Note: Descriptions are shown in the official language in which they were submitted.
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PROTEIN PHOSPHATASES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of protein
phosphatases and
to the use of these sequences in the diagnosis, treatment, and prevention of
immune system disorders,
neurological disorders, developmental disorders, and cell proliferative
disorders, and in the
assessment of the effects of exogenous compounds on the expression of nucleic
acid and amino acid
sequences of protein phosphatases.
l0 BACKGROUND OF THE INVENTION
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. 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. Phosphatases, in contrast, remove these
phosphate groups.
Extracellular signals including hormones, neurotransmitters, and growth and
differentiation factors
can activate kinases, which can occur as cell surface receptors or as the
activator of the final effector
protein, but can also occur along the signal transduction pathway. Cascades of
kinases occur, as well
as kinases sensitive to second messenger molecules. This system allows for the
amplification of weak
signals (low abundance growth factor molecules, for example), as well as the
synthesis of many weak
signals into an all-or-nothing response. Phosphatases, then, 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 generally characterized as either serine/threonine-
or tyrosine-
specific based on their preferred phospho-amino acid substrate. However, 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. Another family of phosphatases is the acid
phosphatase or histidine acid
phosphatase (HAP) family whose members hydrolyze phosphate esters at acidic pH
conditions.
PSPs are found in the cytosol, nucleus, and mitochondria and in association
with cytoskeletal
and membranous structures in most tissues, especially the brain. Some PSPs
require divalent cations,
such as Ca2+ or Mnz+, for activity. PSPs play important roles in glycogen
metabolism, muscle
contraction, protein synthesis, T cell function, neuronal activity, oocyte
maturation, and hepatic
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metabolism (reviewed in Cohen, P. (1989) Annu. Rev. Biochem. 58:453-508). PSPs
can be separated
into two classes. The PPP class includes PPl, PP2A, PP2B/calcineurin, PP4,
PPS, PP6, and PP7.
Members of this class are composed of a homologous catalytic subunit bearing a
very highly
conserved signature sequence, coupled with one or more regulatory subunits
(PROSITE
PDOC00115). Further interactions with scaffold and anchoring molecules
determine the intracellular
localization of PSPs and substrate specificity. The PPM class consists of
several closely related
isoforms of PP2C and is evolutionarily unrelated to the PPP class.
PP1 dephosphorylates many of the proteins phosphorylated by cyclic AMP-
dependent protein
kinase (PKA) and is an important regulator of many cAMP-mediated hormone
responses in cells. A
number of isoforms have been identified, with the alpha and beta forms being
produced by alternative
splicing of the same gene. Both ubiquitous and tissue-specific targeting
proteins for PP1 have been
identified. In the brain, inhibition of PPl activity by the dopamine and
adenosine 3',5'-
monophosphate-regulated phosphoprotein of 32kDa (DARPP-32) is necessary for
normal dopamine
response in neostriatal neurons (reviewed in Price, N.E. and M.C. Mumby (1999)
Curr. Opin.
Neurobiol. 9:336-342). PP1, along with PP2A, has been shown to limit motility
in microvascular
endothelial cells, suggesting a role for PSPs in the inhibition of
angiogenesis (Gabel, S. et al. (1999)
Otolaryngol. Head Neck Surg.121:463-468).
PP2A is the main serine/threonine phosphatase. The core PP2A enzyme consists
of a single
36 kDa catalytic subunit (C) associated with a 65 kDa scaffold subunit (A),
whose role is to recruit
additional regulatory subunits (B). Three gene families encoding B subunits
are known (PR55, PR6I,
and PR72), each of which contain multiple isoforms, and additional families
may exist (Millward,
T.A et al. (1999) Trends Biosci. 24:186-191). These "B-type" subunits are cell
type- and tissue-
specific and determine the substrate specificity, enzymatic activity, and
subcellular localization of the
holoenzyme. The PR55 family is highly conserved and bears a conserved motif
(PROSITE
PDOC00785). PR55 increases PP2A activity toward mitogen-activated protein
kinase (MAPK) and
MAPK kinase (MEK). PP2A dephosphorylates the MAPK active site, inhibiting the
cell's entry into
mitosis. Several proteins can compete with PR55 for PP2A core enzyme binding,
including the CKII
kinase catalytic subunit, polyomavirus middle and small T antigens, and SV40
small t antigen.
Viruses may use this mechanism to commandeer PP2A and stimulate progression of
the cell through
the cell cycle (Pallas, D.C. et al. (1992) J. Virol. 66:886-893). Altered MAP
kinase expression is also
implicated in a variety of disease conditions including cancer, inflammation,
immune disorders, and
disorders affecting growth and development. PP2A, in fact, can dephosphorylate
and modulate the
activities of more than 30 protein kinases in vitro, and other evidence
suggests that the same is true in
vivo for such kinases as PKB, PKC, the calmodulin-dependent kinases, ERK
family MAP kinases,
cyclin-dependent kinases, and the IoB kinases (reviewed in Millward et al.,
supra). PP2A is itself a
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substrate for CKI and CKII kinases, and can be stimulated by polycationic
macromolecules. A PP2A-
like phosphatase is necessary to maintain the Gl phase destruction of
mammalian cyclins A and B
(Bastians, H. et al. (1999) Mol. Biol. Cell 10:3927-3941). PP2A is a major
activity in the brain and is
implicated in regulating neurofilament stability and normal neural function,
particularly the
phosphorylation of the microtubule-associated protein tau.
Hyperphosphorylation of tau has been
proposed to lead to the neuronal degeneration seen in Alzheimer's disease
(reviewed in Price and
Mumby, s_upra).
PP2B, or calcineurin, is a Caz+-activated dimeric phosphatase and is
particularly abundant in
the brain. It consists of catalytic and regulatory subunits, and is activated
by the binding of the
calcium/calmodulin complex. Calcineurin is the target of the immunosuppresant
drugs cyclosporine
and FK506. Along with other cellular factors, these drugs interact with
calcineurin and inhibit
phosphatase activity. In T cells, this blocks the calcium dependent activation
of the NF-AT family of
transcription factors, leading to immunosuppression. This family is widely
distributed, and it is likely
that calcineurin regulates gene expression in other tissues as well. In
neurons, calcineurin modulates
functions which range from the inhibition of neurotransmitter release to
desensitization of
postsynaptic NMDA-receptor coupled calcium channels to long term memory
(reviewed in Price and
Mumby, s_ unra).
Other members of the PPP class have recently been identified (Cohen, P.T.
(1997) Trends
Biochem. Sci. 22:245-251). One of them, PPS, contains regulatory domains with
tetratricopeptide
repeats. It can be activated by polyunsaturated fatty acids and anionic
phospholipids in vitro and
appears to be involved in a number of signaling pathways, including those
controlled by atrial
natriuretic peptide or steroid hormones (reviewed in Andreeva, A.V. and M.A.
Kutuzov (1999) Cell
Signal. 11:555-562).
PP2C is a ~42kDa monomer with broad substrate specificity and is dependent on
divalent
cations (mainly Mnz+ or Mgz+) for its activity. PP2C proteins share a
conserved N-terminal region
with an invariant DGH motif, which contains an aspartate residue involved in
cation binding
(PROSITE PDOC00792). Targeting proteins and mechanisms regulating PP2C
activity have not
been identified. PP2C has been shown to inhibit the stress-responsive p38 and
Jun kinase (JNK)
pathways (Takekawa, M. et al. (1998) EMBO J. 17:4744-4752).
In contrast to PSPs, tyrosine-specific phosphatases (PTPs) are generally
monomeric proteins
of very diverse size (from 20kDa to greater than 100kDa) and structure that
function primarily in the
transduction of signals across the plasma membrane. PTPs are categorized as
either soluble
phosphatases or transmembrane receptor proteins that contain a phosphatase
domain. All PTPs share
a conserved catalytic domain of about 300 amino acids which contains the
active site. The active site
consensus sequence includes a cysteine residue which executes a nucleophilic
attack on the phosphate
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moiety during catalysis (Neel, B.G. and N.K. Tonks (1997) Curr. Opin. Cell
Biol. 9:193-204).
Receptor PTPs are made up of an N-terminal extracellular domain of variable
length, a
transmembrane region, and a cytoplasmic region that generally contains two
copies of the catalytic
domain. Although only the first copy seems to have enzymatic activity, the
second copy apparently
affects the substrate specificity of the first.. The extracellular domains of
some receptor PTPs contain
fibronectin-like repeats, immunoglobulin-like domains, MAM domains (an
extracellular motif likely
to have an adhesive function), or carbonic anhydrase-like domains (PROSITE
PDOC 00323). This
wide variety of structural motifs accounts for the diversity in size and
specificity of PTPs.
PTPs play important roles in biological processes such as cell adhesion,
lymphocyte
IO activation, and cell proliferation. PTPs p, and x are involved in cell-cell
contacts, perhaps regulating
cadherin/catenin function. A number of PTPs affect cell spreading, focal
adhesions, and cell motility,
most of them via the integrinltyrosine kinase signaling pathway (reviewed in
Neel and Tonks, supra).
CD45 phosphatases regulate signal transduction and lymphocyte activation
(Ledbetter, J.A. et al.
(1988) Proc. Natl. Aced. Sci. USA 85:8628-8632). Soluble PTPs containing Src-
homology-2
domains have been identified (SHPs), suggesting that these molecules might
interact with receptor
tyrosine kinases. SHP-1 regulates cytokine receptor signaling by controlling
the Janus family PTKs
in hematopoietic cells, as well as signaling by the T-cell receptor and c-Kit
(reviewed in Neel and
Tonks, sera). M-phase inducer phosphatase plays a key role in the induction of
mitosis by
dephosphorylating and activating the PTK CDC2, leading to cell division
(Sadhu, K. et al. (1990)
Proc. Natl. Aced. Sci. USA 87:5139-5143). In addition, the genes encoding at
least eight PTPs have
been mapped to chromosomal regions that are translocated or rearranged in
various neoplastic
conditions, including lymphoma, small cell lung carcinoma, leukemia,
adenocarcinoma, and
neuroblastoma (reviewed in Charbonneau, H. and N.K. Tonks (1992) Annu. Rev.
Cell Biol. 8:463-
493). The PTP enzyme active site comprises the consensus sequence of the MTM1
gene family. The
MTMl gene is responsible for X-linked recessive myotubular myopathy, a
congenital muscle disorder
that has been linked to Xq28 (Kioschis, P. et al., (1998) Genomics 54:256-266.
Many PTKs are
encoded by oncogenes, and it is well known that oncogenesis is often
accompanied by increased
tyrosine phosphorylation activity. It is therefore possible that PTPs may
serve to prevent or reverse
cell transformation and the growth of various cancers by controlling the
levels of tyrosine
phosphorylation in cells. This is supported by studies showing that
overexpression of PTP can
suppress transformation in cells and that specific inhibition of PTP can
enhance cell transformation
(Charbonneau and Tonks, supra).
Dual specificity phosphatases (DSPs) are structurally more similar to the PTPs
than the PSPs.
DSPs bear an extended PTP active site motif with an additional 7 amino acid
residues. DSPs are
primarily associated with cell proliferation and include the cell cycle
regulators cdc25A, B, and C.
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The phosphatases DUSPl and DUSPZ inactivate the MAPK family members ERK
(extracellular
signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38 on both
tyrosine and threonine
residues (PROSITE PDOC 00323, su ra). In the activated state, these kinases
have been implicated
in neuronal differentiation, proliferation; oncogenic transformation, platelet
aggregation, and
apoptosis. Thus, DSPs are necessary for proper regulation of these processes
(Muda, M. et al. (1996)
J. Biol. Chem. 271:27205-27208). The tumor suppressor PTEN is a DSP that also
shows lipid
phosphatase activity. It seems to negatively regulate interactions with the
extracellular matrix and
maintains sensitivity to apoptosis. PTEN has been implicated in the prevention
of angiogenesis (Girl,
D. and M. Ittmann (1999) Hum. Pathol. 30:419-424) and abnormalities in its
expression are
associated with numerous cancers (reviewed in Tamara, M. et al. ( 1999) J.
Natl. Cancer Inst.
91:1820-1828).
Histidine acid phosphatase (HAP; EXPASY EC 3.1.3.2), also known as acid
phosphatase,
hydrolyzes a wide spectrum of substrates including alkyl, aryl, and acyl
orthophosphate monoesters
and phosphorylated proteins at low pH. HAPs share two regions of conserved
sequences, each
centered around a histidine residue which is involved in catalytic activity.
Members of the HAP
family include lysosomal acid phosphatase (LAP) and prostatic acid phosphatase
(PAP), both
sensitive to inhibition by L-tartrate (PROSTTE PDOC00538).
LAP, an orthophosphoric rnonoester of the endosomal/lysosomal compartment is a
housekeeping gene whose enzymatic activity has been detected in all tissues
examined (Geier, C. et
al. (1989) Eur. J. Biochem. 183:611-616). LAP-deficient mice have progressive
skeletal disorder and
an increased disposition toward generalized seizures (Saftig, P. et al. (1997)
J. Biol. Chem.
272:18628-18635). LAP-deficient patients were found to have the following
clinical features:
intermittent vomiting, hypotonia, lethargy, opisthotonos, terminal bleeding,
seizures, and death in
early infancy (Online Mendelian Inheritance in Man (OMIM) *200950).
PAP, a prostate epithelium-specific differentiation antigen produced by the
prostate gland,
has been used to diagnose and stage prostate cancer. In prostate carcinomas,
the enzymatic activity of
PAP was shown to be decreased compared with normal or benign prostate
hypertrophy cells (Foti,
A.G. et al. (1977) Cancer Res. 37:4120-4124). Two forms of PAP have been
identified, secreted and
intracellular. Mature secreted PAP is detected in the seminal fluid and is
active as a glycosylated
homodimer with a molecular weight of approximately 100-kilodalton.
Intracellular PAP is found to
exhibit endogenous phosphotyrosyl protein'phosphatase activity and is involved
in regulating prostate
cell growth (Meng, T.C. and M.F. Lin (1998) J. Biol. Chem. 34:22096-22104).
Synaptojanin, a polyphosphoinositide phosphatase, dephosphorylates
phosphoinositides at
positions 3, 4 and 5 of the inositol ring. Synaptojanin is a major presynaptic
protein found at clathrin-
. coated endocytic intermediates in nerve terminals, and binds the clathrin
coat-associated protein,
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EPS15, which is mediated by the C-terminal region of synatojanin-170, which
has 3 Asp-Pro-Phe
amino acid repeats. Further, this 3 residue repeat had been found to be the
binding site for the EH
domains of EPS15 (Haffner, C. et al. (1997) FEBS Lett. 419:175-180).
Additionally, synaptojanin
may potentially regulate interactions of endocytic proteins with the plasma
membrane, and be
involved in synaptic vesicle recycling (Brodin, L. et al. (2000) Curr. Opin.
Neurobiol. 10:312-320).
Studies in mice with a targeted disruption in the synaptojanin 1 gene (Synj 1)
were shown to support
coat formation of endocytic vesicles more effectively than was seen in wild-
type mice, suggesting that
Synj 1 can act as a negative regulator of membrane-coat protein interactions.
These findings provide
genetic evidence for a crucial role of phosphoinositide metabolism in synaptic
vesicle recycling
(Cremona, O. et al. (1999) Cell 99:179-188).
The discovery of new protein phosphatases, and the polynucleotides encoding
them, satisfies
a need in the art by providing new compositions which are useful in the
diagnosis, prevention, and
treatment of immune system disorders, neurological disorders, developmental
disorders, and cell
proliferative disorders, and in the assessment of the effects of exogenous
compounds on the
expression of nucleic acid and amino acid sequences of protein phosphatases.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, protein phosphatases, referred
to collectively as
"PP" and individually as "PP-1," "PP-2," "PP-3," "PP-4," "PP-5," "PP-6," "PP-
7," "PP-8," "PP-9,"
and "PP-10." In one aspect, the invention provides an isolated polypeptide
selected from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ 1D NO:l-10, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ll~ NO:1-10, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m NO:1-10, and d) an immunogenic fragment of a polypeptide
having an annino
acid sequence selected from the group consistingoof SEQ ID NO:1-10. In one
alternative, the
invention provides an isolated polypeptide comprising the amino acid sequence
of SEQ 1T3 NO: l-10.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected
from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ m NO:1-10, b) a polypeptide comprising a naturally
occurring amino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
m NO:1-10, c) a biologically active fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-10, and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ m NO: l-10.
In one alternative, the polynucleotide encodes a polypeptide selected from the
group consisting of
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SEQ ID N0:1-10. In another alternative, the polynucleotide is selected from
the group consisting of
SEQ ID N0:11-20.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-10, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ 1D NO:1-10, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m N0:1-10, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ )D N0:1-10. In one
alternative, the
invention provides a cell transformed with the recombinant polynucleotide. In
another alternative, the
invention provides a transgenic organism comprising the recombinant
polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-10, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ )D N0:1-10, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-10, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-10. The method
comprises a)
culturing a cell under conditions suitable for expression of the polypeptide,
wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ >D NO:1-10, b) a
polypeptide comprising a
naturally occurring amino acid sequence at Ieast 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-10, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-10, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-10.
The invention further provides an isolated polynucleotide selected from the
group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ m NO:11-20, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ )D
N0:11-20, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
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complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
In one alternative, the
polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
selected from the group
consisting of a) a polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of SEQ m NO:11-20, b) a polynucleotide comprising a naturally
occurring polynucleotide
sequence at least 90% identical to a polynucleotide sequence selected from the
group consisting of
SEQ m NO:11-20, c) a polynucleotide complementary to the polynucleotide of a),
d) a
polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). The
,.
method comprises a) hybridizing the sample with a probe comprising at least 20
contiguous
nucleotides comprising a sequence complementary to said target polynucleotide
in the sample, and
which probe specifically hybridizes to said target polynucleotide, under
conditions whereby a
hybridization complex is formed between said probe and said target
polynucleotide or fragments
thereof, and b) detecting the presence or absence of said hybridization
complex, and optionally, if
present, the amount thereof. In one alternative, the probe comprises at least
60 contiguous
nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ )D NO:11-20, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ m
NO:11-20, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method
comprises a) amplifying said target polynucleotide or fragment thereof using
polymerase chain
reaction amplification, and b) detecting the presence or absence of said
amplified target
polynucleotide or fragment thereof, and, optionally, if present, the amount
thereof.
The invention further provides a composition comprising an effective amount of
a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ >D NO: l-10, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-10, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ >D
NO:1-10, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ 11? N0:1-10, and a pharmaceutically acceptable excipient. In
one embodiment, the
composition comprises an amino acid sequence selected from the group
consisting of SEQ m NO:1-
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I0. The invention additionally provides a method of treating a disease or
condition associated with
decreased expression of functional PP, comprising administering to a patient
in need of such
treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-10, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-10, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ 1D
NO:1-10, and d) an
imrnunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-10. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional PP, comprising
administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1-10, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ ID NO: l-10, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-I0,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID N0:1-10. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a composition comprising an antagonist compound identified
by the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
PP, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide selected from the gxoup consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-10, b) a
polypeptide comprising a
naturally occurnng amino acid sequence at least 90% identical to an amino acid
sequence selected
from the group consisting of SEQ ID N0:1-10, c) a biologically active fragment
of a polypeptide
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having an amino acid sequence selected from the group consisting of SEQ ID NO:
l-10, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ )D NO:1-10. The method comprises a) combining the
polypeptide with at least
one test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ 117 NO:1-10, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ )D NO:1-10, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ )D
NO:1-10, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m NO:1-10. The method comprises a) combining the polypeptide
with at least
one test compound under conditions permissive for the activity of the
polypeptide, b) assessing the
activity of the polypeptide in the presence of the test compound, and c)
comparing the activity of the
polypeptide in the presence of the test compound with the activity of the
polypeptide in the absence
of the test compound, wherein a change in the activity of the polypeptide in
the presence of the test
compound is indicative of a compound that modulates the activity of the
polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, Wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of SEQ III N0:11-
20, the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, and b)
detecting altered expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
)D NO:l 1-20, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:11-20,
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
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polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
NO:11-20, ii) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID NO:l 1-20,
iii) a polynucleotide complementary to the polynucleotide of i), iv) a
polynucleotide complementary
to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
Alternatively, the target
polynucleotide comprises a fragment of a polynucleotide sequence selected from
the group consisting
of i)-v) above; c) quantifying the amount of hybridization complex; and d)
comparing the amount of
hybridization complex in the treated biological sample with the amount of
hybridization complex in
an untreated biological sample, wherein a difference in the amount of
hybridization complex in the
treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability score for the match
between each
polypeptide and its GenBank homolog is also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
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will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a,". "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection With the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"PP" refers to the amino acid sequences of substantially purified PP 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
PP. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of PP either by directly
interacting with PP or
by acting on components of the biological pathway in which PP participates.
An "allelic variant" is an alternative form of the gene encoding PP. 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 PP include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as PP or a
polypeptide with at least one functional characteristic of PP. Included within
this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding PP, and improper or unexpected hybridization to
allelic variants, with
a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding PP. The
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encoded protein may also be "altered," and may contain deletions, insertions,
or substitutions of
amino acid residues which produce a silent change and result in a functionally
equivalent PP.
Deliberate amino acid substitutions may be made on the basis of similarity in
polarity, charge,
solubility, hydrophobicity, hydrophilicity, andlor the amphipathic nature of
the residues, as long as
the biological or immunological activity of PP is retained. For example,
negatively charged amino
acids may include aspartic acid and glutamic acid, and positively charged
amino acids may include
lysine and arginine. Amino acids with uncharged polar side chains having
similar hydrophilicity
values may include: asparagine and glutamine; and serine and threonine. Amino
acids with
uncharged side chains having similar hydrophilicity values may include:
leucine, isoleucine, and
valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid
sequence to the complete native amino acid sequence associated with the
recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerise chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of PP. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of PP either by
directly interacting with PP or by acting on components of the biological
pathway in which PP
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 PP polypeptides can be prepared using intact polypeptides
or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide
used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived
from the translation of
RNA, or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly
used carriers that are chemically coupled to peptides include bovine serum
albumin, thyroglobulin,
and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to
immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
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on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic PP, or of
any oligopeptide thereof, to
induce a specific immune response in appropriate animals or cells and to bind
with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide
or amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding PP or fragments of
PP 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., NaCI), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI] or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended and
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assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala rt Gly, Sex
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, V al
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
V al 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, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which
retains at least one biological or immunological function of the natural
molecule. A derivative
polypeptide is one modified by glycosylation, pegylation, or any similar
process that retains at least
one biological or immunological function of the polypeptide from which it was
derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
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"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of PP or the polynucleotide encoding Pl~
which is identical
in sequence to but shorter in length than the parent sequence. A fragment may
comprise up to the
entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID NO:11-20 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID NO:11-20, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:11-20 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID NO:11-20 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:11-20 and the region of SEQ ID N0:11-20 to which the fragment
corresponds are routinely
determinable by one of ordinary skill in the art based on the intended purpose
fox the fragment.
A fragment of SEQ m NO:1-10 is encoded by a fragment of SEQ 1D N0:11-20. A
fragment
of SEQ ID NO: l-10 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:1-10. For example, a fragment of SEQ ID NO:1-10 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:1-10.
The precise length of
a fragment of SEQ m NO:1-10 and the region of SEQ ID NO:1-10 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation
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codon (e.g., methionine) followed by an open reading frame and a translation
termination codon. A
"full length" polynucleotide sequence encodes a "full length" polypeptide
sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between
two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.govlBLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatclz: -2
Open Gap: 5 and Extension Gap: 2 penalties
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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 residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the charge
and hydrophobicity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 and Extensiorz Gap: 1 penalties
Gap x drop-off.- 50
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Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ m number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1% (w/v) SDS, and about 100 ~,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 (Tin) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.
(1989) Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring
Harbor Press,
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Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present
invention include wash conditions of 68°C in the presence of about 0.2
x SSC and about 0.1% SDS,
for 1 hour. Alternatively, temperatures of about 65°C,
60°C,'S5°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present
at about 0.1%.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents
include, for instance, sheared and denatured salmon sperm DNA at about 100-200
~.g/ml. Organic
solvent, such as formamide at a concentration of about 35-50% v/v, may also be
used under particular
circumstances, such as for RNA:DNA hybridizations. Useful variations on these
wash conditions
will be readily apparent to those of ordinary skill in the art. Hybridization,
particularly under high
stringency conditions, may be suggestive of evolutionary similarity between
the nucleotides. Such
similarity is strongly indicative of a similar role for the nucleotides and
their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of PP
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment
of PP which is useful in any of the antibody production methods disclosed
herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of PP. For example,
modulation may
cause an increase or a decrease in protein activity, binding characteristics,
or any other biological,
functional, or immunological properties of PP.
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The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an PP 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 PP.
"Probe" refers to nucleic acid sequences encoding PP, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable Iabel or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
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Bioloay, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
aI. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
Institute/MIT 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 UI~ Human
Genome Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, 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
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transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing PP,
nucleic acids encoding PP, or fragments thereof may comprise a bodily fluid;
an extract from a cell,
chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA,
RNA, or cDNA, in
solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at Ieast 75% free, and most preferably at Ieast 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,
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microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" refers to the collective pattern of gene expression by a
particular cell
type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or 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. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook et al. (1989),
supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2,Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
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polynucleotides due to alternate splicing of exons duxing mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides will generally have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, fox example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
I999) set at default parameters. Such a pair of polypeptides may show, for
example, at Ieast 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at Ieast 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human protein phosphatases
(PP), the
polynucleotides encoding PP, and the use of these compositions for the
diagnosis, treatment, or
prevention of immune system disorders, neurological disorders, developmental
disorders, and cell
proliferative disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide m) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ m NO:) and
an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID)
as shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the
invention. Column 3
shows the GenBank identification number (Genbank ID NO:) of the nearest
GenBank homolog.
Column 4 shows the probability score fox the match between each polypeptide
and its GenBank
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homolog. Column 5 shows the annotation of the GenBank homolog along with
relevant citations
where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of 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 ~) for each polypeptide
of the invention.
Column 3 shows the number of amino acid residues in each polypeptide. Column 4
shows potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the
MOTIFS program of the GCG sequence analysis software package (Genetics
Computer Group,
Madison WI). Column 6 shows amino acid residues comprising signature
sequences, domains, and
motifs. Column 7 shows analytical methods for protein structure/function
analysis and in some cases,
searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are protein phosphatases.
For example, SEQ ID
N0:2 is 98% identical to mouse putative protein phosphatase type 2C (GenBank
ID g43250S1) as
determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
The BLAST
probability score is 1.0e-89, which indicates the probability of obtaining the
observed polypeptide
sequence alignment by chance. SEQ ID N0:2 also contains a protein phosphatase
2C 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 BLIMPS,
MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that
SEQ m N0:2 is
a protein phosphatase 2C. In an alternative example, SEQ ID N0:4 is 46%
identical to human protein
phosphatase (GenBank m g6692782) as determined by BLAST. (See Table 2.) The
BLAST
probability score is 2.0e-33. SEQ ID N0:4 also contains a dual specificity
phosphatase, catalytic
domain as determined by searching for statistically significant matches in the
HMM-based PFAM
database. (See Table 3.) Data from BLAST-DOMO analysis provides further
corroborative evidence
that SEQ ID N0:4 is a dual specificity protein phosphatase. In an alternative
example, SEQ ID N0:6
is 45% identical to marine lysosomal acid phosphatase (GenBank ID g52871) as
determined by
BLAST. (See Table 2.) The BLAST probability score is 2.3e-83. SEQ ID N0:6 also
contains a
histidine acid phosphatase domain as determined by searching for statistically
significant matches in
the HMM-based PFAM database. (See Table 3.) Data from BLIMPS, MOTIFS, and
PROFILESCAN
analyses provide further corroborative evidence that SEQ ID N0:6 is an acid
phosphatase. In an
alternative example, SEQ ID N0:7 is 52% identical to mouse neuronal tyrosine
threonine
phosphatase 1 (GenBank ID g1781037) as determined by BLAST. (See Table 2.) The
BLAST
probability score is 1.3e-131. SEQ ID N0:7 also contains a dual specificity
phosphatase active site
domain as determined by searching for statistically significant matches in the
HMM-based PFAM
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database. (See Table 3.) Data from BLTMl'S, MOTIFS, and PROFIL,ESCAN analyses
provide
further corroborative evidence that SEQ ID N0:7 is a tyrosine phosphatase. In
an alternative
example, SEQ ID N0:8 is 61% identical to human tyrosine phosphatase (GenBank
ID g6650693) as
determined by BLAST. (See Table 2.) The BLAST probability score is 1.0e-89.
SEQ ll~ N0:8 also
contains a transmembrane domain as determined by searching for statistically
significant matches in
the HMM-based PFAM database. (See Table 3.) Data from MOTIFS analyses provide
further
corroborative evidence that SEQ m N0:8 is tyrosine specific protein
phosphatase. Tyrosine
phosphatases are one of two general categories of protein phosphatases. In an
alternative example,
SEQ ID NO:10 is 55% identical to human mitogen-activated protein kinase
phosphatase (GenBank ID
g9294745) as determined by BLAST. (See Table 2.) The BLAST probability score
is 1.3e-50. SEQ
ID NO:10 also contains a dual specificity phosphatase catalytic domain as
determined by searching
for statistically significant matches in the HMM-based PFAM database. (See
Table 3.) Data from
BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative
evidence that SEQ
ID NO:10 is a protein kinase phosphatase. SEQ ID NO: l, SEQ ID N0:3, SEQ ID
N0:5, and SEQ ID
N0:9 were analyzed and annotated in a similar manner. The algorithms and
parameters for the
analysis of SEQ >D N0:1-10 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Columns 1 and 2 list the
polynucleotide sequence
identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte
polynucleotide
consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide
of the invention.
Column 3 shows the length of each polynucleotide sequence in basepairs. Column
4 lists fragments
of the polynucleotide sequences which are useful, for example, in
hybridization or amplification
technologies that identify SEQ ID N0:11-20 or that distinguish between SEQ ID
NO:11-20 and
related polynucleotide sequences. Column 5 shows identification numbers
corresponding to cDNA
sequences, coding sequences (exons) predicted from genomic DNA, and/or
sequence assemblages
comprised of both cDNA and genomic DNA. These sequences were used to assemble
the full length
polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the
nucleotide start (5')
and stop (3') positions of the cDNA and/or genomic sequences in column 5
relative to their respective
full length sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
6024861H1 is the
identification number of an Incyte cDNA sequence, and TESTNOT11 is the cDNA
library from
which it is derived. Incyte cDNAs for which cDNA libraries are not indicated
were derived from
pooled cDNA libraries (e.g., 71907683V1). Alternatively, the identification
numbers in column 5
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may refer to GenBank cDNAs or ESTs (e.g., g2114900) which contributed to the
assembly of the
full
length polynucleotide sequences. In addition, the identification numbers in
column 5 may identify
sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database
(i.e., those
sequences including the designation "ENST"). Alternatively, the identification
numbers in column 5
may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.
e., those sequences
including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence
Records (i.e., those
sequences including the designation "NP"). Alternatively, the identification
numbers in column 5
may refer to assemblages of both cDNA and Genscan-predicted exons brought
together by an "exon
stitching" algorithm. For example, FL XXXXXX_NI 1VZ YYYYY_N3 N4 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
Nl,Z,3..., if present, represent specific exons that may have been manually
edited during analysis (See
Example V). Alternatively, the identification numbers in column 5 may refer to
assemblages of
exons brought together by an "exon-stretching" algorithm. For example,
FLXXXXXX_gAAA.AA~BBBBB_1 1V is the identification number of a "stretched"
sequence, with
XXXXXX being the Incyte project identification number, gAAAA,A 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).
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,
ENST for example,
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).
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INCY Full length transcript and exon prediction from mapping of EST
sequences to the genome. Genomic location and EST composition
data are combined to predict the exons and resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in
column 5 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant
Incyte cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library is
the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences which
were used to assemble and confirm the above polynucleotide sequences. The
tissues and vectors
which were used to construct the cDNA libraries shown in Table 5 are described
in Table 6.
The invention also encompasses PP variants. A preferred PP variant is one
which has at least
about 80%, or alternatively at least about 90%, or even at least about 95%
amino acid sequence
identity to the PP amino acid sequence, and which contains at least one
functional or structural
characteristic of PP.
The invention also encompasses polynucleotides which encode PP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:11-20, which encodes PP. The
polynucleotide sequences of
SEQ ID N0:11-20, as presented in the Sequence Listing, embrace the equivalent
RNA sequences,
wherein occurrences of the nitrogenous base thymine are replaced with uracil,
and the sugar backbone
is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
PP. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at
least about 85%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding PP. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID
NO:11-20 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:11-20. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of PP.
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 PP, 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
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be made by selecting combinations based on possible colon choices. These
combinations are made
in accordance with the standard triplet genetic code as applied to the
polynucleotide sequence of
naturally occurring PP, and all such variations are to be considered as being
specifically disclosed.
Although nucleotide sequences which encode PP and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring PP under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding PP or its
derivatives possessing a substantially different colon usage, e.g., inclusion
of non-naturally occurring
colons. Colons may be selected to increase the rate at which expression of the
peptide occurs in a
particular prokaryotic or eukaryotic host in accordance with the frequency
with which particular
colons are utilized by the host. Other reasons for substantially altering the
nucleotide sequence
encoding PP and its derivatives without altering the encoded amino acid
sequences include the
production of RNA transcripts having more desirable properties, such as a
greater half life, than
transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode PP and
PP
derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the synthetic
sequence may be inserted into any of the many available expression vectors and
cell systems using
reagents well known in the art. Moreover, synthetic chemistry may be used to
introduce mutations
into a sequence encoding PP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:11-20 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Applied
Biosystems), thermostable T7 polymerise (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerises and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
CA 02417359 2003-O1-27
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are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology and Biotechnoloay, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding PP may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments
adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme
digestions and legations may be used to insert an engineered double-stranded
sequence into a region
of unknown sequence before performing PCR. Other methods which may be used to
retrieve
unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991)
Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries
(Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need
to screen libraries
and is useful in 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
emitted wavelengths. Outpudlight intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
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process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences ox fragments
thereof
which encode PP may be cloned in recombinant DNA molecules that direct
expression of PP, or
fragments ox functional equivalents thereof, in appropriate host cells. Due to
the inherent degeneracy
of the genetic code, other DNA sequences which encode substantially the same
or a functionally
equivalent amino acid sequence may be produced and used to express PP.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter PP-encoding sequences for a variety of
purposes. including, but not
limited to, modification of the cloning, processing, andlor expression of the
gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of PP, such as its biological ox enzymatic
activity or its ability to
bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding PP may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Aeids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.)
Alternatively, PP itself or a fragment thereof may be synthesized using
chemical methods. For
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example, peptide synthesis can be performed using various solution-phase or
solid-phase techniques.
(See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular
Properties, WH Freeman, New
York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.)
Automated synthesis
may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the
amino acid sequence of PP, or any part thereof, may be altered during direct
synthesis andlor
combined with sequences from other proteins, or any part thereof, to produce a
variant polypeptide or
a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active PP, the nucleotide sequences
encoding PP or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in
a suitable host. These elements include regulatory sequences, such as
enhancers, constitutive and
inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotide sequences
encoding PP. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
PP. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding PP and its initiation codon and upstream regulatory
sequences are inserted into
the appropriate expression vector, no additional transcriptional or
translational control signals may be
needed. However, in cases where only coding sequence, or a fragment thereof,
is inserted, exogenous
translational control signals including an in-frame ATG initiation codon
should be provided by the
vector. Exogenous translational elements and initiation codons may be of
various origins, both
natural and synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers
appropriate for the particular host cell system used. (See, e.g., Scharf, D.
et al. (1994) Results Probl.
Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding PP and appropriate transcriptional and
translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. (1995)
Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, ch. 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding PP. These include, but are not limited to, microorganisms sash as
bacteria transformed with
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recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast
transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV,
or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti
or pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw
Hill, New
York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and
Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993)
Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.)
The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding PP. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding PP can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding PP into the
vector's multiple cloning
site disrupts the lacZ gene, allowing a colorimetric screening procedure for
identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of PP are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of PP may be used. For
example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of PP. A number of vectors
containing
constitutive or inducible promoters, such as alpha factor, alcohol oxidase,
and PGH promoters, may
be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition,
such vectors direct
either the secretion or intracellular retention of expressed proteins and
enable integration of foreign
sequences into the host genome fox stable propagation. (See, e.g., Ausubel,
1995, supra; Bitter, G.A.
et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et al. (1994)
Bio/Technology 12:181-
184.)
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Plant systems may also be used for expression of PP. Transcription of
sequences encoding
PP may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV
used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO
J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.)
These constructs can be~introduced into plant cells by direct DNA
transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of
Science and Technoloay
(1992) McGraw Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding PP may
be ligated into an
adenovirus transcription/translation complex consisting of the late promoter
and tripartite leader
sequence. Insertion in a non-essential El or E3 region of the viral genome may
be used to obtain
infective virus which expresses PP in host cells. (See, e.g., Logan, J. and T.
Shenk (1984) Proc. Natl.
Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such as
the Rous sarcoma virus
(RSV) enhancer, may be used to increase expression in mammalian host cells.
SV40 or EBV-based
vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposornes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of PP in cell lines is preferred. For example, sequences encoding PP can be
transformed into cell
lines using expression vectors which may contain viral origins of replication
and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media
before being switched to selective media. The purpose of the selectable marker
is to confer resistance
to a selective agent, and its presence allows growth and recovery of cells
which successfully express
the introduced sequences. Resistant clones of stably transformed cells may be
propagated using
tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk- and apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
CA 02417359 2003-O1-27
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or herbicide resistance can be used as the basis for selection. For example,
dhff- confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding PP is inserted within a marker gene sequence, transformed
cells containing
sequences encoding PP can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding PP under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually indicates
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding PP and
that express PP
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 fox the detection and/or quantification of nucleic acid or
protein sequences.
Immunological methods for detecting and measuring the expression of PP using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FAGS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on PP is
preferred, but a competitive
binding assay may be employed. These and other assays are well known in the
art. (See, e.g.,
Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press,
St. Paul MN, Sect.
1V; Coligan, J.E. et al. (1997) Current Protocols in Immunoloey, Greene Pub.
Associates and Wiley-
Tnterscience, New York NY; and 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
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WO 02/10363 PCT/USO1/23716
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding PP include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, the sequences encoding PP, or any fragments thereof, may be
cloned into a vector for
the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerise
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding PP may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors
containing polynucleotides which encode PP may be designed to contain signal
sequences which
direct secretion of PP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, andlor activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding PP 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 PP
protein containing a
heterologous moiety that can be recognized by a commercially available
antibody may facilitate the
screening of peptide libraries for inhibitors of PP activity. Heterologous
protein and peptide moieties
may also facilitate purification of fusion proteins using commercially
available affinity matrices.
Such moieties include, but are not limited to, glutathione S-transferase
(GST), maltose binding
protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His,
FLAG, c-myc, and
hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their
cognate fusion
proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
and metal-chelate
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WO 02/10363 PCT/USO1/23716
resins, respectively. FLAG, c-rnyc, 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 PP encoding sequence and the heterologous
protein sequence, so
that PP may be cleaved away from the heterologous moiety following
purification. Methods for
fusion protein expression and purification are discussed in Ausubel (1995,
supra, ch. 10). A variety
of commercially available kits may also be used to facilitate expression and
purification of fusion
proteins.
In a further embodiment of the invention, synthesis of radiolabeled PP may be
achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
PP of the present invention or fragments thereof may be used to screen for
compounds that
specifically bind to PP. At least one and up to a plurality of test compounds
may be screened for
specific binding to PP. Examples of test compounds include antibodies,
oligonucleotides, proteins
(e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
PP, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which PP binds,
or to at least a fragment of the receptor, e.g., the ligand binding site. In
either case, the compound
can be rationally designed using known techniques. In one embodiment,
screening for these
compounds involves producing appropriate cells which express PP, either as a
secreted protein or on
the cell membrane. Preferred cells include cells from mammals, yeast,
Drosophila, or E. coli. Cells
expressing PP or cell membrane fractions which contain PP are then contacted
with a test compound
and binding, stimulation, or inhibition of activity of either PP 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 rnay comprise the steps of combining at least one test compound with
PP, either in solution
or affixed to a solid support, and detecting the binding of PP 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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PP of the present invention or fragments thereof may be used to screen for
compounds that
modulate the activity of PP. Such compounds may include agonists, antagonists,
or partial or inverse
agonists. In one embodiment, an assay is performed under conditions permissive
for PP activity,
wherein PP is combined with at least one test compound, and the activity of PP
in the presence of a
test compound is compared with the activity of PP in the absence of the test
compound. A change in
the activity of PP in the presence of the test compound is indicative of a
compound that modulates the
activity of PP. Alternatively, a test compound is combined with an in vitro or
cell-free system
comprising PP under conditions suitable for PP activity, and the assay is
performed. In either of these
assays, a test compound which modulates the activity of PP 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 PP or their mammalian homologs
rnay be
"knocked out" in an animal model system using homologous recombination in
embryonic stem (ES)
cells. Such techniques are well known in the art and are useful for the
generation of animal models of
human disease. (See, e.g., U.S. Patent Number 5,175,383 and U.S. Patent Number
5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker genes e.g., the neomycin phosphotransferase
gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding
region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-IoxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding PP may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
Iineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding PP 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 PP is injected into animal ES cells, and the injected
sequence integrates into
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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 PP, e.g., by secreting PP in its milk, may also
serve as a convenient
source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-
74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of PP and protein phosphatases. In addition, the expression of
PP is closely
associated with thalamus, pancreas, testis, brain, vascular, and fetal lung
tissues, as well as colon
tissue pseudopolyps associated with multiple tubuvillous adenomas. Therefore,
PP appears to play a
role in immune system disorders, neurological disorders, developmental
disorders, and cell
proliferative disorders. In the treatment of disorders associated with
increased PP expression or
activity, it is desirable to decrease the expression or activity of PP. In the
treatment of disorders
associated with decreased PP expression or activity, it is desirable to
increase the expression or
activity of PP.
Therefore, in one embodiment, PP 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 PP.
Examples of such disorders include, but axe not limited to, an immune system
disorder, such as
acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of
Bruton, common
vaxiable immunodeficiency (CVn, DiGeorge's syndrome (thymic hypoplasia),
thymic dysplasia,
isolated IgA deficiency, severe combined immunodeficiency disease (SC)D),
immunodeficiency with
thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi
syndrome, chronic
granulomatous diseases, hereditary angioneurotic edema, immunodeficiency
associated with
Cushing's disease, Addison's disease, adult respiratory distress syndrome,
allergies., ankylosing
spondylitis, amyloidosis, anemia, asthma, atherosclerosis, 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, 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 extracozporeal circulation, viral,
bacterial, fungal,
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parasitic, protozoal, and helminthic infections, and trauma; 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, prior 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; a developmental disorder, such as renal tubular
acidosis, anemia, Cushing's
syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy,
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, and
sensorineural hearing loss; and 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.
In another embodiment, a vector capable of expressing PP 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 PP including, but not limited to, those described above.
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In a further embodiment, a composition comprising a substantially purified PP
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 PP including, but not
limited to, those provided
above.
In still another embodiment, an agonist which modulates the activity of PP may
be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of PP including, but not limited to, those listed above.
In a further embodiment, an antagonist of PP may be administered to a subject
to treat or
prevent a disorder associated with increased expression or activity of PP.
Examples of such disorder
include, but are not limited to, those immune system disorders, neurological
disorders, developmental
disorders, and cell proliferative disorders described above. In one aspect, an
antibody which
specifically binds PP 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
expiess PP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding PP may be administered to a subject to treat or prevent a disorder
associated with increased
expression or activity of PP including, but not limited to, those described
above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of PP may be produced using methods which are generally known in
the art. In
particular, purified PP may be used to produce antibodies or to screen
libraries of pharmaceutical
agents to identify those which specifically bind PP. Antibodies to PP may also
be generated using
methods that are well known in the art. Such antibodies may include, but are
not limited to,
polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments,
and fragments
produced by a Fab expression library. Neutralizing antibodies (i.e., those
which inhibit dimer
formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with PP or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adZuvants 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
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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 PP
have an amino acid sequence consisting of at least about 5 amino acids, and
generally will consist of
at least about 10 amino acids. It is also preferable that these oligopeptides,
peptides, or fragments are
identical to a portion of the amino acid sequence of the natural protein.
Short stretches of PP amino
acids may be fused with those of another protein, such as KLH, and antibodies
to the chimeric
molecule may be produced.
Monoclonal antibodies to PP may be prepared using any technique which provides
for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Aced. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
I5 In addition, techniques developed for the production of "chimeric
antibodies," such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Aced. Sci. USA 81:6851-6855; Neuberger, M.S, et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce PP-
specific single chain
antibodies. Antibodies with related specificity, but of distinct idiotypic
composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g.,
Burton, D.R. (1991) Proc. Natl. Aced. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Aced. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for PP may also be
generated. For
example, such fragments include, but are not limited to, F(ab')Z fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by xeducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
43
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polyclonal or monoclonal antibodies with established specificities axe well
known in the art. Such
immunoassays typically involve the measurement of complex formation between PP
and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to
two non-interfering PP 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 PP. Affinity
is expressed as an
association constant, Ka, which is defined as the molar concentration of PP-
antibody complex divided
by the molar concentrations of free antigen and free antibody under
equilibrium conditions. The Ka
determined fox a preparation of polyclonal antibodies, which are heterogeneous
in their affinities for
multiple PP epitopes, represents the average affinity, or avidity, of the
antibodies for PP. The Ka
determined for a preparation of monoclonal antibodies, which are monospecific
for a particular PP
epitope, represents a true measure of affinity. High-affinity antibody
preparations with Ka ranging
from about 109 to 10'2 L/mole are preferred for use in immunoassays in which
the PP-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 PP, 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 (I991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is generally employed in procedures
requiring precipitation
of PP-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity, and
guidelines for antibody quality and usage in various applications, are
generally available. (See, e.g.,
Catty, supra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding PP, 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
PP. 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 PP.
(See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
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WO 02/10363 PCT/USO1/23716
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, I~.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding PP 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)-Xl disease
characterized by X-
linked inheritance (Cavazzana-Calva, 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; Bardignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cel175:207-216; Crystal, R.G. et al.
(1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Samia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites
(e.g., against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA. 93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trypanosoma cruzi). In the
case where a genetic deficiency in PP expression or regulation causes disease,
the expression of PP
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 PP
are treated by constructing mammalian expression vectors encoding PP and
introducing these vectors
by mechanical means into PP-deficient cells. Mechanical transfer technologies
for use with cells in
vivo or ex vitro include (i) direct DNA microinjection into individual cells,
(ii) ballistic gold particle
delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene
transfer, and (v) the use of
DNA transposans (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem.
62:191-217; Ivics,
CA 02417359 2003-O1-27
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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 PP 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), and
PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). PP may
be
expressed using (i) a constitutively active promoter, (e.g., from
cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes),
(ii) an inducible
promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard
(1992) Proc. Natl.
Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769;
Rossi, F.M.V. and
H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in
the T-REX plasmid
(Invitrogen)); the ecdysone-inducible promoter (available in the plasmids
PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone
inducible promoter
(Rossi, F.M.V. and Blau, H.M. su ra ), or (iii) a tissue-specific promoter or
the native promoter of the
endogenous gene encoding PP 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 PP expression are treated by constructing a retrovirus vector
consisting of (i) the
. polynucleotide encoding PP under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
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discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
cells), and the return of transduced cells to a patient are procedures well
known to persons skilled in
the art of gene therapy and have been well documented (Ranga, U. et al. (1997)
J. Virol. 71:7020-
7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)
Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding PP to cells which have one or more genetic
abnormalities with respect to
the expression of PP. The construction and packaging of adenovirus-based
vectors are well known to
those with ordinary skill in the art. Replication defective adenovirus vectors
have proven to be
versatile for importing genes encoding immunoregulatory proteins into intact
islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999)
Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature
18:389:239-242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding PP to target cells which have one or more genetic
abnormalities with
respect to the expression of PP. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing PP to cells of the central nervous system,
for which HSV has a
tropism. The construction and packaging of herpes-based vectors axe well known
to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is
hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use
of recombinant
HSV d92 which consists of a genome containing at least one exogenous gene to
be transferred to a
cell under the control of the appropriate promoter for purposes including
human gene therapy. Also
taught by this patent are the construction and use of recombinant HSV strains
deleted for ICP4, ICP27
and ICP22. Fox HSV vectors, see also Goins, W.F. et al. (1999) J. Virol.
73:519-532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The
manipulation of cloned
herpesvirus sequences, the generation of recombinant virus following the
transfection of multiple
plasmids containing different segments of the large herpesvirus genomes, the
growth and propagation
of herpesvirus, and the infection of cells with herpesvirus are techniques
well known to those of
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ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding PP to target cells. The biology of the
prototypic alphavirus, Semliki
Forest Virus (SFV), has been studied extensively and gene transfer vectors
have been based on the
SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol. 9:464-469).
During alphavirus
RNA replication, a subgenomic RNA is generated that normally encodes the viral
capsid proteins.
This subgenomic RNA replicates to higher levels than the full length genomic
RNA, resulting in the
overproduction of capsid proteins relative to the viral proteins with
enzymatic activity (e.g., protease
and polymerase). Similarly, inserting the coding sequence for PP into the
alphavirus genome in place
of the capsid-coding region results in the production of a large number of PP-
coding RNAs and the
synthesis of high levels of PP 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 (S1N) 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 PP
into a variety of cell types. The specific transduction of a subset of cells
in a population may require
the sorting of cells prior to transduction. The methods of manipulating
infectious cDNA clones of
alphaviruses, performing alphavirus cDNA and RNA transfections, and performing
alphavirus
infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions
-10 and +10 from the start site, may also be employed to inhibit gene
expression. Similarly,
inhibition can be achieved using triple helix base-pairing methodology. Triple
helix pairing is useful
because it causes inhibition of the ability of the double helix to open
sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using
triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et
al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and Immunolo~ic Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-
177.) A complementary sequence or antisense molecule may also be designed to
block translation of
mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding PP.
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,
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GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding PP. Such DNA sequences may be incorporated into a wide
variety of vectors
with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these
cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines,
cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine,
queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine,
cytidine, guanine, thymine, and uridine which are not as easily recognized by
endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding PP. 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 PP
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding PP may be therapeutically useful, and in the treatment of disorders
associated with
decreased PP expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding PP may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
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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-occurnng 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 PP is exposed to at least one test compound thus
obtained. The sample may
comprise, for example, an intact or permeabilized cell, or an in vitro cell-
free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
PP are assayed by
any method commonly known in the art. Typically, the expression of a specific
nucleotide is
detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
of the polynucleotide encoding PP. 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 earned out, for example, using a Schizosaccharomyces pombe gene
expression system
(Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000)
Nucleic Acids Res.
28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000)
Biochem. Biophys. Res.
Commun. 268:8-13). A particular embodiment of the present invention involves
screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition
which generally comprises an active ingredient formulated with a
pharmaceutically acceptable
excipient. Excipients may include, for example, sugars, starches, celluloses,
gums, and proteins.
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Various formulations are commonly known and are thoroughly discussed in the
latest edition of
Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
compositions may
consist of PP, antibodies to PP, and mimetics, agonists, antagonists, or
inhibitors of PP.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, intra-
axterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of
fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g. larger peptides
and proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the
lung have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton,
J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage
of administration
without needle injection, and obviates the need 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 PP or fragments thereof. For example, liposome
preparations containing
a cell-impermeable macromolecule may promote cell fusion and intracellular
delivery of the
macromolecule. Alternatively, PP or a fragment thereof may be joined to a
short cationic N-terminal
portion from the HIV Tat-1 protein. Fusion proteins thus generated have been
found to transduce into
the cells of all tissues, including the brain, in a mouse model system
(Schwarze, S.R. et al. (1999)
Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs,
monkeys, or pigs. An animal model may also be used to determine the
appropriate concentration
range and route of administration. Such information can then be used to
determine useful doses and
routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example PP or
fragments thereof, antibodies of PP, and agonists, antagonists or inhibitors
of PP, which ameliorates
the symptoms or condition. Therapeutic efficacy and toxicity may be determined
by standaxd
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
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the population) statistics. The dose ratio of toxic to therapeutic effects is
the therapeutic index, which
can be expressed as the LDSO/EDSO ratio. Compositions which exhibit large
therapeutic indices are
preferred. The data obtained from cell culture assays and animal studies are
used to formulate a range
of dosage for human use. The dosage contained in such compositions is
preferably within a range of
circulating concentrations that includes the EDSO with little or no toxicity.
The dosage varies within
this range depending upon the dosage form employed, the sensitivity of the
patient, and the route of
administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting compositions may be administered every 3 to 4
days, every week,
or biweekly depending on the half-life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about O. l ,ug to 100,000 ,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 PP may be used for
the diagnosis
of disorders characterized by expression of PP, or in assays to monitor
patients being treated with PP
or agonists, antagonists, or inhibitors of PP. Antibodies useful for
diagnostic purposes may be
prepaxed in the same manner as described above for therapeutics. Diagnostic
assays for PP include
methods which utilize the antibody and a label to detect PP 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 PP, including ELISAs, RIAs, and FAGS, are
known in
the art and provide a basis for diagnosing altered or abnormal levels of PP
expression. Normal or
standard values for PP expression are established by combining body fluids or
cell extracts taken
from normal mammalian subjects, for example, human subjects, with antibodies
to PP under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of PP
expressed in subject,
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control, and disease samples from biopsied tissues are compared with the
standard values. Deviation
between standard and subject values establishes the parameters for diagnosing
disease.
In another embodiment of the invention, the polynucleotides encoding PP may be
used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of PP may
be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
PP, and to monitor regulation of PP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding PP or closely related
molecules may be used to
identify nucleic acid sequences which encode PP. The specificity of the probe,
whether it is made
from a highly specific region, e.g., the 5'regulatory region, or from a less
specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurnng sequences encoding PP, 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 PP encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:11-20 or from
genomic sequences including promoters, enhancers, and introns of the PP gene.
Means for producing specific hybridization probes for DNAs encoding PP include
the cloning
of polynucleotide sequences encoding PP or PP derivatives into vectors for the
production of mRNA
probes. Such vectors are known~in the art, are commercially available, and may
be used to synthesize
RNA probes in vitro by means of the addition of the appropriate RNA
polymerases and the
appropriate labeled nucleotides. Hybridization probes may be labeled by a
variety of reporter groups,
for example, by radionuclides such as 32P or 355, or by enzymatic labels, such
as alkaline phosphatase
coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotide sequences encoding PP may be used for the diagnosis of
disorders associated
with expression of PP. Examples of such disorders include, but are not limited
to, an immune system
disorder, such as acquired immunodeficiency syndrome (AIDS), X-linked
agammaglobinemia of
Bruton, common variable immunodeficiency (CVI], DiGeorge's syndrome (thymic
hypoplasia),
thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency
disease (SLID),
immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome),
Chediak-Higashi
syndrome, chronic granulomatous diseases, hereditary angioneurotic edema,
immunodeficiency
associated with Cushing's disease, Addison's disease, adult respiratory
distress syndrome, allergies,
ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,
autoimmune hemolytic anemia,
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autoimmune thyxoiditis, 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, 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 helrninthic infections, and trauma; 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; a developmental disorder, such as renal tubular
acidosis, anemia, Cushing's
syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy,
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, and
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sensorineural hearing loss; and 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.
The polynucleotide sequences encoding PP 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 PP expression.
Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding PP may be useful in
assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding PP maybe labeled by standard methods and added to a fluid
or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a
suitable incubation period, the sample is washed and the signal is quantified
and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to
a control sample then the presence of altered levels of nucleotide sequences
encoding PP 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 PP, 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 PP, 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 PP
may involve the use of PCR. These oligomers may be chemically synthesized,
genexated
enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a polynucleotide
encoding PP, or a fragment of a polynucleotide complementary to the
polynucleotide encoding PP,
and will be employed under optimized conditions for identification of a
specific gene or condition.
Oligomers may also be employed under less stringent conditions for detection
or quantification of
closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding PP may be used to detect single nucleotide polymorphisms (SNPs). SNPs
are substitutions,
insertions and deletions that are a frequent cause of inherited or acquired
genetic disease in humans.
Methods of SNP detection include, but are not limited to, single-stranded
conformation
polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP,
oligonucleotide primers
derived from the polynucleotide sequences encoding PP are used to amplify DNA
using the
polymerase chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the
secondary and tertiary structures of PCR products in single-stranded form, and
these differences are
detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers
are fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such
as DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico
SNP (isSNP), are capable of identifying polymorphisms by comparing the
sequence of individual
overlapping DNA fragments which assemble into a common consensus sequence.
These computer-
based methods filter out sequence variations due to laboratory preparation of
DNA and sequencing
errors using statistical models and automated analyses of DNA sequence
chromatograms. In the
alternative, SNPs may be detected and characterized by mass spectrometry
using, for example, the
high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
Methods which may also be used to quantify the expression of PP include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. hnmunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
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accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives
rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information rnay be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and
effective treatment regimen for that patient. For example, therapeutic agents
which are highly
effective and display the fewest side effects may be selected for a patient
based on his/her
pharmacogenomic profile.
In another embodiment, PP, fragments of PP, or antibodies specific for PP may
be used as
elements on a microarray. The microarray may be used to monitor or measure
protein-protein
interactions, drug-target interactions, and gene expression profiles, as
described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines,
biopsies, or other biological samples. The transcript image may thus reflect
gene expression in vivo,
as in the case of a tissue or biopsy sample, or in vitro, as in the case of a
cell line.
Transcript images which profile the expression of the polynucleotides of the
present
invention may also be used in conjunction with in vitro model systems and
preclinical evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
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compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share
those toxic properties. These fingerprints or signatures are most useful and
refined when they contain
expression information from a large number of genes and gene families.
Ideally, a genome-wide
measurement of expression provides the highest quality signature. Even genes
whose expression is
not altered by any tested compounds are important as well, as the levels of
expression of these genes
are used to normalize the rest of the expression data. The normalization
procedure is useful for
comparison of expression data after treatment with different compounds. While
the assignment of
gene function to elements of a toxicant signature aids in interpretation of
toxicity mechanisms,
knowledge of gene function is not necessary for the statistical matching of
signatures which leads to
prediction of toxicity. (See, for example, Press Release 00-02 from the
National Institute of
Environmental Health Sciences, released February 29, 2000, available at
http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue ox cell type. Each
protein component of a
proteome can be subjected individually to further analysis. Proteome
expression patterns, or profiles,
are analyzed by quantifying the number of expressed proteins and their
relative abundance under
given conditions and at a given time. A profile of a cell's proteome may thus
be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample are
separated by isoelectric focusing in the first dimension, and then according
to molecular weight by
sodium dodecyl sulfate slab gel electrophoresis in the second dimension
(Steiner and Anderson,
su ra). The proteins are visualized in the gel as discrete and uniquely
positioned spots, typically by
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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, fox example, from
biological samples either treated or untreated with a test compound or
therapeutic agent, are
compared to identify any changes in protein spot density related to the
treatment. The proteins in the
spots are partially sequenced using, for example, standard methods employing
chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein in a spot
may be determined by
comparing its partial sequence, preferably of at least 5 contiguous amino acid
residues, to the
polypeptide sequences of the present invention. In some cases, further
sequence data may be
obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for PP to
quantify the
levels of PP expression. In one embodiment, the antibodies are used as
elements on a microarray, and
protein expression levels are quantified by exposing the microarray to the
sample and detecting the
levels of protein bound to each array element (Lueking, A. et al. (1999) Anal.
Biochem. 270:103-111;
Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a variety of
methods known in the art, for example, by reacting the proteins in the sample
with a thiol- or amino-
reactive fluorescent compound and detecting the amount of fluorescence bound
at each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to
rapid degradation of mRNA, so proteomic profiling may be more reliable and
informative in such
cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological
sample. A difference in the amount of protein between the two samples is
indicative of a toxic
response to the test compound in the treated sample. Individual proteins are
identified by sequencing
the amino acid residues of the 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
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incubated with antibodies specific to the polypeptides of the present
invention. The amount of
protein recognized by the antibodies is quantified. The amount of protein in
the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount of
protein between the two samples is indicative of a toxic response to the test
compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W0951251116;
Shalom D. et al.
(1995) PCT application W095135505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are
well known and thoroughly described in DNA Microarrays: A Practical Approach,
M. Schena, ed.
(1999) Oxford University Press, London, hereby expressly incorporated by
reference.
In another embodiment of the invention, nucleic acid sequences encoding PP may
be used to
generate hybridization probes useful in mapping the naturally occurnng genomic
sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a multi-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
(See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding PP 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,
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may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to 11q22-23,
any sequences mapping to that area may represent associated or regulatory
genes for further
investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The
nucleotide sequence of
the instant invention may also be used to detect differences in the
chromosomal location due to
translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, PP, 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 PP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with PP, or
fragments thereof, and
washed. Bound PP is then detected by methods well known in the art. Purified
PP 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 PP specifically compete with a test compound for
binding PP. In this
manner, antibodies can be used to detect the presence of any peptide which
shares one or more
antigenic determinants with PP.
Iu additional embodiments, the nucleotide sequences which encode PP may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are,
therefore, to be construed as merely illustrative, and not limitative of the
remainder of the disclosure
in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below,
including U.S. Ser. No. 60/221,679, U.S. Ser. No. 60/223,272, U.S. Ser. No.
60/224,309, U.S. Ser.
No. 60/226,728, U.S. Ser. No. 60/229,254, and U.S. Ser. No. 60/231,366, are
expressly incorporated
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by reference herein.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database
(Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues
were homogenized
and lysed in guanidinium isothiocyanate, while others were homogenized and
lysed in phenol or in a
suitable mixture of denaturants, such as TRIZOL (Life Technologies), a
monophasic solution of
phenol and guanidine isothiocyanate. The resulting lysates were centrifuged
over CsCI cushions or
extracted with chloroform. RNA was precipitated from the lysates with either
isopropanol or sodium
acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo Alto
CA), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli cells
including XL1-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DH10B, or
ElectroMAX
DHlOB from Life Technologies.
II. Isolation of cDNA Clones
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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 plasnnid DNA was
quantified fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-
200 thermal
cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, sue, unit 7.7). Some of the cDNA sequences were selected for extension
using the techniques
disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein
family
databases such as PFAM. (HMM is a probabilistic approach which analyzes
consensus primary
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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, BL1MPS, and
HIVBVIER. The
Incyte cDNA sequences were assembled to produce full length polynucleotide
sequences.
Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched
sequences, or
Genscan-predicted coding sequences (see Examples IV and V) were used to extend
Incyte cDNA
assemblages to full length. Assembly was performed using programs based on
Phred, Phrap, and
Consed, and cDNA assemblages were screened for open reading frames using
programs based on
GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were
translated to derive
the corresponding full length polypeptide sequences. Alternatively, a
polypeptide of the invention
may begin at any of the methionine residues of the full length translated
polypeptide. Full length
polypeptide sequences were subsequently analyzed by querying against databases
such as the
GenBank protein databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM,
Prosite,
and hidden Markov model (HMM)-based protein family databases such as PFAM.
Full length
polynucleotide sequences are also analyzed using MACDNASIS PRO software
(Hitachi Software
Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide
and polypeptide sequence alignments are generated using default parameters
specified by the
CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment
program
(DNASTAR), which also calculates the percent identity between aligned
sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and
threshold parameters. The first column of Table 7 shows the tools, programs,
and algorithms used,
the second column provides brief descriptions thereof, the third column
presents appropriate
references, all of which are incorporated by reference herein in their
entirety, and the fourth column
presents, where applicable, the scores, probability values, and other
parameters used to evaluate the
strength of a match between two sequences (the higher the score or the lower
the probability value,
the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide
and polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ
ID NO:11-20. Fragments from about 20 to about 4000 nucleotides which are
useful in hybridization
and amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative protein phosphatases were initially identified by running the Genscan
gene
identification program against public genomic sequence databases (e.g., gbpri
and gbhtg). Genscan is
a general-purpose gene identification program which analyzes genomic DNA
sequences from a
variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-
94, and Burge, C. and
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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 phosphatases, the encoded polypeptides
were analyzed by
querying against PFAM models for protein phosphatases. Potential protein
phosphatases were also
identified by homology to Incyte cDNA sequences that had been annotated as
protein phosphatases.
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 Ineyte cDNA or
public cDNA coverage of the Genscan-predicted sequences, thus providing
evidence for transcription.
When Incyte cDNA coverage was available, this information was used to correct
or confirm the
Genscan predicted sequence. Full length polynucleotide sequences were obtained
by assembling
Genscan-predicted coding sequences with Incyte cDNA sequences and/or public
cDNA sequences
using the assembly process described in Example III. Alternatively, full
length polynucleotide
sequences were derived entirely from edited or unedited Genscan-predicted
coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
III were mapped 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
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by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original'GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for
homologous genomic sequences from the public human genome databases. Partial
DNA sequences
were therefore "stretched" or extended by the addition of homologous genomic
sequences. The
resultant stretched sequences were examined to determine whether it contained
a complete gene.
VI. Chromosomal Mapping of PP Encoding Polynucleotides
The sequences which were used to assemble SEQ ~ NO:11-20 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 N0:11-20 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.} The cM
distances are based on genetic markers mapped by Genethon which provide
boundaries for radiation
hybrid markers whose sequences were included in each of the clusters. Human
genome maps and
other resources available to the public, such as the NCBI "GeneMap'99" World
Wide Web site
(http://www.ncbi.nlin.nih.gov/genemap/), can be employed to determine if
previously identified
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disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook, s
bra, ch. 7; Ausubel
(1995) supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the
computer search can be modified to determine whether any particular match is
categorized as exact or
similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding PP are analyzed with respect
to the tissue
sources from which they were derived. For example, some full length sequences
are assembled, at
least in part, with overlapping Incyte cDNA sequences (see Example 111). Each
cDNA sequence is
derived from a cDNA library constructed from a human tissue. Each human tissue
is classified into
one of the following organ/tissue categories: cardiovascular system;
connective tissue; digestive
system; embryonic structures; endocrine system; exocrine glands; genitalia,
female; genitalia, male;
germ cells; hemic and immune system; liver; musculoskeletal system; nervous
system; pancreas;
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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 PP. cDNA sequences and cDNA
library/tissue
information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto
CA).
VIII. Extension of PP Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mgz+, (NH4)ZS04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68 ° C, 5 min; Step 7: storage at 4 ° C.
The concentration of DNA in each well was determined by dispensing 100 ~,1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~,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 ,u1 to 10 ,u1 aliquot of the reaction mixture was
analyzed by
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electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerise (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 polymerise
(Amersham Pharmacia Biotech) and Pfu DNA polymerise (Stratagene) with the
following
parameters: Step l: 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°Io dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC
energy transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5'regulatory sequences using the above procedure along with
oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID NO:l 1-20 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ,uCi of
[y 3zP~ adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextrin bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
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Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared. .
X. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena (1999),
s-unra). Suggested substrates include silicon, silica, glass slides, glass
chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to
arrange and link
elements to the surface of a substrate using thermal, W, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinary skill in the art and may contain any appropriate number of
elements. (See, e.g.,
Schena, M. et aI. (I995) Science 270:467-470; Shalom D. et al. (I996) Genome
Res. 6:639-645;
Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
far ease of defection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element
on the microarray may be assessed. In one embodiment, microarray preparation
and usage is
described in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/pl oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/~tl RNase inhibitor, 500 ~.M dATP, 500 ~.M
dGTP, 500 p,M dTTP, 40
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~.M dCTP, 40 ~.M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 xnI volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of O.SM sodium
hydroxide and
incubated for 20 minutes at 85° C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
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.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
~,g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water,
and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides
are cured in a
110°C oven.
' Array elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522, incorporated herein by reference. 1 ~.l of the array
element DNA, at an average
concentration of 100 ng/pl, is loaded into the open capillary printing element
by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in
0.2% SDS and distilled water as before.
Hybridization
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Hybridization reactions contain 9 ~.l of sample mixture consisting of 0.2 ~,g
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered
with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just
slightly larger than a microscope slide. The chamber is kept at 100% humidity
internally by the
addition of 140 ~,1 of SX SSC in a corner of the chamber. The chamber
containing the arrays is
incubated for about 6.5 hours at 60° C. The arrays are washed for 10
min at 45 ° C in a first wash
buffer (1X SSC, O.I% SDS), three times for 10 minutes each at 45°C in a
second wash buffer (0.1X
SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
I5 containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are
differentially expressed, the calibration is done by labeling samples of the
calibrating cDNA with the
two fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
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computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores. using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each
spot is centered in each element of the grid. The fluorescence signal within
each element is then
integrated to obtain a numerical value corresponding to the average intensity
of the signal. The
software used for signal analysis is the GEMTOOLS gene expression analysis
program (Incyte).
XI. Complementary Polynucleotides
Sequences complementary to the PP-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring PP. 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 PP. 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 PP-encoding transcript.
XII. Expression of PP
Expression and purification of PP is achieved using bacterial or virus-based
expression
systems. For expression of PP in bacteria, cDNA is subcloned into an
appropriate vector containing
an antibiotic resistance gene and an inducible promoter that directs high
levels of cDNA transcription.
Examples of such promoters include, but are not limited to, the trp-lac (tac)
hybrid promoter and the
TS or T7 bacteriophage promoter in conjunction with the lac operator
regulatory element.
Recombinant vectors are transformed into suitable bacterial hosts, e.g.,
BL21(DE3). Antibiotic
resistant bacteria express PP upon induction with isopropyl beta-D-
thiogalactopyranoside (IPTG).
Expression of PP in eukaryotic cells is achieved by infecting insect or
mammalian cell lines with
recombinant Auto.~raphica californica nuclear polyhedrosis virus (AcMNPV),
commonly known as
baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with
cDNA encoding PP by
either homologous recombination or bacterial-mediated transposition involving
transfer plasmid
intermediates. Viral infectivity is maintained and the strong polyhedrin
promoter drives high levels
of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera
frugiperda (Sf9) insect
cells in most cases, or human hepatocytes, in some cases. Infection of the
latter requires additional
genetic modifications to baculovirus. (See Engelhard, E.I~. et al. (1994)
Proc. Natl. Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.)
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In most expression systems, PP is synthesized as a fusion protein with, e.g.,
glutathione S-
transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-
kilodalton enzyme from Schistosoma japonicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from PP
at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffmity purification
using commercially available monoclonal and polyclonal anti-FLAG antibodies
(Eastman Kodak). 6-
His, a stretch of six consecutive histidine residues, enables purification on
metal-chelate resins
(QIAGEN). Methods for protein expression and purification are discussed in
Ausubel (1995, supra,
ch. 10 and 16). Purified PP obtained by these methods can be used directly in
the assays shown in
Examples XVI, XVII, XVIB, and XIX where applicable.
XIII. Functional Assays
PP function is assessed by expressing the sequences encoding PP at
physiologically elevated
levels in mammalian cell culture systems, cDNA is subcloned into a mammalian
expression vector
containing a strong promoter that drives high levels of cDNA expression.
Vectors of choice include
PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of
which contain
the cytomegalovirus promoter. 5-10 ~g of recombinant vector are transiently
transfected into a
human cell line, for example, an endothelial or hematopoietic cell line, using
either liposome
formulations or electroporation. 1-2 ,ug of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
The influence of PP on gene expression can be assessed using highly purified
populations of
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cells transfected with sequences encoding PP 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 PP and other genes of interest can be analyzed by
northern analysis or
microarray techniques.
XIV. Production of PP Specific Antibodies
PP substantially purified using polyacrylamide gel electrophoresis (PAGE; see,
e.g.,
Harrington, M.G. (1990) Methods Enzymol, 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the PP amino acid sequence is analyzed using LASERGENE software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide and anti-PP activity by, for example, binding the peptide or PP to
a substrate, blocking
with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-
iodinated goat anti-
rabbit IgG.
XV. Purification of Naturally Occurring PP Using Specific Antibodies
Naturally occurring or recombinant PP is substantially purified by
immunoaffmity
chromatography using antibodies specific for PP. An immunoaffinity column is
constructed by
covalently coupling anti-PP antibody to an activated chromatographic resin,
such as CNBr-activated
SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is
blocked and washed
according to the manufacturer's instructions.
Media containing PP are passed over the immunoaffinity column, and the column
is washed
under conditions that allow the preferential absorbance of PP (e.g., high
ionic strength buffers in the
presence of detergent). The column is eluted under conditions that disrupt
antibody/PP 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
PP is collected.
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XVI. Identification of Molecules Which Interact with PP
PP, or biologically active fragments thereof, are labeled with'z5I Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a mufti-well plate are incubated with the
labeled PP, washed, and
any wells with labeled PP complex are assayed. Data obtained using different
concentrations of PP
are used to calculate values for the number, affinity, and association of PP
with the candidate
molecules.
Alternatively, molecules interacting with PP 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 MATCHT~1AT~FR system
(Clontech).
PP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT)
which
employs the yeast two-hybrid system in a high-throughput manner to determine
all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
XVII. Demonstration of PP Activity
PP activity is measured by the hydrolysis of para-nitrophenyl phosphate
(PNPP). PP is
incubated together with PNPP in HEPES buffer pH 7.5, in the presence of 0.1%
(3-mercaptoethanol at
37°C for 60 min. The reaction is stopped by the addition of 6 ml of 10
N NaOH (Diamond, R.H. et
al. (1994) Mol. Cell. Biol. 14:3752-62). Alternatively, acid phosphatase
activity of PP is
demonstrated by incubating PP-containing extract with 100 ~,1 of 10 mM PNPP in
0.1 M sodium
citrate, pH 4.5, and 50 ~.l of 40 mM NaCI at 37°C for 20 min. The
reaction is stopped by the addition
of 0.5 ml of 0.4 M glycine/NaOH, pH 10.4 (Saftig, P. et al. (1997) J. Biol.
Chem. 272:18628-18635).
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 PP in the assay.
In the alternative, PP 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% (3-
mercaptoethanol
and 10 ~,M substrate, 3zP-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 ~tl of
4% (w/v) activated charcoal in 0.6 M HCI, 90 mM Na4P20~, and 2 mM NaH2P04,
then centrifuged at
12,000 x g for 5 min. Acid-soluble 3zPi is quantified by liquid scintillation
counting (Sinclair, C. et
al. (1999) J. Biol. Chem. 274:23666-23672).
XVIII. Identification of PP Inhibitors
Compounds to be tested are arrayed in the wells of a 384-well plate in varying
concentrations
along with an appropriate buffer and substrate, as described in the assays in
Example XVII. PP
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activity is measured for each well and the ability of each compound to inhibit
PP activity can be
determined, as well as the dose-response kinetics. This assay could also be
used to identify molecules
which enhance PP activity.
XIX. Identification of PP Substrates
A PP "substrate-trapping" assay takes advantage of the increased substrate
affinity that may
be conferred by certain mutations in the PTP signature sequence. PP bearing
these mutations form a
stable complex with their substrate; this complex may be isolated
biochemically. Site-directed
mutagenesis of invariant residues in the PTP signature sequence in a clone
encoding the catalytic
domain of PP is performed using a method standard in the art or a commercial
kit, such as the
MUTA-GENE kit from BIO-RAD. For expression of PP mutants in Escherichia coli,
DNA fragments
containing the mutation are exchanged with the corresponding wild-type
sequence in an expression
vector bearing the sequence encoding PP or a glutathione S-transferase (GST)-
PP fusion protein. PP
mutants are expressed in E. coli and purified by chromatography.
The expression vector is transfected into COS 1 or 293 cells via calcium
phosphate-mediated
transfection with 20 ~,g of CsCI-purified DNA per 10-cm dish of cells or 8 ~,g
per 6-cm dish. Forty-
eight hours after transfection, cells are stimulated with 100 ng/ml epidermal
growth factor to increase
tyrosine phosphorylation in cells, as the tyrosine kinase EGFR is abundant in
COS cells. Cells are
lysed in 50 mM Tris~HCl, pH 7.5/5 mM EDTA/150 mM NaCI/1% Triton X-100/5 mM
iodoacetic
acid/10 mM sodium phosphate/10 mM NaF/5 ~,g/ml leupeptin/5 ~g/ml aprotinin/1
mM benzamidine
(1 ml per 10-cm dish, 0.5 ml per 6-cm dish). PP is immunoprecipitated from
lysates with an
appropriate antibody. GST-PP fusion proteins are precipitated with glutathione-
Sepharose, 4 ~,g of
mAb or 10 ~,1 of beads respectively per mg of cell lysate. Complexes can be
visualized by PAGE or
further purified to identify substrate molecules (Flint, A.J. et al. (1997)
Proc. Natl. Acad. Sci. USA
94:1680-1685). '
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the
invention. Although the invention has been described in connection with
certain embodiments, it
should be understood that the invention as claimed should not be unduly
limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying
out the invention
which are obvious to those skilled in molecular biology or related fields are
intended to be within the
scope of the following claims.
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CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
<110> INCYTE GENOMICS, INC.
TANG, Y.Tom
ELLIOTT, Vicki S.
RAMKUMAR, Jayala~ni
YAO, Monique G.
BURFORD, Neil
WANG, Yumei E.
STEWART, Elizabeth A.
GANDHI, Ameena R.
PATTERSON, Chandra
LEE, Ernestine A.
HAFALIA, April J.A.
LU, Dyung Aina M.
TRIBOULEY, Catherine M.
GRIFFIN, Jennifer A.
BAUGHN, Mariah R.
YUE, Henry
WARREN, Bridget A.
NGUYEN, Danniel B.
WALIA, Narinder K.
KEARNEY, Liam
<120> PROTEIN PHOSPHATASES
<130> PI-0173 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/221,679; 60/223,272; 60/224,309; 60/226,728; 60/229,254;
60/231,366
<160> 2000-07-28; 2000-08-03; 2000-08-10; 2000-08-18; 2000-08-30;
2000-09-08
<170> PERL Program
<210> 1
<211> 545
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1905692CD1
<400> 1
Met Asn Glu Ser Pro Asp Pro Thr Asp Leu Ala Gly Val Ile Ile
1 5 10 15
Glu Leu Gly Pro Asn Asp Ser Pro Gln Thr Ser Glu Phe Lys Gly
20 25 30
A1a Thr Glu Glu Ala Pro Ala Lys Glu Ser Pro His Thr Ser Glu
35 40 45
Phe Lys Gly Ala Ala Arg Val Ser Pro Ile Ser Glu Ser Val Leu
50 55 60
Ala Arg Leu Ser Lys Phe Glu Asp Glu Asp Ala Glu Asn Val Ala
65 70 75
Ser Tyr Asp Ser Lys Ile Lys Lys Ile Va1 His Ser Ile Va1 Ser
80 85 90
Ser Phe Ala Phe Gly Leu Phe Gly Val Phe Leu Val Leu Leu Asp
95 100 105
Val Thr Leu Ile Leu Ala Asp Leu Ile Phe Thr Asp Ser Lys Leu
110 115 120
Tyr Ile Pro Leu Glu Tyr Arg Ser Ile Ser Leu Ala Ile Ala Leu
1/I8
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
125 130 135
Phe Phe Leu Met Asp Val Leu Leu Arg Val Phe Val Glu Arg Arg
140 145 150
Gln Gln Tyr Phe Ser Asp Leu Phe Asn Ile Leu Asp Thr Ala Tle
155 160 165
Ile Val Ile Leu Leu Leu Val Asp Val Val Tyr Ile Phe Phe Asp
170 175 180
Ile Lys Leu Leu Arg Asn Ile Pro Arg Trp Thr His Leu Leu Arg
185 190 195
Leu Leu Arg Leu Ile Ile Leu Leu Arg Ile Phe His Leu Phe His
200 205 210
Gln Lys Arg Gln Leu Glu Lys Leu Ile Arg Arg Arg Val Ser Glu
215 220 225
Asn Lys Arg Arg Tyr Thr Arg Asp Gly Phe Asp Leu Asp Leu Thr
230 235 240
Tyr Val Thr G1u Arg Ile Ile Ala Met Ser Phe Pro Ser Ser Gly
245 250 255
Arg Gln Ser Phe Tyr Arg Asn Pro Ile Lys Val Ile Pro Tyr Arg
260 265 270
Asp Met Thr Tyr Ile Leu Phe Ile Leu Gly Glu Arg Ala Tyr Asp
275 280 285
Pro Lys His Phe His Asn Arg Val Va1 Arg Ile Met Ile Asp Asp
290 295 300
His Asn Val Pro Thr Leu His Gln Met Val Val Phe Thr Lys Glu
305 310 315
Val Asn Glu Trp Met Ala Gln Asp Leu Glu Asn Ile Val Ala Ile
320 325 330
His Cys Lys Gly Gly Thr Asp Arg Thr Gly Thr Met Val Cys Ala
335 340 345
Phe Leu Ile Ala Ser Glu Ile Cys Ser Thr Ala Lys Glu Ser Leu
350 355 360
Tyr Tyr Phe Gly Glu Arg Arg Thr Asp Lys Thr His Ser Glu Lys
365 370 375
Phe Gln Gly Val Lys Thr Pro Ser G1n Lys Arg Tyr Val Ala Tyr
380 385 390
Phe Ala Gln Val Lys His Leu Tyr Asn Trp Asn Leu Pro Pro Arg
395 400 405
Arg Ile Leu Phe Ile Lys His Phe Ile Ile Tyr Ser Ile Pro Arg
410 415 420
Tyr Val Arg Asp Leu Lys Ile Gln Ile Glu Met Glu Lys Lys Val
425 430 435
Va1 Phe Ser Thr Ile Ser Leu Gly Lys Cys Ser Val Leu Asp Asn
440 445 450
Ile Thr Thr Asp Lys Ile Leu Ile Asp Val Phe Asp Gly Pro Pro
455 460 465
Leu Tyr Asp Asp Val Lys Val Gln Phe Phe Ser Ser Asn Leu Pro
470 475 480
Thr Tyr Tyr Asp Asn Cys Ser Phe Tyr Phe Trp Leu His Thr Ser
485 490 495
Phe Ile Glu Asn Asn Arg Leu Tyr Leu Pro Lys Asn Glu Leu Asp
500 505 510
Asn Leu His Lys Gln Lys Ala Arg Arg Ile Tyr Pro Ser Asp Phe
515 520 525
Ala Val Glu Ile Leu Phe Gly Glu Lys Met Thr Ser Ser Asp Val
530 535 540
Val Ala Gly Ser Asp
545
<210> 2
<211> 360
<212> PRT
<213> Homo Sapiens
2/18
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
<220>
<221> misc_feature
<223> Incyte ID No: 7476908CD1
<400> 2
Met Ile Glu Asp Thr Met Thr Leu Leu Ser Leu Leu Gly Arg Ile
1 5 10 15
Met Arg Tyr Phe Leu Leu Arg Pro Glu Thr Leu Phe Leu Leu Cys
20 25 30
Ile Ser Leu Ala Leu Trp Ser Tyr Phe Phe His Thr Asp Glu Val
35 40 45
Lys Thr Ile Va1 Lys Ser Ser Arg Asp Ala Val Lys Met Val Lys
50 55 60
Ser Lys Val Ala Glu Thr Met Gln Asn Asp Arg Leu Gly G1y Leu
65 70 75
Asp Val Leu Glu Ala Glu Phe Ser Lys Thr Trp Glu Phe Lys Asn
80 85 90
His Asn Val Ala Val Tyr Ser Ile Gln Gly Arg Arg Asp His Met
95 100 105
Glu Asp Arg Phe Glu Val Leu Thr Asp Leu A1a Asn Lys Thr His
110 115 120
Pro Ser Ile Phe Gly Ile Phe Asp Gly His Gly Gly Glu Thr Ala
125 130 135
Ala Glu Tyr Val Lys Ser Arg Leu Pro Glu Ala Leu Lys Gln His
140 145 150
Leu Gln Asp Tyr Glu Lys Asp Lys Glu Asn Ser Val Leu Ser Tyr
155 160 165
Gln Thr Ile Leu Glu Gln Gln Ile Leu Ser Ile Asp Arg Glu Met
170 175 180
Leu Glu Lys Leu Thr Val Ser Tyr Asp Glu Ala Gly Thr Thr Cys
185 190 195
Leu Ile Ala Leu Leu Ser Asp Lys Asp Leu Thr Val Ala Asn Val
200 205 210
Gly Asp Ser Arg Gly Val Leu Cys Asp Lys Asp Gly Asn Ala Ile
215 220 225
Pro Leu Ser His Asp His Lys Pro Tyr Gln Leu Lys Glu Arg Lys
230 235 240
Arg Ile Lys Arg Ala Gly Gly Phe Ile Ser Phe Asn Gly Ser Trp
245 250 255
Arg Val Gln Gly Ile Leu Ala Met Ser Arg Ser Leu Gly Asp Tyr
260 265 270
Pro Leu Lys Asn Leu Asn Val Val Ile Pro Asp Pro Asp I1e Leu
275 280 285
Thr Phe Asp Leu Asp Lys Leu Gln Pro Glu Phe Met Ile Leu Ala
290 295 300
Ser Asp Gly Leu Trp Asp Ala Phe Ser Asn Glu Glu Ala Val Arg
305 310 315
Phe Ile Lys Glu Arg Leu Asp Glu Pro His Phe Gly Ala Lys Ser
320 325 330
Ile Val Leu Gln Ser Phe Tyr Arg Gly Cys Pro Asp Asn Ile Thr
335 340 345
Val Met Val Val Lys Phe Arg Asn Ser Ser Lys Thr Glu Glu Gln
350 355 360
<210> 3
<211> 355
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7708162CD1
3/18
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
<400> 3
Met Asp Phe Leu His Arg Asn Gly Val Leu Ile Ile Gln His Leu
1 5 10 15
Gln Lys Asp Tyr Arg Ala Tyr Tyr Thr Phe Leu Asn Phe Met Ser
20 25 30
Asn Val Gly Asp Pro Arg Asn Ile Phe Phe Ile Tyr Phe Pro Leu
35 40 45
Cys Phe Gln Phe Asn Gln Thr Val Gly Thr Lys Met Ile Trp Val
50 55 60
Ala Val Ile Gly Asp Trp Leu Asn Leu Ile Phe Lys Trp Ile Leu
65 70 75
Phe Gly His Arg Pro Tyr Trp Trp Val Gln Glu Thr Gln Ile Tyr
80 85 90
Pro Asn His Ser Ser Pro Cys Leu Glu Gln Phe Pro Thr Thr Cys
95 100 105
Glu Thr Gly Pro Gly Ser Pro Ser Gly His Ala Met Gly Ala Ser
110 115 120
Cys Val Trp Tyr Val Met Val Thr Ala Ala Leu Ser His Thr Val
125 130 135
Cys Gly Met Asp Lys Phe Ser Ile Thr Leu His Arg Leu Thr Trp
140 145 150
Ser Phe Leu Trp Ser Val Phe Trp Leu Ile Gln Ile Ser Val Cys
155 160 165
Ile Ser Arg Val Phe Ile Ala Thr His Phe Pro His Gln Val Ile
170 175 180
Leu Gly Val Ile Gly Gly Met Leu Val Ala Glu Ala Phe G1u His
185 190 195
Thr Pro Gly Ile Gln Thr Ala Ser Leu G1y Thr Tyr Leu Lys Thr
200 205 210
Asn Leu Phe Leu Phe Leu Phe Ala Val Gly Phe Tyr Leu Leu Leu
215 220 225
Arg Val Leu Asn Ile Asp Leu Leu Trp Ser Val Pro Ile Ala Lys
230 235 240
Lys Trp Cys Ala Asn Pro Asp Trp Ile His Ile Asp Thr Thr Pro
245 250 255
Phe Ala Gly Leu Val Arg Asn Leu Gly Val Leu Phe Gly Leu Gly
260 265 270
Phe Ala Ile Asn Ser Glu Met Phe Leu Leu Ser Cys Arg Gly Gly
275 280 285
Asn Asn Tyr Thr Leu Ser Phe Arg Leu Leu Cys Ala Leu Thr Ser
290 295 300
Leu Thr Ile Leu Gln Leu Tyr His Phe Leu Gln Ile Pro Thr His
305 310 315
Glu Glu His Leu Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Ser
320 325 330
Ile Pro Leu Thr Val Val Ala Phe Ile Pro Tyr Ser Val His Met
335 340 345
Leu Met Lys Gln Ser Gly Lys Lys Ser Gln
350 355
<210> 4
<211> 493
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473603CD1
<400> 4
Met Leu Glu Ser Ala Glu G1n Leu Leu Val Glu Asp Leu Tyr Asn
1 5 10 15
Arg Val Arg Glu Lys Met Asp Asp Thr Ser Leu Tyr Asn Thr Pro
4/18
~~M~~
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
20 25 30
Cys Val Leu Asp Leu Gln Arg Ala Leu Val Gln Asp Arg Gln Glu
35 40 45
Ala Pro Trp Asn Glu Val Asp Glu Val Trp Pro Asn Val Phe Ile
50 55 60
Ala Asp Arg Ser Val Ala Val Asn Lys Gly Arg Leu Lys Arg Leu
65 70 75
Gly Ile Thr His Ile Leu Asn Ala Ala His G1y Thr Gly Val Tyr
80 85 90
Thr Gly Pro Glu Phe Tyr Thr Gly Leu Glu Ile Gln Tyr Leu Gly
95 100 105
Val Glu Val Asp Asp Phe Pro Glu Val Asp Ile Ser Gln His Phe
110 115 120
Arg Lys Ala Tyr Cys His Tyr Ile Ile Phe Ser Cys Va1 Phe Ile
125 130 135
Ser Gly Lys Val Leu Val Ser Ser Glu Met Gly Ile Ser Arg Ser
140 145 150
Ala Va1 Leu Val Val Ala Tyr Leu Met Ile Phe His Asn Met Ala
155 160 165
Ile Leu Glu Ala Leu Met Thr Val Arg Lys Lys Arg Ala Ile Tyr
170 175 180
Pro Asn Glu Gly Phe Leu Lys Gln Leu Arg Glu Leu Asn Glu Lys
185 190 195
Leu Met Glu G1u Arg Glu Glu Asp Tyr Gly Arg Glu Gly Gly Ser
200 205 210
Ala Glu Ala Glu Glu Gly Glu G1y Thr Gly Ser Met Leu Gly Ala
215 220 225
Arg Val His Ala Leu Thr Val Glu Glu Glu Asp Asp Ser Ala Ser
230 235 240
His Leu Ser Gly Ser Ser Leu Gly Lys Ala Thr Gln A1a Ser Lys
245 250 255
Pro Leu Thr Leu Ile Asp Glu Glu Glu Glu Glu Lys Leu Tyr Glu
260 265 270
Gln Trp Lys Lys Gly Gln Gly Leu Leu Ser Asp Lys Val Pro Gln
275 280 285
Asp Gly Gly Gly Trp Arg Ser Ala Ser Ser Gly Gln Gly Gly Glu
290 295 300
Glu Leu Glu Asp Glu Asp Val Glu Arg Ile Ile Gln Glu Trp Gln
305 310 325
Ser Arg Asn Glu Arg Tyr Gln Ala Glu Gly Tyr Arg Arg Trp Gly
320 325 ' 330
Arg Glu Glu Glu Lys Glu Glu Glu Ser Asp Ala Gly Ser Ser Val
335 340 345
G1y Arg Arg Arg Arg Thr Leu Ser Glu Ser Ser Ala Trp Glu Ser
350 355 360
Val Ser Ser His Asp Ile Trp Val Leu Lys Gln Gln Leu Glu Leu
365 370 375
Asn Arg Pro Asp His Gly Arg Arg Arg Arg Ala Asp Ser Met Ser
380 385 390
Ser Glu Ser Thr Trp Asp Ala Trp Asn Glu Arg Leu Leu Glu Ile
395 400 405
Glu Lys Glu Ala Ser Arg Arg Tyr His Ala Lys Ser Lys Arg Glu
410 415 420
Glu Ala Ala Asp Arg Ser Ser G1u Ala Gly Ser Arg Val Arg Glu
425 430 435
Asp Asp Glu Asp Ser Va1 Gly Ser Glu Ala Ser Ser Phe Tyr Asn
440 445 450
Phe Cys Ser Arg Asn Lys Asp Lys Leu Thr Ala Trp Lys Asp Gly
455 ~ 460 465
Arg Ser Arg Glu Ser Asn Leu Asp Phe Thr Arg Lys Thr Trp Glu
470 475 480
Arg Glu Thr Ala Ala Val Ser Pro Val Gln Arg Arg Gln
485 490
5/18
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
<210> 5
<211> 321
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476687CD1
<400> 5
Met Pro Leu Leu Pro Ala Ala Leu Thr Ser Ser Met Leu Tyr Phe
1 5 10 15
Gln Met Val Ile Met Ala Gly Thr Val Met Leu Ala Tyr Tyr Phe
20 25 30
Glu Tyr Thr Asp Thr Phe Thr Val Asn Val Gln Gly Phe Phe Cys
35 40 45
His Asp Ser Ala Tyr Arg Lys Pro Tyr Pro Gly Pro Glu Asp Ser
50 55 60
Ser Ala Val Pro Pro Val Leu Leu Tyr Ser Leu Ala A1a Gly Val
65 70 75
Pro Val Leu Val Ile Ile Val Gly Glu Thr Ala Val Phe Cys Leu
80 85 90
Gln Leu Ala Thr Arg Asp Phe Glu Asn Gln Glu Lys Thr Ile Leu
95 100 105
Thr G1y Asp Cys Cys Tyr Ile Asn Pro Leu Val Arg Arg Thr Val
110 115 120
Arg Phe Leu Gly Ile Tyr Thr Phe Gly Leu Phe Ala Thr Asp Ile
125 130 135
Phe Va1 Asn Ala Gly Gln Val Val Thr Gly Asn Leu Ala Pro His
140 145 150
Phe Leu Ala Leu Cys Lys Pro Asn Tyr Thr Ala Leu Gly Cys Gln
155 160 165
Gln Tyr Thr Gln Phe Ile Ser Gly Glu Glu Ala Cys Thr Gly Asn
170 175 180
Pro Asp Leu Ile Met Arg Ala Arg Lys Thr Phe Pro Ser Lys Glu
185 190 195
Ala Ala Leu Ser Val Tyr Ala Ala Met Tyr Leu Thr Met Tyr Ile
200 205 210
Thr Asn Thr Ile Lys Ala Lys Gly Thr Arg Leu Ala Lys Pro Val
215 220 225
Leu Cys Leu Gly Leu Met Cys Leu Ala Phe Leu Thr Gly Leu Asn
230 235 240
Arg Val Ala Glu Tyr Arg Asn His Trp Ser Asp Val Ile Ala Gly
245 250 255
Phe Leu Val G1y Ile Ser Ile Ala Val Phe Leu Val Val Cys Val
260 265 270
Val Asn Asn Phe Lys Gly Arg Gln Ala Glu Asn Glu His Ile His
275 280 285
Met Asp Asn Leu Ala Gln Met Pro Met Ile Ser Ile Pro Arg Val
290 295 300
Glu Ser Pro Leu Glu Lys Val Thr Ser Val Gln Asn His Ile Thr
305 310 315
Ala Phe Ala Glu Val Thr
320
<210> 6
<211> 426
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7480440CD1
6/18
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
<400> 6
Met Ala Gly Leu Gly Phe Trp Gly His Pro Ala G1y Pro Leu Leu
1 5 10 15
Leu Leu Leu Leu Leu Val Leu Pro Pro Arg Ala Leu Pro Glu Gly
20 25 30
Pro Leu Val Phe Val Ala Leu Val Phe Arg His Gly Asp Arg Ala
35 40 45
Pro Leu Ala Ser Tyr Pro Met Asp Pro His Lys Glu Val Ala Ser
50 55 60
Thr Leu Trp Pro Arg Gly Leu Gly Gln Leu Thr Thr Glu Gly Val
65 70 75
Arg Gln Gln Leu Glu Leu Gly Arg Phe Leu Arg Ser Arg Tyr Glu
80 85 90
Ala Phe Leu Ser Pro Glu Tyr Arg Arg Glu Glu Val Tyr Ile Arg
95 100 105
Ser Thr Asp Phe Asp Arg Thr Leu Glu Ser Ala Gln Ala Asn Leu
110 115 120
Ala Gly Leu Phe Pro Glu Ala Ala Pro Gly Ser Pro Glu Ala Arg
125 130 135
Trp Arg Pro Ile Pro Val His Thr Val Pro Val A1a Glu Asp Lys
140 145 150
Leu Leu Arg Phe Pro Met Arg Ser Cys Pro Arg Tyr His Glu Leu
155 160 165
Leu Arg Glu Ala Thr Glu Ala A1a Glu Tyr Gln Glu Ala Leu Glu
170 175 180
Gly Trp Thr Gly Phe Leu Ser Arg Leu Glu Asn Phe Thr Gly Leu
185 190 195
Ser Leu Val Gly Glu Pro Leu Arg Arg Ala Trp Lys Val Leu Asp
200 205 210
Thr Leu Met Cys Gln Gln Ala His Gly Leu Pro Leu Pro Ala Trp
215 220 225
Ala Ser Pro Asp Val Leu Arg Thr Leu A1a Gln Ile Ser Ala Leu
230 235 240
Asp Ile Gly Ala His Val Gly Pro Pro Arg Ala Ala Glu Lys Ala
245 250 255
Gln Leu Thr Gly Gly Ile Leu Leu Asn Ala Ile Leu Ala Asn Phe
260 265 270
Ser Arg Val Gln Arg Leu Gly Leu Pro Leu Lys Met Val Met Tyr
275 280 285
Ser Ala His Asp Ser Thr Leu Leu Ala Leu Gln Gly Ala Leu Gly
290 295 300
Leu Tyr Asp Gly His Thr Pro Pro Tyr Ala Ala Cys Leu G1y Phe
305 310 315
Glu Phe Arg Lys His Leu Gly Asn Pro Ala Lys Asp Gly Gly Asn
320 325 330
Val Thr Val Ser Leu Phe Tyr Arg Asn Asp Ser Ala His Leu Pro
335 340 345
Leu Pro Leu Ser Leu Pro Gly Cys Pro Ala Pro Cys Pro Leu Gly
350 355 360
Arg Phe Tyr Gln Leu Thr Ala Pro Ala Arg Pro Pro Ala His Gly
365 370 375
Val Ser Cys fiis Gly Pro Tyr Glu Ala Ala Ile Pro Pro Ala Pro
380 385 390
Va1 Val Pro Leu Leu Ala Gly Ala Val Ala Val Leu Val Ala Leu
395 400 405
Ser Leu Gly Leu Gly Leu Leu Ala Trp Arg Pro Gly Cys Leu Arg
410 415 420
Ala Leu Gly Gly Pro Val
425
<210> 7
<211> 665
<212> PRT
7/18
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7480570CD1
<400> 7
Met Ala His Glu Met Ile Gly Thr Gln Ile Val Thr G1u Arg Leu
1 5 10 15
Val Ala Leu Leu Glu Ser Gly Thr Glu Lys Val Leu Leu Ile Asp
20 25 30
Ser Arg Pro Phe Val Glu Tyr Asn Thr Ser His Ile Leu Glu Ala
35 40 45
Ile Asn Ile Asn Cys Ser Lys Leu Met Lys Arg Arg Leu Gln Gln
50 55 60
Asp Lys Val Leu Ile Thr Glu Leu Ile Gln His Ser Ala Lys His
65 70 75
Lys Val Asp Ile Asp Cys Ser Gln Lys Val Val Val Tyr Asp Gln
80 85 90
Ser Ser Gln Asp Val Ala Ser Leu Ser Ser Asp Cys Phe Leu Thr
95 100 105
Val Leu Leu Gly Lys Leu Glu Lys Ser Phe Asn Ser Val His Leu
110 115 120
Leu Ala Gly Gly Phe Ala Glu Phe Ser Arg Cys Phe Pro Gly Leu
125 130 135
Cys Glu Gly Lys Ser Thr Leu Val Pro Thr Cys Ile Ser Gln Pro
140 145 150
Cys Leu Pro Val Ala Asn Ile Gly Pro Thr Arg Ile Leu Pro Asn
-155 160 165
Leu Tyr Leu Gly Cys Gln Arg Asp Val Leu Asn Lys Glu Leu Met
170 175 180
Gln Gln Asn Gly Ile Gly Tyr Val Leu Asn Ala Ser Asn Thr Cys
185 190 195
Pro Lys Pro Asp Phe Ile Pro Glu Ser His Phe Leu Arg Val Pro
200 205 210
Val Asn Asp Ser Phe Cys Glu Lys Ile Leu Pro Trp Leu Asp Lys
215 220 225
Ser Val Asp Phe Ile Glu Lys Ala Lys Ala Ser Asn Gly Cys Val
230 235 240
Leu Val His Cys Leu Ala Gly Ile Ser Arg Ser Ala Thr Ile Ala
245 250 255
Ile Ala Tyr Ile Met Lys Arg Met Asp Met Ser Leu Asp Glu Ala
260 265 270
Tyr Arg Phe Val Lys G1u Lys Arg Pro Thr Ile Ser Pro Asn Phe
275 280 285
Asn Phe Leu Gly Gln Leu Leu Asp Tyr Glu Lys Lys Ile Lys Asn
290 295 300
Gln Thr Gly Ala Ser Gly Pro Lys Ser Lys Leu Lys Leu Leu His
305 310 315
Leu Glu Lys Pro Asn Glu Pro Val Pro Ala Val Ser Glu Gly Gly
320 325 330
Gln Lys Ser Glu Thr Pro Leu Ser Pro Pro Cys Ala Asp Ser Ala
335 340 345
Thr Ser Glu Ala Ala Gly Gln Arg Pro Val His Pro Ala Ser Val
350 355 360
Pro Ser Val Pro Ser Va1 Gln Pro Ser Leu Leu Glu Asp Ser Pro
365 . 370 375
Leu Val Gln Ala Leu Ser Gly Leu His Leu Ser Ala Asp Arg Leu
380 385 390
Glu Asp Ser Asn Lys Leu Lys Arg Ser Phe Ser Leu Asp Ile Lys
395 400 405
Ser Val Ser Tyr Ser Ala Ser Met Ala Ala Ser Leu His Gly Phe
410 415 420
8/18
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
Ser Ser Ser Glu Asp A1a Leu Glu Tyr Tyr Lys Pro Ser Thr Thr
425 430 435
Leu Asp GIy Thr Asn Lys Leu Cys Gln Phe Ser Pro Va1 Gln Glu
440 445 450
Leu Ser Glu Gln Thr Pro Glu Thr Ser Pro Asp Lys Glu Glu Ala
455 460 465
Ser Ile Pro Lys Lys Leu Gln Thr Ala Arg Pro Ser Asp Ser Gln
470 475 480
Ser Lys Arg Leu His Ser Val Arg Thr Ser Ser Ser Gly Thr Ala
485 490 495
Gln Arg Ser Leu Leu Ser Pro Leu His Arg Ser Gly Ser Val Glu
500 505 510
Asp Asn Tyr His Thr Ser Phe Leu Phe Gly Leu Ser Thr Ser Gln
515 520 525
Gln His Leu Thr Lys Ser Ala Gly Leu Gly Leu Lys Gly Trp His
530 535 540
Ser Asp Ile Leu Ala Pro Gln Thr Ser Thr Pro Ser Leu Thr Ser
545 550 555
Ser Trp Tyr Phe Ala Thr Glu Ser Ser His Phe Tyr Ser Ala Ser
560 565 570
Ala Ile Tyr Gly Gly Ser Ala Ser Tyr Ser Ala Tyr Ser Cys Ser
575 580 585
Gln Leu Pro Thr Cys Gly Asp Gln Val Tyr Ser Val Arg Arg Arg
590 595 600
Gln Lys Pro Ser Asp Arg Ala Asp Ser Arg Arg Ser Trp His Glu
605 610 615
Glu Ser Pro Phe Glu Lys Gln Phe Lys Arg Arg Ser Cys Gln Met
620 625 630
Glu Phe Gly Glu Ser Ile Met Ser Glu Asn Arg Ser Arg Glu Glu
635 640 645
Leu Gly Lys Val G1y Ser Gln Ser Ser Phe Ser Gly Ser Met Glu
650 655 660
Ile Ile Glu Val Ser
665
<210> 8
<211> 254
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4555838CD1
<400> 8
Met Ala Ala Val Ala Ala Thr Ala Ala Ala Lys Gly Asn Gly Gly
1 5 10 15
Gly Gly Gly Arg Ala Gly Ala Gly Asp Ala Ser Gly Thr Arg Lys
20 25 30
Lys Lys Gly Pro Gly Pro Pro Ala Thr Ala Tyr Leu Val Ile Tyr
35 40 45
Asn Val Va1 Met Thr Ala Gly Trp Leu Val Ile Ala Val Gly Leu
50 55 60
Val Arg Ala Tyr Leu Ala Lys Gly Ser Tyr His Ser Leu Tyr Tyr
65 70 75
Ser Ile Glu Lys Pro Leu Lys Phe Phe Gln Thr Gly Ala Leu Leu
80 85 90
Glu Ile Leu His Cys Ala Ile Gly Ile Val Pro Ser Ser Val Val
95 100 105
Leu Thr Ser Phe Gln Val Met Ser Arg Val Phe Leu Ile Trp Ala
110 115 120
Val'Thr His Ser Val Lys Glu Val Gln Ser Glu Asp Ser Val Leu
125 130 135
9/18
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
Leu Phe Val Ile Ala Trp Thr Ile Thr Glu Ile Ile Arg Tyr Ser
140 145 150
Phe Tyr Thr Phe Ser Leu Leu Asn His Leu Pro Tyr Leu Ile Lys
155 160 165
Trp Ala Arg Tyr Thr Leu Phe Ile Val Leu Tyr Pro Met Gly Val
170 175 180
Ser Gly Glu Leu Leu Thr Ile Tyr A1a Ala Leu Pro Phe Val Arg
185 190 195
Gln Ala Gly Leu Tyr Ser Ile Ser Leu Pro Asn Lys Tyr Asn Phe
200 205 210
Ser Phe Asp Tyr Tyr Ala Phe Leu Ile Leu Ile Met Ile Ser Tyr
215 220 225
Ile Pro Ile Phe Pro Gln Leu Tyr Phe His Met Ile His Gln Arg
230 235 240
Arg Lys Ile Leu Ser His Thr Glu Glu His Lys Lys Phe G1u
245 250
<210> 9
<211> 267
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 636866CD1
<400> 9
Met Ser Gly Cys Phe Pro Va1 Ser Gly Leu Arg Cys Leu Ser Arg
1 5 10 15
Asp Gly Arg Met Ala Ala Gln Gly Ala Pro Arg Phe Leu Leu Thr
20 25 30
Phe Asp Phe Asp Glu Thr Ile Val Asp Glu Asn Ser Asp Asp Ser
35 40 45
Ile Val Arg Ala Ala Pro Gly Gln Arg Leu Pro Glu Ser Leu Arg
50 55 60
Ala Thr Tyr Arg Glu Gly Phe Tyr Asn Glu Tyr Met Gln Arg Val
65 70 75
Phe Lys Tyr Leu Gly Glu Gln Gly Val Arg Pro Arg Asp Leu Ser
80 85 90
Ala Ile Tyr Glu Ala Ile Pro Leu Ser Pro Gly Met Ser Asp Leu
95 100 105
Leu Gln Phe Val Ala Lys G1n Gly Ala Cys Phe G1u Val Ile Leu
110 115 120
Tle Ser Asp Ala Asn Thr Phe Gly Val Glu Ser Ser Leu Arg Ala
125 130 135
Ala Gly His His Ser Leu Phe Arg Arg Ile Leu Ser Asn Pro Ser
140 145 150
Gly Pro Asp Ala Arg Gly Leu Leu Ala Leu Arg Pro Phe His Thr
155 160 165
His Ser Cys Ala Arg Cys Pro Ala Asn Met Cys Lys His Lys Val
170 175 180
Leu Ser Asp Tyr Leu Arg G1u Arg Ala His Asp Gly Va1 His Phe
185 190 195
Glu Arg Leu Phe Tyr Val Gly Asp Gly Ala Asn Asp Phe Cys Pro
200 205 210
Met Gly Leu Leu Ala Gly Gly Asp Val Ala Phe Pro Arg Arg Gly
215 220 225
Tyr Pro Met His Arg Leu Ile Gln Glu Ala Gln Lys Ala Glu Pro
230 235 240
Ser Ser Phe Arg Ala Ser Val Val Pro Trp Glu Thr Ala Ala Asp
245 250 255
Val Arg Leu His Leu Gln Gln Val Leu Lys Ser Cys
260 265
10/18
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
<210> 10
<211> 329
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475576CD1
<400> 10
Met Gln Gly Gln Thr Val Val Pro Lys Asp Ser Tyr Thr Ile Ser
1 5 ' 10 15
Leu Ile G1n Arg Leu Arg Gly Arg Glu Ala Ala Arg Arg Thr His
20 25 30
Glu Asn Leu Leu Arg Leu Ser Ala Leu Val Arg Ser Pro Gln Thr
3S 40 45
Ala Ser Ile Asp Cys His Thr Trp Ser Val Ser Ser Gly Thr Asn
50 55 60
Thr Ser Leu Gln Ala Ser Gly Leu Gly Arg Gln Gly Ser Cys Asp
65 70 75
Arg Ile A1a Ser Arg Ala Ala Ser Trp Gly Cys Thr Arg Thr Ala
80 85 90
Ala Pro Gly Ile Met Gly Asn Gly Met Thr Lys Val Leu Pro Gly
95 100 105
Leu Tyr Leu Gly Asn Phe I1e Asp Ala Lys Asp Leu Asp Gln Leu
110 115 120
Gly Arg Asn Lys Ile Thr His Ile Ile Ser Ile His Glu Ser Pro
125 130 135
Gln Pro Leu Leu Gln Asp Ile Thr Tyr Leu Arg Ile Pro Val Ala
140 145 150
Asp Thr Pro Glu Val Pro Ile Lys Lys His Phe Lys Glu Cys Ile
155 160 165
Asn Phe Ile His Cys Cys Arg Leu Asn G1y Gly Asn Cys Leu Val
170 175 180
His Cys Phe Ala Gly Ile Ser Arg Ser Thr Thr Ile Val Thr Ala
185 190 195
Tyr Val Met Thr Val Thr Gly Leu Gly Trp Arg Asp Val Leu Glu
200 205 210
Ala Ile Lys Ala Thr Arg Pro Ile Ala Asn Pro Asn Pro Gly Phe
215 220 225
Arg Gln Gln Leu Glu Glu Phe Gly Trp Ala Ser Ser G1n Lys Leu
230 235 240
Arg Arg Gln Leu Glu Glu Arg Phe G1y Glu Ser Pro Phe Arg Asp
245 250 255
Glu Glu Glu Leu Arg Ala Leu Leu Pro Leu Cys Lys Arg Cys Arg
260 265 270
Gln Gly Ser Ala Thr Ser Ala Ser Ser Ala G1y Pro His Ser Ala
275 280 285
Ala Ser Glu Gly Thr Leu Gln Arg Leu Val Pro Arg Thr Pro Arg
290 295 300
Glu Ala His Arg Pro Leu Pro Leu Leu Ala Arg Val Lys Gln Thr
305 310 315
Phe Ser Cys Leu Pro Arg Cys Leu Ser Arg Lys Gly Gly Lys
320 325
<210> 11
<211> 1845
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 1905692CB1
11/18
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
<400> 11
cacaaatgaa ttatcaggag tgaacccaga ggcacgtatg aatgaaagtc ctgatccgac 60
tgacctggcg ggagtcatca ttgagctcgg ccccaatgac agtccacaga caagtgaatt 120
taaaggagca accgaggagg cacctgcgaa agaaagccca cacacaagtg aatttaaagg 180
agcagcccgg gtgtcaccta tcagtgaaag tgtgttagca cgactttcca agtttgaaga 240
tgaagatgct gaaaatgttg cttcatatga cagcaagatt aagaaaattg tgcattcaat 300
tgtatcatcc tttgcatttg gactatttgg agttttcctg gtcttactgg atgtcactct 360
catccttgcc gacctaattt tcactgacag caaactttat attcctttgg agtatcgttc 420
tatttctcta gctattgcct tattttttct catggatgtt cttcttcgag tatttgtaga 480
aaggagacag cagtattttt ctgacttatt taacatttta gatactgcca ttattgtgat 540
tcttctgctg gttgatgtcg tttacatttt ttttgacatt aagttgctta ggaatattcc 600
cagatggaca catttacttc gacttctacg acttattatt ctgttaagaa tttttcatct 660
gtttcatcaa aaaagacaac ttgaaaagct gataagaagg cgggtttcag aaaacaaaag 720
gcgatacaca agggatggat ttgacctaga cctcacttac gttacagaac gtattattgc 780
tatgtcattt ccatcttctg gaaggcagtc tttctataga aatccaatca aggttattcc 840
ctatagagat atgacataca tattatttat tttaggtgaa agagcttacg atcctaagca 900
cttccataat agggtcgtta gaatcatgat tgatgatcat aatgtcccca ctctacatca 960
gatggtggtt ttcaccaagg aagtaaatga gtggatggct caagatcttg aaaacatcgt 1020
agcgattcac tgtaaaggag gcacagatag aacaggaact atggtttgtg ccttccttat 1080
tgcctctgaa atatgttcaa ctgcaaagga aagcctgtat tattttggag aaaggcgaac 1140
agataaaacc cacagcgaaa aatttcaggg agtaaaaact ccttctcaga agagatatgt 1200
tgcatatttt gcacaagtga aacatctcta caactggaat ctccctccaa gacggatact 1260
ctttataaaa cacttcatta tttattcgat tcctcgttat gtacgtgatc taaaaatcca 1320
aatagaaatg gagaaaaagg ttgtcttttc cactatttca ttaggaaaat gttcggtact 1380
tgataacatt acaacagaca aaatattaat tgatgtattc gacggtccac ctctgtatga 1440
tgatgtgaaa gtgcagtttt tctcttcgaa tcttcctaca tactatgaca attgctcatt 1500
ttacttctgg ttgcacacat cttttattga aaataacagg ctttatctac caaaaaatga 1560
attggataat ctacataaac aaaaagcacg gagaatttat ccatcagatt ttgccgtgga 1620
gatacttttt ggcgagaaaa tgacttccag tgatgttgta gctggatccg attaagtata 1680
gctccccctt ccccttctgg gaaagaatta tgttctttcc aaccctgcca catgttcata 1740
tatcctaaat ctatcctaaa tgttccttga agtatttatt tatgtttata tatgtttata 1800
tatgttcttc ataaatctat tacatatata tagataaaaa aaaaa 1845
<210> 12
<211> 2451
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476908CB1
<400> 12
ccggacccgg cgagccttcg gggcgcgcgt cgctggtggt ggttgaggct ctagcgataa 60
taaatgatag aggatacaat gactttgctg tctctgctgg gtcgcatcat gcgctacttc 120
ttgctgagac ccgagacgct tttcctgctg tgcatcagct tggctctatg gagttacttc 180
ttccacaccg acgaggtgaa gaccatcgtg aagtccagcc gggacgccgt gaagatggtg 240
aagagcaagg tagccgagac catgcagaac gatcgactcg gggggcttga tgtgctcgag 300
gccgagtttt ccaagacctg ggagttcaag aaccacaacg tggcggtgta ctccatccag 360
ggccggagag accacatgga ggaccgcttc gaagttctca cggatctggc caacaagacg 420
cacccgtcca tcttcgggat cttcgacggg cacgggggag agactgcagc tgaatatgta 480
aaatctcgac tcccagaggc tcttaaacag catcttcagg actacgagaa agacaaagaa 540
aatagtgtat tatcttacca gaccatcctt gaacagcaga ttttgtcaat tgaccgagaa 600
atgctagaaa aattgactgt atcctatgat gaagcaggca caacgtgttt gattgctctg 660
ctatcagata aagacctcac tgtggccaac gtgggtgact cgcgcggggt cctgtgtgac 720
aaagatggga acgctattcc tttgtctcat gatcacaagc cttaccagtt gaaggaaaga 780
aagaggataa agagagcagg tggtttcatc agtttcaatg gctcctggag ggtccaggga 840
atcctggcca tgtctcggtc cctgggggat tatccgctga aaaatctcaa cgtggtcatc 900
ccagacccag acatcctgac ctttgacctg gacaagcttc agcctgagtt catgatcttg 960
gcatcagatg gtctctggga tgctttcagc aatgaagaag cagttcgatt catcaaggag 1020
cgcttggatg aacctcactt tggggccaag agcatagttt tacagtcatt ttacagaggc 1080
tgccctgaca atataacagt catggtggtg aagttcagaa atagcagcaa aacagaagag 1140
cagtgaaccc ttcaggggtc tcagctgcct tagactaaag gactttcaac acactggtct 1200
12/18
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
cttttaattt agtgaaaagt gtgggagttg taattaggat catccacccc agacatggaa 1260
tcccccctcc ctggtggtct taggtctata atcagtgacg aacagagggt gcccttggcc 1320
aatgtagtta agaaactgga aaatggtttc ttcatgtttt cccaactctt tcatccagtg 1380
tccaaaatat ataagtaaat agctgtagag tcacatatat gaagtgaata gcatatgtgt 1440
catttagtct ccctgaagat tcttttcaag atcctgttca gggtcctcca ggcatcagct 1500
gttgtgtcct ctctttgtaa cagtggacag gacagaccac ccagtgctgc aggagacagg 2560
ccactgcgtc acctgtgagt ggtcaggggc tgatgtggca acaccctctg ccaagagaca 1620
gagctgtcct gagaatgctt tgtccttctg agcccatgtt ttctgctcag tagcagcttg 1680
gaagcagatt tggaatggtt tattattttg gctgctcttg gggactgcga gaagcagaga 1740
gaatgagaga ccagtggcaa ctgcctgcac agcagagata accctcttcc cttgcttcct 1800
ttaatagtta aatagacttt gtataccacc tgaccagcct ttgtgcattt atcctaatca 1860
tgcatgaccg ttaacctttt gcttagtcct taccatatgt aataggcagc tgttaaattc 1920
accaacagat accctgattt ttcatcttac gtgaccaaga aaccacgtta ggggaaatga 1980
aaaaagcaag ccacaatacc atgattcctt ccattttcaa cagtagatga aggaaatgat 2040
actgaatgag tcacagtgtt ccctggcaag taagctgttt gcattgagaa aggagtgagc 2100
tggtgaggtt accaccctga attgagctcc agctgccagt ttttgtgttt ttccttgccc 2160
ctttccaagt ggttttcaag tgtcaggcag tgttctgaga agcagcagcc tataactgta 2220
tgtgtgttcc ttgaagccag gtgcagagtt cccagctact gcagcttggg atttggtggg 2280
aaactactgg gataagcttc tccttgacaa tggaaaggca gcagtcttca acatttggtt 2340
gcaaatctcc atccacatca gggagctttc cccaggcaaa tacaaaccgc cccgtggcct 2400
gcaggcctgc aggggaggca gcaaagggac ctggcagttg caacacagta a 2451
<210> 13
<211> 1105
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7708162CB1
<400> 13 .
agtgtgctgg aaaggtttag aatgcctctt tttcaagatg gatttccttc acaggaatgg 60
agtgctcata attcagcatt tgcagaagga ctaccgagct tactacactt ttctaaattt 120
tatgtccaat gttggagacc ccaggaatat ctttttcatt tattttccac tttgttttca 180
atttaatcag acagttggaa ccaagatgat atgggtagca gtcattgggg attggttaaa 240
tcttatattt aaatggatat tatttggtca tcgaccttac tggtgggtcc aagaaactca 300
gatttaccca aatcactcaa gtccatgcct tgaacagttc cctactacat gtgaaacagg 360
tccaggaagt ccatctggcc atgcaatggg cgcatcctgt gtctggtatg tcatggtaac 420
cgctgccctg agccacactg tctgtgggat ggataagttc tctatcactc tgcacagact 480
gacctggtca tttctttgga gtgttttttg gttgattcaa atcagtgtct gcatctccag 540
agtattcata gcaacacatt ttcctcatca agttattctt ggagtaattg gtggcatgct 600
ggtggcagag gcctttgaac acactccagg catccaaacg gccagtctgg gcacatacct 660
gaagaccaac ctctttctct tcctgtttgc agttggcttt tacctgcttc ttagggtgct 720
caacattgac ctgctgtggt ccgtgcccat agccaaaaag tggtgtgcta accccgactg 780
gatccacatt gacaccacgc cttttgctgg actcgtgaga aaccttgggg tcctctttgg 840
cttgggcttt gcaatcaact cagagatgtt cctcctgagc tgccgagggg gaaataacta 900
cacactgagc ttccggttgc tctgtgcctt gacctcattg acaatactgc agctctacca 960
tttcctccag atcccgactc acgaagagca tttattttat gtgctgtctt tttgtaaaag 1020
tgcatccatt cccctaactg tggttgcttt cattccctac tctgttcata tgttaatgaa 1080
acaaagcgga aagaagagtc agtag 1105
<210> 14
<211> 1730
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 7473603CB1
<400> 14
atgctggagt ctgctgaaca gctgctggtg gaggacctgt acaaccgcgt cagggagaag 60
13/18
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
atggatgaca ccagcctcta taatacgccc tgtgtcctgg acctacagcg ggccctggtt 120
caggatcgcc aagaggcgcc ctggaatgag gtggatgagg tctggcccaa tgtcttcata 180
gctgacagga gtgtggctgt gaacaagggg aggctgaaga ggctgggaat cacccacatt 240
ctgaatgctg cgcatggcac cggcgtttac actggccccg aattctacac tggcctggag 300
atccagtacc tgggtgtaga ggtggatgac tttcctgagg tggacatttc ccagcatttc 360
cggaaggcgt actgtcatta catcattttc tcttgtgttt tcatttcagg gaaagtcctg 420
gtcagcagcg aaatgggcat cagccggtca gcagtgctgg tggtcgccta cctgatgatc 480
ttccacaaca tggccatcct ggaggctttg atgaccgtgc gtaagaagcg ggccatctac 540
cccaatgagg gcttcctgaa gcagctgcgg gagctcaatg agaagttgat ggaggagaga 600
gaagaggact atggccggga ggggggatca gctgaggctg aggagggcga gggcactggg 660
agcatgctcg gggccagagt gcacgccctg acggtggaag aggaggacga cagcgccagc 720
cacctgagtg gctcctccct ggggaaggcc acccaggcct ccaagcccct caccctcata 780
gacgaggagg aggaggagaa actgtacgag cagtggaaga aggggcaggg cctcctctca 840
gacaaggtcc cccaggatgg aggtggctgg cgctcagcct cctctggcca gggtggggag 900
gagctcgagg acgaggacgt ggagaggatc atccaggagt ggcagagccg aaacgagagg 960
taccaagcag aagggtaccg gaggtgggga agggaggagg agaaggagga ggagagcgac 1020
gctggctcct cggtggggag gcggcggcgc accctgagcg agagcagcgc ctgggagagc 1080
gtgagcagcc acgacatctg ggtcctgaag cagcagctgg agctgaaccg cccggaccac 1140
ggcaggaggc gccgcgcaga ctcgatgtcc tcggagagca cctgggacgc atggaacgag 1200
aggctgctgg agattgagaa ggaggcttcc cggaggtacc acgccaagag caagagagag 1260
gaggcggcag acaggagctc agaagcaggg agcagggtgc gggaggatga tgaggacagc 1320
gtgggctctg aggccagttc cttctacaac ttctgcagca ggaacaagga caagctcact 1380
gcctggaaag atggaagatc aagagaatcc aatttggatt tcacaagaaa gacttgggag 1440
cgggagacag cagcggtgag cccggtgcag aggaggcagt aggggagaag aacccctccg 2500
acgtcagcct gacagcctac caggccctgg aagctgaaac accagaagaa ggtggggcag 1560
tgagaaccag gaggaggtgg tggagctcag caggggggag gacttggcct tggctaagaa 1620
gagacgacgg aggctggagc tgctggagag aagccggaga acctggagga gagccagtct 1680
attgcagctg ggaggcggac agtccagcgc ggggagattc cctgttggtt 1730
<210> 15
<211> 2145
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476687CB1
<400> 15
tcctctgcag cctgccggcg cctcgaagcc cggacctgcc tccgcctctt cctccagcgg 60
ctccatcccg CCtCCCgtgC CtCgtCCtCC CgCCgCCtCC gccgccgcgc ccccggtggc 120
tgcctcggcg gaccggggag ggggcccacc gcgtcggccg cccgctcggc tcggctcggc 180
ccggcccggg aggcgtgcat gcccctgctg cccgcggcgc tcaccagcag catgctctat 240
ttccagatgg tgatcatggc agggacggtg atgctggcgt actacttcga gtatacggac 300
acgttcaccg tgaacgtgca gggcttcttc tgccacgaca gcgcctaccg caaaccctac 360
ccgggcccgg aggacagcag cgccgtgccc cccgtgctcc tctactcgct ggccgccggg 420
gtccccgtgc tcgtgataat agttggagaa actgctgtct tttgcctaca actagccaca 480
agggattttg aaaaccagga aaaaactatt ttaactggag actgttgcta tataaacccg 540
ctggtgcgcc gaactgtccg atttcttgga atttatacat ttggactgtt tgctacagat 600
atctttgtaa atgctggaca agtagtcaca ggaaatctgg ccccacattt ccttgccctg 660
tgtaagccca attatacagc acttggatgt cagcagtata cacaattcat cagtggggaa 720
gaggcctgta ctggcaaccc agatctcatc atgagagccc gaaaaacctt tccatccaaa 780
gaagcagctc tcagtgtcta tgcagctatg tatctgacca tgtacatcac caacacaatc 840
aaagccaagg gaaccagact tgctaagcca gttctatgct tgggcttaat gtgtttggca 900
tttcttactg gactcaacag agtagcagaa tatcgaaatc attggtcaga tgttatagca 960
ggctttctgg ttggaatatc tatagcagta tttctggttg tgtgcgtggt gaataatttc 1020
aaagggagac aagcagaaaa tgagcatata cacatggata atctggcaca gatgccaatg 1080
atcagcattc ctcgagtaga aagtcctttg gaaaaggtaa catctgtaca gaaccacatc 1140
actgccttcg cagaagtcac atgatatcga agcagatggt ttttcactgc attggacatc 1200
atcccttttt accatccatt cataacaccc aaagtttgtt tgattgcaag tgaagtttat 1260
aaagttgttt aataattttt ataattttaa aatcaatgtc aacctatctg tttcccccac 1320
tagactgtga gctcctcgag ggcaggattg tctttttttt tctgtgtccc cagcacttac 1380
aacaaagcct agcacaaagt aggtgttcaa caaaaatgtg atgaccaaat gaaaaaaatt 1440
14/18
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
Cttaaataaa acattcactt tagtttctca cagaatcatt gcaattatgt taaaagaaat 1500
ctctacataa atcgtatttg tgtatgaaaa ccttctattt tgggctagtt atttttttaa 1560
tcttcatata tctattcagc agtatgccat atttaatttg aagtggactt tgaaagtcat 1620
gggggttttt attttgttat tcagcatgac attatttcca ttcgtaacat ttcagtgtgt 1680
gaaattactt tatttttaga aagtatgttc tatagtaaaa taatgtttcc acattatatt 1740
atgttatatt tcacttaaaa tactattcat actatacatt ctaagactgg tgcttctgct 1800
tttgaagggg aaaatgccaa tttttactgt aataagtaat gtatcataat taaaaattat 1860
ttatttggac ttcttctcct gacaattgtg gcttaattca tgacttgttt ttgaatgcag 1920
gcagtattta gggtagttta aatgagtaaa ttcagcactg gtaccttatt attgagtaat 2980
ttcccatggg tagcagtgtc tcaagagtgg tcaaaagctc cactcttagg cttttttact 2040
actaaagatt ccacataatt taaatgggaa agaactatac cctgacacat aatttaaatt 2200
atataaatgc tagaaatatg gttaatgtat tttactttgc atttg 2145
<210> 16
<211> 1352
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7480440CB1
<400> 16
atggccggcc tggggttttg gggccaccct gctggacctc tcctgctgct gctgctgctg 60
gtgctgccac cccgggccct gccagaagga cccctggtgt tcgtggctct ggtattccgc 120
catggcgacc gggccccgct ggcctcctac cccatggacc cacacaagga ggtggcctcc 180
accctgtggc cacgaggcct gggccagctg accacggagg gggtccgcca gcagctggag 240
ctgggccgct tcctgaggag ccgctacgag gccttcctga gtccggagta ccggcgggag 300
gaggtgtaca tccgcagcac ggactttgac cgcacgctgg agagtgccca ggccaacctt 360
gccgggctgt ttcccgaggc tgctccaggg agccccgagg cccgctggag gccgatcccg 420
gtgcacacgg tgcccgtggc tgaggataag ctgctgaggt tccccatgcg cagctgtccc 480
cgataccacg agctgctgcg ggaggccacc gaggccgccg agtaccagga ggccctggag 540
ggctggacgg gcttcctgag tcgcctggag aacttcacgg gactgtcgct ggttggagag 600
ccactgcgca gggcatggaa ggttctggac accctcatgt gccagcaagc ccacggtctt 660
ccactaccag cctgggcctc cccagatgtc ctgcggactc ttgcccagat ctcggctttg 720
gatattggag cccacgtggg cccaccccgg gcagcagaga aggcccagct gacagggggg 780
atcctgctga atgctatcct tgcaaacttc tcccgggtcc agcgcctggg gctgcccctc 840
aagatggtca tgtactcagc tcatgacagc accctgctgg ccctccaggg ggccctgggc 900
ctctatgatg gacacacccc gccatatgct gcctgcctcg gctttgagtt ccggaagcac 960
ctggggaatc ccgccaaaga tggagggaat gtcaccgtct ccctcttcta ccgcaatgac 1020
tccgcccacc tgcccctgcc tctcagcctc cccgggtgcc cggccccctg tccactaggc 1080
cgcttctacc agctgactgc cccggcccgg cctcccgccc atggggtctc ctgccatggc 1140
ccctatgagg CtgCCatCCC CCCagCtCCa gtggtgcccc tgctggccgg agctgtagct 1200
gtgctggtgg cactcagctt ggggctgggc ctgctggcct ggagaccagg gtgcctgcgg 1260
gccttggggg gccccgtgtg agccagaaac cagggcttcc ctacccccag ctgacactgg 1320
accccaacat gtatgetcag tagctgcaaa as 1352
<210> 17
<211> 3766
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7480570CB1
<400> 17
gaaaagaaga tgaggaggag agcgacggga cgggacgcga gcgggagcgc agccgccctc 60
tcggctccgc ggcggcgcct cgcaagtccg ggaggcgagg ggggcccgag gggagacgcc 120
gtgacaactt tcgtttccct ctgagggaat tgggaggtcg gcggccccaa aagctttcag 180
tccagtgtaa agctgttgga gcgcgggagc aaaggtaaag aatgatgtaa tgcgctggct 240
gctccaaagc atcttttgtt gtggaatggt tattccagtc atctctttat gaatcaaatg 300
tgaggggctg ctttgtggac ggagtccttt gcaagagcac atcaacggga aagagaaaga 360
15/28
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
gacattcact tggagggctc ttgctgaaaa tgggtttaac tctccttttg ccagtcacca 420
ccagcctgac ctcatacact tttagtacaa tggagtggct gagcctttga gcacaccacc 480
attacatcat cgtggcaaat taaagaagga ggtgggaaaa gaggacttat tgttgtcatg 540
gcccatgaga tgattggaac tcaaattgtt actgagaggt tggtggctct gctggaaagt 600
ggaacggaaa aagtgctgct aattgatagc cggccatttg tggaatacaa tacatcccac 660
attttggaag ccattaatat caactgctcc aagcttatga agcgaaggtt gcaacaggac 720
aaagtgttaa ttacagagct catccagcat tcagcgaaac ataaggttga cattgattgc 780
agtcagaagg ttgtagttta cgatcaaagc tcccaagatg ttgcctctct ctcttcagac 840
tgttttctca ctgtacttct gggtaaactg gagaagagct tcaactctgt tcacctgctt 900
gcaggtgggt ttgctgagtt CtCtCgttgt ttCCCtggCC tctgtgaagg aaaatccact 960
ctagtcccta cctgcatttc tcagccttgc ttacctgttg ccaacattgg gccaacccga 2020
attcttccca atctttatct tggctgccag cgagatgtcc tcaacaagga gctgatgcag 1080
cagaatggga ttggttatgt gttaaatgcc agcaatacct gtccaaagcc tgactttatc 1140
cccgagtctc atttcctgcg tgtgcctgtg aatgacagct tttgtgagaa aattttgccg 1200
tggttggaca aatcagtaga tttcattgag aaagcaaaag cctccaatgg atgtgttcta 1260
gtgcactgtt tagctgggat ctcccgctcc gccaccatcg ctatcgccta catcatgaag 1320
aggatggaca tgtctttaga tgaagcttac agatttgtga aagaaaaaag acctactata 1380
tctccaaact tcaattttct gggccaactc ctggactatg agaagaagat taagaaccag 1440
actggagcat cagggccaaa gagcaaactc aagctgctgc acctggagaa gccaaatgaa 1500
cctgtccctg ctgtctcaga gggtggacag aaaagcgaga cgcccctcag tccaccctgt 1560
gccgactctg ctacctcaga ggcagcagga caaaggcccg tgcatcccgc cagcgtgccc 1620
agcgtgccca gcgtgcagcc gtcgctgtta gaggacagcc cgctggtaca ggcgctcagt 1680
gggctgcacc tgtccgcaga caggctggaa gacagcaata agctcaagcg ttccttctct 1740
ctggatatca aatcagtttc atattcagcc agcatggcag catccttaca tggcttctcc 1800
tcatcagaag atgctttgga atactacaaa ccttccacta ctctggatgg gaccaacaag 1860
ctatgccagt tctcccctgt tcaggaacta tcggagcaga ctcccgaaac cagtcctgat 1920
aaggaggaag ccagcatccc caagaagctg cagaccgcca ggccttcaga cagccagagc 1980
aagcgattgc attcggtcag aaccagcagc agtggcaccg cccagaggtc ccttttatct 2040
ccactgcatc gaagtgggag cgtggaggac aattaccaca ccagcttcct tttcggcctt 2100
tccaccagcc agcagcacct cacgaagtct gctggcctgg gccttaaggg ctggcactcg 2160
gatatcttgg CCCCCCagaC CtCtaCCCCt tCCCtgaCCa gcagctggta ttttgccaca 2220
gagtcctcac acttctactc tgcctcagcc atctacggag gcagtgccag ttactctgcc 2280
tacagctgca gccagctgcc cacttgcgga gaccaagtct attctgtgcg caggcggcag 2340
aagccaagtg acagagctga ctcgcggcgg agctggcatg aagagagccc ctttgaaaag 2400
cagtttaaac gcagaagctg ccaaatggaa tttggagaga gcatcatgtc agagaacagg 2460
tcacgggaag agctggggaa agtgggcagt cagtctagct tttcgggcag catggaaatc 2520
attgaggtct cctgagaaga aagacacttg tgacttctat agacaatttt tttttcttgt 2580
tcacaaaaaa attccctgta aatctgaaat atatatatgt acatacatat atatttttgg 2640
aaaatggagc tatggtgtaa aagcaacagg tggatcaacc cagttgttac tctcttaaca 2700
tctgcatttg agagatcagc taatacttct ctcaacaaaa atggaagggc agatgctagg 2760
atccccccta gacggaggaa aaccatttta ttcagtgaat tacacatcct cttgttctta 2820
aaaaagcaag tgtctttggt gttggaggac aaaatcccct accattttca cgttgtgcta 2880
ctaagagatc tcaaatatta gtctttgtcc ggacccttcc atagtacacc ttagcgctga 2940
gactgagcca gcttgggggt caggtaggta gaccctgtta gggacagagc ctagtggtaa 3000
atccaagaga aatgatccta tccaaagctg attcacaaac ccacgctcac ctgacagccg 3060
agggacacga gcatcactct gctggacgga ccattagggg ccttgccaag gtctacctta 3120
gagcaaaccc agtacctcag acaggaaagt cggggctttg accactacca tatctggtag 3180
cccattttct aggcattgtg aataggtagg tagctagtca cacttttcag accaattcaa 3240
actgtctatg cacaaaattc ccgtgggcct agatggagat aatttttttt tcttctcagc 3300
tttatgaaga gaagggaaac tgtctaggat tcagctgaac caccaggaac ctggcaacat 3360
cacgatttaa gctaaggttg ggaggctaac gagtctacct ccctctttgt aaatcaaaga 3420
attgtttaaa atgggattgt caatccttta aataaagatg aacttggttt caagccaaat 3480
gtgaatttat ttgggttggt agcagagcag cagcaccttc aaattctcag ccaaagcaga 3540
tgtttttgcc ctttctgctt cactgcatgg atacagttgg taaaatgtaa taatatggca 3600
gaattttata ggaaacttcc tagggaggta aattatggga agattaagaa aggtacaaat 3660
tgctgaggag aagcaggaaa cctgtttcct tagtggcttt tatcccctcg gcatgcgatg 3720
gggctgatgt ttctatgatt gcctcagact ttcacattta ctagta 3766
<210> 18
<211> 2656
<212> DNA
<213> Homo Sapiens
16/18
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
<220>
<221> misc_feature
<223> Incyte ID No: 4555838CB1
<220>
<221> unsure
<222> 2532
<223> a, t, c, g, or other
<400> 18
gccgtggacc ctgtcccggc tccgccccgc ggccctggcc ccgcctgccc ctcgtccctt 60
cctccgcccc ctctcctcgc gtagtccggc ccgagccgct cgcgctagga gagcgggctt 120
cgggcacttg acatggcggc agtggcggcg actgcagcag cgaaggggaa tgggggcggc 180
ggtgggaggg ccggggccgg ggacgccagc ggcacgcgga agaagaaggg cccggggccc 240
ccggccacgg cgtacctggt catctacaat gtggtgatga cagccgggtg gctggttata 300
gcggttggtc tggtccgagc atacctggct aagggtagct accatagcct ttattattca 360
attgaaaagc ctttgaaatt ctttcaaact ggagccttat tggagatttt acattgtgct 420
ataggaattg ttccatcttc tgttgtcctg acttctttcc aggtgatgtc aagagttttt 480
ctaatatggg cagtaacaca tagcgtcaaa gaggtacaga gtgaagacag tgtcctcctg 540
tttgttattg catggacgat cacggaaatc atccgttact ccttttatac attcagtcta 600
ttaaaccatc tgccttacct catcaaatgg gccaggtaca cacttttcat tgtgctgtac 660
ccaatgggag tgtcaggaga actgctcaca atatatgcag ctctgccctt tgtcagacaa 720
gctggcctat attccatcag tttacccaac aaatacaatt tctcttttga ctactatgca 780
ttcctgattc taataatgat ctcctacatt ccaatttttc cccagttata cttccacatg 840
atacaccaga gaagaaagat cctttctcat actgaagaac acaagaaatt tgaatagttc 900
ctgctttctg cacctcccac caaaacaaac ttttcaatga tcaaaaaatg ctgcagattt 960
tttgagttcc caatacgttt catagaaaat aagtaagaac tatttttaaa atattcaaac 1020
aaaactaaaa caaaaatcca gtgtcacatg ggcctgagat tttattttag aaaaaggttg 1080
ttacataaaa caccctggcc agttcatttc agcatgctct ttcaaccaga agttcttaat 1140
atttatgatg gcactagaaa gggatttggc attttatgtc cttctgtgtc cttcatgtat 1200
ctgatcaatg aagacctgta acactaagta cttgagagtt acagtctgaa taatgaagtc 1260
gtaccagctg aatagcccag cttgcagtat agttatgttt cagtctgcag tgtgtttagc 1320
attcccttgt caaagtgctt gactgcatgc tggaaacttt gtatttttga agcagcaaac 1380
tctgttctct ggaatgctct gaagttatgg ctgggaccta tcccctcaca tctaatgaat 1440
gaattataaa atgtatatgt ctatgaagct tcggggtagt gcctgtaatc agaaaacaac 1500
ttagaaccct tttgtttgtt tccaattgag tcattactgc ctgccactaa gaaacgtgct 1560
tgaatctaat aagtatgtgt gtaccgtaaa gaatatatct tatctggagc tcagcctcaa 1620
tcatgtctta acaaaatgac aggtctcaga aagggggagc tcaatagctc aaaagtgaca 1680
agtccttttc acagcaccgt tctcagaaca cctctgagta acgtgtttgc cagtagctat 1740
tctcactgat gcactgatgg ccctgaagaa gcggatccag tcacatagga aaggaggctg 1800
tgttagtgaa agcacatgga aggtgttgct ttagaaaggt agtcaggaaa aacattcagg 1860
aatagattta tacaccatta ttgttttatt tttaaatttt cattcactct tctgtttgga 1920
tacttttgct aattaacgtc ctatgttaat ttccaccaag ctataagtcc atagtcagta 1980
aaacattccc cttgggctgt catgagctaa aagcagtgtc atctccgcat gttggagcag 2040
ccaagaaata gtttggtact accgacatcg tctaatccat gtcacatcct catacaattt 2100
aattgctcaa ccatgcattt aaaactcctc aagaaaggat tggtactgca actgtaggta 2160
aactgaaaaa aaataagaaa gaaagagttg gatgaaaatg tgaaagccca agtttagatg 2220
tgcattaagt attaaatagc acagtatctt cttcatggag ccttttttcc tcccccatcc 2280
cctgcagctg cctttttttg ggggcagggt gggggttgat gttgaacttt aagagtttaa 2340
aagtttagct tattgagtag ttgtcattta aaatataatt gcgaatatca gaaaactcat 2400
actggaaaac taaatttttt tttttctctt gagacggagt ctcgctctgt tgcccaggct 2460
ggagtgcagt ggcgcgatct cggctcactg caagctccac ctcccgggtt cacgccatcc 2520
tcctgactca gnctcctgag tagctgggac tacaggtgcc tggcaacaaa cccagctaat 2580
ttttttgttt ttaagaaaaa cggggttcac cgggttaccc agatggtctg atttctgacc 2640
cttgacaccc gctaag 2656
<210> 19
<211> 1292
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
17/18
CA 02417359 2003-O1-27
WO 02/10363 PCT/USO1/23716
<223> Incyte ID No: 636866CB1
<400> 19
cgctgctgca gcagccgcag cgccggccgc ggctccggct ccggctccgg ctcccgggca 60
tttaaagggg acgcggcggc tgcccggggg ggatgagggg caagtggagg ggacggctca 120
gacgcacatc atcctcagtc cctcgggact ggagggactc gtgagccgga gcccagaaat 180
ccgggggtgg ataagacacc gCgtCCCCtC CaattCCCgt aagcacccct tgctccatcc 240
tgcgccccaa tacctcagct agCCCCCttC CCCa.CttCtt aCaCtCCaaa CtCagCCggg 3OO
acagacctct gctgccgccg cccccacgaa cgtgtgacga cggctggagg ccaacagagt 360
ccctacaggt ggtgctcacg gtaatgcacc gacaatgagt ggctgttttc cagtttctgg 420
cctccgctgc ctatctaggg acggcaggat ggccgcgcag ggcgcgccgc gcttcctcct 480
gaccttcgac ttcgacgaga ctatcgtgga cgaaaacagc gacgattcga tcgtgcgcgc 540
cgcgccgggc cagcggctcc cggagagcct gcgagccacc taccgcgagg gcttctacaa 600
cgagtacatg cagcgcgtct tcaagtacct gggcgagcag ggcgtgcggc cgcgggacct 660
gagcgccatc tacgaagcca tccctttgtc gccaggcatg agcgacctgc tgcagtttgt 720
ggcaaaacag ggcgcctgct tcgaggtgat tctcatctcc gatgccaaca cctttggcgt 780
ggagagctcg ctgcgcgccg ccggccacca cagcctgttc cgccgcatcc tcagcaaccc 840
gtcggggccg gatgcgcggg gactgctggc tctgcggccg ttccacacac acagctgcgc 900
gcgctgcccc gccaacatgt gcaagcacaa ggtgctcagc gactacctgc gcgagcgggc 960
ccacgacggc gtgcacttcg agcgcctctt ctacgtgggc gacggcgcca acgacttctg 2020
ccccatgggg ctgctggcgg gcggcgacgt ggccttcccg cgccgcggct accccatgca 1080
ccgcctcatt caggaggccc agaaggccga gcccagctcg ttccgcgcca gcgtggtgcc 1140
ctgggaaacg gctgcagatg tgcgcctcca cctgcaacag gtgctgaagt cgtgctgagt 1200
ctggccgcct gcaggggggt acccgggcca acggcggagg gggcggggaa gggagattcg 1260
gcaaagacag ctttactact cccttaaaaa as 1292
<210> 20
<211> 1325
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475576CB1
<400> 20
atgcaggggc agactgtagt tccaaaagat tcctacacta tatcccttat ccagaggctg 60
cggggccgtg aggccgcaag gagaacccat gagaaccttc ttcggctgtc tgccctagtg 120
agatccccac agacagctag catcgactgc cacacgtggt cagtttctag tggaaccaat 180
acttcgctgc aggcgtcggg cctgggccgt cagggcagct gtgaccggat cgcttcccgg 240
gcggcgagct gggggtgcac ccggaccgcc gcccccggga tcatgggcaa tggcatgacc 300
aaggtacttc ctggactcta cctcggaaac ttcattgatg ccaaagacct ggatcagctg 360
ggccgaaata agatcacaca catcatctct atccatgagt caccccagcc tctgctgcag 420
gatatcacct accttcgcat cccggtcgct gatacccctg aggtacccat caaaaagcac 480
ttcaaagaat gtatcaactt catccactgc tgccgcctta atggggggaa ctgccttgtg 540
cactgctttg caggcatctc tcgcagcacc acgattgtga cagcgtatgt gatgactgtg 600
acggggctag gctggcggga cgtgcttgaa gccatcaagg ccaccaggcc catcgccaac 660
cccaacccag gctttaggca gcagcttgaa gagtttggct gggccagttc ccagaagctt 720
cgccggcagc tggaggagcg cttcggcgag agccccttcc gcgacgagga ggagttgcgc 780
gcgctgctgc cgctgtgcaa gcgctgccgg cagggctccg CgaCCtCggC CtCCtCCgCC 840
gggccgcact cagcagcctc cgagggaacc ctgcagcgcc tggtgccgcg cacgccccgg 900
gaagcccacc ggCCgCtgCC gctgctggcg cgcgtcaagc agactttctc ttgcctcccc 960
cggtgtctgt cccgcaaggg cggcaagtga ggatgcagtc cagccgtggc tccccacttc 1020
cgactggctc ccttcggggg ctgtctgcgc cttccacgcc ccccaggacg ggcccagagg 1080
ctgggggagc cccgcggcgg cctgaaccct gcctcccgcg cccgccctgc tcgtccgcgt 1140
ctgcagtcag cgtccccaac ctgtgcgtct ctgtgtccgg gccggcctgc tgcagccacc 1200
tggtgcctta gtccttgggc tgggggaggg ggcccaccct taaaggcggc gggaggggag 1260
ggagggagag tggagggttt gacgggcctg gagggtatta aagagacaca gaagaaaaaa 1320
aaaaa 1325
18/18
