thr Asn Ala Phe Gln Tyr Val Gln Lys
140 145 150
Asn Arg Gly Ile Asp Ser Glu Asp Ala Tyr Pro Tyr Val Gly Gln
155 160 165
Glu Glu Ser Cys Met Tyr Asn Pro Thr Gly Lys Ala Ala Lys Cys
170 175 180
Arg Gly Tyr Arg Glu Ile Pro Glu Gly Asn Glu Lys Ala Leu Lys
185 190 195
Arg Ala Val Ala Arg Val Gly Pro Val Ser Val Ala Ile Asp Ala
200 205 210
Ser Leu Thr Ser Phe Gln Phe Tyr Ser Lys Gly Val Tyr Tyr Asp
215 220 225
Glu Ser Cys Asn Ser Asp Asn Leu Asn His Ala Val Leu Ala Val
230 235 240
Gly Tyr Gly Ile Gln Lys Gly Asn Lys His Trp Ile Ile Lys Asn
245 250 255
Ser Trp Gly Glu Asn Trp Gly Asn Lys Gly Tyr Ile Leu Met Ala
260 265 270
Arg Asn Lys Asn Asn Ala Cys Gly Ile Ala Asn Leu Ala Ser Phe
275 280 285
Pro Lys Met
<210> 3
<211> 608
<212> PRT
<213> Homo Sapiens
<220>
<221> mist-feature
<223> Incyte ID No: 7500273CD1
<400> 3
3/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
Met Arg Pro Val Ser Val Trp Gln Trp Ser Pro Trp Gly Leu Leu
1 5 10 15
Leu Cys Leu Leu Cys Ser Ser Cys Leu Gly Ser Pro Ser Pro Ser
20 25 30
Thr Gly Pro Glu Lys Lys Ala Gly Ser Gln Gly Leu Arg Phe Arg
35 40 45
Leu Ala Gly Phe Pro Arg Lys Pro Tyr Glu Gly Arg Val Glu I1e
50 55 60
Gln Arg Ala Gly Glu Trp Gly Thr Ile Cys Asp Asp Asp Phe Thr
65 70 75
Leu Gln Ala Ala His Ile Leu Cys Arg Glu Leu Gly Phe Thr Glu
80 85 90
Ala Thr Gly Trp Thr His Ser Ala Lys Tyr Gly Pro Gly Thr Gly
95 100 105
Arg Ile Trp Leu Asp Asn Leu Ser Cys Ser Gly Thr Glu Gln Ser
110 115 120
Val Thr Glu Cys Ala Ser Arg Gly Trp Gly Asn Ser Asp Cys Thr
125 130 135
His Asp Glu Asp Ala Gly Val Ile Cys Lys Asp Gln Arg Leu Pro
140 145 150
Gly Phe Ser Asp Ser Asn Val Ile Glu Ala Arg Val Arg Leu Lys
155 160 165
Gly Gly Ala His Pro Gly Glu Gly Arg Val Glu Val Leu Lys Ala
170 175 180
Ser Thr Trp Gly Thr Val Cys Asp Arg Lys Trp Asp Leu His Ala
185 190 195
Ala Ser Val Val Cys Arg Glu Leu Gly Phe Gly Ser Ala Arg Glu
200 205 210
Ala Leu Ser Gly Ala Arg Met Gly Gln Gly Met Gly Ala Ile His
215 220 225
Leu Ser Glu Val Arg Cys Ser Gly Gln Glu Leu Ser Leu Trp Lys
230 235 240
Cys Pro His Lys Asn Ile Thr Ala Glu Asp Cys Ser His Ser Gln
245 250 255
Asp Ala G1y Val Arg Cys Asn Leu Pro Tyr Thr Gly Ala Glu Thr
260 265 270
Arg Ile Arg Leu Ser Gly Gly Arg Ser Gln His Glu Gly Arg Val
275 280 285
Glu Val Gln Ile Gly Gly Pro Gly Pro Leu Arg Trp Gly Leu Ile
290 295 300
Cys Gly Asp Asp Trp Gly Thr Leu Glu Ala Met Val Ala Cys Arg
305 310 315
Gln Leu Gly Leu Gly Tyr Ala Asn His Gly Leu Gln Glu Thr Trp
320 325 330
Tyr Trp Asp Ser Gly Asn Ile Thr Glu Va1 Val Met Ser Gly Val
335 340 345
Arg Cys Thr Gly Thr Glu Leu Ser Leu Asp Gln Cys Ala His His
350 355 360
Gly Thr His Ile Thr Cys Lys Arg Thr Gly Thr Arg Phe Thr Ala
365 370 375
Gly Val Ile Cys Ser G1u Thr Ala Ser Asp Leu Leu Leu His Ser
380 385 390
Ala Leu Val Gln G1u Thr Ala Tyr Ile Glu Asp Arg Pro Leu His
395 400 405
Met Leu Tyr Cys Ala Ala Glu Glu Asn Cys Leu Ala Ser Ser Ala
410 415 420
4/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
Arg Ser Ala Asn Trp Pro Tyr Gly His Arg Arg Leu Leu Arg Phe
425 430 435
Ser Ser Gln Ile His Asn Leu Gly Arg Ala Asp Phe Arg Pro Lys
440 445 450
Ala Gly Arg His Ser Trp Val Trp His Glu Cys His Gly His Tyr
455 460 465
His Ser Met Asp Ile Phe Thr His Tyr Asp Ile Leu Thr Pro Asn
470 475 480
Gly Thr Lys Val Ala Glu Gly His Lys Ala Ser Phe Cys Leu Glu
485 . 490 ~ 495
Asp Thr Glu Cys Gln Glu Asp Val Ser Lys Arg Tyr Glu Cys Ala
500 505 510
Asn Phe Gly Glu Gln Gly Ile Thr Val Gly Cys Trp Asp Leu Tyr
515 520 525
Arg His Asp Ile Asp Cys Gln Trp Ile Asp Ile Thr Asp Val Lys
530 535 540
Pro Gly Asn Tyr Ile Leu Gln Val Val Ile Asn Pro Asn Phe Glu
545 550 555
Val Ala Glu Ser Asp Phe Thr Asn Asn Ala Met Lys Cys Asn Cys
560 565 570
Lys Tyr Asp Gly His Arg Ile Trp Val His Asn Cys His Ile Gly
575 580 585
Asp Ala Phe Ser Glu Glu Ala Asn Arg Arg Phe Glu Arg Tyr Pro
590 595 600
Gly Gln Thr Ser Asn Gln Ile I'le
605
<210> 4
<211> 218
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500183CD1
<400> 4
Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile
1 5 10 15
Gly Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu
20 25 30
Cys Glu Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg
35 40 45
Gly Arg Ala Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val
50 55 60
Leu Thr Ala Ala His Cys Ile Arg Lys Pro Gly Asp Asp Ser Ser
65 70 75
His Asp Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Glu Leu Thr
80 85 90
Asp Ala Val Lys Val Met Asp Leu Pro Thr Gln Glu Pro Ala Leu
g5 100 105
Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Glu
110 115 ' 120
Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu His Val
125 130 135
Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr
5/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
140 145 150
Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr
155 160 165
Cys Ser Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu
170 175 180
Gln Gly Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu
185 190 195
Arg Pro Ser Leu Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile
200 205 210
Lys Asp Thr Ile Val Ala Asn Pro
215
<210> 5
<211> 172
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7499957CD1
<400> 5
Met Ala Glu Leu Thr Ala Leu Glu Ser Leu Ile Glu Met Gly Phe
1 5 10 15
Pro Arg Gly Arg Ala Glu Lys Ala Leu Ala Leu Thr Gly Asn Gln
20 25 30
Gly Ile Glu Ala Ala Met Asp Trp Leu Met Glu His Glu Asp Asp
35 40 45
Pro Asp Val Asp Glu Pro Leu Glu Thr Pro Leu Gly His Ile Leu
50 55 60
Gly Arg Glu Pro Thr Ser Ser Glu Pro Gly Pro Val Pro Ser Ser
65 70 75
Pro Ser Gln Glu Pro Pro Thr Lys Arg Glu Tyr Asp Gln Cys Arg
80 85 90
Ile Gln Val Arg Leu Pro Asp Gly Thr Ser Leu Thr Gln Thr Phe
95 100 105
Arg Ala Arg Glu Gln Leu Ala Ala Val Arg Leu Tyr Val Glu Leu
110 115 120
His Arg Gly Glu Glu Leu Gly Gly Gly Gln Asp Pro Val Gln Leu
125 130 135
Leu Ser Gly Phe Pro Arg Arg Ala Phe Ser Glu Ala Asp Met Glu
140 145 150
Arg Pro Leu Gln Glu Leu Gly Leu Val Pro Ser Ala Val Leu Ile
155 160 165
Val Ala Lys Lys Cys Pro Ser
170
<210> 6
<211> 831
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500001CD1
6/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
~400> 6
Met Ala Ala Ala Met Glu Thr Glu Gln Leu Gly Val Glu Ile Phe
1 ~ 5 10 15
Glu Thr Ala Asp Cys Glu Glu Asn Ile Glu Ser Gln Asp Arg Pro
20 25 30
Lys Leu Glu Pro Phe Tyr Val Glu Arg Tyr Ser Trp Ser Gln Leu
35 40 45
Lys Lys Leu Leu Ala Asp Thr Arg Lys Tyr His Gly Tyr Met Met
50 55 60
Ala Lys Ala Pro His Asp Phe Met Phe Val Lys Arg Asn Asp Pro
65 70 75
Asp Gly Pro His Ser Asp Arg Ile Tyr Tyr Leu Ala Met Ser Gly
80 85 90
Glu Asn Arg Glu Asn Thr Leu Phe Tyr Ser Glu Ile Pro Lys Thr
95 l00 105
Ile Asn Arg Ala Ala Val Leu Met Leu Ser Trp Lys Pro Leu Leu
110 115 120
Asp Leu Phe Gln Ala Thr Leu Asp Tyr Gly Met Tyr Ser Arg Glu
125 130 135
Glu Glu Leu Leu Arg Glu Arg Lys Arg Ile Gly Thr Val Gly Ile
140 145 150
Ala Ser Tyr Asp Tyr His Gln Gly Ser Gly Thr Phe Leu Phe Gln
155 160 165
Ala Gly Ser Gly Ile Tyr His Val Lys Asp Gly Gly Pro Gln Gly
170 175 180
Phe Thr Gln Gln Pro Leu Arg Pro Asn Leu Val Glu Thr Ser Cys
185 190 195
Pro Asn Ile Arg Met Asp Pro Lys Leu Cys Pro Ala Asp Pro Asp
200 205 210
Trp Ile Ala Phe Ile His Ser Asn Asp Ile Trp Ile Ser Asn Ile
215 220 225
Val Thr Arg Glu Glu Arg Arg Leu Thr Tyr Val His Asn Glu Leu
230 235 240
Ala Asn Met Glu Glu Asp Ala Arg Ser Ala Gly Val Ala Thr Phe
245 250 255
Val Leu Gln Glu Glu Phe Asp Arg Tyr Ser Gly Tyr Trp Trp Cys
260 265 270
Pro Lys Ala Glu Thr Thr Pro 5er Gly Gly Lys Ile Leu Arg Ile
275 280 285
Leu Tyr Glu Glu Asn Asp Glu Ser Glu Val Glu Ile Ile His Val
290 295 300
Thr Ser Pro Met Leu Glu Thr Arg Arg Ala Asp Ser Phe Arg Tyr
305 310 315
Pro Lys Thr Gly Thr Ala Asn Pro Lys Val Thr Phe Lys Met Ser
320 325 330
G1u Ile Met Ile Asp Ala Glu Gly Arg Ile Ile Asp Val Ile Asp
335 340 345
Lys Glu Leu Ile Gln Pro Phe Glu Ile Leu Phe Glu Gly Val Glu
350 355 360
Tyr Ile Ala Arg Ala Gly Trp Thr Pro Glu Gly Lys Tyr Ala Trp
365 370 375
Ser Ile Leu Leu Asp Arg Ser Gln Thr Arg Leu Gln Ile Val Leu
380 385 390
Ile Ser Pro Glu Leu Phe Ile Pro Val Glu Asp Asp Val Met Glu
395 400 405
Arg Gln Arg Leu Ile Glu Ser Val Pro Asp Ser Val Thr Pro Leu
7/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
410 415 420
Ile Ile Tyr Glu Glu Thr Thr Asp Ile Trp Ile Asn Ile His Asp
425 430 435
Ile Phe His Val Phe Pro Gln Ser His Glu G1u Glu Ile Glu Phe
440 445 450
Ile Phe Ala Ser Glu Cys Lys Thr Gly Phe Arg His Leu Tyr Lys
455 460 465
Ile Thr Ser Ile Leu Lys Glu Ser Lys Tyr Lys Arg Ser Ser Gly
470 475 480
Gly Leu Pro Ala Pro Ser Asp Phe Lys Cys Pro Ile Lys Glu Glu
485 490 495
Ile Ala Ile Thr Ser Gly Glu Trp Glu Val Leu Gly Arg His Gly
500 505 510
Ser Asn Ile Gln Val Asp Glu Val Arg Arg Leu Val Tyr Phe Glu
515 520 525
Gly Thr Lys Asp Ser Pro Leu GTu His His Leu Tyr Val Val Ser
530 535 540
Tyr Val Asn Pro Gly Glu Val Thr Arg Leu Thr Asp Arg Gly Tyr
545 550 555
Ser His Ser Cys Cys Ile Ser Gln His Cys Asp Phe Phe Ile Ser
560 565 570
Lys Tyr Ser Asn Gln Lys Asn Pro His Cys Val Ser Leu Tyr Lys
575 580 585
Leu Ser Ser Pro Glu Asp Asp Pro Thr Cys Lys Thr Lys Glu Phe
590 595 600
Trp Ala Thr Ile Leu Asp Ser Ala Gly Pro Leu Pro Asp Tyr Thr
605 610 615
Pro Pro Glu Ile Phe Ser Phe Glu Ser Thr Thr Gly Phe Thr Leu
620 625 630
Tyr Gly Met Leu Tyr Lys Pro His Asp Leu Gln Pro Gly Lys Lys
635 640 645
Tyr Pro Thr Val Leu Phe Ile Tyr Gly Gly Pro Gln Val Gln Leu
650 655 660
Val Asn Asn Arg Phe Lys Gly Val Lys Tyr Phe Arg Leu Asn Thr
665 670 675
Leu Ala Ser Leu Gly Tyr Val Val Val Val Ile Asp Asn Arg Gly
680 685 690
Ser Cys His Arg Gly Leu Lys Phe Glu Gly Ala Phe Lys Tyr Lys
695 700 705
Met Val Ala Ile Ala Gly Ala Pro Val Thr Leu Trp Ile Phe Tyr
710 715 720
Asp Thr Gly Tyr Thr Glu Arg Tyr Met Gly His Pro Asp Gln Asn
725 730 735
Glu Gln Gly Tyr Tyr Leu Gly Ser Val Ala Met Gln Ala Glu Lys
740 745 750
Phe Pro Ser Glu Pro Asn Arg Leu Leu Leu Leu His Gly Phe Leu
755 760 765
Asp Glu Asn Val His Phe Ala His Thr Ser Ile Leu Leu Ser Phe
770 775 780
Leu Val Arg Ala Gly Lys Pro Tyr Asp Leu Gln Ile Tyr Pro Gln
785 790 795
Glu Arg His Ser Ile Arg Val Pro Glu Ser Gly Glu His Tyr Glu
800 805 810
Leu His Leu Leu His Tyr Leu Gln Glu Asn Leu Gly Ser Arg Ile
g15 820 825
Ala Ala Leu Lys Val Ile
8/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
830
<210> 7
<211> 936
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503245CD1
<400> 7
Met Thr Leu Leu Ala Pro Trp Tyr Thr Gly Pro Met Ile Pro Met
1 5 10 15
Asp Val Asn Glu Pro Ser Ser Val Thr Thr Ala Pro Thr Leu Ser
20 25 30
Ser Ser Leu Gln His Ile Ser Ser Phe Leu Ala Thr Gly Lys Lys
35 40 45
Leu Ser'Leu His Phe Gly His Pro Arg Glu Cys Glu Val Thr Arg
50 55 60
Ile Asp Asp Lys Asn Arg Arg Gly Leu Glu Asp Ser Glu Pro Gly
65 70 75
Ala Lys Leu Phe Asn Asn Asp Gly Val Cys Cys Cys Leu Gln Lys
80 85 90
Arg Gly Pro Val Asn Ile Thr Ser Val Cys Val Ser Pro Arg Thr
g5 100 105
Leu Gln Ile Ser Val Phe Val Leu Ser Glu Lys Tyr Glu Gly Ile
110 115 120
Val Lys Phe Glu Ser Asp Glu Leu Pro Phe Gly Val Ile Gly Ser
125 130 135
Asn Ile Gly Asp Ala His Phe Gln Glu Phe Arg Ala Gly Ile Ser
140 145 150
Trp Lys Pro Val Val Asp Pro Asp Asp Pro Ile Pro Gln Phe Pro
155 160 165
Asp Cys Cys Ser Ser Ser Ser Ser Arg Ile Pro Ser Val Ser Val
1T0 175 180
Leu Val Ala Val Pro Leu Val Ala Gly His Lys Gly Gln Ala Phe
185 190 195
Ile Glu Arg Met Leu Gly Cys Phe Lys Glu Leu Lys Gln Glu Leu
200 205 210
Thr Gln Glu Gly Pro Gly Gly Gly His Pro Arg Ser Ala Trp Pro
215 220 225
Pro Arg Arg His Ala Gln Trp Pro Pro Glu Pro Cys Glu Gln Gly
230 235 240
Glu Glu Pro Pro Pro Val Glu Ala Glu Glu Val Glu Glu Ala Glu
245 250 255
Thr Ala Glu Lys Ala Glu Arg Lys Val Glu Ala Glu Ala Lys Val
260 265 270
Glu Gly Lys Ala Glu Ala Ala Gly Lys Ala Glu Ala Ala Gly Lys
275 280 285
Val Asp Ala Thr Glu Lys Val Glu Thr Ala Gly Lys Val Asp Ala
290 295 300
Ala Gly Lys Val Glu Thr Ala Glu Gly Pro Gly Arg Arg Ala Glu
305 310 315
Leu Lys Leu Glu Pro Glu Pro Glu Pro Va1 Arg Glu Ala Glu Gln
320 325 330
9/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
Glu Pro Lys Gln Glu Leu Glu Asp Glu Asn Pro Ala Arg Ser Gly
335 340 345
Gly Gly Gly Asn Ser Asp Glu Val Pro Pro Pro Thr Leu Pro Ser
350 355 360
Asp Pro Pro Arg Pro Pro Asp Pro Ser Pro Arg Arg Ser Arg Ala
365 370 375
Pro Arg Arg Arg Pro Arg Pro Arg Pro Gln Thr Arg Leu Arg Thr
380 385 390
Pro Pro Gln Pro Arg Pro Arg Pro Pro Pro Arg Pro Arg Pro Arg
395 ~ 400 405
Arg Gly Pro Gly Gly Gly Cys Leu Asp Val Asp Phe Ala Val Gly
410 415 420
Pro Pro Gly Cys Ser His Val Asn Ser Phe Lys Val Gly Glu Asn
425 430 435
Trp Arg Gln Glu Leu Arg Val Ile Tyr Gln Cys Phe Val Trp Cys
440 445 450
Gly Thr Pro Glu Thr Arg Lys Ser Lys Ala Lys Ser Cys Ile Cys
455 460 465
His Val Cys Gly Thr His Leu Asn Arg Leu His Ser Cys Leu Ser
470 475 480
Cys Val Phe Phe Gly Cys Phe Thr Glu Lys His Ile His Glu His
485 490 495
Ala Glu Thr Lys Gln His Asn Leu Ala Val Asp Leu Tyr Tyr Gly
500 505 510
Gly Ile Tyr Cys Phe Met Cys Lys Asp Tyr Val Tyr Asp Lys Asp
515 520 525
Ile Glu Gln Ile Ala Lys Glu Glu Gln Gly Glu Ala Leu Lys Leu
530 535 540
Gln Ala Ser Thr Ser Thr Glu Val Ser His Gln Gln Cys Ser Val
545 550 ~ 555
Pro Gly Leu Gly Glu Lys Phe Pro Thr Trp Glu Thr Thr Lys Pro
560 565 570
Glu Leu Glu Leu Leu Gly His Asn Pro Arg Arg Arg Arg Ile Thr
575 580 585
Ser Ser Phe Thr Ile Gly Leu Arg Gly Leu Ile Asn Leu Gly Asn
590 595 600
Thr Cys Phe Met Asn Cys Ile Val Gln Ala Leu Thr His Thr Pro
605 610 615
Ile Leu Arg Asp Phe Phe Leu Ser Asp Arg His Arg Cys Glu Met
620 625 630
Pro Ser Pro Glu Leu Cys Leu Val Cys Glu Met Ser Lys Leu Leu
635 640 645
His Leu Val Trp Ile His Ala Arg His Leu Ala Gly Tyr Arg Gln
650 655 660
Gln Asp Ala His Glu Phe Leu Ile Ala Ala Leu Asp Val Leu His
665 670 675
Arg His Cys Lys Gly Asp Asp Val Gly Lys Ala Ala Asn Asn Pro
680 685 690
Asn His Cys Asn Cys Ile Ile Asp Gln Ile Phe Thr Gly Gly Leu
695 700 705
Gln Ser Asp Val Thr Cys Gln Ala Cys His Gly Val Ser Thr Thr
710 715 720
Ile Asp Pro Cys Trp Asp Ile Ser Leu Asp Leu Pro Gly Ser Cys
725 730 735
Thr Ser Phe Trp Pro Met Ser Pro Gly Arg Glu Ser Ser Val Asn
740 745 750
10/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
Gly Glu Ser His Ile Pro Gly Ile Thr Thr Leu Thr Asp Cys Leu
755 760 765
Arg Arg Phe Thr Arg Pro Glu His Leu Gly Ser Ser Ala Lys Ile
770 775 780
Lys Cys Gly Ser Cys Gln Ser Tyr Gln Glu Ser Thr Lys Gln Leu
785 790 795
Thr Met Asn Lys Leu Pro Val Val Ala Cys Phe His Phe Lys Arg
800 805 810
Phe Glu His Ser Ala Lys Gln Arg Arg Lys Ile Thr Thr Tyr Ile
815 820 825
Ser Phe Pro Leu Glu Leu Asp Met Thr Pro Phe Met Ala Ser Ser
830 835 840
Lys Glu Ser Arg Met Asn Gly Gln Leu Gln Leu Pro Thr Asn Ser
845 850 855
Gly Asn Asn Glu Asn Lys Tyr Ser Leu Phe Ala Val Val Asn His
860 865 870
Gln Gly Thr Leu Glu Ser Gly His Tyr Thr Ser Phe Ile Arg His
875 880 885
His Lys Asp Gln Trp Phe Lys Cys Asp Asp A1a Val Ile Thr Lys
890 895 900
Ala Ser Ile Lys Asp Val Leu Asp Ser Glu Gly Tyr Leu Leu Phe
905 910 915
Tyr His Lys Gln Val Leu Glu His Glu Ser Glu Lys Val Lys Glu
920 925 930
Met Asn Thr Gln Ala Tyr
935
<2l0> 8
<211> 456
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503237CD1
<400> 8
Met Leu Ser Ser Arg Ala Glu Ala Ala Met Thr Ala Ala Asp Arg
10 15
Ala Ile Gln Arg Phe Leu Arg Thr Gly Ala Ala Val Arg Tyr Lys
20 25 30
Val Met Lys Asn Trp Gly Val Ile Gly Gly Ile Ala Ala Ala Leu
35 40 45
Ala Ala Gly Ile Tyr Val Ile Trp Gly Pro Ile Thr Glu Arg Lys
50 55 60
Lys Arg Arg Lys Ala Leu Ser Cys Gln Glu Val Thr Asp Asp Glu
65 70 75
Val Leu Asp Ala Ser Cys Leu Leu Asp Val Leu Arg Met Tyr Arg
80 85 90
Trp Gln Ile Ser Ser Phe Glu Glu Gln Asp Ala His Glu Leu Phe
g5 100 105
His Val Ile Thr Ser Ser Leu Glu Asp Glu Arg Asp Arg Gln Pro
110 115 ~.2 0
Arg Val Thr His Leu Phe Asp Val His Ser Leu Glu Gln Gln Ser
125 130 135
Glu Ile Thr Pro Lys Gln Ile Thr Cys Arg Thr Arg Gly Ser Pro
1l/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
140 145 150
His Pro Thr Ser Asn His Trp Lys Ser Gln His Leu Phe His Gly
155 160 165
Arg Leu Thr Ser Asn Met Val Cys Lys His Cys Glu His Gln Ser
170 175 180
Pro Val Arg Phe Asp Thr Phe Asp Ser Leu Ser Leu Ser Ile Pro
185 190 195
Ala Ala Thr Trp Gly His Pro Leu Thr Leu Asp His Cys Leu His
200 205 210
His Phe Ile Ser Ser Glu Ser Val Arg Asp Val Val Cys Asp Asn
215 220 225
Cys Thr Lys Ile Glu Ala Lys Gly Thr Leu Asn Gly Glu Lys Val
230 235 240
Glu His Gln Arg Thr Thr Phe Val Lys Gln Leu Lys Leu Gly Lys
245 250 255
Leu Pro Gln Cys Leu Cys Ile His Leu Gln Arg Leu Ser Trp Ser
260 265 270
Ser His Gly Thr Pro Leu Lys Arg His Glu His Va1 Gln Phe Asn
275 280 285
Glu Phe Leu Met Met Asp Ile Tyr Lys Tyr His Leu Leu Gly His
290 295 300
Lys Pro Ser Gln His Asn Pro Lys Leu Asn Lys Asn Pro Gly Pro
305 310 315
Thr Leu Glu Leu Gln Asp Gly Pro Gly Ala Pro Thr Pro Val Leu
320 325 330
Asn Gln Pro Gly Ala Pro Lys Thr Gln Ile Phe Met Asn Gly Ala
335 340 345
Cys Ser Pro Ser Leu Leu Pro Thr Leu Ser Ala Pro Met Pro Phe
350 355 360
Pro Leu Pro Val Val Pro Asp Tyr Ser Ser Ser Thr Tyr Leu Phe
365 370 375
Arg Leu Met Ala Val Val Val His His Gly Asp Met His Ser Gly
380 385 390
His Phe Val Thr Tyr Arg Arg Ser Pro Pro Ser Ala Arg Asn Pro
395 400 405
Leu Ser Thr Ser Asn Gln Trp Leu Trp Val Ser Asp Asp Thr Val
410 415 420
Arg Lys Ala Ser Leu Gln Glu Val Leu Ser Ser Ser Ala Tyr Leu
425 430 435
Leu Phe Tyr Glu Arg Val Leu Ser Arg Met Gln His Gln Ser Gln
440 445 450
Glu Cys Lys Ser Glu Glu
455
<210> 9
<211> 516
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503230CD1
<400> 9
Met Pro Asp Gln Leu Glu Ser Leu Pro Leu Phe Ser Lys Lys Asn
1 5 10 15
12/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
Leu Tle Asp Ile Leu A1a Ile Gly Gly Val Gln Lys Leu Lys Gln
20 25 30
Ile Ile Asp Val Ile Asp Lys Glu Leu Ile Gln Pro Phe Glu Ile
35 40 45
Leu Phe Glu Gly Val Glu Tyr Ile Ala Arg Ala Gly Trp Thr Pro
50 55
Glu Gly Lys Tyr Ala Trp Ser Ile Leu Leu Asp Arg Ser Gln Thr
70 75
Arg Leu Gln Ile Val Leu Ile Ser Pro Glu Leu Phe Ile Pro Val
80 85 90
Glu Asp Asp Val Met Glu Arg Gln Arg Leu Ile Glu Ser Val Pro
95 100 105
Asp Ser Val Thr Pro Leu Ile Ile Tyr Glu Glu Thr Thr Asp Ile
110 115 120
Trp Ile Asn Ile His Asp Ile Phe His Val Phe Pro Gln Ser His
125 130 135
Glu Glu Glu Ile Glu Phe Ile Phe Ala Ser Glu Cys Lys Thr Gly
140 145 150
Phe Arg His Leu Tyr Lys Ile Thr 5er Ile Leu Lys Glu Ser Lys
155 160 l65
Tyr Lys Arg Ser Ser Gly Gly Leu Pro Ala Pro Thr Val Thr Trp
170 l75 180
Met Ile Thr Phe Met Arg Ser Leu Gly Thr Pro Ser Cys Met Cys
185 190 195
Val Thr His Ile Val Glu Ile Gln Val Asp Glu Val Arg Arg Leu
200 205 210
Val Tyr Phe Glu Gly Thr Lys Asp Ser Pro Leu Glu His His Leu
215 220 225
Tyr Val Val Ser Tyr Val Asn Pro Gly Glu Val Thr Arg Leu Thr
230 235 240
Asp Arg Gly Tyr Ser His Ser Cys Cys Ile Ser Gln His Cys Asp
245 250 255
Phe Phe Ile Ser Lys Tyr Ser Asn Gln Lys Asn Pro His Cys Val
260 265 270
Ser Leu Tyr Lys Leu Ser Ser Pro Glu Asp Asp Pro Thr Cys Lys
275 280 285
Thr Lys Glu Phe Trp Ala Thr Ile Leu Asp Ser Ala Gly Pro Leu
290 295 300
Pro Asp Tyr Thr Pro Pro Glu Ile Phe Ser Phe Glu Ser Thr Thr
305 310 315
Gly Phe Thr Leu Tyr Gly Met Leu Tyr Lys Pro His Asp Leu Gln
320 325 330
Pro Gly Lys Lys Tyr Pro Thr Val Leu Phe Ile Tyr Gly Gly Pro
335 340 345
Gln Val Gln Leu Val Asn Asn Arg Phe Lys Gly Val Lys Tyr Phe
350 355 360
Arg Leu Asn Thr Leu Ala Ser Leu Gly Tyr Val Val Val Val Ile
365 370 375
Asp Asn Arg Gly Ser Cys His Arg Gly Leu Lys Phe Glu Gly Ala
380 385 390
Phe Lys Tyr Lys Met Val Ala Ile Ala Gly Ala Pro Val Thr Leu
395 400 405
Trp Ile Phe Tyr Asp Thr Gly Tyr Thr Glu Arg Tyr Met Gly His
410 415 420
Pro Asp Gln Asn Glu Gln Gly Tyr Tyr Leu Gly Ser Val Ala Met
425 430 435
13/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
Gln Ala Glu Lys Phe Pro Ser Glu Pro Asn Arg Leu Leu Leu Leu
440 445 450
His Gly Phe Leu Asp Glu Asn Val His Phe Ala His Thr Ser Ile
455 460 465
Leu Leu Ser Phe Leu Val Arg Ala Gly Lys Pro Tyr Asp Leu Gln
470 475 480
Glu Arg His Ser Ile Arg Val Pro Glu Ser Gly Glu His Tyr Glu
485 490 495
Leu His Leu Leu His Tyr Leu Gln Glu Asn Leu Gly Ser Arg Ile
500 505 510
Ala Ala Leu Lys Val Ile
515
<210> 10
<211> 824
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503231CD1
<400> 10
Met Ala Ala Ala Met Glu Thr Glu Gln Leu Gly Val Glu Ile Phe
1 5 10 15
Glu Thr Ala Asp Cys Glu Glu Asn Ile Glu Ser Gln Asp Arg Pro
20 25 30
Lys Leu Glu Pro Phe Tyr Val Glu Arg Tyr Ser Trp Ser Gln Leu
35 40 45
Lys Lys Leu Leu Ala Asp Thr Arg Lys Tyr His Gly Tyr Met Met
50 55 60
Ala Lys Ala Pro His Asp Phe Met Phe Val Lys Arg Asn Asp Pro
65 70 75
Asp Gly Pro His Ser Asp Arg Ile Tyr Tyr Leu Ala Met Ser Gly
80 85 90
Glu Asn Arg Glu Asn Thr Leu Phe Tyr Ser Glu Ile Pro Lys Thr
95 100 105
Ile Asn Arg Ala Ala Val Leu Met Leu Ser Trp Lys Pro Leu Leu
110 115 120
Asp Leu Phe Gln Gln Gln Pro Leu Arg Pro Asn Leu Val Glu Thr
125 130 135
Ser Cys Pro Asn Ile Arg Met Asp Pro Lys Leu Cys Pro Ala Asp
140 145 150
Pro Asp Trp Ile Ala Phe Ile His Ser Asn Asp Ile Trp Ile Ser
155 160 165
Asn Ile Val Thr Arg Glu Glu Arg Arg Leu Thr Tyr Val His Asn
170 175 180
Glu Leu Ala Asn Met Glu Glu Asp Ala Arg Ser Ala Gly Val Ala
185 190 195
Thr Phe Val Leu Gln Glu Glu Phe Asp Arg Tyr Ser G1y Tyr Trp
200 205 210
Trp Cys Pro Lys Ala Glu Thr Thr Pro Ser Gly Gly Lys Ile Leu
215 220 225
Arg Ile Leu Tyr Glu Glu Asn Asp Glu Ser Glu Val Glu Ile Ile
230 235 240
His Val Thr Ser Pro Met Leu Glu Thr Arg Arg Ala Asp Ser Phe
14/76
CA 02460476 2004-03-15
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245 250 255
Arg Tyr Pro Lys Thr Gly Thr Ala Asn Pro Lys Val Thr Phe Lys
260 265 270
Met Ser Glu Ile Met Ile Asp Ala Glu Gly Arg Ile Ile Asp Val
275 280 285
Ile Asp Lys Glu Leu Ile Gln Pro Phe Glu Ile Leu Phe Glu Gly
290 295 300
Val Glu Tyr Ile Ala Arg Ala Gly Trp Thr Pro Glu Gly Lys Tyr
305 310 315
Ala Trp Ser Ile Leu Leu Asp Arg Ser Gln Thr Arg Leu Gln Ile
320 325 330
Val Leu Ile Ser Pro Glu Leu Phe Ile Pro Val Glu Asp Asp Val
335 340 345
Met Glu Arg Gln Arg Leu Ile Glu Ser Val Pro Asp Ser Val Thr
350 355 360
Pro Leu Ile Ile Tyr Glu Glu Thr Thr Asp Ile Trp Ile Asn Ile
365 370 375
His Asp Ile Phe His Val Phe Pro Gln Ser His Glu Glu Glu Ile
380 385 390
Glu Phe Ile Phe Ala Ser Glu Cys Lys Thr Gly Phe Arg His Leu
395 400 405
Tyr Lys Ile Thr Ser Ile Leu Lys Glu Ser Lys Tyr Lys Arg Ser
410 415 420
Ser Gly Gly Leu Pro Ala Pro Ser Asp Phe Lys Cys Pro Ile Lys
425 430 435
Glu Glu Ile Ala Ile Thr Ser Gly Glu Trp Glu Val Leu Gly Arg
440 445 450
His Gly Ser Asn Ile Gln Val Asp Glu Val Arg Arg Leu Val Tyr
455 460 465
Phe Glu Gly Thr Lys Asp Ser Pro Leu Glu His His Leu Tyr Val
470 475 480
Va1 Ser Tyr Val Asn Pro Gly Glu Val Thr Arg Leu Thr Asp Arg
485 490 495
G1y Tyr Ser His Ser Cys Cys Ile Ser Gln His Cys Asp Phe Phe
500 505 510
Ile Ser Lys Tyr Ser Asn Gln Lys Asn Pro His Cys Val Ser Leu
515 520 525
Tyr Lys Leu Ser Ser Pro Glu Asp Asp Pro Thr Cys Lys Thr Lys
530 535 540
Glu Phe Trp Ala Thr Ile Leu Asp Ser Ala Gly Pro Leu Pro Asp
545 550 555
Tyr Thr Pro Pro Glu Ile Phe Ser Phe Glu Ser Thr Thr Gly Phe
560 565 570
Thr Leu Tyr Gly Met Leu Tyr Lys Pro His Asp Leu Gln Pro Gly
575 580 585
Lys Lys Tyr Pro Thr Val Leu Phe Ile Tyr Gly Gly Pro Gln Val
590 595 600
Gln Leu Val Asn Asn Arg Phe Lys Gly Val Lys Tyr Phe Arg Leu
605 610 615
Asn Thr Leu Ala Ser Leu Gly Tyr Val Val Val Val Ile Asp Asn
620 625 630
Arg Gly Ser Cys His Arg Gly Leu Lys Phe Glu Gly Ala Phe Lys
635 640 645
Tyr Lys Met Gly Gln Ile Glu Ile Asp Asp Gln Val Glu Gly Leu
650 655 660
Gln Tyr Leu Ala Ser Arg Tyr Asp Phe Ile Asp Leu Asp Arg Val
15/76
CA 02460476 2004-03-15
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665 670 675
Gly Ile His Gly Trp Ser Tyr Gly Gly Tyr Leu Ser Leu Met Ala
680 685 690
Leu Met Gln Arg Ser Asp Ile Phe Arg Val Ala Ile Ala Gly Ala
695 700 705
Pro Val Thr Leu Trp Ile Phe Tyr Asp Thr Gly Tyr Thr Glu Arg
710 715 720
Tyr Met Gly His Pro Asp Gln Asn Glu Gln Gly Tyr Tyr Leu Gly
725 730 735
Ser Val Ala Met Gln Ala Glu Lys Phe Pro Ser Glu Pro Asn Arg
740 745 750
Leu Leu Leu Leu His Gly Phe Leu Asp Glu Asn Val His Phe Ala
755 760 765
His Thr Ser Ile Leu Leu Ser Phe Leu Val Arg Ala Gly Lys Pro
770 775 780
Tyr Asp Leu Gln Ile Tyr Pro Gln Glu Arg His Ser Ile Arg Val
785 790 795
Pro Glu Ser Gly Glu His Tyr Glu Leu His Leu Leu His Tyr Leu
800 805 810
Gln Glu Asn Leu Gly Ser Arg Ile Ala Ala Leu Lys Val Ile
8l5 820
<210> 11
<211> 188
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 71592234CD1
<400> 11
Met Thr Thr Pro Phe Phe Leu Leu Arg Thr Ala Gly Phe Ala Asp
1 5 10 15
Ala Thr Thr Thr Gly Ser Pro Ile Leu Ser Ala Thr Val Asn Leu
20 25 30
Thr Met Phe Phe Asp Ile Ala Val Asp Gly Glu Pro Leu Gly His
35 40 45
Val Ser Phe Gly Ile Leu Ser Ala Arg Ile Pro Lys Thr Ala Glu
50 55 60
Asn Leu Trp Cys Thr Gly Glu Lys Gly Phe Gly Tyr Lys Gly Ser
65 70 75
Cys Phe His Arg Tle Ile Pro Gly Phe Met Cys Gln Cys Gly Lys
80 85 90
Phe Thr Arg His Asn Gly Thr Gly Gly Lys Ser Ile Cys Gly Glu
g5 100 105
Lys Phe Asp Asp Lys Asn Val Ile Leu Lys His Thr Gly Arg Gly
110 115 120
Ile Leu Ser Met Glu Thr Gly Gly Pro Asn Thr Asn Gly Ser Gln
125 130 135
Phe Phe Ile Cys Thr Gly Lys Thr Glu Trp Leu Asp Gly Lys Tyr
140 145 150
Met Val Phe Ser Lys Val Lys Glu Gly Lys Asn Ile Val Glu Ala
155 160 165
Met Glu Arg Phe Gly Ser Arg Asn Gly Lys Ile Ser Lys Lys Ile
170 175 180
16/76
CA 02460476 2004-03-15
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Thr Ile Ala Asp Cys Gly Gln Leu
185
<210> 12
<211>=552
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 90035161CD1
<400> 12
Met Arg Pro Val Ser Val Trp Gln Trp Ser Pro Trp Gly Leu Leu
1 5 10 15
Leu Cys Leu Leu Cys Ser Ser Cys Leu Gly Ser Pro Ser Pro Ser
20 25 30
Thr Gly Pro Glu Lys Lys Ala Gly Ser Gln Gly Leu Arg Phe Arg
35 40 45
Leu Ala Gly Phe Pro Arg Lys Pro Tyr Glu Gly Arg Val Glu Ile
50 55 60
Gln Arg Ala Gly Glu Trp Gly Thr Ile Cys Asp Asp Asp Phe Thr
65 70 75
Leu Gln Ala Ala His Ile Leu Cys Arg Glu Leu Gly Phe Thr Glu
80 ' 85 90
Ala Thr Gly Trp Thr His Ser Ala Lys Tyr Gly Pro Gly Thr Gly
95 100 105
Arg Ile Trp Leu Asp Asn Leu Ser Cys Ser Gly Thr Glu Gln Ser
110 115 120
Val Thr Glu Cys Ala Ser Arg Gly Trp Gly Asn Ser Asp Cys Thr
125 130 135
His Asp Glu Asp Ala Gly Val Ile Cys Lys Asp Gln Arg Leu Pro
140 145 150
Gly Phe Ser Asp Ser Asn Val Ile Glu Ala Arg Val Arg Leu Lys
155 160 165
Gly Gly Ala His Pro Gly Glu Gly Arg Val Glu Val Leu Lys Ala
170 175 180
Ser Thr Trp Gly Thr Val Cys Asp Arg Lys Trp Asp Leu His Ala
185 190 195
Ala Ser Val Val Cys Arg Glu Leu Gly Phe Gly Ser Ala Arg Glu
200 205 210
Ala Leu Ser Gly Ala Arg Met Gly Gln Gly Met Gly Ala Ile His
215 220 225
Leu Ser Glu Val Arg Cys Ser Gly Gln Glu Leu Ser Leu Trp Lys
230 235 240
Cys Pro His Lys Asn Ile Thr Ala Glu Asp Cys Ser His Ser Gln
245 250 255
Asp Ala Gly Val Arg Cys Asn Leu Pro Tyr Thr Gly Ala Glu Thr
260 265 270
Arg Glu Thr Trp Tyr Trp Asp Ser Gly Asn Ile Thr Glu Val Val
275 280 285
Met Ser Gly Val Arg Cys Thr Gly Thr Glu Leu Ser Leu Asp Gln
290 295 300
Cys Ala His His Gly Thr His Ile Thr Cys Lys Arg Thr Gly Thr
305 310 315
Arg Phe Thr Ala Gly Val Ile Cys Ser Glu Thr Ala Ser Asp Leu
17/76
CA 02460476 2004-03-15
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320 325 330
Leu Leu His Ser Ala Leu Val Gln Glu Thr Ala Tyr Ile Glu Asp
335 340 345
Arg Pro Leu His Met Leu Tyr Arg Ala Ala Glu Glu Asn Cys Leu
350 355 360
Ala Ser Ser Ala Arg Ser Ala Asn Trp Pro Tyr Gly His Arg Arg
365 370 375
Leu Leu Arg Phe Ser Ser Gln Ile His Asn Leu Gly Arg Ala Asp
380 385 390
Phe Arg Pro Lys Ala Gly Arg His Ser Trp Val Trp His Glu Cys
395 400 405
His Gly His Tyr His Ser Met Asp Ile Phe Thr His Tyr Asp Ile
410 415 420
~Leu Thr Pro Asn Gly Thr Lys Val Ala Glu Gly His Lys Ala Ser
425 430 435
Phe Cys Leu Glu Asp Thr Glu Cys Gln Glu Asp Val Ser Lys Arg
440 445 450
Tyr Glu Cys Ala Asn Phe Gly Glu Gln Gly Ile Thr Val Gly Cys
455 460 465
Trp Asp Leu Tyr Arg His Asp Ile Asp Cys Gln Trp Ile Asp Ile
470 475 480
Thr Asp Val Lys Pro Gly Asn Tyr Ile Leu Gln Val Val Ile Asn
485 490 495
Pro Asn Phe Glu Val Ala Glu Ser Asp Phe Thr Asn Asn Ala Met
500 505 510
Lys Cys Asn Cys Lys Tyr Asp Gly His Arg Ile Trp Val His Asn
515 520 525
Cys His Ile Gly Asp Ala Phe Ser Glu Glu Ala Asn Arg Arg Phe
530 535 540
Glu Arg Tyr Pro Gly Gln Thr Ser Asn Gln Ile Ile
545 550
<210> 13
<211> 303
<212> PRT
<213> Homo Sapiens
<220>
<221> mist-feature
<223> Incyte ID No: 55141453CD1
<400> 13
Met Gly Ser Ala Gly Arg Leu His Tyr Leu Ala Met Thr Ala Glu
1 5 10 15
Asn Pro Thr Pro Gly Asp Leu Ala Pro Ala Pro Leu Ile Thr Cys
20 25 ~ 30
Lys Leu Cys Leu Cys Glu Gln Ser Leu Asp Lys Met Thr Thr Leu
35 40 45
Gln Glu Cys Gln Cys Ile Phe Cys Thr Ala Cys Leu Lys Gln Tyr
50 55 60
Met Gln Leu Ala Ile Arg Glu Gly Cys Gly Ser Pro Ile Thr Cys
65 70 75
Pro Asp Met Val Cys Leu Asn His Gly Thr Leu G1n Glu Ala Glu
80 85 90
Ile Ala Cys Leu Val Pro Val Asp Gln Phe Gln Leu Tyr Gln Arg
95 100 105
18/76
CA 02460476 2004-03-15
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Leu Lys Phe Glu Arg Glu Val His Leu Asp Pro Tyr Arg Thr Trp
110 115 120
Cys Pro Val Ala Asp Cys Gln Thr Val Cys Pro Val Ala Ser Ser
125 130 135
Asp Pro Gly Gln Pro Val Leu Val Glu Cys Pro Ser Cys His Leu
140 145 150
Lys Phe Cys Ser Cys Cys Lys Asp Ala Trp His Ala Glu Val Ser
155 160 165
Cys Arg Asp Ser Gln Pro Ile Val Leu Pro Thr Glu His Arg Ala
170 175 180
Leu Phe Gly Thr Asp Ala Glu Ala Pro Ile Lys Gln Cys Pro Val
185 190 195
Cys Arg Val Tyr Ile Glu Arg Asn Glu Gly Cys Ala Gln Met Met
200 205 210
Cys Lys Asn Cys Lys His Thr Phe Cys Trp Tyr Cys,Leu Gln Asn
215 220 225
Leu Asp Asn Asp Ile Phe Leu Arg His Tyr Asp Lys Gly Pro Cys
230 235 240
Arg Asn Lys Leu Gly His Ser Arg Ala Ser Val Met Trp Asn Arg
245 250 255
Thr Gln Val Val Gly Ile Leu Val Gly Leu Gly Ile Ile Ala Leu
260 265 ~ 270
Val Thr Ser Pro Leu Leu Leu Leu Ala Ser Pro Cys Ile Ile Cys
275 280 285
Cys Val Cys Lys Ser Cys Arg Gly Lys Lys Lys Lys His Asp Pro
290 295 300
Ser Thr Thr
<210> 14
<211> 945
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503135CD1
<400> 14
Met Ala Glu Gly Gly Gly Cys Arg Glu Arg Pro Asp Ala Glu Thr
1 5 10 15
Gln Lys Ser Glu Leu Gly Pro Leu Met Arg Thr Thr Leu Gln Arg
20 25 30
Gly Ala Gln Trp Tyr Leu Ile Asp Ser Arg Trp Phe Lys Gln Trp
35 40 45
Lys Lys Tyr Val Gly Phe Asp Ser Trp Gly Met Tyr Asn Val Gly
50 55 60
Glu His Asn Leu Phe Pro Gly Pro Ile Asp Asn Ser Gly Leu Phe
65 70 75
Ser Asp Pro Glu Ser Gln Thr Leu Lys Glu His Leu Ile Asp Glu
g0 85 90
Leu Asp Tyr Val Leu Val Pro Thr Glu Ala Trp Asn Lys Leu Leu
g5 100 105
Asn Trp Tyr Gly Cys Val Glu Gly Gln Gln Pro Ile Val Arg Lys
110 115 120
Val Val Glu His Gly Leu Phe Val Lys His Cys Lys Val Glu Val
19/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
125 130 135
Tyr Leu Leu Glu Leu Lys Leu Cys Glu Asn Ser Asp Pro Thr Asn
140 145 150
Val Leu Ser Cys His Phe Ser Lys Ala Asp Thr Ile Ala Thr Ile
155 160 165
Glu Lys Glu Met Arg Lys Leu Phe Asn Ile Pro Ala Glu Arg Glu
170 175 180
Thr Arg Leu Trp Asn Lys Tyr Met Ser Asn Thr Tyr Glu Gln Leu
185 190 195
Ser Lys Leu Asp Asn Thr Val Gln Asp Ala Gly Leu Tyr Gln Gly
200 205 210
Gln Val Leu Val Ile Glu Pro Gln Asn Glu Asp Gly Thr Trp Pro
215 220 225
Arg Gln Thr Leu Gln Ser Lys Ser Ser Thr Ala Pro Ser Arg Asn
230 235 240
Phe Thr Thr Ser Pro Lys Ser Ser Ala Ser Pro Tyr Ser Ser Val
245 250 255
Ser Ala Ser Leu Ile Ala Asn Gly Asp Ser Thr Ser Thr Cys Gly
260 265 270
Met His Ser Ser Gly Val Ser Arg Gly Gly Ser Gly Phe Ser Ala
275 280 285
Ser Tyr Asn Cys Gln Glu Pro Pro Ser Ser His Ile Gln Pro Gly
290 295 300
Leu Cys Gly Leu Gly Asn Leu Gly Asn Thr Cys Phe Met Asn Ser
305 310 315
Ala Leu Gln Cys Leu Ser Asn Thr Ala Pro Leu Thr Asp Tyr Phe
320 325 330
Leu Lys Asp Glu Tyr Glu Ala Glu Ile Asn Arg Asp Asn Pro Leu
335 340 345
Gly Met Lys Gly Glu Ile Ala Glu Ala Tyr Ala Glu Leu Ile Lys
350 355 360
Gln Met Trp Ser Gly Arg Asp Ala His Val Ala Pro Arg Met Phe
365 370 375
Lys Thr Gln Val Gly Arg Phe Ala Pro Gln Phe Ser Gly Tyr Gln
380 385 390
Gln Gln Asp Ser Gln Glu Leu Leu Ala Phe Leu Leu Asp Gly Leu
395 400 405
His Glu Asp Leu Asn Arg Val Val Ala Lys Glu Ala Trp Glu Asn
410 415 420
His Arg Leu Arg Asn Asp Ser Val Ile Val Asp Thr Phe His Gly
425 430 435
Leu Phe Lys Ser Thr Leu Val Cys Pro Glu Cys Ala Lys Val Ser
440 445 450
Val Thr Phe Asp Pro Phe Cys Tyr Leu Thr Leu Pro Leu Pro Leu
455 460 465
Lys Lys Asp Arg Val Met Glu Val Phe Leu Val Pro Ala Asp Pro
470 475 480
His Cys Arg Pro Thr Gln Tyr Arg Val Thr Val Pro Leu Met Gly
485 490 495
Ala Val Ser Asp Leu Cys Glu Ala Leu Ser Arg Leu Ser Gly Ile
500 505 510
Ala Ala Glu Asn Met Val Val Ala Asp Val Tyr Asn His Arg Phe
515 520 525
His Lys Ile Phe Gln Met Asp Glu Gly Leu Asn His Ile Met Pro
530 535 540
Arg Asp Asp Ile Phe Val Tyr Glu Val Cys Ser Thr Ser Val Asp
20/76
CA 02460476 2004-03-15
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545 550 555
Gly Ser Glu Cys Val Thr Leu Pro Val Tyr Phe Arg Glu Arg Lys
560 565 570
Ser Arg Pro Ser Ser Thr Ser Ser Ala Ser Ala Leu Tyr Gly Gln
575 580 585
Pro Leu Leu Leu Ser Val Pro Lys His Lys Leu Thr Leu Glu Ser
590 595 600
Leu Tyr Gln Ala Val Cys Asp Arg Ile Ser Arg Tyr Val Lys Gln
605 610 615
Pro Leu Pro Asp Glu Phe Gly Ser Ser Pro Leu Glu Pro Gly Ala
620 625 630
Cys Asn Gly Ser Arg Asn Ser Cys Glu Gly Glu Asp Glu Glu Glu
635 640 645
Met Glu His Gln Glu Glu Gly Lys Glu Gln Leu Ser Glu Thr Glu
650 655 660
Gly Ser Gly Glu Asp Glu Pro Gly Asn Asp Pro Ser Glu Thr Thr
665 670 675
Gln Lys Lys Ile Lys Gly Gln Pro Cys Pro Lys Arg Leu Phe Thr
680 685 690
Phe Ser Leu Val Asn Ser Tyr C~~ly Thr Ala Asp Ile Asn Ser Leu
695 700 705
Ala Ala Asp Gly Lys Leu Leu Lys Leu Asn Ser Arg Ser Thr Leu
710 715 720
Ala Met Asp Trp Asp Ser Glu Thr Arg Arg Leu Tyr Tyr Asp Glu
725 730 735
Gln Glu Ser Glu Ala Tyr Glu Lys His Val Ser Met Leu Gln Pro
740 745 750
Gln Lys Lys Lys Lys Thr Thr Val Ala Leu Arg Asp Cys Ile Glu
755 760 765
Leu Phe Thr Thr Met Glu Thr Leu Gly Glu His Asp Pro Trp Tyr
770 775 780
Cys Pro Asn Cys Lys Lys His Gln Gln Ala Thr Lys Lys Phe Asp
785 790 795
Leu Trp Ser Leu Pro Lys Ile Leu Val Val His Leu Lys Arg Phe
800 805 810
Ser Tyr Asn Arg Tyr Trp Arg Asp Lys Leu Asp Thr Val Val Glu
815 820 825
Phe Pro Ile Arg Gly Leu Asn Met Ser Glu Phe Val Cys Asn Leu
830 835 840
Ser Ala Arg Pro Tyr Val Tyr Asp Leu Ile Ala Val Ser Asn His
845 850 855
Tyr Gly Ala Met Gly Val Gly His Tyr Thr Ala Tyr Ala Lys Asn
860 865 870
Lys Leu Asn Gly Lys Trp Tyr Tyr Phe Asp Asp Ser Asn Val Ser
875 880 885
Leu Ala Ser Glu Asp Gln Ile Val Thr Lys Ala Ala Tyr Val Leu
890 895 900
Phe Tyr Gln Arg Arg Asp Asp Glu Phe Tyr Lys Thr Pro Ser Leu
905 910 915
Ser Ser Ser Gly Ser Ser Asp Gly Gly Thr Arg Pro Ser Ser Ser
920 925 930
Gln Gln Gly Phe Gly Asp Asp Glu Ala Cys Ser Met Asp Thr Asn
935 940 945
<210> 15
21/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
<211> 315
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503242CD1
<400> 15
Met Lys Arg Ala Ala Met Ala Leu His Ser Pro Gln Tyr Ile Phe
1 5 10 15
Gly Asp Phe Ser Pro Asp Glu Phe Asn Gln Phe Phe Val Thr Pro
20 25 30
Arg Ser Ser Val Glu Gly Arg Gln Glu Asp Ala Glu Glu Tyr Leu
35 40 ~ 45
Gly Phe Ile Leu Asn Gly Leu His Glu Glu Met Leu Asn Leu Lys
50 55 60
Lys Leu Leu Ser Pro Ser Asn Glu Lys Leu Thr Ile Ser Asn Gly
65 70 75
Pro Lys Asn His Ser Val Asn Glu Glu Glu Gln Glu Glu Gln Gly
80 85 90
Glu Gly Ser Glu Asp Glu Trp Glu Gln Val Gly Pro Arg Asn Lys
95 100 105
Thr Ser Val Thr Arg Gln Ala Asp Phe Val Gln Thr Pro Ile Thr
110 115 120
Gly Ile Phe Gly Gly His Ile Arg Ser Val Val Tyr Gln Gln Ser
125 130 135
Ser Lys Glu Ser Ala Thr Leu Gln Pro Phe Phe Thr Leu Gln Leu
140 145 150
Asp Ile Gln Ser Asp Lys Ile Arg Thr Val Gln Asp Ala Leu Glu
155 160 165
Ser Leu Val Ala Arg Glu Ser Val Gln Gly Tyr Thr Thr Lys Thr
170 175 180
Lys Gln Glu Val Glu Ile Ser Arg Arg Val Thr Leu Glu Lys Leu
185 190 195
Pro Pro Val Leu Val Leu His Leu Lys Arg Phe Val Tyr Glu Lys
200 205 210
Thr Gly Gly Cys Gln Lys Leu Ile Lys Asn Ile Glu Tyr Pro Val
215 220 225
Asp Leu Glu Ile Ser Lys Glu Leu Leu Ser Pro Gly Val Lys Asn
230 235 240
Lys Asn Phe Lys Cys His Arg Thr Tyr Arg Leu Phe Ala Val Val
245 250 255
Tyr His His Gly Asn Ser Ala Thr Gly Gly His Tyr Thr Thr Asp
260 265 270
Val Phe Gln Ile Gly Leu Asn Gly Trp Leu Arg Ile Asp Asp Gln
275 280 285
Thr Val Lys Val Ile Asn Gln Tyr Gln Val Val Lys Pro Thr Ala
290 295 300
Glu Arg Thr Ala Tyr Leu Leu Tyr Tyr Arg Arg Val Asp Leu Leu
305 310 315
<210> 16
<211> 204
<212> PRT
22/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7504774CD1
<400> 16
Met Lys Arg Leu Thr Cys Phe Phe Ile Cys Phe Phe Leu Ser Glu
1 5 10 15
Val Ser Gly Phe Glu Ile Pro Ile Asn Gly Leu Ser Glu Phe Val
20 25 30
Asp Tyr Glu Asp Leu Val Glu Leu Ala Pro Gly Lys Phe Gln Leu
35 40 45
Val Ala Glu Asn Arg Arg Tyr Gln Arg Ser Leu Pro Gly Glu Ser
50 55 60
Glu Glu Met Met Glu Glu Val Asp Gln Val Thr Leu Tyr Ser Tyr
65 70 75
Lys Val Gln Ser Thr Ile Thr Ser Arg Met Ala Thr Thr Met Ile
80 85 90
Gln Ser Lys Val Val Asn Asn Ser Pro Gln Pro Gln Asn Val Val
95 100 105
Phe Asp Val Gln Ile Pro Lys Gly Ala Phe Ile Ser Asn Phe Ser
110 ~ 115 120
Met Thr Val Asp Gly Lys Thr Phe Arg Ser Ser Ile Lys Glu Lys
125 130 135
Thr Val Gly Arg Ala Leu Tyr Ala Gln Ala Arg Ala Lys Gly Lys
140 145 150
Thr Ala Gly Leu Val Arg Gly Leu Gln Lys Asp Tyr Arg Thr Asp
155 160 165
Leu Val Phe Gly Thr Asp Val Thr Cys Trp Phe Val His Asn Ser
170 175 180
Gly Lys Gly Phe Tle Asp Gly His Tyr Lys Asp Tyr Phe Val Pro
185 190 195
Gln Leu Tyr Ser Phe Leu Lys Arg Pro
200
<210> 17
<211> 228
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503166CD1
<400> 17
Met Phe Leu Val Asn Ser Phe Leu Lys Gly Gly Gly Gly Gly Gly
1 5 10 15
Gly Gly Gly Gly Arg Ile Leu Gly Gly Val Ile Ser Ala Ile Ser
20 25 30
Glu Ala Ala Ala Gln Tyr Asn Pro Glu Pro Pro Pro Pro Arg Thr
35 40 45
His Tyr Ser Asn Ile Glu Ala Asn Glu Ser Glu Glu Val Arg Gln
50 55 60
Phe Arg Arg Leu Phe Ala Gln Leu Ala Gly Asp Asp Met Glu Val
65 70 75
23/76
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Ser Ala Thr Glu Leu Met Asn Ile Leu Asn Lys Val Val Thr Arg
80 85 90
His Pro Asp Leu Lys Thr Asp Gly Phe Gly Ile Asp Thr Cys Arg
95 100 105
Ser Met Val Ala Val Met Asp Ser Asp Thr Thr Gly Lys Leu Gly
110 115 120
Phe Glu Glu Phe Lys Tyr Leu Trp Asn Asn Ile Lys Arg Trp Gln
125 130 135
Ala Ile Tyr Lys Gln Phe Asp Thr Asp Arg Ser Gly Thr Ile Cys
140 145 150
Ser Ser Glu Leu Pro Gly Ala Phe Glu Ala Ala Gly Phe His Leu
155 160 165
Asn Glu His Leu Tyr Asn Met Ile Ile Arg Arg Tyr Ser Asp Glu
170 175 180
Ser Gly Asn Met Asp Phe Asp Asn Phe Ile Ser Cys Leu Val Arg
185 190 195
Leu Asp Ala Met Phe Arg Ala Phe Lys Ser Leu Asp Lys Asp Gly
200 205 210
Thr Gly Gln Ile Gln Val Asn Ile Gln Glu Trp Leu Gln Leu Thr
215 220 225
Met Tyr Ser
<210> 18
<211> 160
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503171CD1
<400> 18
Met Glu Tyr Leu Ile Gly Ile Gln Gly Pro Asp Tyr Val Leu Val
1 5 10 15
Ala Ser Asp Arg Val Ala Ala Ser Asn Ile Val Gln Met Lys Asp
20 25 30
Gly Tyr Glu Leu Ser Pro Thr Ala Ala Ala Asn Phe Thr Arg Arg
35 40 45
Asn Leu Ala Asp Cys Leu Arg Ser Arg Thr Pro Tyr His Val Asn
50 55 60
Leu Leu Leu Ala Gly Tyr Asp Glu His Glu Gly Pro Ala Leu Tyr
65 70 75
Tyr Met Asp Tyr Leu Ala Ala Leu Ala Lys Ala Pro Phe A1a Ala
80 85 90
His Gly Tyr Gly Ala Phe Leu Thr Leu Ser Ile Leu Asp Arg Tyr
95 100 105
Tyr Thr Pro Thr Ile Ser Arg Glu Arg Ala Val Glu Leu Leu Arg
110 115 120
Lys Cys Leu Glu Glu Leu Gln Lys Arg Phe Ile Leu Asn Leu Pro
125 130 135
Thr Phe Ser Val Arg Ile Ile Asp Lys Asn Gly Ile His Asp Leu
140 145 150
Asp Asn Ile Ser Phe Pro Lys Gln Gly Ser
155 160
24/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
<210> 19
<211> 139
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503220CD1
<400> 19
Met Leu Ser Leu Asp Phe Leu Asp Asp Val Arg Arg Met Asn Lys
1 5 10 15
Arg Gln Leu Tyr Tyr Gln Val Leu Asn Phe Gly Met Ile Val Ser
20 25 30
Ser Ala Leu Met Ile Trp Lys Gly Leu Met Val Ile Thr Gly Ser
35 40 45
Glu Ser Pro Ile Val Val Val Leu Ser Gly Ser Met Glu Pro Ala
50 55 60
Phe His Arg Gly Asp Leu Leu Phe Leu Thr Asn Arg Val Glu Asp
65 70 75
Pro Ile Arg Val Gly Glu Ile Val Val Phe Arg Ile Glu Gly Arg
80 85 90
Glu Ile Pro Ile Val His Arg Val Leu Lys Ile His Glu Lys Phe
95 100 105
Val Pro Tyr Ile Gly Ile Val Thr Ile Leu Met Asn Asp Tyr Pro
110 115 120
Lys Phe Lys Tyr Ala Va1 Leu Phe Leu Leu Gly Leu Phe Val Leu
125 130 135
Val His Arg Glu
<210> 20
<211> 216
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6145631CD1
<400> 20
Met Lys Gly Lys Glu Glu Lys Glu Gly Gly Ala Arg Leu Gly Ala
1 5 10 15
Gly Gly Gly Ser Pro Glu Lys Ser Pro Ser Ala Gln Glu Leu Lys
20 25 30
Glu Gln Gly Asn Arg Leu Phe Val Gly Arg Lys Tyr Pro Glu Ala
35 40 45
Ala Ala Cys Tyr Gly Arg Ala Ile Cys Gln Leu Glu Met Glu Ser
50 55 60
Tyr Asp Glu Ala Ile Ala Asn Leu Gln Arg Ala Tyr Ser Leu Ala
65 70 75
Lys Glu Gln Arg Leu Asn Phe Gly Asp Asp Ile Pro Ser Ala Leu
80 85 90
Arg Ile Ala Lys Lys Lys Arg Trp Asn Ser Ile Glu Glu Arg Arg
95 100 105
Ile His Gln Glu Ser Glu Leu His Ser Tyr Leu Ser Arg Leu Ile
25/76
CA 02460476 2004-03-15
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110 115 120
Ala Ala Glu Arg Glu Arg Glu Leu Glu Glu Cys Gln Arg Asn His
125 130 135
Glu Gly Asp Glu Asp Asp Ser His Val Arg Ala Gln Gln Ala Cys
140 145 150
Ile Glu Ala Lys His Val Arg Val Pro Pro Thr His Met Trp Val
155 160 165
Cys Val Cys Ala Arg Gly Val Gly Ala Ser Pro Pro Cys Val Gly
170 175 180
Ser Val Pro His Gly Gly Gly Arg Trp Gly Val Ser Pro Lys His
185 190 195
Ser Thr Gln Leu Phe Thr Gly Gln Val His Gly Gly His Gly Arg
200 205 210
Ala Phe Phe Ser Gly Gly
215
<210> 21
<211> 194
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7504980CD1
<400> 21
Met Val Ser Arg Met Val Ser Thr Met Leu Ser Gly Leu Leu Phe
1 5 10 15
Trp Leu Ala Ser Gly Trp Thr Pro Ala Phe Ala Tyr Ser Pro Arg
20 25 30
Thr Pro Asp Arg Val Ser Glu Ala Asp Ile Gln Arg Leu Leu His
35 40 45
Gly Val Met Glu Gln Leu Gly Ile Ala Arg Pro Arg Val Glu Tyr
50 55 60
Pro Ala His Gln Ala Met Asn Leu Val Gly Pro Gln Ser Ile Glu
65 70 75
Gly Gly Ala His Glu Gly Leu Gln His Leu Gly Pro Phe Gly Asn
80 85 90
Ile Pro Asn Ile Val Ala Glu Leu Thr Gly Asp Asn Ile Pro Lys
95 100 105
Asp Phe Ser Glu Asp Gln Gly Tyr Pro Asp Pro Pro Asn Pro Cys
110 115 120
Pro Val Gly Lys Thr Ala Asp Asp Gly Cys Leu Glu Asn Thr Pro
125 130 135
Asp Thr Ala Glu Phe Ser Arg Glu Phe Gln Leu His Gln His Leu
140 145 150
Phe Asp Pro G1u His Asp Tyr Pro Gly Leu Gly Lys Trp Ser Val
155 160 165
Asn Pro Tyr Leu Gln Gly Gln Arg Leu Asp Asn Val Val Ala Lys
170 175 180
Lys Ser Val Pro His Phe Ser Asp Glu Asp Lys Asp Pro Glu
185 190
<210> 22
<211> 361
<212> PRT
26/76
CA 02460476 2004-03-15
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<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3118830CD1
<400> 22
Met Ala Gln Arg Cys Val Cys Val Leu Ala Leu Val Ala Met Leu
1 5 10 15
Leu Leu Val Phe Pro Thr Val Ser Arg Ser Met Gly Pro Arg Ser
20 25 30
Gly Glu His Gln Arg Ala Ser Arg Tle Pro Ser Gln Phe Ser Lys
35 40 45
Glu Glu Arg Val Ala Met Lys Glu Ala Leu Lys Gly Ala Ile Gln
50 55 60
Ile Pro Thr Val Thr Phe Ser Ser Glu Lys 5er Asn Thr Thr Ala
65 70 75
Leu Ala Glu Phe Gly Lys Tyr Ile His Lys Val Phe Pro Thr Val
80 85 90
Val Ser Thr Ser Phe Ile Gln His Glu Val Val Glu Glu Tyr Ser
95 100 105
His Leu Phe Thr Ile Gln Gly Ser Asp Pro Ser Leu Gln Pro Tyr
110 115 120
Leu Leu Met Ala His Phe Asp Val Val Pro Ala Pro Glu Glu Gly
125 130 135
Trp Glu Val Pro Pro Phe Ser Gly Leu Glu Arg Asp Gly Val Ile
140 145 150
Tyr Gly Arg Gly Thr Leu Asp Asp Lys Asn Ser Va1 Met Ala Leu
155 160 165
Leu Gln Ala Leu Glu Leu Leu Leu Ile Arg Lys Tyr Ile Pro Arg
170 175 180
x
Arg Ser Phe Phe Ile Ser Leu Gly His Asp Glu Glu Ser Ser Gly
185 190 195
Thr Gly Ala Gln Arg Ile Ser Ala Leu Leu Gln Ser Arg Gly Val
200 205 210
Gln Leu Ala Phe Ile Val Asp Glu Gly Gly Phe Ile Leu Asp Asp
215 220 225
Phe Ile Pro Asn Phe Lys Lys Pro Ile Ala Leu Ile Ala Val Ser
230 235 240
Glu Lys Gly Ser Met Asn Leu Met Leu Gln Val Asn Met Thr Ser
245 250 255
Gly His Ser Ser Ala Pro Pro Lys Glu Thr Ser Ile Gly Ile Leu
260 265 270
Ala Ala Ala Val Ser Arg Leu Glu Gln Thr Pro Met Pro Ile Ile
275 280 285
Phe Gly Ser Gly Thr Val Val Thr Val Leu Gln Gln Leu Ala Asn
290 295 300
Glu Val Tyr Gly Glu Lys Ser Leu Asn Gln Cys Asn Asn Gln Asp
305 310 315
His His Gly Thr His His Ile Gln Ser Arg Gly Gln Val Gln Cys
320 325 330
His Pro Pro Ser G1y Pro Gly His Ser Gln Leu Pro Asp Ser Pro
335 340 345
Trp Thr Asp Ser Pro Arg Gly Pro Arg Thr His Glu Glu His Cys
350 355 360
Gly
27/76
CA 02460476 2004-03-15
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<210> 23
<211> 487
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7505187CD1
<400> 23
Met Ala Pro Ala Arg Thr Met Ala Arg Ala Arg Leu Ala Pro Ala
1 5 10 15
Gly Ile Pro Ala Val Ala Leu Trp Leu Leu Cys Thr Leu Gly Leu
20 25 30
Gln Gly Thr Gln Ala Gly Pro Pro Pro Ala Pro Pro Gly Leu Pro
35 40 45
Ala Gly Ala Asp Cys Leu Asn Ser Phe Thr Ala Gly Val Pro Gly
50 55 60
Phe Val Leu Asp Thr Asn Ala Ser Val Ser Asn Gly Ala Thr Phe
65 70 75
Leu Glu Ser Pro Thr Val Arg Arg Gly Trp Asp Cys Val Arg Ala
gp g5 90
Cys Cys Thr Thr Gln Asn Cys Asn Leu Ala Leu Val Glu Leu Gln
95 100 105
Pro Asp Arg Gly Glu Asp Ala Ile Ala Ala Cys Phe Leu Ile Asn
110 115 120
Cys Leu Tyr Glu Gln Asn Phe Val Cys Lys Phe Ala Pro Arg Glu
125 130 135
Gly Phe Ile Asn Tyr Leu Thr Arg Glu Val Tyr Arg Ser Tyr Arg
140 145 150
Gln Leu Arg Thr Gln Gly Phe Gly Gly Ser Gly Ile Pro Lys Ala
155 160 165
Trp Ala Gly Ile Asp Leu Lys Val Gln Pro Gln Glu Pro Leu Val
170 175 180
Leu Lys Asp Val Glu Asn Thr Asp Trp Arg Leu Leu Arg Gly Asp
185 190 195
Thr Asp Val Arg Val Glu Arg Lys Asp Pro Asn Gln Val Glu Leu
200 205 210
Trp Gly Leu Lys Glu Gly Thr Tyr Leu Phe Gln Leu Thr Val Thr
215 220 225
Ser Ser Asp His Pro Glu Asp Thr Ala Asn Val Thr Val Thr Val
230 235 240
Leu Ser Thr Lys Gln Thr Glu Asp Tyr Cys Leu Ala Ser Asn Lys
245 250 255
Val Gly Arg Cys Arg Gly Ser Phe Pro Arg Trp Tyr Tyr Asp Pro
260 265 270
Thr Glu Gln Ile Cys Lys Ser Phe Val Tyr Gly Gly Cys Leu Gly
275 280 285
Asn Lys Asn Asn Tyr Leu Arg Glu Glu Glu Cys Ile Leu Ala Cys
290 295 300
Arg Gly Val Gln Gly Gly Pro Leu Arg Gly Ser Ser Gly Ala Gln
305 310 315
Ala Thr Phe Pro Gln Gly Pro Ser Met Glu Arg Arg His Pro Asp
320 325 330
28/76
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Thr Ser Gly Phe Asp Glu Leu Gln Arg Ile His Phe Pro Ser Asp
335 340 345
Lys Gly His Cys Val Asp Leu Pro Asp Thr Gly Leu Cys Lys Glu
350 355 360
Ser Ile Pro Arg Trp Tyr Tyr Asn Pro Phe Ser Glu His Cys Ala
365 370 375
Arg Phe Thr Tyr Gly Gly Cys Tyr Gly Asn Lys Asn Asn Phe Glu
380 385 390
Glu Glu Gln Gln Cys Leu Glu Ser Cys Arg Gly Ile Ser Lys Lys
395 400 405
Asp Val Phe Gly Leu Arg Arg Glu Ile Pro Ile Pro Ser Thr Gly
410 415 420
Ser Val Glu Met Ala Val Ala Val Phe Leu Val Ile Cys Ile Val
425 430 435
Val Val Val Ala Ile Leu Gly Tyr Cys Phe Phe Lys Asn Gln Arg
440 445 450
Lys Asp Phe His Gly His His His His Pro Pro Pro Thr Pro Ala
455 460 465
Ser Ser Thr Val Ser Thr Thr Glu Asp Thr Glu His Leu Val Tyr
470 475 480
Asn His Thr Thr Arg Pro Leu
485
<210> 24
<211> 329
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7506567CD1
<400> 24
Met Lys Leu Ile Thr Ile Leu Phe Leu Cys Ser Arg Leu Leu Leu
1 5 10 15
Ser Leu Thr Gln Glu Ser Gln Ser Glu Glu Ile Asp Cys Asn Asp
20 25 30
Lys Asp Leu Phe Lys Ala Val Val Thr Ala Gln Tyr Asp Cys Leu
35 40 45
Gly Cys Val His Pro Ile Ser Thr Gln Ser Pro Asp Leu Glu Pro
50 55 60
Ile Leu Arg His Gly Ile Gln Tyr Phe Asn Asn Asn Thr Gln His
65 70 75
Ser Ser Leu Phe Met Leu Asn Glu Val Lys Arg Ala Gln Arg Gln
80 85 90
Val Val Ala Gly Leu Asn Phe Arg Ile Thr Tyr Ser Ile Val Gln
95 100 105
Thr Asn Cys Ser Lys Glu Asn Phe Leu Phe Leu Thr Pro Asp Cys
110 115 120
Lys Ser Leu Trp Asn Gly Asp Thr Gly Glu Cys Thr Asp Asn Ala
125 130 135
Tyr Ile Asp Ile Gln Leu Arg Ile Ala Ser Phe Ser Gln Asn Cys
140 145 150
Asp Ile Tyr Pro Gly Lys Asp Phe Val Gln Pro Pro Thr Lys Ile
155 160 165
Cys Val Gly Cys Pro Arg Asp Ile Pro Thr Asn Ser Pro Glu Leu
29/76
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170 175 180
Glu Glu Thr Leu Thr His Thr Ile Thr Lys Leu Asn Ala Glu Asn
185 190 195
Asn Ala Thr Phe Tyr Phe Lys Ile Asp Asn Val Lys Lys Ala Arg
200 205 210
Val Gln Val Val Ala Gly Lys Lys Tyr Phe Ile Asp Phe Val Ala
215 220 225
Arg Glu Thr Thr Cys Ser Lys Glu Ser Asn Glu Glu Leu Thr Glu
230 235 240
Ser Cys Glu Thr Lys Lys Leu Gly Gln Ser Leu Asp Cys Asn Ala
245 250 255
Glu Val Tyr Val Val Pro Trp Glu Lys Lys Ile Tyr Pro Thr Val
260 265 270
Asn Cys Gln Pro Leu Gly Met Ile Ser Leu Met Lys Arg Pro Pro
275 280 285
Gly Phe Ser Pro Phe Arg Ser Ser Arg Ile Gly Glu Tle Lys Glu
290 295 300
Glu Thr Thr Ser His Leu Arg Ser Cys Glu Tyr Lys Gly Arg Pro
305 310 315
Pro Lys Ala Gly Ala Glu Pro Ala Ser Glu Arg Glu Val Ser
320 325
<210> f5
<211> 151
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503673CD1
<400> 25
Met Pro Gly Gly Ala Gly Ala Ala Arg Leu Cys Leu Leu Ala Phe
1 5 10 15
Ala Leu Gln Pro Leu Arg Pro Arg Ala Ala Arg Glu Pro Gly Trp
20 25 30
Thr Arg Gly Ser Glu Glu Gly Ser Pro Lys Leu Gln His Glu Leu
35 40 45
Ile Ile Pro G1n Trp Lys Thr Ser Glu Ser Pro Va1 Arg Glu Lys
50 55 60
His Pro Leu Lys Ala Glu Leu Arg Val Met Ala Glu Gly Arg Glu
65 70 75
Leu Ile Leu Asp Leu Glu Lys Asn Glu Gln Leu Phe Ala Pro Ser
80 85 90
Tyr Thr Glu Thr His Tyr Thr Ser Ser Gly Asn Pro Gln Thr Thr
95 100 105
Thr Arg Lys Leu Glu Asp His Cys Phe Tyr His Gly Thr Val Arg
110 115 120
Glu Thr Glu Leu Ser Ser Val Thr Leu Ser Thr Cys Arg Gly Ile
125 130 135
Ser Phe Arg Arg Ile Asp Glu Thr Arg Thr Pro Pro Asn Thr Ser
140 145 150
Ser
<210> 26
30/76
CA 02460476 2004-03-15
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<211> 341
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 90123303CD1
<400> 26
Met Gln Gly Thr Pro Gly Gly Gly Thr Arg Pro Gly Pro Ser Pro
1 5 10 15
Val Asp Arg Arg Thr Leu Leu Val Phe Ser Phe Ile Leu Ala Ala
20 25 30
Ala Leu Gly Gln Met Asn Phe Thr Gly Asp Gln Val Leu Arg Val
35 40 45
Leu Ala Lys Asp Glu Lys Gln Leu Ser Leu Leu Gly Asp Leu Glu
50 55 60
Gly Leu Lys Pro Gln Lys Val Asp Phe Trp Arg Gly Pro Ala Arg
65 70 75
Pro Ser Leu Pro Val Asp Met Arg Val Pro Phe Ser Glu Leu Lys
80 85 90
Asp Ile Lys Ala Tyr Leu Glu Ser His Gly Leu Ala Tyr Ser Ile
95 100 105
Met Ile Lys Asp Ile Gln Val Leu Leu Asp Glu Glu Arg Gln Ala
110 115 120
Met Ala Lys Ser Arg Arg Leu Glu Arg Ser Thr Asn Ser Phe Ser
125 130 135
Tyr Ser Ser Tyr His Thr Leu Glu Glu Ile Tyr Ser Trp Ile Asp
140 145 150
Asn Phe Val Met Glu His Ser Asp Ile Val Ser Lys Ile Gln Ile
155 160 165
Gly Asn Ser Phe Glu Asn Gln Ser Ile Leu Val Leu Lys Phe Ser
170 175 180
Thr Gly Gly Ser Arg His Pro Ala Ile Trp Ile Asp Thr Gly Ile
185 190 195
His Ser Arg Glu Trp Ile Thr His Ala Thr Gly Ile Trp Thr Ala
200 205 210
Asn Lys Ile Val Ser Asp Tyr Gly Lys Asp Arg Val Leu Thr Asp
215 220 225
Ile Leu Asn Ala Met Asp Ile Phe Ile Glu Leu Val Thr Asn Pro
230 235 240
Asp Gly Phe Ala Phe Thr His Ser Met Asn Arg Leu Trp Arg Lys
245 250 255
Asn Lys Ser Ile Arg Pro Gly Ile Phe Cys Ile Gly Val Asp Leu
260 265 270
Asn Arg Asn Trp Lys Ser Gly Phe Gly Asp Val Ala Ser Gly Ile
275 280 285
Thr Val Asp Trp Ala Tyr Asp Ser Gly Ile Lys Tyr Ala Phe Ser
290 295 300
Phe Glu Leu Arg Asp Thr Gly Gln Tyr Gly Phe Leu Leu Pro Ala
305 310 315
Thr Gln Ile Ile Pro Thr Ala Gln Glu Thr Trp Met Ala Leu Arg
320 325 330
Thr Ile Met Glu His Thr Leu Asn His Pro Tyr
335 340
31/76
CA 02460476 2004-03-15
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<210> 27
<211> 521
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7505765CD1
<400> 27
Met Met Pro Thr Pro Val Ile Leu Leu Lys Glu Gly Thr Asp Ser
1 5 10 15
Ser Gln Gly Ile Pro Gln Leu Val Ser Asn Ile Ser Ala Cys Gln
20 25 30
Val Ile Ala Glu Ala Val Arg Thr Thr Leu Gly Pro Arg Gly Met
35 40 45
Asp Lys Leu Ile Val Asp Gly Arg Ala Lys Thr Leu Val Asp Ile
50 55 60
Ala Lys Ser Gln Asp Ala Glu Val Gly Asp Gly Thr Thr Ser Val
65 70 75
Thr Leu Leu Ala Ala Glu Phe Leu Lys Gln Val Lys Pro Tyr Val
80 85 90
Glu Glu Gly Leu His Pro Gln Ile Ile Ile Arg Ala Phe Arg Thr
g5 100 105
Ala Thr Gln Leu Ala Val Asn Lys Ile Lys Glu Ile Ala Val Thr
110 115 120
Val Lys Lys Ala Asp Lys Val Glu Gln Arg Lys Leu Leu Glu Lys
125 130 135
Cys Ala Met Thr Ala Leu Ser Ser Lys Leu Tle Ser Gln Gln Lys
140 145 150
Ala Phe Phe Ala Lys Met Val Val Asp Ala Val Met Met Leu Asp
155 160 165
Asp Leu Leu Gln Leu Lys Met Ile Gly Ile Lys Lys Val Gln Gly
170 175 180
Gly Ala Leu Glu Asp Ser Gln Leu Val Ala Gly Val Ala Phe Lys
185 190 195
Lys Thr Phe Ser Tyr Ala Gly Phe Glu Met Gln Pro Lys Lys Tyr
200 205 210
His Asn Pro Lys Ile Ala Leu Leu Asn Val Glu Leu Glu Leu Lys
215 220 225
Ala Glu Lys Asp Asn Ala Glu Ile Arg Val His Thr Val Glu Asp
230 235 240
Tyr Gln Ala Ile Val Asp Ala Glu Trp Asn Ile Leu Tyr Asp Lys
245 250 255
Leu Glu Lys Ile His His Ser Gly Ala Lys Val Val Leu Ser Lys
260 265 270
Leu Pro Ile Gly Asp Val Ala Thr Gln Tyr Phe Ala Asp Arg Asp
275 280 285
Met Phe Cys Ala Gly Arg Val Pro Glu Glu Asp Leu Lys Arg Thr
290 295 300
Met Met Ala Cys Gly Gly Ser Ile Gln Thr Ser Val Asn Ala Leu
305 310 315
Ser Ala Asp Val Leu Gly Arg Cys Gln Val Phe Glu Glu Thr Gln
320 325 330
Ile Gly Gly Glu Arg Tyr Asn Phe Phe Thr Gly Cys Pro Lys Ala
335 340 345
32/76
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Lys Thr Cys Thr Phe Ile Leu Arg Gly Gly Ala Glu Gln Phe Met
350 355 360
Glu Glu Thr Glu Arg Ser Leu His Asp Ala Ile Met Ile Val Arg
365 370 375
Arg Ala Ile Lys Asn Asp Ser Val Val Ala Gly Gly Gly Ala Ile
380 385 390
Glu Met Glu Leu Ser Lys Tyr Leu Arg Asp Tyr Ser Arg Thr Ile
395 400 405
Pro Gly Lys Gln Gln Leu Leu Ile Gly Ala Tyr Ala Lys Ala Leu
410 415 420
Glu Ile Ile Pro Arg Gln Leu Cys Asp Asn Ala Gly Phe Asp Ala
425 430 435
Thr Asn Ile Leu Asn Lys Leu Arg Ala Arg His Ala Gln Gly Gly
440 445 450
Thr Trp Tyr Gly Val Asp Ile Asn Asn Glu Asp Ile Ala Asp Asn
455 460 465
Phe Glu Ala Phe Val Trp Glu Pro Ala Met Val Arg Ile Asn Ala
470 475 480
Leu Thr Ala Ala Ser Glu Ala Ala Cys Leu Ile Val Ser Val Asp
485 490 495
Glu Thr Ile Lys Asn Pro Arg Ser Thr Val Asp Ala Pro Thr Ala
500 505 510
Ala Gly Arg Gly Arg Gly Arg Gly Arg Pro His
515 520
<2l0> 28
<211> 432
<212> PRT
<213> Homo Sapiens
<220>
<221> misc-feature
<223> Incyte ID No: 7505775CD1
<400> 28
Met Asp Ile Asp Lys Asp Leu Glu Ala Pro Leu Tyr Leu Thr Pro
10 15
Glu Gly Trp Ser Leu Phe Leu Gln Arg Tyr Tyr Gln Val Val His
20 25 30
Glu Gly Ala Glu Leu Arg His Leu Asp Thr Gln Val Gln Arg Cys
35 40 45
Glu Asp Ile Leu Gln Gln Leu Gln Ala Val Val Pro Gln Ile Asp
50 55 60
Met Glu Gly Asp Arg Asn Ile Trp Ile Val Lys Pro Gly Ala Lys
65 70 75
Ser Arg Gly Arg Gly Ile Met Cys Met Asp His Leu Glu Glu Met
80 85 90
Leu Lys Leu Val Asn Gly Asn Pro Val Val Met Lys Asp Gly Asn
g5 100 105
Ser Val His Leu Cys Asn Asn Ser Ile Gln Lys His Leu Glu Asn
110 115 l20
Ser Cys His Arg His Pro Leu Leu Pro Pro Asp Asn Met Trp Ser
125 130 135
Ser Gln Arg Phe Gln Ala His Leu Gln Glu Met Gly Ala Pro Asn
140 145 150
Ala Trp Ser Thr Ile Ile Val Pro Gly Met Lys Asp Ala Val Ile
33/76
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155 160 165
His Ala Leu Gln Thr Ser Gln Asp Thr Val Gln Cys Arg Lys Ala
170 175 180
Ser Phe Glu Leu Tyr Gly Ala Asp Phe Val Phe Gly Glu Asp Phe
185 190 195
Gln Pro Trp Leu Ile Glu Ile Asn Ala Ser Pro Thr Met Ala Pro
200 205 210
Ser Thr Ala Val Thr Ala Arg Leu Cys Ala Gly Val Gln Ala Asp
215 220 225
Thr Leu Arg Val Val Ile Asp Arg Met Leu Asp Arg Asn Cys Asp
230 235 240
Thr Gly Ala Phe Glu Leu Ile Tyr Lys Gln Pro Ala Val Glu Val
245 250 255
Pro Gln Tyr Val Gly Ile Arg Leu Leu Val Glu Gly Phe Thr Ile
260 265 270
Lys Lys Pro Met Ala Met Cys His Arg Arg Met Gly Val Arg Pro
275 280 285
Ala Val Pro Leu Leu Thr Gln Arg Gly Ser Gly Glu Gly Lys Asp
290 295 300
Ser Gly Thr Pro Thr His Arg Ser Ala Ser Arg Lys Gly Thr Gly
305 310 315
Ala Arg Ser Leu Gly His Ser Glu Lys Pro Val Ser Thr Ala Thr
320 325 330
Thr Ser Ala Pro Gly Lys Gly Lys Lys Gly Lys Ala Lys Arg Ala
335 340 345
Thr Ala Leu Val Cys Pro Asn Leu Trp Glu Trp Asp Ala Pro Ser
350 355 360
Thr Arg Met Gly Cys Ile Phe Thr Met Thr Phe Ser Ser Gly Asp
365 370 375
Arg Gln Pro His His Leu Asn Arg Leu Pro Leu Ser Pro Lys Asn
380 385 390
Pro Gln Ala Leu Gly Lys Thr Ile Pro Pro Lys His Pro Ser Val
395 400 405
Pro Arg Arg Phe Ile Pro Ala Leu Gln Ala Pro Pro Asn His Leu
410 415 420
Asp Gln Pro Pro His Gln Arg Ala Thr Ser Ser Lys
425 430
<210> 29
<211> 220
<212> PRT
<213> Homo Sapiens
<220>
<221> misc-feature
<223> Incyte ID No: 7500181CD1
<400> 29
Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile
1 5 10 15
Gly Ala A1a Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu
20 25 30
Cys Glu Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg
35 40 45
Gly Arg Ala Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val
50 55 60
34/76
CA 02460476 2004-03-15
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Leu Thr Ala Ala His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu
65 70 75
Gly Arg His Ser Leu Phe His Pro Glu Asp Thr Gly Gln Val Phe
80 85 90
Gln Val Ser His Ser Phe Pro His Pro Leu Tyr Asp Met Ser Leu
95 100 105
Leu Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser Ile Glu
110 115 120
Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu
125 130 135
His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln Lys
140 145 150
Val Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys
155 160 165
Ser Thr Cys Ser Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly
170 175 180
Val Leu Gln Gly Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu
185 190 195
Pro Glu Arg Pro Ser Leu Tyr Thr Lys Val Val His Tyr Arg Lys
200 205 210
Trp Ile Lys Asp Thr Ile Val Ala Asn Pro
215 220
<210> 30
<211> 195
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503799CD1
<400> 30
Met Glu Ala Phe Leu Gly Ser Arg Ser Gly Leu Trp Ala Gly Gly
1 5 10 15
Pro Ala Pro Gly Gln Phe Tyr Arg Ile Pro Ser Thr Pro Asp Ser
20 25 30
Phe Met Asp Pro Ala Ser Ala Leu Tyr Arg Gly Pro Ile Thr Arg
35 40 45
Thr Gln Ile Asp Glu Glu Leu Leu Gly Asp Gly His Ser Tyr Ser
50 55 60
Pro Arg Ala Ile His Ser Trp Leu Thr Arg Ala Met Tyr Ser Arg
65 70 75
Arg Ser Lys Met Asn Pro Leu Trp Asn Thr Met Val Ile Gly Gly
g0 g5 90
Tyr Ala Asp Gly Glu Ser Phe Leu Gly Tyr Val Asp Met Leu Gly
95 100 105
Val Ala Tyr Glu Ala Pro Ser Leu Ala Thr Gly Tyr Gly Ala Tyr
110 115 120
Leu Ala Gln Pro Leu Leu Arg Glu Val Leu Glu Lys Gln Pro Va1
125 130 135
Leu Ser Gln Thr Glu Ala Arg Asp Leu Val Glu Arg Cys Met Arg
140 145 150
Val Leu Tyr Tyr Arg Asp Ala Arg Ser Tyr Asn Arg Phe Gln Thr
155 160 165
Ala Thr Val Thr Glu Lys Gly Val Glu Ile Glu Gly Pro Leu Ser
35/76
CA 02460476 2004-03-15
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170 175 180
Thr Glu Thr Asn Trp Asp Ile Ala His Met Ile Ser Gly Phe Glu
185 190 195
<210> 31
<211> 372
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7504602CD1
<400> 31
Met Val Trp Lys Val Ala Val Phe Leu Ser Val Ala Leu Gly Ile
1 5 10 15
Gly Ala Val Pro Ile Asp Asp Pro Glu Asp Gly Gly Lys His Trp
20 25 30
Val Val Ile Val Ala Gly Ser Asn Gly Trp Tyr Asn Tyr Arg His
35 40 45
Gln Ala Asp Ala Cys His Ala Tyr Gln Ile Ile His Arg Asn Gly
50 55 60
Ile Pro Asp Glu Gln Ile Val'Val Met Met Tyr Asp Asp Ile Ala
65 70 75
Tyr Ser Glu Asp Asn Pro Thr Pro Gly Ile Val Ile Asn Arg Pro
80 85 90
Asn Gly Thr Asp Val Tyr Gln Gly Val Pro Lys Asp Tyr Thr Gly
95 100 105
Glu Asp Val Thr Pro Gln Asn Phe Leu Ala Val Leu Arg Gly Asp
110 115 120
Ala Glu Ala Val Lys Gly Ile Gly Ser Gly Lys Val Leu Lys Ser
125 130 135
Gly Pro Gln Asp His Val Phe Ile Tyr Phe Thr Asp His Gly Ser
140 145 150
Thr Gly Ile Leu Val Phe Pro Asn Glu Asp Leu His Val Lys Asp
155 160 165
Leu Asn Glu Thr Ile His Tyr Met Tyr Lys His Lys Met Tyr Arg
170 175 180
Lys Met Val Phe Tyr Ile Glu Ala Cys Glu Ser Gly Ser Met Met
185 190 195
Asn His Leu Pro Asp Asn Ile Asn Val Tyr Ala Thr Thr Ala Ala
200 205 210
Asn Pro Arg Glu Ser Ser Tyr Ala Cys Tyr Tyr Asp Glu Lys Arg
215 220 225
Ser Thr Tyr Leu Gly Asp Trp Tyr Ser Val Asn Trp Met Glu Asp
230 235 240
Ser Asp Val Glu Asp Leu Thr Lys Glu Thr Leu His Lys G1n Tyr
245 250 255
His Leu Val Lys Ser His Thr Asn Thr Ser His Val Met Gln Tyr
260 265 270
Gly Asn Lys Thr Ile Ser Thr Met Lys Val Met Gln Phe Gln Gly
275 280 285
Met Lys Arg Lys Ala Ser Ser Pro Val Pro Leu Pro Pro Val Thr
290 295 300
His Leu Asp Leu Thr Pro Ser Pro Asp Val Pro Leu Thr Ile Met
36/76
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305 310 315
Lys Arg Lys Leu Met Asn Thr Asn Asp Leu Glu Glu Ser Arg Gln
320 325 330
Leu Thr Glu Glu Ile Gln Arg His Leu Asp Asp Lys Ile Val His
335 340 345
Gly Pro Arg Val Pro Trp Ser Leu Leu Lys Ser Cys Leu Leu Glu
350 355 360
Ala Phe Pro Ser Val Ser Ala Pro Pro Thr Val Cys
365 370
<210> 32
<211> 410
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5873533CD1
<400> 32
Met His Leu Thr Asn Tyr Ser Ile Asn Lys His Ser Ser Asn Phe
1 5 10 15
Ser Arg Asp Ala His Ser Gly Ser Lys Arg Lys Leu Ser Thr Phe
20 25 30
Ser Ala Tyr Leu Glu Asp His Ser Tyr Asn Val Glu Gln Ile Trp
35 40 45
Arg Asp Ile Glu Asp Val Ile Ile Lys Thr Leu Ile Ser Ala His
50 55 60
Pro Ile Ile Arg His Asn Tyr His Thr Cys Phe Pro Asn His Thr
65 70 75
Leu Asn Ser Ala Cys Phe Glu Ile Leu Gly Phe Asp Ile Leu Leu
80 85 90
Asp His Lys Leu Lys Pro Trp Leu Leu Glu Val Asn His Ser Pro
g5 100 105
Ser Phe Ser Thr Asp Ser Arg Leu Asp Lys Glu Val Lys Asp Gly
110 115 120
Leu Leu Tyr Asp Thr Leu Val Leu Ile Asn Leu Glu Ser Cys Asp
125 130 135
Lys Lys Lys Val Leu Glu Glu Glu Arg Gln Arg Gly Gln Phe Leu
140 145 150
Gln Gln Cys Cys Ser Arg Glu Met Arg Ile Glu Glu Ala Lys Gly
155 160 165
Phe Arg Ala Val Gln Leu Lys Lys Thr Glu Thr Tyr Glu Lys Glu
170 175 180
Asn Cys Gly Gly Phe Arg Leu Ile Tyr Pro Ser Leu Asn Ser Glu
185 190 195
Lys Tyr Glu Lys Phe Phe Gln Asp Asn Asn Ser Leu Phe Gln Asn
200 205 210
Thr Val Ala Ser Arg Ala Arg Glu Glu Tyr Ala Arg Gln Leu Ile
215 220 225
Gln Glu Leu Arg Leu Lys Arg Glu Lys Lys Pro Phe Gln Met Lys
230 235 240
Lys Lys Val Glu Met Gln Gly Glu Ser Ala Gly Glu Gln Val Arg
245 250 255
Lys Lys Gly Met Arg Gly Trp Gln Gln Lys Gln Gln Gln Lys Asp
260 265 270
37/76
CA 02460476 2004-03-15
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Lys Ala Ala Thr Gln Ala Ser Lys Gln Tyr Ile Gln Pro Leu Thr
275 280 285
Leu Val Ser Tyr Thr Pro Asp Leu Leu Leu Ser Val Arg Gly Glu
290 295 300
Arg Lys Asn Glu Thr Asp Ser Ser Leu Asn Gln Glu Ala Pro Thr
305 310 315
Glu Lys Ala Ser Ser Val Phe Pro Lys Leu Thr Ser Ala Lys Pro
320 325 330
Phe Ser Ser Leu Pro Asp Leu Arg Asn Ile Asn Leu Ser Ser Ser
335 340 345
Lys Leu Glu Pro Ser Lys Pro Asn Phe Ser Ile Lys Glu Ala Lys
350 355 360
Ser Ala Ser Ala Val Asn Val Phe Thr Gly Thr Val Lys Thr Pro
365 370 375
Pro Cys Leu Gly Ser Ala Thr Pro Ala Val Thr Ala Leu Ala Arg
380 385 390
Arg Gly Ser Trp Met Cys Pro Pro Ser Ser Cys Arg Val Leu Arg
395 400 405
Ala Ile Met Leu Leu
410
<210> 33
<211> 265
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> 2ncyte ID No: 71033239CD1
<400> 33
Met Ala Glu Asp Lys Glu Thr Lys His Gly Gly His Lys Asn Gly
1 5 10 . l5
Arg Lys Gly Gly Leu Ser Gly Thr Ser Phe Phe Thr Trp Phe Met
20 25 30
Val Ile Ala Leu Leu Gly Val Trp Thr Ser Val Ala Val Val Trp
35 40 45
Phe Asp Leu Val Asp Tyr Glu Glu Val Leu Gly Lys Leu Gly Ile
50 55 60
Tyr Asp Ala Asp Gly Asp Gly Asp Phe Asp Val Asp Asp Ala Lys
65 70 75
Val Leu Leu Gly Leu Lys Glu Arg Ser Thr Ser Glu Pro Ala Val
80 85 90
Pro Pro Glu Glu Ala Glu Pro His Thr Glu Pro Glu Glu Gln Val
95 100 105
Pro Val Glu Ala Glu Pro Gln Asn Ile Glu Asp Glu Ala Lys Glu
110 115 120
Gln Ile Gln Ser Leu Leu His Glu Met Val His Ala Glu His Val
125 130 135
Glu Gly Glu Asp Leu Gln Gln Glu Asp Gly Pro Thr Gly Glu Pro
140 145 150
Gln Gln Glu Asp Asp Glu Phe Leu Met Ala Thr Asp Val Asp Asp
155 160 165
Arg Phe Glu Thr Leu Glu Pro Glu Val Ser His Glu Glu Thr Glu
170 175 180
His Ser Tyr His Val Glu Glu Thr Asp 5er Ser Glu Pro Val Val
38/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
185 190 195
Glu Asp Glu Arg Leu His His Asp Thr Asp Asp Val Thr Tyr Gln
200 205 210
Val Tyr Glu Glu Gln Ala Val Tyr Glu Pro Leu Glu Asn Glu Gly
215 220 225
Ile Glu Ile Thr Glu Val Thr Ala Pro Pro Glu Asp Asn Pro Val
230 235 240
Glu Asp Ser Gln Val Ile Val Glu Glu Val Ser Ile Phe Pro Val
245 250 255
Glu Glu Gln Gln Glu Val Pro Pro Asp Thr
260 265
<210> 34
<211> 146
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7506001CD1
<400> 34
Met Ala Val Ala Asn Ser Ser Pro Val Asn Pro Val Val Phe Phe
1 5 10 15
Asp Val Ser Ile Gly Gly Gln Glu Val Gly Arg Met Lys Ile Glu
20 25 30
Leu ~Phe Ala Asp Val Val Pro Lys Thr Ala Glu Asn Phe Arg Gln
35 40 45
Phe Cys Thr Gly Glu Phe Arg Lys Asp Gly Val Pro Ile Gly Tyr
50 55 60
Lys Gly Ser Thr Phe His Arg Val Ile Lys Asp Phe Met Ile Gln
65 70 75
Gly Gly Asp Phe Val Asn Ala Asn Ser Gly Pro Ser Thr Asn Gly
80 85 90
Cys Gln Phe Phe Ile Thr Cys Ser Lys Cys Asp Trp Leu Asp Gly
g5 100 105
Lys His Val Val Phe Gly Lys Ile Ile Asp Gly Leu Leu Val Met
110 115 120
Arg Lys Ile Glu Asn Val Pro Thr Gly Pro Asn Asn Lys Pro Lys
125 130 135
Leu Pro Val Val Ile Ser Gln Cys Gly Glu Met
140 145
<210> 35
<211> 196
<212> PRT
<213> Homo Sapiens
<220>
<221> mist-feature
<223> Incyte ID No: 7506026CD1
<400> 35
Met Glu Asp Ser Met Asp Met Asp Met Ser Pro Leu Arg Pro Gln
1 5 10 15
Asn Tyr Leu Phe Ala Val Glu Glu Asp Ala Glu Ser Glu Asp Glu
39/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
20 25 30
Glu Glu Glu Asp Val Lys Leu Leu Ser Ile Ser Gly Lys Arg Ser
35 40 45
Ala Pro Gly Gly Gly Ser Lys Val Pro Gln Lys Lys Val Lys Leu
50 55 60
Ala Ala Asp Glu Asp Asp Asp Asp Asp Asp Glu Glu Asp Asp Asp
65 70 75
Glu Asp Asp Asp Asp Asp Asp Phe Asp Asp Glu Glu Ala Glu Glu
80 85 90
Lys Ala Pro Val Lys Lys Ser Ile Arg Asp Thr Pro Ala Lys Asn
95 100 105
Ala Gln Lys Ser Asn Gln Asn Gly Lys Asp Ser Lys Pro Ser Ser
110 115 120
Thr Pro Arg Ser Lys Gly Gln Glu Ser Phe Lys Lys Gln Glu Lys
125 130 135
Thr Pro Lys Thr Pro Lys Gly Pro Ser Ser Val Glu Asp Ile Lys
140 145 150
Ala Lys Met Gln Ala Ser Ile Glu Lys Gly Gly Ser Leu Pro Lys
155 160 165
Val Glu Ala Lys Phe Ile Asn Tyr Val Lys Asn Cys Phe Arg Met
170 175 180
Thr Asp Gln Glu Ala Ile Gln Asp Leu Trp Gln Trp Arg Lys Ser
185 190 195
Leu
<210> 36
<211> 431
<212> PRT
<213> Homo sapiens
<220>
<221> misc-feature
<223> Incyte ID No: 8167924CD1
<400> 36
Met Ser Asp Glu Gly Ser Arg Gly Ser Arg Leu Pro Leu Ala Leu
10 15
Pro Pro Ala Ser Gln Gly Cys Ser Ser Gly Gly Gly Gly Gly Gly
20 25 30
Ser Ser Ala Gly Gly~Ser Gly Asn Ser Arg Pro Pro Arg Asn Leu
35 40 45
Gln Gly Leu Leu Gln Met Ala Ile Thr Ala Gly Ser Glu Glu Pro
50 55 60
Asp Pro Pro Pro Glu Pro Met Ser Glu Glu Arg Arg Gln Trp Leu
65 70 75
Gln Glu Ala Met Ser Ala Ala Phe Arg Gly Gln Arg Glu Glu Val
g0 85 90
Glu Gln Met Lys Ser Cys Leu Arg Val Leu Ser Gln Pro Met Pro
g5 100 105
Pro Thr Ala Gly Glu Ala Glu Gln Ala Ala Asp Gln Gln Glu Arg
110 115 120
Glu Gly Ala Leu Glu Leu Leu Ala Asp Leu Cys Glu Asn Met Asp
125 130 135
Asn Ala Ala Asp Phe Cys Gln Leu Ser Gly Met His Leu Leu Val
140 145 150
40/76
CA 02460476 2004-03-15
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Gly Arg Tyr Leu Glu Ala Gly Ala Ala Gly Leu Arg Trp Arg Ala
155 160 165
Ala Gln Leu Ile Gly Thr Cys Ser Gln Asn Val Ala Ala Ile Gln
170 175 180
Glu Gln Val Leu Gly Leu Gly Ala Leu Arg Lys Leu Leu Arg Leu
185 190 195
Leu Asp Arg Asp Ala Cys Asp Thr Val Arg Val Lys Ala Leu Phe
200 205 210
Ala Ile Ser Cys Leu Val Arg Glu Gln Glu Ala Gly Leu Leu Gln
215 220 225
Phe Leu Arg Leu Asp Gly Phe Ser Val Leu Met Arg Ala Met Gln
230 235 240
Gln Gln Val Gln Lys Leu Lys Val Lys Ser Ala Phe Leu Leu Gln
245 250 255
Asn Leu Leu Val Gly His Pro Glu His Lys Gly Thr Leu Cys Ser
260 265 270
Met Gly Met Val Gln Gln Leu Val Ala Leu Val Arg Thr Glu His
275 280 285
Ser Pro Phe His Glu His Val Leu Gly Ala Leu Cys Arg Val Cys
290 295 300
Ala Ser Val Gly Ser Arg Asn Trp Ala Trp Arg Ser Ser Ser Ala
305 310 315
Thr Ala Val Ser Cys Cys Ser Ser Met Arg Ser Thr Arg Arg Ser
320 325 330
Trp Ser Ser Val Lys Ser Cys Tyr Arg Pro Val Ser Pro Ala Gln
335 340 345
Arg Thr Thr Ala Trp Ile Gly Glu Thr Arg Trp Leu Leu Ala Pro
350 355 360
Phe Ser Val Gly Thr Pro Gly Leu Leu Pro Pro Ser Pro Pro Thr
365 370 375
Arg Pro Ser Pro Lys Gly Ser Gln Gly Leu Gly Ala Trp Thr Gln
380 385 390
Gly Val Pro Ala Arg Leu Cys Ala Val Pro Gly Arg Gly Ala Glu
395 400 405
Lys Gly Thr Ser Ser Leu Asp Pro Thr Ser His Ala Leu Thr Leu
410 415 420
Ile Pro Val Leu Leu Ser Thr Gln Leu Phe Gln
425 430
<210> 37
<211> 300
<212> PRT
<213> Homo Sapiens
<220>
<221> mist-feature
<223> Incyte ID No: 2365313CD1
<400> 37
Met Glu Pro Pro Met Glu Pro Ser G1y Gly Glu Gln Glu Pro Gly
10 15
Ala Val Arg Phe Leu Asp Leu Pro Trp Glu Asp Val Leu Leu Pro
20 25 30
His Val Leu Asn Arg Val Pro Leu Arg Gln Leu Leu Arg Leu Gln
35 40 45
Arg Val Ser Arg Ala Phe Arg Ser Leu Val Gln Leu His Leu Ala
41/76
CA 02460476 2004-03-15
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50 55 60
Gly Leu Arg Arg Phe Asp Ala Ala Gln Val Gly Pro Gln Ile Pro
65 70 75
Arg Ala Ala Leu Ala Arg Leu Leu Arg Asp Ala Glu Gly Leu Gln
80 85 90
Glu Leu Ala Leu Ala Pro Cys His Glu Trp Leu Ser Asp Glu Asp
g5 100 105
Leu Val Pro Val Leu Ala Arg Asn Pro Gln Leu Arg Ser Val Ala
110 115 120
Leu Gly Gly Cys Gly Gln Leu Ser Arg Arg Ala Leu Gly Ala Leu
125 130 135
Ala Glu G1y Cys Pro Arg Leu Gln Arg Leu Ser Leu Ala His Cys
140 145 150
Asp Trp Val Asp Gly Leu Ala Leu Arg Gly Leu Ala Asp Arg Cys
155 160 165
Pro Ala Leu Glu Glu Leu Asp Leu Thr Ala Cys Arg Gln Leu Lys
170 175 180
Asp Glu Ala Ile Val Tyr Leu Ala Gln Arg Arg Gly Ala Gly Leu
185 190 195
Arg Ser Leu Ser Leu Ala Val Asn Ala Asn Val Gly Asp Ala Ala
200 205 210
Val Gln Glu Leu Ala Arg Asn Cys Pro Glu Leu His His Leu Asp
215 220 225
Leu Thr Gly Cys Leu Arg Val Gly Ser Asp Gly Val Arg Thr Leu
230 235 240
Ala Glu Tyr Cys Pro Val Leu Arg Ser Leu Arg Val Arg His Cys
245 250 255
His His Val Ala Glu Ser Ser Leu Ser Arg Leu Arg Lys Arg Gly
260 265 270
Val Asp Ile Asp Val Glu Pro Pro Leu His Gln Ala Leu Val Leu
275 280 285
Leu Gln Asp Met Ala Gly Phe Ala Pro Phe Va1 Asn Leu Gln Val
2g0 295 300
<210> 38
<211> 554
<212> PRT
<213> Homo sapiens
<220>
<221> misc-feature
<223> Incyte ID No: 7503156CD1
<400> 38
Met Ala Glu Leu Ser Glu Glu Ala Leu Leu Ser Val Leu Pro Thr
1 5 10 15
Ile Arg Val Pro Lys Ala Gly Asp Arg Val His Lys Asp Glu Cys
20 25 30
Ala Phe Ser Phe Asp Thr Pro Glu Ser Glu Gly Gly'Leu Tyr Ile
35 40 45
Cys Met Asn Thr Phe Leu Gly Phe Gly Lys Gln Tyr Val Glu Arg
50 55 60
His Phe Asn Lys Thr Gly Gln Arg Val Tyr Leu His Leu Arg Arg
65 70 75
Thr Arg Arg Pro Lys Glu Glu Asp Pro Ala Thr Gly Thr Gly Asp
42/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
80 85 90
Pro Pro Arg Lys Lys Pro Thr Arg Leu Ala Ile Gly Val Glu Gly
95 100 105
Gly Phe Asp Leu Ser Glu Glu Lys Phe Glu Leu Asp Glu Asp Val
110 115 120
Lys Ile Val Ile Leu Pro Asp Tyr Leu Glu Ile Ala Arg Asp Gly
125 130 135
Leu Gly Gly Leu Pro Asp Ile Val Arg Asp Arg Val Thr Ser Ala
140 145 150
Val Glu Ala Leu Leu Ser Ala Asp Ser Ala Ser Arg Lys Gln Glu
155 160 165
Val Gln Ala Trp Asp Gly Glu Val Arg Gln Val Ser Lys His Ala
170 175 180
Phe Ser Leu Lys Gln Leu Asp Asn Pro Ala Arg Ile Pro Pro Cys
185 190 195
Gly Trp Lys Cys Ser Lys Cys Asp Tyr Ile Met Gln Leu Pro Val
200 205 210
Pro Met Asp Ala Ala Leu Asn Lys Glu Glu Leu Leu Glu Tyr Glu
215 220 225
Glu Lys Lys Arg Gln Ala Glu Glu Glu Lys Met Ala Leu Pro Glu
230 235 240
Leu Val Arg Ala Gln Val Pro Phe Ser Ser Cys Leu Glu Ala Tyr
245 250 255
Gly Ala Pro Glu Gln Val Asp Asp Phe Trp Ser Thr Ala Leu Gln
260 265 270
Ala Lys Ser Val Ala Val Lys Thr Thr Arg Phe Ala Ser Phe Pro
275 280 285
Asp Tyr Leu Val Tle Gln Ile Lys Lys Phe Thr Phe Gly Leu Asp
290 295 300
Trp Val Pro Lys Lys Leu Asp Val Ser Ile Glu Met Pro Glu Glu
305 310 315
Leu Asp Ile Ser Gln Leu Arg Gly Thr Gly Leu Gln Pro Gly Glu
320 325 ~ 330
Glu Glu Leu Pro Asp Ile Ala Pro Pro Leu Val Thr Pro Asp Glu
335 340 345
Pro Lys Ala Pro Met Leu Asp Glu Ser Val Ile Tle Gln Leu Val
350 355 360
Glu Met Gly Phe Pro Met Asp Ala Cys Arg Lys Ala Val Tyr Tyr
365 370 375
Thr Gly Asn Ser Gly Ala Glu Ala Ala Met Asn Trp Val Met Ser
380 385 390
His Met Asp Asp Pro Asp Phe Ala Asn Pro Leu Ile Leu Pro Gly
395 400 405
Ser Ser Gly Pro Gly Ser Thr Ser Ala Ala Ala Asp Pro Pro Pro
410 415 420
Glu Asp Cys Val Thr Thr Ile Val Ser Met Gly Phe Ser Arg Asp
425 430 435
Gln Ala Leu Lys Ala Leu Arg Ala Thr Asn Asn Ser Leu Glu Arg
440 445 450
Ala Val Asp Trp Ile Phe Ser His Ile Asp Asp Leu Asp Ala Glu
455 460 465
Ala Ala Met Asp Ile Ser Glu Gly Arg Ser Ala Ala Asp Ser Ile
470 475 480
Ser Glu Ser Val Pro Val Gly Pro Lys Val Arg Asp Gly Pro Gly
485 490 495
Lys Tyr Gln Leu Phe Ala Phe Ile Ser His Met Gly Thr Ser Thr
43/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
500 505 510
Met Cys Gly His Tyr Val Cys His Ile Lys Lys Glu G1y Arg Trp
515 520 525
Val Ile Tyr Asn Asp Gln Lys Val Cys Ala Ser Glu Lys Pro Pro
530 535 540
Lys Asp Leu Gly Tyr Ile Tyr Phe Tyr Gln Arg Val Ala Ser
545 550
<210> 39
<211> 278
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7505980CD1
<400> 39
Met Glu Thr Arg Tyr Asn Leu Lys Ser Pro Ala Val Lys Arg Leu
1 5 10 15
Met Lys Glu Ala Ala Glu Leu Lys Asp Pro Thr Asp His Tyr His
20 25 30
Ala Gln Pro Leu Glu Asp Asn Leu Phe Glu Trp His Phe Thr Val
35 40 ' 45
Arg Gly Pro Pro Asp Ser Asp Phe Asp Gly Gly Val Tyr His Gly
50 55 60
Arg Ile Val Leu Pro Pro Glu Tyr Pro Met Lys Pro Pro Ser Ile
65 70 75
Ile Leu Leu Thr Ala Asn Gly Arg Phe Glu Val Gly Lys Lys Ile
80 85 90
Cys Leu Ser Ile Ser Gly His His Pro Glu Thr Trp Gln Pro Ser
95 100 105
Trp Ser Ile Arg Thr Ala Leu Leu Ala Ile Ile Gly Phe Met Pro
110 115 120
Thr Lys Gly Glu Gly Ala Ile Gly Ser Leu Asp Tyr Thr Pro Glu
125 130 135
Glu Arg Arg Ala Leu Ala Lys Lys Ser Gln Asp Phe Cys Cys Glu
140 145 150
Gly Cys Gly Ser Ala Met Lys Asp Val Leu Leu Pro Leu Lys Ser
155 160 165
Gly Ser Asp Ser Ser Gln Ala Asp Gln Glu Ala Lys Glu Leu Ala
170 175 180
Arg Gln Ile Ser Phe Lys Tyr Gly Leu Gln Asn Ser Ser Ala Ala
185 190 195
Ser Phe His Gln Pro Thr Gln Pro Val Ala Lys Asn Thr Ser Met
200 205 210
Ser Pro Arg Gln Arg Arg Ala Gln Gln Gln Ser Gln Arg Arg Leu
215 220 225
Ser Thr Ser Pro Asp Val Ile Gln Gly His Gln Pro Arg Asp Asn
230 235 240
His Thr Asp His Gly Gly Ser Ala Val Leu Ile Val Ile Leu Thr
245 250 255
Leu Ala Leu Ala Ala Leu Ile Phe Arg Arg Ile Tyr Leu Ala Asn
260 265 270
Glu Tyr Ile Phe Asp Phe Glu Leu
275
44/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
<210> 40
<211> 688
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7646577CD1
<400> 40
Met Asp Arg Cys Lys His Val Gly Arg Leu Arg Leu Ala Gln Asp
1 5 10 15
His Ser Ile Leu Asn Pro Gln Lys Trp Cys Cys Leu Glu Cys Ala
20 25 30
Thr Thr Glu Ser Val Trp Ala Cys Leu Lys Cys Ser His Val Ala
35 40 45
Cys Gly Arg Tyr Ile Glu Asp His Ala Leu Lys His Phe Glu Glu
50 55 60
Thr Gly His Pro Leu Ala Met Glu Val Arg Asp Leu Tyr Val Phe
65 70 75
Cys Tyr Leu Cys Lys Asp Tyr Val Leu Asn Asp Asn Pro Glu Gly
80 85 90
Asp Leu Lys Leu Leu Arg Ser Ser Leu Leu Ala Val Arg Gly Gln
g5 100 105
Lys Gln Asp Thr Pro Val Arg Arg Gly Arg Thr Leu Arg Ser Met
110 115 120
Ala Ser Gly Glu Asp Val Val Leu Pro Gln Arg Ala Pro Gln Gly
125 130 135
Gln Pro Gln Met Leu Thr Ala Leu Trp Tyr Arg Arg Gln Arg Leu
140 145 150
Leu Ala Arg Thr Leu Arg Leu Trp Phe Glu Lys Ser Ser Arg Gly
155 160 165
Gln Ala Lys Leu Glu Gln Arg Arg Gln Glu Glu Ala Leu Glu Arg
170 175 180
Lys Lys Glu Glu Ala Arg Arg Arg Arg Arg Glu Val Lys Arg Arg
185 190 195
Leu Leu Glu Glu Leu Ala Ser Thr Pro Pro Arg Lys Ser Ala Arg
200 205 210
Leu Leu Leu His Thr Pro Arg Asp Ala Gly Pro Ala Ala Ser Arg
215 220 225
Pro Ala Ala Leu Pro Thr Ser Arg Arg Val Pro Ala Ala Thr Leu
230 235 240
Lys Leu Arg Arg Gln Pro Ala Met Ala Pro Gly Val Thr Gly Leu
245 250 255
Arg Asn Leu Gly Asn Thr Cys Tyr Met Asn Ser Ile Leu Gln Val
260 265 270
Leu Ser His Leu Gln Lys Phe Arg Glu Cys Phe Leu Asn Leu Asp
275 280 285
Pro Ser Lys Thr Glu His Leu Phe Pro Lys Ala Thr Asn Gly Lys
290 295 300
Thr Gln Leu Ser Gly Lys Pro Thr Asn Ser Ser Ala Thr Glu Leu
305 310 315
Ser Leu Arg Asn Asp Arg Ala Glu Ala Cys Glu Arg Glu Gly Phe
320 325 330
Cys Trp Asn Gly Arg Ala Ser Ile Ser Arg Ser Leu Glu Leu Ile
335 340 345
45/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
Gln Asn Lys Glu Pro Ser Ser Lys His Ile Ser Leu Cys Arg Glu
350 355 360
Leu His Thr Leu Phe Arg Val Met Trp Ser Gly Lys Trp Ala Leu
365 370 375
Val Ser Pro Phe Ala Met Leu His Ser Val Trp Ser Leu Ile Pro
380 385 390
Ala Phe Arg Gly Tyr Asp Gln Gln Asp Ala Gln Glu Phe Leu Cys
395 400 405
Glu Leu Leu His Lys Val Gln Gln Glu Leu Glu Ser Glu Gly Thr
410 415 420
Thr Arg Arg Ile Leu Tle Pro Phe Ser Gln Arg Lys Leu Thr Lys
425 430 435
Gln Val Leu Lys Val Val Asn Thr Ile Phe His Gly Gln Leu Leu
440 445 450
Ser Gln Val Thr Cys Ile Ser Cys Asn Tyr Lys Ser Asn Thr Ile
455 460 465
Glu Pro Phe Trp Asp Leu Ser Leu Glu Phe Pro Glu Arg Tyr His
470 475 480
Cys Ile Glu Lys Gly Phe Val Pro Leu Asn Gln Thr Glu Cys Leu
485 490 495
Leu Thr Glu Met Leu Ala Lys Phe Thr Glu Thr Glu Ala Leu Glu
500 505 510
Gly Arg Ile Tyr Ala Cys Asp Gln Cys Asn Ser Lys Arg Arg Lys
515 520 525
Ser Asn Pro Lys Pro Leu Val Leu Ser Glu Ala Arg Lys Gln Leu
530 535 540
Met Ile Tyr Arg Leu Pro Gln Val Leu Arg Leu His Leu Lys Arg
545 550 555
Phe Arg Trp Ser Gly Arg Asn His Arg Glu Lys Ile Gly Val His
560 565 570
Val Val Phe Asp Gln Val Leu Thr Met Glu Pro Tyr Cys Cys Arg
575 580 585
Asp Met Leu Ser Ser Leu Asp Lys Glu Thr Phe Ala Tyr Asp Leu
590 595 600
Ser Ala Val Val Met His His Gly Lys Gly Phe Gly Ser Gly His
605 610 615
Tyr Thr Ala Tyr Cys Tyr Asn Thr Glu Gly Gly Phe Trp Val His
620 625 630
Cys Asn Asp Ser Lys Leu Asn Val Cys Ser Val Glu Glu Val Cys
635 640 645
Lys Thr Gln Ala Tyr Ile Leu Phe Tyr Thr Gln Arg Thr Val Gln
650 655 660
Gly Asn Ala Arg Ile Ser Glu Thr His Leu Gln Ala Gln Val Gln
665 670 675
Ser Ser Asn Asn Asp Glu Gly Arg Pro Gln Thr Phe Ser
680 685
<210> 41
<211> 1544
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500287CB1
46/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
<400>, 41
aggaactcac acagcttttg gcctgagccc ccgttaccaa gagaaaggag gtttttgcca 60
aggactccaa ggggagtgca cttgatgctg gtcgggaccc aaagcgccca gccctccctg 120
agacattgtg tgagtcgggc tgggcctcaa acacggcccc cactgcccca ccccagccag 180
ggtggtgctt gtgtgggtag gactttaaat ccagctgcca gacccctgga cgggagaagg 240
agagacggct ggccaccatg cacggctcct gcagtttcct gatgcttctg ctgccgctac 300
tgctactgct ggtggccacc acaggccccg ttggagccct cacagatgag gagaaacgtt 360
tgatggtgga gctgcacaac ctctaccggg cccaggtatc cccgccggcc tcagacatgc 420
tgcacatgag atgggacgag gagctggccg ccttcgccaa ggcctacgca cggcagtgcg 480
tgtggggcca caacaaggag cgcgggcgcc gcggcgagaa tctgttcgcc atcacagacg 540
agggcatgga cgtgccgctg gccatggagg agtggcacca cgagcgtgag cactacaacc 600
tcagcgccgc cacctgcagc ccaggccaga tgtgcggcca ctacacgcag gtggtatggg 66.0
ccaagacaga gaggatcggc tgtggttccc acttctgtga gaagctccag ggtgttgagg 720
agaccaacat cgaattactg gtgtgcaact atgagcctcc ggggaacgtg aaggggaaac 780
ggccctacca ggaggggact ccgtgctccc aatgtccctc tggctaccac tgcaagaact 840
ccctctgtga acccatcgga agcccggaag atgctcagga tttgccttac ctggtaactg 900
aggccccatc cttccgggcg actgaagcat cagactctag gaaaatgggt gcagagggcc 960
ctgacaagcc tagcgtcgtg tcagggctga actcgggccc tggtcatgtg tggggccctc 1020
tcctgggact actgctcctg cctcctctgg tgttggctgg aatcttctga aggggatacc 1080
actcaaaggg tgaagaggtc agctgtcctc ctgtcatctt ccccaccctg tccccagccc 1140
ctaaacaaga tacttcttgg ttaaggccct ccggaaggga aaggctacgg ggcatgtgcc 1200
tcatcacacc atccatcctg gaggcacaag gcctggctgg ctgcgagctc aggaggccgc 1260
ctgaggactg cacaccgggc ccacacctot cctgcccctc cctcctgagt cctgggggtg 1320
ggaggatttg agggagctca ctgcctacct ggcctggggc tgtctgccca cacagcatgt 1380
gcgctctccc tgagtgcctg tgtagctggg gatggggatt cctaggggca gatgaaggac 1440
aagccccact ggagtggggt tctttgagtg ggggaggcag ggacgaggga aggaaagtaa 1500
ctcctgactc tccaataaaa acctgtccaa cctgtgaaaa aaaa 1544
<210> 42
<211> 1586
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500511CB1
<400> 42
caaattttcc agccgatcac tggagctgac ttccgcaatc ccgatggaat aaatctagca 60
cccctgatgg tgtgcccaca ctttgctgcc gaaacgaagc cagacaacag atttccatca 120
gcaggatgtg ggggctcaag gttctgctgc tacctgtggt gagctttgct ctgtaccctg 180
aggagatact ggacacccac tgggagctat ggaagaagac ccacaggaag caatataaca 240
acaagaccag tgaagaggtg gttcagaaga tgactggact caaagtaccc ctgtctcatt 300
cccgcagtaa tgacaccctt tatatcccag aatgggaagg tagagcccca gactctgtcg 360
actatcgaaa gaaaggatat gttactcctg tcaaaaatca gggtcagtgt ggttcctgtt 420
gggcttttag ctctgtgggt gccctggagg gccaactcaa gaagaaaact ggcaaactct 480
taaatctgag tccccagaac ctagtggatt gtgtgtctga gaatgatggc tgtggagggg 540
gctacatgac caatgccttc caatatgtgc agaagaaccg gggtattgac tctgaagatg 600
cctacccata tgtgggacag gaagagagtt gtatgtacaa cccaacaggc aaggcagcta 660
aatgcagagg gtacagagag atccccgagg ggaatgagaa agccctgaag agggcagtgg 720
cccgagtggg acctgtctct gtggccattg atgcaagcct gacctccttc cagttttaca 780
gcaaaggtgt gtattatgat gaaagctgca atagcgataa tctgaaccat gcggttttgg 840
cagtgggata tggaatccag aagggaaaca agcactggat aattaaaaac agctggggag 900
aaaactgggg aaacaaagga tatatcctca tggctcgaaa taagaacaac gcctgtggca 960
ttgccaacct ggccagcttc cccaagatgt gactccagcc agccaaatcc atcctgctct 1020
'tccatttctt ccacgatggt gcagtgtaac gatgcacttt ggaagggagt tggtgtgcta 1080
47/76
CA 02460476 2004-03-15
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tttttgaagc agatgtggtg atactgagat tgtctgttca gtttccccat ttgtttgtgc 1140
ttcaaatgat ccttcctact ttgcttctct ccacccatga cctttttcac tgtggccatc 1200
aggactttcc ctgacagctg tgtactctta ggctaagaga tgtgactaca gcctgcccct 1260
gactgtgttg tcccagggct gatgctgtac aggtacaggc tggagatttt cacataggtt 1320
agattctcat tcacgggact agttagcttt aagcacccta gaggactagg gtaatctgac 1380
ttctcacttc ctaagttccc ttctatatcc tcaaggtaga aatgtctatg ttttctactc~1440
caattcataa atctattcat aagtctttgg tacaagttta catgataaaa agaaatgtga 1500
tttgtcttcc cttctttgca cttttgaaat aaagtattta tctcctgtct acagtttaat 1560
aaatagcatc tagtacacaa aaaaaa 1586
<210> 43
<211> 2368
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500273CB1
<400> 43
ctaggactga tctccaggac cagcactctt ctcccagccc ttagggtcct gctcggccaa 60
ggccttccct gccatgcgac ctgtcagtgt ctggcagtgg agcccctggg ggctgctgct 120
gtgcctgctg tgcagttcgt gcttggggtc tccgtcccct tccacgggcc ctgagaagaa 180
ggccgggagc caggggcttc ggttccggct ggctggcttc cccaggaagc cctacgaggg 240
ccgcgtggag atacagcgag ctggtgaatg gggcaccatc tgcgatgatg acttcacgct 300
gcaggctgcc cacatcctct gccgggagct gggcttcaca gaggccacag gctggaccca 360
cagtgccaaa tatggccctg gaacaggccg catctggctg gacaacttga gctgcagtgg 420
gaccgagcag agtgtgactg aatgtgcctc ccggggctgg gggaacagtg actgtacgca 480
cgatgaggat gctggggtca tctgcaaaga ccagcgcctc cctggcttct cggactccaa 540
tgtcattgag gcccgtgtcc gtctaaaggg cggcgcccac cctggagagg gccgggtaga 600
agtcctgaag gccagcacat ggggcacagt ctgtgaccgc aagtgggacc tgcatgcagc 660
cagcgtggtg tgtcgggagc tgggcttcgg gagtgctcga gaagctctga gtggcgctcg 720
catggggcag ggcatgggtg ctatccacct gagtgaagtt cgctgctctg gacaggagct 780
ctccctctgg aagtgccccc acaagaacat cacagctgag gattgttcac atagccagga 840
tgccggggtc cggtgcaacc taccttacac tggggcagag accaggatcc gactcagtgg 900
gggccgcagc caacatgagg ggcgagtcga ggtgcaaata gggggacctg ggccccttcg 960
ctggggcctc atctgtgggg atgactgggg gaccctggag gccatggtgg cctgtaggca 1020
actgggtctg ggctacgcca accacggcct gcaggagacc tggtactggg actctgggaa 1080
tataacagag gtggtgatga gtggagtgcg ctgcacaggg actgagctgt ccctggatca 1140
gtgtgcccat catggcaccc acatcacctg caagaggaca gggacccgct tcactgctgg 1200-
agtcatctgt tctgagactg catcagatct gttgctgcac tcagcactgg tgcaggagac 1260
cgcctacatc gaagaccggc ccctgcatat gttgtactgt gctgcggaag agaactgcct 1320
ggccagctca gcccgctcag ccaactggcc ctatggtcac cggcgtctgc tccgattctc 1380
ctcccagatc cacaacctgg gacgagctga cttcaggccc aaggctgggc gccactcctg 144 0
ggtgtggcac gagtgccatg ggcattacca cagcatggac atcttcactc actatgatat 1500
cctcacccca aatggcacca aggtggctga gggccacaaa gctagtttct gtctcgaaga 1560
cactgagtgt caggaggatg tctccaagcg gtatgagtgt gccaactttg gagagcaagg 1620
catcactgtg ggttgctggg atctctaccg gcatgacatt gactgtcagt ggattgacat 1680
cacggatgtg aagccaggaa actacattct ccaggttgtc atcaacccaa actttgaagt 1740
agcagagagt gactttacca acaatgcaat gaaatgtaac tgcaaatatg atggacatag 1800
aatctgggtg cacaactgcc acattggtga tgccttcagt gaagaggcca acaggaggtt 1860
tgaacgctac cctggccaga ccagcaacca gattatctaa gtgccactgc cctctgcaaa 1920
ccaccactgg cccctaatgg caggggtctg aggctgccat tacctcagga gcttaccaag 1980
aaacccatgt cagcaaccgc actcatcaga ccatgcacta tggatgtgga actgtcaagc 2040
agaagttttc accctccttc agaggccagc tgtcagtatc tgtagccaag catgggaatc 2100
tttgctccca ggcccagcac cgagcagaac agaccagagc ccaccacacc acaaagagca 2160
48/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
gcacctgact aactgcccac aaaagatggc agcagctcat tttctttaat aggaggtcag 2220
gatggtcagc tccagtatct cccctaagtt tagggggata cagctttacc tctagccttt 2280
tggtggggga aaagatccag ccctcccacc tcatttttta ctataatatg ttgctaggta 2340
taattttatt ttatataaaa agtgttga 2368
<210> 44
<211> 801
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500183CB1
<400> 44
accacctgca cccggagagc tgtgtcacca tgtgggtccc ggttgtcttc ctcaccctgt 60
ccgtgacgtg gattggtgct gcacccctca tcctgtctcg gattgtggga ggctgggagt 120
gcgagaagca ttcccaaccc tggcaggtgc ttgtggcctc tcgtggcagg gcagtctgcg 180
gcggtgttct ggtgcacccc cagtgggtcc tcacagctgc ccactgcatc aggaagccag 240
gtgatgactc cagccacgac ctcatgctgc tccgcctgtc agagcctgcc gagctcacgg 300
atgctgtgaa ggtcatggac ctgcccaccc aggagccagc actggggacc acctgctacg 360
cctcaggctg gggcagcatt gaaccagagg agttcttgac cccaaagaaa cttcagtgtg 420
tggacctcca tgttatttcc aatgacgtgt gtgcgcaagt tcaccctcag aaggtgacca 480
agttcatgct gtgtgctgga cgctggacag ggggcaaaag cacctgctcg ggtgattctg 540
ggggcccact tgtctgtaat ggtgtgcttc aaggtatcac gtcatggggc agtgaaccat 600
gtgccctgcc cgaaaggcct tccctgtaca ccaaggtggt gcattaccgg aagtggatca 660
aggacaccat cgtggccaac ccctgagcac ccctatcaac cccctattgt agtaaacttg 720
gaaccttgga aatgaccagg ccaagactca agcctcccca gttctactga cctttgtcct 780
801
taggtgtgag gtccagggtt g
<210> 45
<211> 756
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7499957CB1
<400> 45
gctttcttct cgtcggtgtt cccggctgct atagagccgg gtgagagagc gagcgcccgt 60
cggcgggtgt cgagagcggg ttgcctcgcg ctgacccttc ccgccctcct tctcgtcaca 120
caccaggtcc ccgcggaagc cgcggtgtcg gcgccatggc ggagctgacg gctcttgaga 180
gtctcatcga gatgggcttc cccaggggac gcgcggagaa ggctctggcc ctcacaggga 240
accagggcat cgaggctgcg atggactggc tgatggagca cgaagacgac cccgatgtgg 300
acgagccttt agagactccc cttggacata tcctgggacg ggagcccact tcctcagagc 360
caggtcctgt tccctcttct cccagccagg agcctcccac caagcgggag tatgaccagt 420
gtcgcataca ggtcaggctg ccagatggga cctcactgac ccagacgttc cgggcccggg 480
aacagctggc agctgtgagg ctctatgtgg agctccaccg tggggaggaa ctaggtgggg 540
gccaggaccc tgtgcaattg ctcagtggct tccccagacg ggccttctca gaagctgaca 600
tggagcggcc tctgcaggag ctgggactcg tgccttctgc tgttctcat~ gtggccaaga 660
aatgtcccag ctgagggcct ttgtcccatt gtccctctgt gaccccttca tctttgataa 720
agcactgaca tctccttcct aataaataga ccctga 756
<210> 46
<211> 2952
49/76
CA 02460476 2004-03-15
WO 03/031939 PCT/US02/32850
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500001CB1
<400> 46
gcctccgagg ccaaggccgc tgctactgcc gccgctgctt cttagtgccg cgttcgccgc 60
ctgggttgtc accggcgccg ccgccgagga agccactgca accaggaccg gagtggaggc 120
ggcgcagcat gaagcggcgc aggcccgctc catagcgcac gtcgggacgg tccgggcggg 180
gccgggggga aggaaaatgc aacatggcag cagcaatgga aacagaacag ctgggtgttg 240
agatatttga aactgcggac tgtgaggaga atattgaatc acaggatcgg cctaaattgg 300
agccttttta tgttgagcgg tattcctgga gtcagcttaa aaagctgctt gccgatacca 360
gaaaatatca tggctacatg atggctaagg caccacatga tttcatgttt gtgaagagga 420
atgatccaga tggacctcat tcagacagaa tctattacct tgccatgtct ggtgagaaca 480
gagaaaatac actgttttat tctgaaattc ccaaaactat caatagagca gcagtcttaa 540
tgctctcttg gaagcctctt ttggatcttt ttcaggcaac actggactat ggaatgtatt 600
ctcgagaaga agaactatta agagaaagaa aacgcattgg aacagtcgga attgcttctt 660
acgattatca ccaaggaagt ggaacatttc tgtttcaagc cggtagtgga atttatcacg 720
taaaagatgg agggccacaa ggatttacgc aacaaccttt aaggcccaat ctagtggaaa 780
ctagttgtcc caacatacgg atggatccaa aattatgccc tgctgatcca gactggattg 840
cttttataca tagcaacgat atttggatat ctaacatcgt aaccagagaa gaaaggagac 900
tcacttatgt gcacaatgag ctagccaaca tggaagaaga tgccagatca gctggagtcg 960
ctacctttgt tctccaagaa gaatttgata gatattctgg ctattggtgg tgtccaaaag 1020
ctgaaacaac tcccagtggt ggtaaaattc ttagaattct atatgaagaa aatgatgaat 108 0
ctgaggtgga aattattcat gttacatccc ctatgttgga aacaaggagg gcagattcat 1140
tccgttatcc taaaacaggt acagcaaatc ctaaagtcac ttttaagatg tcagaaataa 1200
tgattgatgc tgaaggaagg atcatagatg tcatagataa ggaactaatt caaccttttg 1260
agattctatt tgaaggagtt gaatatattg ccagagctgg atggactcct gagggaaaat 1320
atgcttggtc catcctacta gatcgctccc agactcgcct acagatagtg ttgatctcac 1380
ctgaattatt tatcccagta gaagatgatg ttatggaaag gcagagactc attgagtcag 1440'
tgcctgattc tgtgacgcca ctaattatct atgaagaaac aacagacatc tggataaata 1500
tccatgacat ctttcatgtt tttccccaaa gtcacgaaga ggaaattgag tttatttttg 1560
cctctgaatg caaaacaggt ttccgtcatt tatacaaaat tacatctatt ttaaaggaaa 1620
gcaaatataa acgatccagt ggtgggctgc ctgctccaag tgatttcaag tgtcctatca 1680
aagaggagat agcaattacc agtggtgaat gggaagttct tggccggcat ggatctaata 174 0
tccaagttga tgaagtcaga aggctggtat attttgaagg caccaaagac tcccctttag 1800
agcatcacct gtacgtagtc agttacgtaa atcctggaga ggtgacaagg ctgactgacc 1860
gtggctactc acattcttgc tgcatcagtc agcactgtga cttctttata agtaagtata 1920
gtaaccagaa gaatccacac tgtgtgtccc tttacaagct atcaagtcct gaagatgacc 1980
caacttgcaa aacaaaggaa ttttgggcca ccattttgga ttcagcaggt cctcttcctg 2040
actatactcc tccagaaatt ttctcttttg aaagtactac tggatttaca ttgtatggga 2100
tgctctacaa gcctcatgat ctacagcctg gaaagaaata tcctactgtg ctgttcatat 2160
atggtggtcc tcaggtgcag ttggtgaata atcggtttaa aggagtcaag tatttccgct 2220
tgaataccct agcctctcta ggttatgtgg ttgtagtgat agacaacagg ggatcctgtc 2280
accgagggct taaatttgaa ggcgccttta aatataaaat ggttgctatt gctggggccc 2340
cagtcactct gtggatcttc tatgatacag gatacacgga acgttatatg ggtcaccctg 2400
accagaatga acagggctat tacttaggat ctgtggccat gcaagcagaa aagttcccct 2460'
ctgaaccaaa tcgtttactg ctcttacatg gtttcctgga tgagaatgtc cattttgcac 2520
ataccagtat attactgagt tttttagtga gggctggaaa gccatatgat ttacagatct 2580
atcctcagga gagacacagc ataagagttc ctgaatctgg agaacattat gaactgcatc 2640
ttttgcacta ccttcaagaa aaccttggat cacgtattgc tgctctaaaa gtgatataat 2700
tttgacctgt gtagaactct ctggtataca ctggctattt aaccaaatga ggaggtttaa 2760
tcaacagaaa acacagaatt gatcatcaca ttttgatacc tgccatgtaa catctactcc 2820
tgaaaataaa tgtggtgcca tgcaggggtc tacggtttgt ggtagtaatc taatacctta 2880
50/76
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 268
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