Note: Descriptions are shown in the official language in which they were submitted.
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
PROTEIN PHOSPHATASES
TECH1VICAL 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 including cancer, and in
the assessment of the effects of exogenous compounds on the expression of
nucleic acid and amino acid
sequences of protein phosphatases.
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 Mn2+, for activity. PSPs play important roles in glycogen
metabolism, muscle
contraction, protein synthesis, T cell function, neuronal activity, oocyte
maturation, and hepatic
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
metabolism (reviewed in Cohen, P. (1989) Annu. Rev. Biochem. 58:453-508). PSPs
canbe separated
into two classes. The PPP class includes PP1, 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 deternune 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 PP1 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, PR61,
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
eycle (Dallas, 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,
2
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
cyclin-dependent kinases, and the IxB kinases (reviewed in Millward et al.,
supra). PP2A is itself a
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
(Basnans, 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
phosphorylanon 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, supra).
PP2B, or calcineurin, is a Ca2+-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 postsynapnc
NMDA-receptor coupled calcium channels to long term memory (reviewed in Price
and Mumby,
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
tetratricopepnde
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
canons (mainly Mn2+ or Mg2+) for its activity. PP2C proteins share a conserved
N-terminal region with
an invariant DGH motif, which contains an aspartate residue involved in canon
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 (JNI~)
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
3
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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
moiety during catalysis (heel, B.G. and N.K. Tonks (1997) Curr. Opin, Cell
Biol. 9:193-204) .
Receptor PTPs axe made up of an N-terminal extracellular domain of variable
length, a transmembrane
region, and a cytoplasnuc 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
d
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 activation,
and cell proliferation. PTPs p arid x are involved in cell-cell contacts,
perhaps regulating
cadherinlcatenin function. A number of PTPs affect cell spreading, focal
adhesions, and cell motility,
most of them via the integrin/tyrosine kinase signaling pathway (reviewed in
Neel and Tonks, su ra).
CD45 phosphatases regulate signal transduction and lymphocyte activation
(Ledbetter, J.A. et al.
(1988) Proc. Natl. Acad. 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 confxolling the Tarsus
family PTKs in
hematopoietic cells, as well as signaling by the T-cell receptor and c-Kit
(reviewed in Neel and Tonks,
su ra). 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. Acad. 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 MTMI 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 (Charbomieau and Tonks,
supra).
Dual specificity phosphatases (DSPs) are structurally more similar to the PTPs
than the PSPs.
4
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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. The
phosphatases DUSP1 and DUSP2 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, supra). 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 (Giri,
D. and M. Ittmann
(1999) Hum. Pathol. 30:419-424) and abnormalities in its expression are
associated with numerous
cancers (reviewed in Tamura, 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 (PROSITE PDOC00538).
LAP, an orthophosphoric monoester 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
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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,
EPS 15, 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 ofimmune system disorders, neurological disorders, developmental
disorders, and cell
proliferative disorders, including cancer 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," and "PP-S." In one
aspect, the invention
provides an isolated polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1-5, b) a
naturally occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID N0:1-5, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-5, and
d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-5. In one alternative, the invention provides an
isolated polypeptide
comprising the amino acid sequence of SEQ ID N0:1-5.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected from
the group consisting of a) a polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-5, b) a naturally occurring polypeptide comprising
an amino acid sequence
at least 90% identical to an amino acid sequence selected from the group
consisting of SEQ ID N0:1-5,
c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
6
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
group consisting of SEQ ID N0:1-5, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID N0:1-5. In
one alternative, the
polynucleotide encodes a polypeptide selected from the group consisting of SEQ
ID N0:1-5. In another
alternative, the polynucleotide is selected from the group consisting of SEQ
ID N0:6-10.
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
N0:1-5, b) a naturally occurring polypeptide comprising an amino acid sequence
at least 90% identical
to an amino acid sequence selected from the group consisting of SEQ ID N0:1-5,
c) a biologically
active fragment of a polypeptide having an amino acid sequence selected from
the group consisting of
SEQ ID N0:1-5, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ ID N0:1-5. In one alternative, the
invention provides a cell
transformed with the recombinant polynucleotide. In another alternative, the
invention provides a
transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting of
SEQ ID N0:1-5, b) a naturally occurring polypeptide comprising an amino acid
sequence at least 90%
identical to an amino acid sequence selected from the group consisting of SEQ
ID N0:1-5, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-5, and d) an immunogenic fragment of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ ID NO:l-5. The method
comprises a) culturing a
cell under conditions suitable for expression of the polypeptide, wherein said
cell is transformed with a
recombinant polynucleotide comprising a promoter sequence operably linked to a
polynucleotide
encoding the polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid sequence
selected from the group consisting of SEQ ID N0:1-5, b) a naturally occurring
polypeptide comprising
an amino acid sequence at least 90% identical to an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-5, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID N0:1-5, and d) an
immunogenic fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-5.
The invention further provides an isolated polynucleotide selected from the
group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ ID
N0:6-10, b) a naturally occurring polynucleotide comprising a polynucleotide
sequence at least 90%
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:6-10, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). In one alternative,
the polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ ID
N0:6-10, b) a naturally occurring polynucleotide comprising a polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:6-10, 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 ID
N0:6-10, b) a naturally occurring polynucleotide comprising a polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:6-10, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) amplifying said
target polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b)
detecting the presence or absence of said amplified target polynucleotide or
fragment thereof, and,
optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of
a polypeptide
selected from the group consisting of a) a polypeptide comprising an amino
acid sequence selected from
the group consisting of SEQ ID NO:1-5, b) a naturally occurring polypeptide
comprising an amino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
ID N0:1-5, c) a biologically active fragment of a polypeptide having an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-5, and d) an immunogenic fragment of
a polypeptide having
an amino acid sequence selected from the group consisting of SEQ ID N0:1-5,
and a pharmaceutically
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
acceptable excipient. In one embodiment, the composition comprises an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-5. 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 N0:1-5, b) a
naturally occw-ring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID N0:1-5, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-5, and
d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-5. 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 N0:1-5, b) a
naturally occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-5, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-5, and
d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-5. 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 group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID N0:1-5, b) a naturally
occurring polypeptide
cmoprising an amino acid sequence at least 90% identical to an amino acid
sequence selected from the
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
group consisting of SEQ ID NO:1-5, c) a biologically active fragment of a
polypeptide having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-5, and d) an
immunogenic fragment
of a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:I-5.
The method comprises a) combining the polypeptide with at least one test
compound under suitable
conditions, and b) detecting binding of the polypeptide to the test compound,
thereby identifying a
compound that specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID N0:1-5, b) a
naturally occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID N0:1-5, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-5, and
d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-5. 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
sequence selected from the group consisting of SEQ ID N0:6-10, the method
comprising a) exposing
a sample comprising the target polynucleotide to a compound, and b) detecting
altered expression of
the target polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:6-10, ii) a
naturally occurring polynucleotide comprising a polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:6-10,
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
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
sample, said target polynucleotide selected from the group consisting of i) a
polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:6-10, ii) a
naturally occurring polynucleotide comprising a polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:6-10,
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 fragments which were used to assemble polynucleotide
sequences of the
invention, along with selected fragments of the polynucleotide sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
11
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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
12
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
locus other than the normal chromosomal locus for the polynucleotide sequence
encoding PP. The
encoded protein may also be "altered," and may contain deletions, insertions,
or substitutions of amino
acid residues which produce a silent change and result in a functionally
equivalent PP. Deliberate
amino acid substitutions may be made on the basis of similarity in polarity,
charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues,
as long as the biological
or immunological activity of 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 polymerase chain reaction (PCR)
technologies well known
in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity of
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')2, 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 (KL,H). The coupled peptide is then used to immunize the
animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies which
13
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
bind specifically to antigenic determinants (particular regions or three-
dimensional structures on the
protein). An antigenic determinant may compete with the intact antigen (i.e.,
the immunogen used to
elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as ~2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic 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
Wn or Phrap
14
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
(University of Washington, Seattle WA). Some sequences have been both extended
and assembled to
produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, GIn, Isis
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Tle
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid 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
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
A "fragment" is a unique portion of PP or the polynucleotide encoding PP which
is identical
in sequence to but shorter in length than the parent sequence. A fragment may
comprise up to the
entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25 % or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:6-10 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:6-10, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:6-10 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:6-10 from related polynucleotide sequences. The precise length of a
fragment of SEQ ID
N0:6-10 and the region of SEQ ID N0:6-10 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 fragment of SEQ ID N0:1-5 is encoded by a fragment of SEQ ID N0:6-10. A
fragment of
SEQ ID NO:1-5 comprises a region of unique amino acid sequence that
specifically identifies SEQ ID
NO:1-5. For example, a fragment of SEQ ID NO:1-5 is useful as an immunogenic
peptide for the
development of antibodies that specifically recognize SEQ ID N0:1-5. The
precise length of a
fragment of SEQ ID NO:1-5 and the region of SEQ ID N0:1-5 to which the
fragment corresponds are
routinely determinable by one of ordinary skill in the art based on the
intended purpose for the
fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full
length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two ox 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
16
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
the sequences being compared in order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence
alignment program. This program is part of the LASERGENE software package, a
suite of molecular
biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in
Higgins, D.G.
and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992)
CABIOS 8:189-191.
For pairwise alignments of polynucleotide sequences, the default parameters
are set as follows:
Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted"
residue weight table is
selected as the default. Percent identity is reported by CLUSTAL V as the
"percent similarity" between
aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms is
provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment Search
Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which
is available from several
sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm,nih,gov/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for bofih 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: BLOSUMcS2
Reward for match: 1
Penalty for mismatch: -2
Opera Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off. 50
Expect: l0
Wm°d Si.~e: 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
17
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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 Extension Gap: 1 penalties
Gap x drop-off. SO
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence, for
example, as defined by a particular SEQ ID number, or may be measured over a
shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for instance,
18
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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, fox example, at 68°C in the presence of
about 6 x SSC, about 1 % (w/v)
SDS, and about 100 ~~ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (T~ for the
specific sequence at a defined ionic
strength and pH. The 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 T~ and conditions
for nucleic acid hybridization are well known and can be found in Sambrook, J.
et al. (1989) Molecular
Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Press,
Plainview NY; specifically
see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1 % SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used. SSC concentration may
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking
19
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
reagents are used to block non specific hybridization. Such blocking reagents
include, for instance,
sheared and denatured salmon sperm DNA at about 100-200 ~ ~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., Cpt 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 systenuc 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.
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.
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
"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
S 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 label or
reporter molecule. Typical
labels include radioactive isotopes, ligands, chemiluminescent agents, and
enzymes. "Primers" are
short nucleic acids, usually DNA oligonucleotides, which may be annealed to a
target polynucleotide by
complementary base-pairing. The primer may then be extended along the target
DNA strand by a DNA
polymerase enzyme. Primer pairs can be used for amplification (and
identification) of a nucleic acid
sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
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, fox
example Sambrook, J. et aI. (1989) Molecular Cloning: A Laboratory Manual, 2nd
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
21
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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 aeids, e.g., by
genetie engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid, Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for
example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
22
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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 ox 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 axe
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides by
different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
23
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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,
txansfection,
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),
sue.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having at
least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of the
nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
set at default parameters. Such a pair of nucleic acids may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at
least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% or greater sequence
identity over a certain defined length. A variant may be described as, for
example, an "allelic" (as
defined above), "splice," "species," or "polymorphic" variant. A splice
variant may have significant
identity to a reference molecule, but will generally have a greater or lesser
number of polynucleotides
due to alternative splicing of exons during mRNA processing. 'The
corresponding polypeptide may
24
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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 nave significant amino acid identity relative to
each other. A polymorphic
variant is a variation in the polynucleotide sequence of a particular gene
between individuals of a given
species. Polymorphic variants also may encompass "single nucleotide
polymorphisms" (SNPs) in
which the polynucleotide sequence varies by one nucleotide base. The presence
of SNPs may be
indicative of, for example, a certainpopulation, a disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having at
least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of the
polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
set at default parameters. Such a pair of polypeptides may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, at least 91 %, at least 92%, at
least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or
greater sequence identity over a
certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human 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, including cancer.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ ID NO:) and an
Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as
shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBankprotein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the
invention. Column 3
shows the GenBank identification number (Genbank ID NO:) of the nearest
GenBank homolog.
Column 4 shows the probability score for the match between each polypeptide
and its GenBank
homolog. Column 5 shows the annotation of the GenBank homolog along with
relevant citations where
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and 2
show the polypeptide sequence identification number (SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention. Column 3
shows the number of amino acid residues in each polypeptide. Column 4 shows
potential
phosphorylataon sites, and column S shows potential glycosylation sites, as
determined by the MOTIFS
program of the GCG sequence analysis software package (Genetics Computer
Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structurelfunction analysis and in some
cases, searchable
databases to which the analytical methods were applied.
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
NO:S is 98% identical to rat dual specificity phosphatase (GenBank ID
g1220173) as determined by the
Basic Local Alignment Search Tool (BLAST). (See Table 2.) In The BLAST
probability score is
7.9e-150, which indicates the probability of obtaining the observed
polypeptide sequence alignment by
chance. SEQ ID NO:S also contains a dual specificity phophatase catalytic
domain as determined by
searching for statistically significant matches in the hidden Maxkov model
(HMM)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from BLIMPS
and
PROFILESCAN analyses provide further corroborative evidence that SEQ ID NO:S
is dual specificity
phosphatase. In an alternative example, SEQ ID N0:2 is 40% identical to human
dual specificity
phosphatase MKP-5 (g513995) as determined by the Basic Local Alignment Search
Tool (BLAST).
In The BLAST probability score is 1 e-22 (see Table 2), which indicates the
probability of obtaining the
observed polypeptide sequence alignment by chance. SEQ ID N0:2 also contains a
dual specificity
phosphatase catalytic domain as determined by searching for statistically
significant matches in the
hidden Maxkov model (HMM)-based PFAM database of conserved protein family
domains. The
percent identity is 44% and the probability value is 1e-38. Based on BLAST and
HMM analyses, SEQ
ID N0:2 is a dual specificity phosphatase which removes phosphates from
tyrosine, threonine or serine
residues. In an alternative example, SEQ ID N0:4 is 97% identical to rat
pyruvate dehydrogenase
phosphatase isoenzyme 1 (g3298607) as determined by the Basic Local Alignment
Search Tool
(BLAST). The probability score is 7.3 e-292 (see Table 2), indicating that a
match between these two
sequences would occur by chance only once in 7.3 X 10292 times. SEQ ID N0:4
also contains a protein
phosphatase 2c signature sequence as determined by searching against the
hidden Markov model
(HMM)-based PFAM database of conserved protein family domains, the DOMO
database of
homologous protein domain families, the PRODOM database (an automatic
compilation of homologous
26
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
protein domains), the BLOCKS structural fingerprint database and MOTIFS, a
program that searches
amino acid sequences for patterns that matched those defined in Prosite (see
Tables 3 and 7 for details).
Based on BLAST, BLIMPS and HMM-based analyses, the protein of the present
invention is a
phosphatase which removes phosphates from threonine or serine residues. SEQ ID
N0:1 and SEQ ID
N0:3 were analyzed and annotated in a similar manner. The algorithms and
parameters for the analysis
of SEQ ID N0:1-5 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 N0:6-10 or that distinguish between SEQ ID
N0:6-10 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 sequences in column 5 relative to their respective full
length sequences.
The identification numbers in Column S of Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
6954474H1 is the identification number of an Incyte cDNA sequence, and
BLADNOR01 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., 70001461D1). Alternatively, the
identification numbers in
column 5 may refer to GenBank cDNAs or ESTs (e.g., g2141792) which contributed
to the assembly
of the full length polynucleotide sequences. Alternatively, the identification
numbers in column 5 may
refer to coding regions predicted by Genscan analysis of genomic DNA. The
Genscan-predicted coding
sequences may have been edited prior to assembly. (See Example IV.)
Alternatively, the identification
numbers in column 5 may refer to assemblages of both cDNA and Genscan-
predicted exons brought
together by an "exon stitching" algorithm. (See Example V.) Alternatively, the
identification numbers
in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together by
an "exon-stretching" algorithm. (See Example V.) In some cases, Incyte cDNA
coverage redundant
with the sequence coverage shown in column 5 was obtained to confirm the final
consensus
polynucleotide sequence, but the relevant Incyte cDNA identification numbers
are not shown.
27
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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 conf'~rm 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 N0:6-10, which encodes PP. The polynucleotide sequences
of SEQ ID N0:6-10,
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 ttze group consisting of SEQ ID N0:6-10
which has at least about
70%, or alternatively at least about 85%, or even at least about 95%
polynucleotide sequence identity to
a nucleic acid sequence selected from the group consisting of SEQ ID N0:6-10.
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
pxoduced. Thus, the
invention contemplates each and every possible variation of polynucleotide
sequence that could be made
by selecting combinations based on possible codon choices. These combinations
are made in
accordance with the standard triplet genetic code as applied to the
polynucleotide sequence of naturally
occurring 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
28
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
derivatives possessing a substantially different codon usage, e.g., inclusion
of non-naturally occurring
codons. Codons may be selected to increase the rate at which expression of the
peptide occurs in a
particular prokaryotic or eukaryotic host in accordance with the frequency
with which particular codons
are utilized by the host. Other reasons for substantially altering the
nucleotide sequence encoding 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
N0:6-10 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; I~immel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of the
embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment of
DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA sequencing
system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system
(Molecular Dynamics,
Sunnyvale CA), or other systems known in the art. The resulting sequences are
analyzed using a
variety of algorithms which are well known in the art. (See, e.g., Ausubel,
F.M. (1997) Short Protocols
in Molecular Biolo~y, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
(1995) Molecular
Biolo~y and Biotechnology, 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
29
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
promoters and regulatory elements. For example, one method which may be
employed, restriction-site
PCR, uses universal and nested primers to amplify unlmown sequence from
genomic DNA within a
cloning vector. (See, e.g., Sarkax, G. (1993) PCR Methods Applic. 2:318-322.)
Another method,
inverse PCR, uses primers that extend in divergent directions to amplify
unknown sequence from a
circularized template. The template is derived from restriction fragments
comprising a known genomic
locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic
Acids Res. 16:8186.) A
third method, capture PCR, involves PCR amplification of DNA fragments
adjacent to known
sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al. (1991)
PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme
digestions and ligations
may be used to insert an engineered double-stranded sequence into a region of
unknown sequence before
performing PCR. Other methods which may be used to retrieve 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 intxonlexon 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, axe preferable for situations in
which an oligo d(T) library
does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence into 5'
non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze the
size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable fox sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode PP may be cloned in recombinant DNA molecules that direct expression of
PP, or fragments or
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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, and/or expression of the
gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction sites,
alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol, 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of PP, such as its biological or enzymatic
activity or its ability to
bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding 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 Acids 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
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, r.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
31
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
part thereof, may be altered during direct synthesis and/or combined with
sequences from other
proteins, or any part thereof, to produce a variant polypeptide or a
polypeptide having a sequence of a
naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. andF.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, su ra, pp. 28-53.)
In order to express a biologically active 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 colon and adjacent sequences, e.g. the Kozak sequence. In cases
where sequences
encoding PP and its initiation colon 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 colon
should be provided by the
vector. Exogenous translational elements and initiation colons 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-ALaboratory
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 such as
bacteria transformed with
recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast
transformed with yeast
expression vectors; insect cell systems infected with viral expression vectors
(e.g., baculovirus); plant
cell systems transformed with viral expression vectors (e.g., cauliflower
mosaic virus, CaMV, or
32
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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. Nail. 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 fox
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 a1. (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 Saccharonivces cerevisiae or Pichia pastoris. In addition,
such vectors direct either the
secretion or intracellular retention of expressed proteins and enable
integration of foreign sequences into
the host genome for stable propagation. (See, e.g., Ausubel, 1995, supra;
Bitter, G.A. et al. (1987)
Methods Enzymol. 153:516-544; and Scorer, C.A. et al. (1994) Bio/Technology
12:181-184.)
Plant systems may also be used for expression of 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).
33
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
Alternatively, plant promoters such as the small subunit of RUBISCO or heat
shock promoters may be
used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Brogue, R. et
al. (1984) Science
224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-
I05.) 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 ugated into an
adenovirus transcription/translation complex consisting of the late promoter
and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses 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 (liposomes,
polycationic amino polymers,
or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
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 unes.
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, antimetaboute, antibiotic, or
herbicide resistance can be
used as the basis for selection. For example, dhfr confers resistance to
methotrexate; neo confers
resistance to the aminoglycosides neomycin and G-418; and als and pat confer
resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g.,
Wigler, M. et al. (1980)
34
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F, et al. (1982) 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 presencelabsence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
conf'~rmed. 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 for 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 (RTAs), and
fluorescence
activated cell sorting (FACS). 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 Laborator~Manual, APS Press, St. Paul MN, Sect.
IV; Coligan, J.E. et
al. (1997) Current Protocols in Immunolo~y, Greene Pub. Associates and Wiley-
Interscience, New
York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa
NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
Labeled hybridization
or PCR probes for detecting sequences related to polynucleotides encoding 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
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
mRNA probe. Such vectors are known in the art, are commercially available, and
may be used to
synthesize RNA probes in vitro by addition of an appropriate RNA polymerase
such as T7, T3, or SP6
and labeled nucleotides. These procedures may be conducted using a variety of
commercially available
kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison
WZ), 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
andlor 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 eukaxyotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of the
polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which cleaves a
"prepro" or "pro" form of the
protein may also be used to specify protein targeting, folding, and/or
activity. Different host cells
which have specific cellular machinery and characteristic mechanisms for post-
translational activities
(e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type
Culture
Collection (ATCC, Manassas VA) and may be chosen to ensure the correct
modification and processing
of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding 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 resins,
respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
proteins using commercially available monoclonal and polyclonal antibodies
that specifically recognize
36
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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
IO 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, T.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 may 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.
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,
37
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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
may be
"knocked out" in an animal model system using homologous recombination in
embryonic stem (ES)
cells. Such techniques are well known in the art and are useful for the
generation of animal models of
human disease. (See, e.g., U.S. Patent Number 5,175,383 and U.S. Patent Number
5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse embryo
and grown in culture. The ES cells are transformed with a vector containing
the gene of interest
disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo;
Capecchi, M.R. (1989)
Science 244:1288-1292). The vector integrates into the corresponding region of
the host genome by
homologous recombination. Alternatively, homologous recombination takes place
using the Cre-loscP
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 lineages
differentiate into, fox
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 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
38
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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. Anne. 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 tumorous, immunological, neurological, placental,
reproductive, and diseased tissue.
Therefore, PP appears to play a role in immune system disorders, neurological
disorders, developmental
disorders and cell proliferative disorders including cancer. 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 are not limited to, an immune system
disorder, such as
acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of
Breton, common
variable immunodeficiency (CVI), DiGeorge's syndrome (thymic hypoplasia),
thymic dysplasia,
isolated IgA deficiency, severe combined immunodeficiency disease (SCID),
ixnmunodeficiency 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
extracorporeal
circulation, viral, bacterial, fungal, 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
extxapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron
disorders, progressive
39
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple
sclerosis and other
demyelinating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis,
viral central nervous system
disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-Straussler-Scheinker
syndrome, fatal familial insomnia, nutritional and metabolic diseases of the
nervous system,
neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal
syndrome, mental retardation and other developmental disorders of the central
nervous system including
Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous
system disorders, cranial
nerve disorders, spinal cord diseases, muscular dystrophy and other
neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and polymyositis,
inherited, metabolic, endocrine,
and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders
including mood, anxiety,
and schizophrenic disorders, seasonal affective disorder (SAD), akathesia,
amnesia, catatonia, diabetic
neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic
neuralgia, Tourette's
disorder, progressive supranuclear palsy, corticobasal degeneration, and
familial frontotemporal
dementia; 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.
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
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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 disorders
include, but are not limited to, those immune system disorders, neurological
disorders, developmental
disorders and cell pxoliferative disorders, including cancer 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 Bells or tissues which
express PP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding PP may be administered to a subject to treat ox 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 adjuvants
may be used to increase
immunological response. Such adjuvants include, but are not limited to,
Fxeund's, mineral gels such as
aluminum hydroxide, and surface active substances such as lysolecithin,
pluronic polyols, polyanions,
41
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
peptides, oiI 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. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morxison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce 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. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter,
G, et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for PP may also be
generated. For
example, such fragments include, but are not limited to, F(ab')2 fragments
produced by pepsin digestion
of the antibody molecule and Fab fragments generated by reducing the disulfide
bridges of the F(ab')2
fragments. Alternatively, Fab expression libraries may be constructed to allow
rapid and easy
identification of monoclonal Fab fragments with the desired specificity. (See,
e.g., Huse, W.D. et al.
(1989) Science 246:1275-1281.)
42
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities axe well
known in the art. Such
immunoassays typically involve the measurement of complex formation between 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, sera).
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 I~ determined for 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 aff nity. High-affinity antibody preparations with I~ ranging
from about 109 to 1012
L/mole are preferred for use in immunoassays in which the PP-antibody complex
must withstand
rigorous manipulations. Low-affinity antibody preparations with I~ ranging
from about 106 to 10'
L/mole are preferred for use in immunopurification and similar procedures
which ultimately require
dissociation of 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
(1991) A Practical
Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
the quality and suitability of such preparations for certain downstream
applications. For example, a
polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of 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
43
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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
S intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Cli. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intxacellularly
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.)
1S 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)-X1 disease characterized
by X-linked
inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe
combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
2S 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.
44
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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
transposons (Morgan, R.A, and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-
217; Ivics, Z.
(1997) Cell 91:501-510; Boulay, J-L. and H. Recipon (1998) Curr. Opin.
Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of PP include, but
are not limited
to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad
CA),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). PP may be
expressed using
(i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous
sarcoma virus (RSV),
SV40 virus, thymidine kinase (TIC), 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
FI~506/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 eneoding 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
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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 5,920,434 to Rigg ("Method fox obtaining
retrovirus packaging
cell lines producing high transducing efficiency retroviral supernatant")
discloses a method for
obtaining retrovirus packaging cell lines and is hereby incorporated by
reference. Propagation of
retrovirus vectors, transduction of a population of cells (e.g., CD4+ T-
cells), and the return of
transduced cells to a patient are procedures well known to persons skilled in
the art of gene therapy and
have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029;
Bauer, G, et al. (1997)
Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716; Ranga, U. et
al. (1998) Proc.
Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding 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
46
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
appropriate promoter for purposes including human gene therapy. Also taught by
this patent are the
construction and use of recombinant HSV strains deleted for ICP4, ICP27 and
ICP22. For HSV
vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et
al. (1994) Dev. Biol.
163:152-161, hereby incorporated by reference. The manipulation of cloned
herpesvirus sequences, the
generation of recombinant virus following the transfection of multiple
plasmids containing different
segments of the large herpesvirus genomes, the growth and propagation of
herpesvirus, and the
infection of cells with herpesvirus are techniques well known to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding 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 (SIN) indicates
that the lytic replication
of alphaviruses can be altered to suit the needs of the gene therapy
application (Dryga, S.A. et al.
(1997) Virology 228:74-83). The wide host range of alphaviruses will allow the
introduction of 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. I63-
177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA by
preventing the transcript from binding to ribosomes.
47
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the xegion of the target gene containing the cleavage site,
may be evaluated for
20 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' andlor 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
br promoters of
polynucleotide expression. Thus, in the treatment of disorders associated with
increased PP
48
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
expression ox activity, a compound which specifically inhibits expression of
the polynucleotide
encoding PP may be therapeutically useful, and in the treament 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
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
palynucleotide 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 carried out, for example, using a Schizosaccharom,~ces 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.)
49
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
Any of the therapeutic methods described above may be applied to any subject
in need of such
therapy, including, for example, mammals such as humans, dogs, cats, cows,
horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition which
generally comprises an active ingredient formulated with a pharmaceutically
acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and
proteins. Various
formulations are commonly known and are thoroughly discussed in the latest
edition of Remin~ton's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of 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, infra-
arterial, intramedullary, intrathecal,
intxaventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, ox 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 fox 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 ox 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
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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
standard
pharmaceutical procedures in cell cultures or with experimental animals, such
as by calculating the
EDso (the dose therapeutically effective in 50% of the population) or LDSO
(the dose lethal to 50% of the
population) statistics. The dose ratio of toxic to therapeutic effects is the
therapeutic index, which can
be expressed as the LDso/EDSO ratio. Compositions which exhibit large
therapeutic indices are
preferred. The data obtained from cell culture assays and animal studies are
used to formulate a range
of dosage fox human use. The dosage contained in such compositions is
preferably within a range of
circulating concentrations that includes the EDSO with little or no toxicity.
The dosage varies within this
range depending upon the dosage form employed, the sensitivity of the patient,
and the route of
administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the subject
requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the active
moiety or to maintain the desired effect. Factors which may be taken into
account include the severity
of the disease state, the general health of the subject, the age, weight, and
gender of the subject, time
and frequency of administration, drug combination(s), reaction sensitivities,
and response to therapy.
Long-acting compositions may be administered every 3 to 4 days, every week, or
biweekly depending
on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ,ug to 100,000 ,ug, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind 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 prepared in
the same manner as described above for therapeutics. Diagnostic assays fox 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
51
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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 FACS, 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, 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 occurring 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
N0:6-10 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.
52
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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
(SCID),
inununodeficiency 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 extracoyoreal circulation, viral, bacterial, fungal,
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, prion diseases including
kuru, Creutzfeldt-Jakob
disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,
nutritional and
metabolic diseases of the nervous system, neurofibromatosis, tuberous
sclerosis, cerebelloretinal
hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and
other developmental
disorders of the central nervous system including Down syndrome, cerebral
palsy, neuroskeletal
disorders, autonomic nervous system disorders, cranial nerve disorders, spinal
cord diseases, muscular
dystrophy and other neuromuscular disorders, peripheral nervous system
disorders, dermatomyositis
and polymyositis, inherited, metabolic, endocrine, and toxic myopathies,
myasthenia gravis, periodic
paralysis, mental disorders including mood, anxiety, and schizophrenic
disorders, seasonal affective
53
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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. 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 may be labeled by standard methods and added to a fluid or tissue
sample from a patient
under conditions suitable for the formation of hybridization complexes. After
a suitable incubation
period, the sample is washed and the signal is quantified and compared with a
standard value. If the
amount of signal in the patient sample is significantly altered in comparison
to a control sample then the
presence of altered levels of nucleotide sequences encoding 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
54
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
hybridization may be quantified by comparing the values obtained from normal
subjects with values
from an experiment in which a known amount of a substantially purified
polynucleotide is used.
Standard values obtained in this manner may be compared with values obtained
from samples from
patients who are symptomatic for a disorder. Deviation from standard values is
used to establish the
presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding PP may
involve the use of PCR. These oligomers may be chemically synthesized,
generated enzymatically, or
produced in vitro. Oligomers will preferably contain a fragment of a
polynucleotide encoding 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.
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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. Immunol. Methods
159:235-244; Duplaa, C. et
al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple
samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic profile.
In another embodiment, 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
56
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
.Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a
signature similar to that of a compound with known toxicity, it is likely to
share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain
expression information
from a large number of genes and gene families. Ideally, a genome-wide
measurement of expression
provides the highest quality signature. Even genes whose expression is not
altered by any tested
compounds are important as well, as the levels of expression of these genes
are used to normalize the
rest of the expression data. The normalization procedure is useful for
comparison of expression data
after treatment with different compounds. While the assignment of gene
function to elements of a
toxicant signature aids in interpretation of toxicity mechanisms, knowledge of
gene function is not
necessary for the statistical matching of signatures which leads to prediction
of toxicity. (See, for
example, Press Release 00-02 from the National Institute of Environmental
Health Sciences, released
February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.)
Therefore, it is
important and desirable in toxicological screening using toxicant signatures
to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological sample
containing nucleic acids with the test compound. Nucleic acids that are
expressed in the treated
biological sample are hybridized with one or more probes specific to the
polynucleotides of the
present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
57
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a proteome
can be subjected individually to further analysis. Proteome expression
patterns, or profiles, are
analyzed by quantifying the number of expressed proteins and their relative
abundance under given
conditions and at a given time. A profile of a cell's proteome may thus be
generated by separating and
analyzing the polypeptides of a particular tissue or cell type. Tn one
embodiment, the separation is
achieved using two-dimensional gel electrophoresis, in which proteins from a
sample are separated by
isoelectric focusing in the first dimension, and then according to molecular
weight by sodium dodecyl
sulfate slab gel electrophoresis in the second dimension (Steiner and
Anderson, supra). The proteins are
visualized in the gel as discrete and uniquely positioned spots, typically by
staining the gel with an agent
such as Coomassie Blue or silver or fluorescent stains. The optical density of
each protein spot is
generally proportional to the level of the protein in the sample. The optical
densities of equivalently
positioned protein spots from different samples, for example, from biological
samples either treated or
untreated with a test compound or therapeutic agent, are compared to identify
any changes in protein
spot density related to the treatment. The proteins in the spots are partially
sequenced using, for
example, standard methods employing chemical or enzymatic cleavage followed by
mass spectrometry.
The identity of the protein in a spot may be determined by comparing its
partial sequence, preferably of
at least 5 contiguous amino acid residues, to the polypeptide sequences of the
present invention. In
some cases, further sequence data may be obtained for definitive protein
identification.
A proteomic profile may also be generated using antibodies specific for 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 aI. (1999) Anal.
Biochem. 270:103-111;
Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a variety of
methods known in the art, for example, by reacting the proteins in the sample
with a thiol- or amino-
reactive fluorescent compound and detecting the amount of fluorescence bound
at each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and should
be analyzed in parallel with toxicant signatures at the transcript level.
There is a poor correlation
between transcript and protein abundances for some proteins in some tissues
(Anderson, N.L. and J.
Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures
may be useful in the
analysis of compounds which do not significantly affect the transcript image,
but which alter the
58
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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 aanount of each protein can be quantified The
amount of each protein
is compared to the amount of the corresponding protein in an untreated
biological sample. A difference
in the amount of protein between the two samples is indicative of a toxic
response to the test compound
in the treated sample. Individual proteins are identified by sequencing the
amino acid residues of the
individual proteins and comparing these partial sequences to the polypeptides
of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared with
the amount in an untreated biological sample. A difference in the amount of
protein between the two
samples is indicative of a toxic response to the test compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are well
known and thoroughly described in DNA Microarrays: A Practical Ap rp oach, 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 occurring
genomic sequence. Either
2S 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
59
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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, 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 a1. (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 W084103564.) 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
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
manner, antibodies can be used to detect the presence of any peptide which
shares one or more antigenic
determinants with PP.
In 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 preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below,
including U.S. Ser. No. 60/199,010, U.S. Ser. No. 60/202,340, U.S. Ser. No.
60/203,424, U.S. Ser.
No. 60/205,642, and U.S. Ser. No. 60/208,854 are expressly incorporated by
reference herein.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database
(Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues
were homogenized
and lysed in guanidinium isothiocyanate, while others were homogenized and
lysed in phenol or in a
suitable mixture of denaturants, such as TRIZOL (Life Technologies), a
monophasic solution of phenol
and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl
cushions or extracted
with chloroform. RNA was precipitated from the lysates with either isopropanol
or sodium acetate and
ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, su ra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
61
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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 51000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo Alto
CA), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli cells
including XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DHSa, DH10B, or
ElectroMAX
DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo excision
using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were
purified using at least
one of the following: a Magic or WIZARD Minipreps DNA purification system
(Promega); an AGTC
Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8
Plasmid, QIAWELL
8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L.
PREP 96 plasmid
purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1 ml of
distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rio, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in 384-
well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAIV 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
62
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with
standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel, 1997,
supra, unit 7.7). Some of the cDNA sequences were selected fox extension using
the techniques
disclosed in Example VIII.
The polynucleotide sequences dexived from Incyte cDNAs were validated by
removing vector,
linker, and poly(A) sequences and by masking ambiguous bases, using algorithms
and programs based
on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The
Incyte cDNA
sequences or translations thereof were then queried against a selection of
public databases such as the
GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and
BLOCKS, PRINTS,
DOMO, PRODOM, and hidden Markov model (HMM)-based protein family databases
such as PFAM.
(HMM is a probabilistic approach which analyzes consensus primary structures
of gene families.
See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The
queries were
performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte
cDNA
sequences were assembled to produce full length polynucleotide sequences.
Alternatively, GenBank
cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-
predicted coding
sequences (see Examples IV and V) were used to extend Incyte cDNA assemblages
to full length.
Assembly was performed using programs based on Phred, Phrap, and Consed, and
cDNA assemblages
were screened for open reading frames using programs based on GeneMark, BLAST,
and FASTA.
The full length polynucleotide sequences were translated to derive the
corresponding full length
polypeptide sequences. Alternatively, a polypeptide of the invention may begin
at any of the methionine
residues of the full length translated polypeptide. Full length polypeptide
sequences were subsequently
analyzed by querying against databases such as the GenBank protein databases
(genpept), SwissProt,
BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model (HMM)-based
protein
family databases such as PFAM. Full length polynucleotide sequences are also
analyzed using
MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA)
and
LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence
alignments are
generated using default parameters specified by the CLUSTAL algorithm as
incorporated into the
MEGALIGN multisequence alignment program (DNASTAR), which also calculates the
percent
identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and threshold
parameters. The first column of Table 7 shows the tools, programs, and
algorithms used, the second
column provides brief descriptions thereof, the third column presents
appropriate references, all of
63
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
which are incorporated by reference herein in their entirety, and the fourth
column presents, where
applicable, the scores, probability values, and other parameters used to
evaluate the strength of a match
between two sequences (the higher the score or the lower the probability
value, the greater the identity
between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide and
polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:6-10. 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 vaxiety of
organisms (See Burge, C. and S. I~axlin (1997) J. Mol. Biol. 268:78-94, and
Burge, C. and S. Karlin
(1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates
predicted axons 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 axons. BLAST analysis was also used to find any Incyte cDNA or public
cDNA coverage of
the Genscan-predicted sequences, thus providing evidence for transcription.
When Incyte cDNA
coverage was available, this information was used to correct or confirm the
Genscan predicted
sequence. Full length polynucleotide sequences were obtained by assembling
Genscan-predicted coding
sequences with Incyte cDNA sequences and/or public cDNA sequences using the
assembly process
described in Example III. Alternatively, full length polynucleotide sequences
were derived entirely from
edited or unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
Partial cDNA sequences were extended with axons predicted by the Genscan gene
identification
program described in Example IV. Partial cDNAs assembled as described in
Example III were mapped
64
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
to genomic DNA and parsed into clusters containing related cDNAs and Genscan
exon predictions from
one or more genomic sequences. Each cluster was analyzed using an algorithm
based on graph theory
and dynamic programming to integrate cDNA and genomic information, generating
possible splice
variants that were subsequently confirmed, edited, or extended to create a
full length sequence.
Sequence intervals in which the entire length of the interval was present on
more than one sequence in
the cluster were identified, and intervals thus identified were considered to
be equivalent by transitivity.
For example, if an interval was present on a cDNA and two genomic sequences,
then all three intervals
were considered to be equivalent. This process allows unrelated but
consecutive genomic sequences to
be brought together, bridged by cDNA sequence. Intervals thus identified were
then "stitched" together
by the stitching algorithm in the order that they appear along their parent
sequences to generate the
longest possible sequence, as well as sequence variants. Linkages between
intervals which proceed
along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic
sequence) were
given preference over linkages which change parent type (cDNA to genomic
sequence). The resultant
stitched sequences were translated and compared by BLAST analysis to the
genpept and gbpri public
databases. Incorrect exons predicted by Genscan were corrected by comparison
to the top BLAST hit
from genpept. Sequences were further extended with additional cDNA sequences,
or by inspection of
genomic DNA, when necessary.
"Stretched" Seguences
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 GenB ank 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 ID N0:6-10 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
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
SEQ ID N0:6-10 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM
distances are based on genetic markers mapped by Genethon which provide
boundaries for radiation
hybrid markers whose sequences were included in each of the clusters. Human
genome maps and
other resources available to the public, such as the NCBI "GeneMap'99" World
Wide Web site
(http://www.ncbi.nlm.nih.govlgenemapn, can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
In this manner, SEQ ID N0:5 was mapped to chromosome 12 within the interval
from 95.80 to
97.00 centiMorgans.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a gene
and involves the hybridization of a labeled nucleotide sequence to a membrane
on which RNAs from a
particular cell type or tissue have been bound. (See, e.g., Sambrook, sera,
ch. 7; Ausubel (1995)
sue, 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
66
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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 III). Each cDNA
sequence is derived
from a cDNA library constructed from a human tissue. Each human tissue is
classified into one of the
following organ/tissue categories: cardiovascular system; connective tissue;
digestive system;
embryonic structures; endocrine system; exocrine glands; genitalia, female;
genitalia, male; germ cells;
heroic and immune system; liver; musculoskeletal system; nervous system;
pancreas; respiratory
system; sense organs; skin; stomatognathic system; unclassified/mixed; or
urinary tract. The number of
libraries in each category is counted and divided by the total number of
libraries across all categories.
Similarly, each human tissue is classified into one of the following
disease/condition categories: cancer,
cell line, developmental, inflammation, neurological, trauma, cardiovascular,
pooled, and other, and the
number of libraries in each category is counted and divided by the total
number of libraries across all
categories. The resulting percentages reflect the tissue- and disease-specific
expression of cDNA
encoding 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.
67
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
Selected human cDNA libraries were used to extend the sequence. If more than
one extension
was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)2S O4,
and 2-mercaptoethanol, Taq DNA polymerise (Amersham Phaxmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerise (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 20 ~l aliquot of the reaction mixture was
analyzed by electrophoresis
on a 1 % agarose gel to determine which reactions were successful in extending
the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to relegation 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
relegated 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 1:
94 ° C, 3 min; Step 2: 94 ° C, 15 sec; Step 3: 60 ° C, 1
min; Step 4: 72 ° C, 2 min; Step 5 : steps 2, 3, and 4
repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C.
DNA was quantified by PICOGREEN
reagent (Molecular Probes) as described above. Samples with low DNA recoveries
were reamplified
68
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
using the same conditions as described above. Samples were diluted with 20%
dimethysulfoxide (1:2,
vlv), and sequenced using DYENAMIC energy transfer sequencing primers and the
DYENAMIC
DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator
cycle
sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5'regulatory sequences using the above procedure along with
oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:6-10 are employed to screen cDNAs,
genomic
DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about
20 base pairs, is
specifically described, essentially the same procedure is used with larger
nucleotide fragments.
Oligonucleotides are designed using state-of the-art software such as OLIGO
4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~cCi of
[~y-32P] adenosine
triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase
(DuPont NEN, Boston
MA). The labeled oligonucleotides are substantially purified using a SEPHADEX
G-25 superfine size
exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot
containing 10' counts per
minute of the labeled probe is used in a typical membrane-based hybridization
analysis of human
genomic DNA digested with one of the following endonucleases: Ase I, Bgl II,
Eco RI, Pst I, Xba I, or
Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5 %
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
X. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.), mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), supra). Suggested
substrates include silicon, silica, glass slides, glass chips, and silicon
wafers. Alternatively, a procedure
analogous to a dot or slot blot may also be used to arrange and link elements
to the surface of a
substrate using thermal, UV, chemical, or mechanical bonding procedures. A
typical array may be
produced using available methods and machines well known to those of ordinary
skill in the art and may
69
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470;
Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson
(1998) Nat. Biotechnol.
16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The array
elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described in
detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~il oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 unitsl~l RNase inhibitor, 500 pM dATP, 500 pM dGTP, 500 pM
dTTP, 40 ~uM
dCTP, 40 f.iM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)''~ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 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 ail SX SSC/0.2% SDS.
Microarra~paration
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
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
fig. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05 % aminopropyl silane (Sigma) in 95 % ethanol. Coated slides
are cured in a 110 °C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522, incorporated herein by reference. 1 ~.Q of the array
element DNA, at an average
concentration of 100 ng/~l, is loaded into the open capillary printing element
by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 f.Q of sample mixture consisting of 0.2 ~g
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65 ° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of
140 i.il of SX SSC in a corner of the chamber. The chamber containing the
arrays is incubated for
about 6.5 hours at 60° C. The arrays are washed for 10 min at 45
° C in a first wash buffer (1X SSC,
0.1 % SDS), three times for 10 minutes each at 45 ° C in a second wash
buffer (0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
71
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater N~ corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are differentially
expressed, the calibration is done by labeling samples of the calibrating cDNA
with the two
fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
XI. Complementary Polynucleotides
Sequences complementary to the 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.
72
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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 (tic)
hybrid promoter and the
T5 or T7 bacteriophage promoter in conjunction with the lac operator
regulatory element.
Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21
(DE3). Antibiotic
resistant bacteria express PP upon induction with isopropyl beta-D-
thiogalactopyranoside (1PTG).
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
fruy'perda (Sf9) insect
cells in most cases, ox human hepatocytes, in some cases. Infection of the
latter requires additional
genetic modifications to baculovirus. (See Engelhard, E.K. et al. (1994) Proc.
Natl. Acid. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.)
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 immunoaffinity
purification using
commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman
Kodak). 6-His, a
stretch of six consecutive histidine residues, enables purification on metal-
chelate resins (QIAGEN).
Methods for protein expression and purification are discussed in Ausubel
(1995, supra, ch. 10 and 16).
Purified PP obtained by these methods can be used directly in the assays shown
in Examples XVI,
XVII, XVIII, 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
73
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
cytomegalovirus promoter. 5-10 ~cg of recombinant vector are transiently
transfected into a human cell
line, for example, an endothelial or hematopoietic cell line, using either
liposome formulations or
electroporation. 1-2 ,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
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, su ra, ch. 11.)
74
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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 % B SA,
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
immunoaffinity
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.
XVI. Identification of Molecules Which Interact with PP
PP, or biologically active fragments thereof, are labeled with 1'~I Bolton-
Hunter reagent. (See,
e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.) Candidate
molecules previously
arrayed in the wells of a multi-well plate are incubated with the labeled 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 MATCHMAKER 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 %
p-mercaptoethanol at
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
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
~1 of 40 mM NaCl 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 (Saftag, 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 axe performed with 2 or 4
nM enzyme in a final
volume of 30 ~l containing 60 mM Tris, pH 7.6, 1 mM EDTA, 1 mM EGTA, 0.1 % p-
mercaptoethanol
and 10 ~M substrate, 32P-labeled on serine/threonine or tyrosine, as
appropriate. Reactions are initiated
with substrate and incubated at 30° C for 10-15 min. Reactions are
quenched with 450 ~1 of 4% (w/v)
activated charcoal in 0.6 M HCl, 90 mM Na4P20~, and 2 mM NaH2P04, then
centrifuged at 12,000 x g
for 5 min. Acid-soluble 32Pi is quantified by liquid scintillation counting
(Sinclair, C. et al. (1999) J.
Biol. Chem. 274:23666-23672).
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
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 COS1 or 293 cells via calcium
phosphate-mediated
transfection with 20 ~ g of CsCl-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
76
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
tyrosine phosphorylation in cells, as the tyrosine kinase EGFR is abundant in
COS cells. Cells are
lysed in 50 mM Tris~HCl, pH 7.515 mM EDTA/150 mM NaCl/1 % Triton X-100/5 mM
iodoacetic
acid/10 mM sodiumphosphate/10 inM NaF/S ~~ml leupeptin/5 ~g/ml aprotinin/1 mM
benzamidine (1
ml per 10-crim 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.
77
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
a
H
N
H
O r1c-Ic-Ir-1W
U WW Wa1U
H UU UU r1
U ~oM t~tmo
N Ind~OM l0
~
.J,r1l001N 01
~
~y Od~L(1rIM
y
U oo l0tY1d~
r1
~',oL cHW -!
O
H d~to~or1t
W
N
.,1
h
O
N
H
U
H
r--I
O~
O o
W
~ ~~ ~~ H
~
A
H
-~If~Cafaf~U
-I-~UU UU r1
~DM l~L11l0
N L(1d~OM l0
N
-IW--Il001N 01
~
,5yOcrLnr1M
,
U oo ~otnd~
r-i
~, oL dWtW
O -I
H d~totoc-)L
W
N
w1
O
1~
'~,
H
r-I
O<
O
W
W -IN M~ l.n
Cly
q
H
c-~
-L~lflM L~LlllD
N Wd~oM vo
U
J~ c-Il0O1N O1
N I
,5yOd~Lllc-IM
-n
U oo ~oi.nd~
O
~I oL dW <-I
n
H CHl0l0r1L~
W
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
I I
~ o N
o
m oo rn
~o
I c>3 rtS I
~o ~o
~n ~ ~ .u o,
~
rt
~
U -r1 ,L~, ,1~, M
N
-ri i ~ , r-~
~,' ~ ~H
--rl -I~O ~ O
,.~
~
U ~ -rl,~', ..C; U
M N N ~
U U7 U ~ '.' ~ -r-I
~-- '.' ~
, -a b1
~--
U1 4-I~ M N N
.-. M
Ln
a ~ -~I~n m
~
N CJ U (~ tti ~I
M N N l
N
-r1 ~ N ~ ~'
~, Ul ~1 ~7
~7.!-~ ~
N
U -r-Ir-IS-I .~I ".~
O ,.G ~i
~ U U ~
U
,~ ~ 5r ~ .
S~
~ ~ N ~
~ W r~ r-1 .,
p'
N I ~ O '~ ~-'
W O r-I
f~ ~ -.-t -.-I O
~
Cx,
-r-I H N PO N N
N Pa -r-I
~I ~o .L~ ~ m
L'~ l-~
~
N O ~ r~ ~ t0
to
~ ~
~y N ~ ~ (d
O1 I'7
r-. G4
H
~-I
U1 ~f',r5n ~'t
tT oo co
~'
~ N~
U ~ 01 of O
o~
-r-I v-I-, a--I ,.C;
N r-i 01
H
b~ r U1 u1 m-I
U7 W ~-- ~-
rti
~ U U
D .1 ~
1~ f~
b1 ~I r1 -ri -rl J-1
(d I r-I r1
N
O O ,-L.,''~ b1 Y51 -rl
U7 tCi rti r-1
N U ~ ~
r1
N
O ~ f''-,~ O N
.~i U1
x ~ , 4i ~.' ~ U
N N
x ~
~
~
N ~,' . ~i ~ N ~ Ul
> ~'rtT N
f~' ~ ~S7
-r-I .-~
~I
r~ ~ v '-'J-I .l~ r~
U Sir:-i ~,' ~
~' >=,
oo ~
(Y1 .1~ ~ .1-1 .l~ r1
,L,' U1 N N ''~
~ (IS c~ l0
c0 00
~'.,r1 r1 td td fIi
O O O O '',3'
O ~ ;~ N
~ l0 l0
~ ~qN R ~ a ~~d
~x ~~a
~
C~$
C ~ , - ~
7 - -I
-
+~
r1 01 r1 N N O
r1 M O d~ 01 L(1
-r1 c-I v-IN N r1
,iZ f I 1 I I
rt W W W W W
U
,.Q O O O O o
~I
O ~o O ~ M of
O
. . . . .
W tW -I d~ t t~
m
A
H Ll1
r-i l0 O1 L~ M
x N O O O L~
CO M ~O 10 s--1
(a Lll 00 00 ~ O
W O u-I01 01 N
00 M N N N
N M c-IM M v-I
O
C7 t~ b~ b~ b) t3~
~
N ~-I
-I H ~-I r-I Ca
-~I f-~ fa (a Ca U
U U U U ~-I
CL lD M <' LC1 l0
N L(7 d~ O M ~D
N
r! l0 a1 N o1
O d~ Lf1 r1 M
U o o ~o u1 ~H
r1
O 0 ~ W .n .-I
~1
H ~N ~o ~o .-I t~
W
H
N
-rl
O
N
p
H
H
O
W
W r-I N M ~N 111
U1
'79
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
um n cn cn m
x H x H x
Z ~ H
q O O t O O O R
ra ~ ~ I- U ~ x ~ a o
a x
~ o O f~ a r U~ O a W a W LY
u~ (x P~lW Ga <s.,
U
N
-~I w fa a, I I W fa I I ast w
~n
~n
I I I ~n ~ a I ~ ~n I ~ I
?r H E-~ fx w w H H w w r~w H
o
,.sz
.--Ip u~ W ~ ~ fly ~ ~ W y n
~
c~
l~j ~ ~ H H O FC H H H
.!~ ~ x a ~ a a
1.7
a~
ra a a x a a r a~ a a x m a~
~ r~ as m as w ~a w
a
U
r1 U1
N W U
H
~C
r~ ~-t H W
~ ~ o ~
to ~ ,~ ,-i ~J aC
., r1
N U f~ W N ~I C~H H
O
O
~ ~ ~
Ch O O ~ - I U1 U
Ctl
OI
I ~i ~ x o, -~Irt ~nw r~
I
I
~ ~ b H
to td H to ~ 1 d~O
~o ~.1
a ~ ~ ~ H H ~ -~ f~~ W
H
c - - - d~
d
Lf7 ,-C;N N N H r1 CQ IYi
O
to
N p, .N .I-t.u U M U U diW O
N ~ O
M
~., Ul O O O H I N N L~Ul ',.T~,
N M N U1
M
W I O ~I ~t ~I f~ O d~ N ~,
CO M 01
I
I
U1 W C7 ,.G , f~ S~ H ~o ~I N N ~ Z W
W O tn ~ a~ ~ p
.-i
N CI1O .--I, c-tr1 U rl ~ m u1 I W f~
U1 c0 N I U
W
U r.~U - U U U W .N rdrt W C7 H
4a N r.~C7 o W
~,' H W-1 ~y -ri-r1ri P.t (d .I~'.!-1l0O ~
-r1 ~ I I N L~ I~
O M
LC7
N ~ i-l -L~ 4-I4-14-I Cl~ ~',fd(IiN P.,'
.1-~I O -~I ~ t11-~I l0 rn ~-I,~ m W LC1
o ~I ~ ~ -~I-~I OW ~ A o
~n ~n -I a ~I
~,
~o
,~
~r~ wM o U U,-IU~ U a~ao~o -~ s?,s~, ~wa~
v ~nx. ~r -~i a~~o~ a~ aCNMU, m ~nu~ ..xHr~N
. ..
c~~ o r.~~C w ~,..~,..~,mn~wH~C ovoo m waH,-t
x. m - -~I ~n m ~ r~ ~ ~ .~,~ ~ra x
M M w a .u ~, a M
~
N W F(,' U M O ri M M -r1ri SW-I~ ~-IW Ca
US d~ l~ O ~-I M l0 O p., W v-I
~I l0 [-I N N N N W N (d (p a .. UrW '~
U c-I N ~ O U7 N l0 L ' " N
~ W N . x I ~
' a0 ~ ~ H ~
I 0 0 ~ O E-~
W
1 Z ~ d ~ ~i ~I .~ c , . ~ ~..,I ,
.43 CO N ' M L ., 0 O , , N toFf,'
~', H C7 -r1rl -rl ',~f I O -ri-ri N ,."C,'
o U1 o o ~ M 1 L M H I
~I to M
I M
fa W G4 -rl Ul U7 U1 H O ~ ''i~N N M ,~Z.,
-r1 O O In O O ,'~ O d~ N M O ~-IU di
H u1 ~-I O O N O W tn .I-~.u ~ W
la ~ rt a ~ O I ~ n r1r1 O ~
~o -W-I~ ~
is O O fa rti ~I ~t ~I ~-I U C7 O O ~ (x O
N ~ W ~ f~lP-~J-I Ga f~~o H f~q
OI ,~ Ca 1x
f~
r -~I ~ x ~ ~, ~, ~,o ~ ~ ~I~ta ~mH~
o W P.~ o H H H P.~W W H
u1 f-~ ~ Pa ~ u1
f~ ~
I
U7
r1
~S
N
-~ m n
?,
-
.1J N
U1
U1
.!-~O c-I v-I
',~y
O
O 01 N c-i
r-I
-ri
W
t'J
l~
N r-i o
O c-1 l0
O v-I u-I H
~ 01 N
M
H H ~o
o~ o~ N
I-n
N
J-W -I r1 L~
c-I M
l~ c-I
N M
tti o~ to ~
u1 H u1
t~ u1
tn In
c~
r-i ~ oo ~n
~I ~I
u1 H vo 00
~ ~o ~
~ U1
E-m
((S OD v-I l0 01
~I d~ N
l0 d1
-r1 ~-I d~ l0 u-I
O c-I r1 N
N G~
O N
c-i
.t-~.~d~r'ntnd~H Mt~ HcnH~ ~
p, uwn H E-~
<n
N o ~o ~-I o~
tn a~ ~
N co o~
o
.!-~M L~ l0 L~
O l~ O c-1
.L~ r1 c-I
l0 N
LP1
l0
O l0 N ~ v-I
.L~,c-I N c-I
-rl N ~
N M
v-I
N
W cJ~ H u~ cJ~
W u1 u~ c4
c4 H U1
u1 u1
H
v1
N
-~
~, 1.f1
'~
r-I d~ L~
-r1
U7
r5',N N
R.n'
q
f-~ to U
~ ~ o
N L d~
n
0
U o 0
~-t
~ d
w
p
I- ~
i
M I
W I
U1 r1 N
H
',~
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
cn m m
O O U H
CS U '
Z,
~ ~ o ~ ~ a ~ o a
rya
rn
U O P~ O f~ 0.'1O Ix !~ W CJa
N
-a fa W f~ W i fa P.~ I I W
m
m
.L I cn I m n i ~n I I ~n ~n a
r
zs
~a
~r H f~ H H f~ f~ W H H W W H
O
,~
r1 U] H Ul C/.~ H w ,err.,'Ul U1 ~i ~i f~
,i;
(0
rd aC H FC FC H ~ H ~C a,' H H O
J-I
.N
~' Pa ~,'Pq al ~ x a1 al fn W Pa ~4
C-~
I
H
I m N U7
U 1 W N ~I N
~
~ H a
o H ~~ c
d
~ ~
W ~, FC M M O ~' b1 ,.C,'
H H !a u1 W fy, ~ -r1i
M 4-I
W aC I ~ tl~ P i m m m
~ co W ~i
u1 U o -~I O O ~ O
d~ 1
M ~+-iw FC ~ v x ~ ,.t~oo~ ,
N o, s~
O -~Iu1 H N U U .I~ W H f~ Nm S~
W u1 ~o N
O .1.1O (.~ Cla N N O c-I H r-1 ,.5(a Ul
~ I
M
O x ~L Pa ?a ~ U H ~ I.L.~~
W t-1 W r~
N
~ W W o W W R~ H H v -.~~~ -~I
M .N
In
U1 f~ o~ N H w ,'~ N l~.~,'N
,5 o
U U W O U U U U U H W .t~N~L .1-t
O d~ f-~ N
I
N N C11 N N N N H U M O ,'~U7 O
x Ul I ~N M M
FC W d~ ~ f~ W W ~! O 5-I
fx r1 I W o~ m
M
Ul W N Z W W N ~ N H W 'n.~l,$2~01rCif~-t
,.'7 N 00 01 01 N O
LIl
N U1 u1 W f~ cJ~ U7 u1 U7 U u1 LC1S~
U7 U to N d~ N ~"m-I FC
'~-~
U r.~ rd U' H a', cW d (d W f~ U NI U
4-I W ~-I - ~--1'
~ H .t~O fx H +~ -t~.u W ~ W -~if.~N -rl
-~i N In C7 o la m
I
N (!3Qi ~ ~ N (!3(a U~ Ff', 4-!1~','4-1
.!~ " W ,~-r 01 C '' I 01 U] -1 C~ ,~-1
t1 N ~ W i -
~ N l
; '
' H ~
~.,(~ O 1 , ~ ,. 7 ry -. 01-r r
w ~, I w ,- ., 01 a N v ~nN ,
~ w o, s~,ss,N oo q ~ ~n y
a ,-I s~ r~ c~ U
M ,-I ~
~r
N ~n ~n xH~ md~ m m m~-IW~M H~C7 v No aJ
~
~'T3o oarwaH ow oL o~-,oa I-7H W"~,H , (~~-I,N~..~
c-I I
x ~~r~a rx x- ~~nx~ ,~ a- ~ H,~ ~ ~,ar~.u,~
M M ..o
n N W , W f~ W S~ s~ S~ M FC M.l~-rl
(d ~n .-iIn L~ ~ ~ N W o M
L~ c-I U
00
a ~ w z ~M ~ a Ma~ wN H~~~I a~ ~o ~
~ ' ~n ~x
~
~n Z ~ OHd~ ~Md~ t~ ~ ~o~lP-r~ ~FC~ s~ M~~ ~
1~ I I H H I I -I ' ~ s~ Oi I
-I i d O l - - ~ a W l O -
H l0 N x a 01 l l l - - l
- H l ~
- L
. c r , W r r c M r r Or r
~., W N N O OJ M r J G4 r lN U1
(lj !11 M 'Z, I M ~ 4J E-i O U7 O I~
-rl H I U ~-I t -I l~ O O CJ ~ ~ 1.(~
' -I ~ W E-~ U 1 . '' O I = l
S ~ ~ -i l u1 u1 O N
~ 1 0 I ~ O O
1 ~ ~1 -
~
V ~ N J..O L . . . , f W -P r
, r f 1 , c ,- . ~
rt 1
tn o~w o~ xoHVnw oao, oa oa om~ ,-IqM oaHw ~I wow ~I.uol
~ o
-~I r~ ~ ~ ~n x ~I ~I ~I ,~ x ~ ~, ~I 5.,
o ~ H x M N H ~I U
U~ w w w H w t~ w w ~ W x H Pa H
P~ M ~ u1 a ~ U ~ iZ rti
I
~ W o
.-I
r~
N
r as o
1 ~-I
~
z
c~3
~ ~
o
. o
~r
O r-I N
r1
r1
G4 '~, l
C7
-!~
O c-I L~ O
c-I N
-ri L~ L(1 L~ LIW
c-I O --I
.l~ M O1 ~-IM
N M N
td U1 v1 v1 v1
v1 U1 H
r1 00 L(1 O ~
'Jy N rI 01
Id N O L~ M
k1 00 L~ L~
-r1 N In ~-IM
O c-I N v-I
H tn u~ v1
rn u~ H
s~
Pa
~n
N N v-I l0 L(1
U7 t0 d~ L~
N M r1
.1, W ~ L~ l0 c-1
O Lf1 l0 M
.!- lD d~
O c-I r1 M
~i dW N r1
-r1 -I N
d~
W H U] Ul CIA
W U~ Cl~H
U1 H H
U
tb
m
-~1
-~i
m
N H
--I W
~1 U
U
Q3 M
N
N 61
ri M
U Ln d~
r~
W
H -f L
W
H
ttl
L!~ di
H
''Z,
$l
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
rt ro ~
U a~ w
-~ ~n m w
~ o ,~ ~x~
~~ ~sw
m .u .u
~~ax
U
-rl
1~
cd
U
N
rti
1~
O
N u1 ø,
U 4-1
~' .,-I ~y
N 1.~ 1.)
~i U ..
N -ri U
Ul C5 4-I P-I M
-r1 U7 M
N tti U f~ M
V ~, ~ rt ~ w
-~ o ~ o
cn A
r., c~ ,-r .u
.u u~ m
a ~, o
o~-~
w t7 .u
0
-a
-~ o
o ~ a~
.u o ~
o ~ -~
w w m
a~
0
-~ -~ m
0
ov
U r1
~, O ~1
H W H
W ~1 O ~
Cl~ H ''~,
82
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
0
.r.,
m
0
W M N NN O1~NdlLf1 Lll1 l0'd~LOO00N dlM NInO1M l0
01r1MO r1r1L~c-IW-IO1NO L(1Nl0O1l0dlMO ~ 01a0c0CHM 61
L M Nc1dl00Lf1r1NM M N~0dl01CONIll01~OO 01d~O l0c-I01l0
M c-Ic-INw-ILdr1r1r1c-IIllr1u-IL~c-Ic-Iw-Iw-IN r1Nr1l0Nr1c-1r1IIW
1
S~
O
O
W M In c-I O01 c-I01l0 v-I 01 L~
dldlNr-I l0L~N O M 01 N r-Idlc-Id~L L~Op O1~l0r1 M
N N Ldl MLl1M d~ M 01 01MN 00O1N 01dl Q1l0O O lf7
L(W -1L~r1d~r1c-IOld~01r1Oll0c-IL~c-W-Il0r1c-IwidW-1r1d~r1Lf1c--Ir1
u-I L~N N mlN r-1c-IN r-1 r1 r1r1 Nc-1
O c-IO O OO OO O O O O O OO
u7 ~ H fx ~ ~H U W Pa U H ~ W!~
O O ~ H OO ~O W W W O H O
~ H 4a~~ H fl~C~ f~ f~
N ~1 N cry O ~U u~~ H U H ~ W
~
n ~ ~ n n W
-l W H ~ C~ ~H -I-l ~ H
-~
rd ~l H ~ U1p~1 Wx U~W al alO ~.d(.~
A ~
.~x ~r~w oN x xx ~rxr~h x~ x~n~ Mw x ~~r~Ixh
U l0dl00dll0L~01M L(1M c-IdiO1117C~CONl0l~MdlIllc-I111111Mdldl
d~LW-Ic-Idl01L~l0Lf'1dlLf7Mdld~Or1L~M L(7I~~ r101CO~Hl0O 00
N u-Idll000M Mr10101l0dlc-IO v-I0100l~In00c-IM 01r1c-IdlOO l0
O dlOdi00OdlLl701dlLf1Lf10001Lf1Lf1L~N i1)l0O O ~OM N l~M N
O LflOChM Or1dll0O r-Id~r1c-Il00000N 0000l0CO~d~Ch01
N O 01ON O ON M OL~N NO l~d~O Lf7~--IO OL~L~Oc-1c-IMl~r1
Ul L~l0Cl0L~L~isC~Ml0~1L~L~L ~OL~C~l'L~L~dllO<'l~C~l~l0L~
I
U1 N
111 LC7
l0
'~ CO M r1 t!1
.1J 01
N v-I L~ L(7
O lt)
.1~ r1 M r1
O l0 ~-i
U -I . dl r-i 1
y . N I
N I LC) M ! o1
~ I c-i
r-I L 00 01 ~-I N
td L d~ d~
N ~ 1 I M 117
,~I LC1 N lp
Ul l~ c-I ~-I W c-I
fsl c-1 --! H
N
U
N
J~
M L~ N M 01
N N L(1 01 l0 l0
N
Cl~ N r1 a-I N c-I
a
N
l.~ c--I
O c-I v-W -I c-i P4
Pa al Pa W U
r1 U U U U
U ~ M t~ Ln ~o
u) n o M ~o
y -I l0 01 N o1
~r O dl Lfl c-I M
U 0 0 ~o In dl
r-1
~ 0 ~ dl u1 r-1
o
~-1
H dl to l~ c-1 L
W
H
O
1~
O
v
O
H
U
H
H
Oi
O O
W
W o t m o1 r1
clW
83
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
a
m
.r.,
1J l~Nr-IO ~
~, O OO N O
N u1HW H En
U7 N OW O O
N r~"~,'G4
~ N UU u1W
N H ~n
-l
~
~x x aw w as
CC3
W r r1c-I,-IW
i
H pa~W PaU
U UU U r-I
.!.)l4ML~Ll1l0
N tt1d~O M to
U
.1~c-IlD01N 01
N
',?~'I~O d~Lnr1M
U o o~oind~
O
~1 0 rd1tns-i
H d~totor1L
w
N
l~
O
N
O
r-I
'~,
U
q
~
O<
O o
W
w ~o~000,r1
U7
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
0 U
~
O o ~ N . s.'r !!) ~ M '~
4" N ?~, S- ~ ~r
~'
N ~I ~ N ~ ~n ~ o N ~ ~1 -- v m
it
-a U N .1~ U~ ~ O ~' '~ -a .I~ ~ U 'J
N ~ U1
r-I a d, U ~ -a .i~ .1.~ (a u1 N .s.' O -a
~ ~ O O J.) .~., ~
I ~
O ~-, O N U1 ,h r1 (IS I<j 4-I .~,
U U N (0 , O (0 .!.J
-r1 ~'.,
r1 m ~I ~I ~ ~I . .~ ~ -a rt 3 N ~I N N
?, ~I ~ N ~ rti s~ .u rt ~
~4 l 4 ~
~ 1 1
~ ;
N '"
E '
~
r-I -a (d ri - -1
l Ul ,i 1
?-1 ~- .y O O
-1 C7 -~ ~-I .1 ,
~-I r1 U7 ~I r ~
(D N V ~ U
G7 ~,' -a U7 ;j O Q,' f~ O
.-I ~H P=n N (fS ~
U N O G' ~-I U7 '~ O ,5 ,51 .1-~'Z -a ~-I N r1
U7 ,~,' 'C' -r1 -a .~i ~,' N U1 ~.,
.N ~ o ~ a~ -a a~ -a -, ~ o U a ~n ~ o ~ o
.u ~a o m q H ~ ~ ,~ ~
N U O U -a .), -a U7 N S..'' U (d ,Sy-a U1
~'', r-I r1 ~', U ~'', ~ .4~ O .I~ S'.,
.I-1
H ~ ~.I r1 U ~ .1-~ u1
O >~ cd '~ ~ cd ~ U r-I O 4-I ? i .1-~
f Zi ~ ra ~ a -a
o u ~ ~
? --I ~ 'u
~ w ~ ~
a
~
-I U . tIl td ''d J~
ml , 1~ ~' W
t~ -a ri 'O - N ~ Ql
U .
I O O 4-I
~ 4~ ~ N b1 b1 O 'LS
O ~
- . .,
., .
~ ~I N ~ ~ -a ,~ N N ~ cd s~ ~I U ~ ~
~ ~ ~ N O ~ 5
~ U m .N ~ r1 g ,-I ~ o .~ ~ o c~ a~ a~
a~ ~ a~ r1 ra m o -a a~ ~a
(d t17 r1 b1 r-I r-I b1 .N ~.,'' td .N
rtS ~ U1 ~-I r-I (S 'Z3 U ?:31 ~-I '~y-~-I
O ~-1 (d .U ,5
U7 M -a N ~' Q1 RS U -a ~y O O td -a rd
O td ''C5 ~I ~ ~; i.~, -a ~
U
.~., N ~., c~ r-1 ?-I U .U U '-O ~, U1 .1~
~-1 ~ r1 O -1~ O ri ~-I .1~ ~ U1 r-I O
O
O -a o -a t51 rt ~ ~ ~ w U O -a U7 U1
.t-~ m t!i ~C ~ '~ O .I-~
UI
~ .N u1 I ~ N ~I 4-I N N N J.~ !-I O U
N ~ U ~y u7 ~ .~ N u1
,''d
4-I ~-I ,fi J.J r-1 N r-I ,.C, ~,' ~ Q~ ?-I
N ..C; f-1 r1 (d U1 G' N RWi N
O (CS
<v .!, (CS O ~,' ~ ri ~ .~ .~ 41 '~ N ~ .I~
~t,' .J~ ~ O .~ '~C tf~ ~L U7 (~
N W
~ s~,-a -a o ra r1 a~ ra a~ ~I ~ 3 ~a r~
g o o -a ca a~ ~ ra as
a~ 0 3 ~ a~ ~I -a r1 -a ~n ,.~ a~ a~ r1 ~ .
3 ~ ~,~ .r, -a .u .u ~I 3 ~n
zi o
.u ~I o .~ m ~ ,x -a o r1 .u ~u 5 c~ a~
cn ~ ~ ra ~I -a co g o u~
.u
Ii f~ UI S-1 r1 f(S ~I fCS ~I 4-1 O .!W U7
.l~ ~,' .~'.,tr$ 'Z'S U O '~ -1 ~-a O
Ii UI
r-I ,1,4.a (d U N ri r-1 , b14-1-a .~, N t~
O O ~,' ~; -.-1 i-l 'd U S-I
S-~'' ~ S-I
O ''C~ ~ ~ UI ~ I-1 ~ rtf N d Q7 4-I N ,
-a -a ~ O (a O '~ r~ 4-I ~I ~-I O
O >~'
U) U 'd 'Z5 ?d rtS ~ ~-I ~ 'T5 ~-1 N O u1
O .h !~ d~ ~ r-1 ~ ~ U7 r-I tii 4-t
-a .1-~ N O U ~ ~ N O td O O ~ .f~ -a
~-I td -a ~ N - >_; U O >_; r-I
tn
-a v ~ ~I ~ -a .u ~ ~ -a ~., 3 a~ 0 4-I ~ m
N ro o, U ~ a~ rt ~I tr
N -.-I -a '~ .~ t~ ~.,' ~ v .!~ ;~ .~-1U
~; ~-I ~,' tl~ J..1 O ~', q$ r-I
~-I !U N
-a S-I ?-I ~ r--I ~: -a r1 r-I u1 4..1 ~ a
u1 'L$ O O r-i 'i~ ~,' ''d cd Cl~ O
a .1.I ~-I
,.q ~' N N O O ~- b~ U ~ ~ rtf ~ U7 Zi O W-I
~a U o~ N N O ,L1 U ,
5C .u zi ~ ~I I td a ~ U1 ~ O -a ~; ~I
~I r1 p, U1 -a U td (5 ~ -a td
O
.~, N O .fl 4-1 b-I -a fd ~I ~-I .t-I 4-I
-~', -.-I N O .1~ O ~-I O -a r-I ,4
O r.~, N ,i,"
O ~i U1 rl (S I~., .~., O ~ 4-I -a 'ZS .!-~
'Jy O -~ ~i u7 r-I td ~I -a r1
td O
~-1 .!-~ O '~ N O O '~y U1 i ~S .1~ ~-1 'ZS
rd U ,.C, ~ U P.t 'd .t~ r1 S~ t6
-a c-I ' 0.3
~I-I m ~-I a~ ~ ~ ~ ~ s~ -a ~ .u ra a~
3 .I-~ o, .-I ~., ~.,,.~ o w
~ o -a
N td o~ .I-I I ~ i O u7 ~ >~ ~ .~',.L
.-i .~' U ~'.r .t-) ~
tn
'Ti .U ''C1 rti N N f-a N W to
~,'' ~ ,5 O .1.~ O N N '~y $ O r1
N r1 ,5r 41
N rv O o -a ~ U U N 'ZS r-I
-a -- ri r~ ~ U7 1~ ~I ''d ~ -a
fxl ~L U1 ~ ~-I rd
~
.1J to -4.1 O , r1 ni Z51 ~ O ~I
-a 1J ''~ -a td ~ O i ~ ~,' O ~ a
r
U ~ rd .u U7 rt O >_; ~ N r-i ~ r-i rd U7
cn U -a (x U O N -a -a >_; ~
u7
~ O N U rt -a ,~ -a ~ .!-I -a N S~ ~ f~ a r('l
~-I ~C U7 -a ~ .u .I~ ~ -a '~ rt
~-I !~ ~-I 4-t U ~., -!~ U7 r-I U1 r~l r-I
c~ ?-1 ..R (d O ,.R O td r-I !y
',~' ' 4 ~
~ '
'
.l-~ -a .t -I ~ tf~ r1 ~ ~-I U ~. ~ N
N ~-I U7 J-, FC O >=I rtS 4-I O ~ CL
~r " ~, ,~, r1
U7 U 1.7 r-I O ~.,' O N '~ f-I O O N O ~-I
,.Q ,L; O '~ a , .~, S,' N ~ ~
~ ~I --. ,.~ a~ ~I as -a ~ ~, I?, ~I a~ ra
rt ~ it .--I ~ m ~ -a ~ a~
a~ a ~ r-I
l ~ a U
o ~ -a u~ 3 0
~ a ~
a~ d ~ rt ~
~ o
m c - b1
r ,. bt 'i3 ~r
rt - .t~ >_,'
I ~ c
U U O cl~ I
U ,'~ c~ tr a r-I -a ~ O (7,
U N U S'
O u1 4-I -a .~., , N f~ 55~ W ~i 'Z3 ~o S"-,
.1~ ~?t ~-I N ~- ~-I I-r''
-a .ii
cn .u ~, ~, -a u~ .u ~ a~ ~ it -a a~ r1 -a
N O .u .u a~ ~ ~, ~L ~ O ~-I ~
' rt
~
(d rd ~ O u1 ~ ~I O ~I ,.f; u7 .!-~ u1
H U N ~ O '- U7 .1.~ -a
~-1 U 4-I N -- ~y tn
~ U1 tii ?r ~ rtf ?-I ~ U1
~ rt U '~ ~r b7-.-I ~ U7 ?-I ~
-a .tJ '~ U ~ i O
O N 'T5 ?-i N td :~ ~, ~", .1-~ r-I N .!~ ~ O
~, , ~ m N -a cd -a to O
~t J..1 ~-1 ~ U (CS a U7 U7 4-I '~ O .t~ rCS
!y ~.,'' r1 O ~. U7 ~-i ~ ~', ~1 U
+-1 O .U J..1
a
~-I i ,.R N -a 'c; ''i~ N O O U7 4-I N ~
~ ~ .R O O U ''d O O N 4-I N U1 (d
.. U
f!3 ~.,'' .l~ ',~ Id .!.) N .1.~ -.-I -L~
O N O ~ ~-1 b1 ~I ~, ~' U1 f-2~ rd .U ''Cj rd
.-I ~,' ri ~
~-1
S-i rti r-i U ~I ~ t; -a N ~ U ~ ~; -I U U
~-i ~ ~-i cd a Ql rd O .u ,-~ O
U~ ~ o~ ra
>~,~ ~ ca ~, ~ a~ a~ 0 3 ~ o ~ ~ ~C ~ ,-I ~ ~I
~a 3 ~ ~ .u .u a~ ~ FC U
O-a ~ !_,' ?.1 U '~ -a ,.Q U ~I N td ~I
~ N U~ N O U1 r! r-! W -a O 5y ''d
~; ~ ,L~
O
-ar1 .s: O ~; .1.1 N u7 ~ b~.~ .U .1~
~-I N 5r N td N O -a ~ 1.~ r-I
to ,t; rI a
~-I
.t-~~I O .1J S-I 1 Q1 ~ N .-1 S-1 U7 ~ ca Ul
?-I 4-W td f.~-a N f~ -a U7 -a O O
4..1
N
C~,'~ US ~ -a ?-I b~ ~ .I-~ s,: b~ ~ s~
~ ~ rt O ~ .~ ~ ~ .>, td N W ~ s~ I
-- ~ O
''d
-aO N .N ~ .N ~ O ~ O rd O ~I
~-to O -I~ ~-1 4-! ~,' N to O ~
~ ~ ~I N O ct3 td
~-1J..~ ~ ~,' U r0 O N -a -a !-1 U -a .s-i U to
t~ ,S~ U1 r-I b1 ~ ~ -.-I -a ~.,'' N
0~ f~ ''d
UU O N ~ O ~ ~ U1 .t~ r-I O U1 $ U7 N '~
-~-i N of ?-I ?-I N cd 'zf ~ .!~
U1(~ ~-I '~ U7 O O ri N U1 ~ u7 ~ rtS Ul
a O .1..> ~-I ~ td r1 .1J u7 f~ 7y
r-I -a ~-I Iti tti
td
N~I 4-I N ~ rd ~I ~ (d ?-I N td U -a rd
~-I b7 'w N r-i U1 ,.c; O ~ ~t', I
~ S-1 a
A.u .u-aa-Was 3~~-a t~.u ~~ a~ 3zi--~ ~ 3~~~
o GNU ~
.~ ''o .I-~ ~ '~ ~ O U U1 O ~ N FC rtS ~o
?-I N N r1 I '~ N rtf S-1 N
.~ ~; N
~r~ N -a S~ ~r ~ W-I .1-~ -a ~r .t~ U ~
~r .!~ .1-~ -a '~ ~ tJ~ .~'., 'Lf O O
O U N N
?-Im p ~ 5y ~-I ~-I N tti s=; O .1-1~-I td S-I W 6
U N ''d ~-I -a ~-I O td O U -- ~ -a ttf
',~ 16
US-a ,~ cd N rt ~ N U N ~-I U ccf ~-I ~ U
~ O td O N -a ~ U rd --I O td r-I U
U~ O .1~
~-IU7 ~I O ~-I ~I O .1-~ -a -a .1J ~-I N ~''.,~i
-n S-1 ,~' N ~1 r-I 'J ~ ~', rd ~ 'Z~ .~.,
Ql QJ !-I -a
-a
,.Q-a N U N ,~ ~ ~, ',~ ~-I J-1 ~ ~,' O Q O
~ I~ !~ U1 U7 4-I 'Jy r1 N 'Tl .h '~
'~ r-I O U .~, O '~
-arC 'Z~ .t_i -a N U ~ ra td !_,' -a O ~ ~,' -a
-a ~ ~ N .4 -a ~-I ~,' 4-I N -ri (..,'~1
O ~-! N ~ s~
G,'
H7H v ~ H r1 a ~I r~ ~ R, tr-a a tr ~- a ~
~n ~ ~ rx .4.~ ,~ p., rt -- c~ ~u .u -a
w ~I -a rd -a
~IH
OR; T~ JI
aO U U U
UW '~-~ '~ '-7a
NU1 H H H
W ~ s~
l~ N r1 O
O O O N
~,~ E-W l H
S-IN O W O
f~
S-IN U U ~1
~H ~ a
~
ax a a~ w i
85 ..
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
v
~a
U~
U
rd
N
-r-I
(a
v
Q7
.Lt
-r1
N
.!~
~-I
'~
~i
~
x~s~~~a
v
ra
-~,
-~
.1-~
U
1-~
~
''d
S-1
r-I
v
O
~
~
'~
b1
U
O
r~
4-1
'~
;j
r-1
td
.u
ZS
rt5
U
N
v
U
-rl
-r-I
U1
-rl
~,
~I
'C3
-r-I
td
~
~
~;
S-I
O
~
3
.~
.u
~a
0
-~
v
~I
-~
?,
v
~a
~
~a
~
N
~
~
o
~
rt
~
-
o
u~
~
ra
zs
~
ra
v
w
s~
4-I
U
~
(fS
~
0
U
'tJ
t
0
1
.~
~
N
U
S~
O
.u
v
.u
v
~I
r1
o
~
O
O
N
U
r-!
U1
I
.U
O
--I
(~
~I
~',
~-I
~
O
~d
~-I
N
~',
~
,ii
-rl
rd
v
~l
~
4a
rrj
U
,5
O
U
-.-I
CCj ~
-r1
v
.N
~
~
a
~
~
.
-
u
U
td
-rl
b~
O ~
,S~
~
O
-
-I~u7
N
O
m
.~',
~'
~-I
~.,
-~ O
.U
O
O
'
-
U ~.,
-I.J
.1~
-I~
U1 U1
O
(d
U
(d
~~
~
o
3
~
~ 7~~a~~
~ ~ o
v
~-~
~a ~a
~
~
o
-~I
~I
~ ~I
o~
~~
v
,.Q
~
-r1
U
S-I
',~
-rl-.-i
N
cd
-rl
cd
-rl
Hl ~-l
~-I
4-I
~
U
.-I
O 'Jn
.N U
U 'z,
o
I
5r H
~ W
I
86
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
0
°° ~ a ~ a~ o
W ~ .~ ~, .-.
o ~ ~ '~ on ~, ~, ~ o
° W ~ o
p, ~ b ~ ., ,
o '"' ~ o ~ ~ W ~-. °
o ~ v ~ ~ ~ .~o 00 ~ ~ ~ '~ r~
~ °° o ~ o
0
0
H ~n :c ~ , W .~ ~ ,-. ~ P.
o ~ o .~
,~ ~ . ~ o .a W '~ ~ ~ cn a
c'~. ~ H~~~ t~5~°~.~~~ o Wo
W ~ w ~ W ~~'' ~ ~ w ~ w W ~ ~--~ ~ ao
~t
ti ~ ,-, j ~ ~ o
s.
o ~ o p~ ~ '~'' ~, ~ E--H N ~ a N ~ '-'
d as ~ °° M ~ ~' . b
U U U " °~ °° ~ ~ ~ x o o v~ o ,~ "' as
U U U ~ ~ ~ ~ o ~ ~ ~ o ~ so '"
.. ~ N ~'
N ~ ~: oho ~'' ~ ~ ~ :~ ~ .D .b o
~ U ~ ° v~ M ~ N ~ v~ ~ ~ '~ ~ ~ U '~ U
0 0 0 ~ ",~ n? ,~ ~ W ~ x ~ ,-, . ~ ~. ~ c~ ~ ~
N T~ ,-~ O '"'' ~ ~ b ~ N
'b ~ ,s~° ~ N ~ ~ o ~ ~ °~,' ro ~ ~ ,.dap
n ~ ~ ~ ~ ~, ~ ~ ~~ U ~ ~ b
D, ~, P.~ P. v~ U ~ ~ U ~ U ~ M
'p"b ~~O(~,~, ~"U~iN~ N~ U U
U
b b ~ b ~ o ,~ d ~ ø' o .~ b ° ~ ° i
a ~ ~ ~ ate, ~ ~ ~ ~ ~ w b b ~ ~ L7 a ~N ° M
~Nz ~,z~ ~~ xz~w~~ xN~.Az
E-~
U
N
,x '.r'~ '~ U U ~ p a~ O
fn a" t-i O "' U ~ ..~ p ~
N ~,, v~ ~ O by ~~ 4-i
N '~ b v~ c~"' ~ ~ ~ ~ ~ W H by °
b ~ ,"'~,,n U '° ~~ °~ '~, b ~ O " '~ U d
V ~ ~ ~ rUn ~ ~ c~ ~ ~ ~ U
° '~ O ~ b°'A a" w
v~ b O ~ ~ ~ N U ~ cei ~ N p ~ "~ r1,
~ P, V ~ E" ~ ~ '~ -~U. N ~ ~ ~ ~ ~ y~~ ~ c~G
'~ .b ~7 ~ . ~ .~ ~ U ~ ~J N ~ ~ ,~ ~ cON
D
O 'U °U C U ~ '~ Z= ~ ~ ~ w 'n t-~ ~ ~~ U a" iv
U ~ ..~-~ '~ ~ N cd ~ ~ ° v~ w
.~ ~ ;~ a~ Y .~ ~ b ~ ~ ~ ;~ ~ v~ ~ b
~ ~ ~ ~ ~~~ ~U °
y ..-v N U w .r w ~ cc3 ~ N
N ~ ~ ..~~ ~ .~7 ~C, ~ r~ U N ~, ~ ~ .fl 0 Q ~J
't"" cd U a " ,.d ~ ~ py .S"~' ,~ O w > v~
':~ ,n ~ O .~~ ~U ~ ~ ~ '~~' ,p O G ~ ~ ~ O ~ ~~ c~~n
v~ fn ~~ ~ ,s~ '~ c~
:N ~ cd :'~ ~, w .~ G ~, cn °
Ow Ct-~4 O A cd bA ..U.~ ~ U .fl O ...."'-i U tr U ~ U ~O O
V ~~ c~C .r'r ~ cG~U~ ~.~U'~ ~~~U c~U
A
U
U ° v~
w
a
w ~ ~ ~ ~ w
8y_
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
~ v-; r"
0
°*~' o
sue. '~
H ~~ 0 0 0 ~.~
0
P, o en
~t~ ~ .~' ~ ~ m
P~ ~ ~ 1~ o °° .~
~ø'' t~, ~J V1 r-~, U1 '-'
N N
o ~ '~ ~ oNO N av
v0 O ~b t~ ~ v~ °A ~ "'' ~ ~ ~n N O o
O Q~ ~ ~-i ~, ~ ~v' 'd
H ~ ~ U v H
N Z 'b o0 a~ l~ cn V~ Pa O ~, ~ > cd
c/: W ~ ~ ~ v~ .~~-i ~ O O V ~ 4" ø, '
O\ vi ~ N b4 t~ ~ dp
" ~-; ~ YO ~1.! W ~ ~ 0 ° ~ ~,'
o .. ~ a~ H ,~ on a~ _,-, A. ~ ~ U ~ o
o .~ ~ ~ ~ ~ p x .~ ~ iU'-""
a°°, ~ ,-i ~ ~ ~ ~ a\ ° o ~ va o.°o ~ ' ~ ~, ''~ ~
m ~
N oo °° ~j oo ~ a\ ~ ,b ~ ° ~ .~ ~' ~ a1 Q,
Wo o N ~ f~ ~ dy ~ ~ a; ~ c', °' ~ v~ ~ a~ U
c, '~ .s'~' l ~ op ~y ~, ~ .. ~ '-' '-' ~ ~ ~ m i-.a N b v~ e4 v~
-i--' i i GG ~.~'1 ~~ wd d~ ~ .~ '~ ~ ~ b p; m
oy~.7 ~ W O ~ N ~ ~ °) N ~'' c~ N 'n N Y ~ ~ U P, N
v~ V'i N ~: ''~ ~ ~ ~ 'rT~' ~ ~ m W ~ ' V ~~., ~ U
V f~ o0 ~ ~-' d' ~ ~ G 'v~
~ on '~ ~ H ~ ~ ~ o o _~ r' o oNO .~ a °
°~' o ?, U ~' i' ~ o ~ o ~ ~ y ~ n
N P~, U~ U~' °~° ~ W oo ~ ~ ~ ~ L7 C7 ~ so ~ P; N Pte; ri U
C7 ~ f~1 ''~ ~
b ~
o a~
U
G .,~,u ~ H .'~ i'~~' ~ G b N
~., i~ ~ .fl ~ N ~ .i~~ ~' ~O.i U ~ ~' O
V ~ ~ ~ ~ ~ '~ t3~ Pi ~" ~ ~ v~
"C~ N ~ OO b-0 ~y ~ ~ ~ v~ c~ fr' U ~ .,Ø,
~n ~ ,~ G ~-r ~ by , '~ ' ~ ~' ~1 Pa
U ~ 9, ...G, ' V '~ A '.~1- ~ r-~~ ~ ~ > C
U ..~ ..., ~,~,~ ~ ~-i '~ O y ~ O O
N ,.U.i 'N x . ~ U ~
b~ ~ ~ ~ o ,~ ~ Tj ~ ~ o °'
O b4 t~ G
c~ ~ ~ ~ ~ .o~°n ~ ~ Pi N ~~1 O '~
U V 's~pp ~ ~ e~~ cc3 .'..~ .~ U ~ v~ ;d U ~ ~ O
q ~ ., .fl .a .b ~ ~ c",
~ y~ '~ '~ b
C t-~ rn ~ O v! .fl ~ N
w N O .~~ v~ y., ~' ,O ø, a~S .~ U ~'' y ,y~
G ~ c'~ ~ ~ O ~ ~ .-~ ~ N ~ ~ ,~ ~ ~ ~~ c"UC
.? o en ~ ~ a~ ~ ° °o '~ '~ ~ °' ~ a ~
0 0.. ~ ~ .> .~i ~ o '~ '~ o ~ ~ .o
a 'a ''~ ~ ~ °' .~1 ~ °' °' ~ ~1 a~ ° o ~ ~
° p "d ° e>
y 'o ~, .c a, p, o ~ a, on ~ 3 ~ a, ~ °.~...~' c~, ; ; v ~, Y
~1 ~~b ~~' ~Uo~ ~~ ~°~' ebb ~~~ ~'~~,-,
b ~,
o ~
~ o v~
0
0.i~ 0.~ Pte., U ~ H H
$g
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
<110> INCYTE GENOMICS, INC.
TANG, Y. Tom
YUE, Henry
KHAN, Farrah
WANG, Yumei E.
PATTERSON, Chandra
GANDHI, Ameena R.
WALIA, Narinder
STEWART, A. Elizabeth
TRIBOULEY, Catherine
HAFALIA, April
NGUYEN, Danniel
ELLIOTT, Vicki S.
LEE, Ernestine Lee
<120> PROTEIN PHOSPHATASES
<130> PI-0077 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/199,010; 60/202,340; 60/203,424; 60/205,642; 60/208,854
<151> 2000-04-20; 2000-05-05; 2000-05-10; 2000-05-18; 2000-06-02
<160> 10
<170> PERL Program
<210> 1
<211> 284
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte TD No: 400015&CD2
<400> 1
Met Ala Val Asp Ile Glu Tyr Arg Tyr Asn Cys Met Ala Pro Ser
1 5 10 15
Leu Arg Gln Glu Arg Phe Ala Phe Lys Ile Ser Pro Lys Pro Ser
20 25 30
Lys Pro Leu Arg Pro Cys Ile Gln Leu Ser Ser Lys Asn Glu Ala
35 40 45
Ser Gly Met Val Ala Pro Ala Val Gln Glu Lys Lys Val Lys Lys
50 55 60
Arg Val Ser Phe Ala Asp Asn Gln Gly Leu A1a Leu Thr Met Val
65 70 75
Lys Val Phe Ser Glu Phe Asp Asp Pro Leu Asp Met Pro Phe Asn
80 85 90
Ile Thr Glu Leu Leu Asp Asn Ile Val Ser Leu Thr Thr Ala Glu
95 100 105
Ser Glu Ser Phe Val Leu Asp Phe Ser Gln Pro Ser Ala Asp Tyr
110 115 120
Leu Asp Phe Arg Asn Arg Leu Gln Ala Asp His Val Cys Leu Glu
125 130 135
Asn Cys Val Leu Lys Asp Lys Ala Ile Ala Gly Thr Val Lys Val
140 145 150
Gln Asn Leu Ala Phe Glu Lys Thr Val Lys Ile Arg Met Thr Phe
155 160 265
Asp Thr Trp Lys Ser Tyr Thr Asp Phe Pro Cys Gln Tyr Val Lys
170 175 180
Asp Thr Tyr Ala Gly Ser Asp Arg Asp Thr Phe Ser Phe Asp Ile
185 190 195
Ser Leu Pro Glu Lys Ile Gln Ser Tyr Glu Arg Met Glu Phe Ala
200 205 210
Val Tyr Tyr Glu Cys Asn Gly Gln Thr Tyr Trp Asp Ser Asn Arg
1/9
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
215 220 225
Gly Lys Asn Tyr Arg I1e Ile Arg Ala Glu Leu Lys Ser Thr Gln
230 235 240
Gly Met Thr Lys Pro His Ser Gly Pro Asp Leu Gly Ile Ser Phe
245 250 255
Asp Gln Phe Gly Ser Pro Arg Cys Ser Tyr Gly Leu Phe Pro Glu
260 265 270
Trp Pro Ser Tyr Leu G1y Tyr Glu Lys Leu Gly Pro Tyr Tyr
275 280
<210> 2
<211> 217
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6704643CD1
<400> 2
Met Tyr Ser Leu Asn Gln Glu Ile Lys Ala Phe Ser Arg Asn Asn
1 5 10 15
Leu Arg Lys Gln Cys Thr Arg Val Thr Thr Leu Thr Gly Lys Lys
20 25 30
Ile Ile Glu Thr Trp Lys Asp Ala Arg Ile His Val Va1 Glu Glu
35 40 45
Val Glu Pro Ser Ser Gly Gly Gly Cys Gly Tyr Val Gln Asp Leu
50 55 60
Ser Ser Asp Leu Gln Val Gly Val Ile Lys Pro Trp Leu Leu Leu
65 70 75
Gly Ser Gln Asp Ala Ala His Asp Leu Asp Thr Leu Lys Lys Asn
80 85 90
Lys Val Thr His Tle Leu Asn Val Ala Tyr Gly Val Glu Asn Ala
95 100 105
Phe Leu Ser Asp Phe Thr Tyr Lys Ser Ile Ser Ile Leu Asp Leu
110 115 120
Pro Glu Thr Asn Ile Leu Ser Tyr Phe Pro Glu Cys Phe Glu Phe
125 13 0 135
Ile Glu Glu Ala Lys Arg,Lys Asp Gly Val Val Leu Val His Cys
140 145 150
Asn AIa GIy Val Ser Arg Ala Ala AIa I1e Val Ile Gly Phe Leu
155 160 165
Met Asn Ser Glu Gln Thr Ser Phe Thr Ser Ala Phe.Ser Leu Val
170 175 180
Lys Asn Ala Arg Pro Ser Ile Cys Pro Asn Ser G1y Phe Met Glu
185 190 195
Gln Leu Arg Thr Tyr Gln Glu Gly Lys Glu Ser Asn Lys Cys Asp
200 205 210
Arg Ile Gln Glu Asn Ser Ser
215
<210> 3
<211> 529
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6465907CD1
<400> 3
Met Ser Ser Thr Val Ser Tyr Trp Ile Leu Asn Ser Thr Arg Asn
1 5 10 15
Ser Ile Ala Thr Leu Gln Gly Gly Arg Arg Leu Tyr Ser Arg Tyr
20 25 30
Val Ser Asn Arg Asn Lys Leu Lys Trp Arg Leu Phe Ser Arg Val
35 40 45
2/9
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
Pro Pro Thr Leu Asn Ser Ser Pro Cys Gly Gly Phe Thr Leu Cys
50 55 60
Lys Ala Tyr Arg His Thr Ser Thr Glu Glu Asp Asp Phe His Leu
65 70 75
Gln Leu Ser Pro Glu Gln Ile Asn Glu Val Leu Arg Ala Gly Glu
80 85 90
Thr Thr His Lys Ile Leu Asp Leu Glu Ser Arg Val Pro Asn Ser
95 100 105
Val Leu Arg Phe Glu Ser Asn Gln Leu Ala Ala Asn Ser Pro Val
110 115 120
Glu Asp Arg Arg Gly Val Ala Ser Cys Leu Gln Thr Asn Gly Leu
225 130 135
Met Phe Gly Ile Phe Asp Gly His Gly Gly His Ala Cys Ala Gln
140 145 150
Ala Val Ser Glu Arg Leu Phe Tyr Tyr Val Ala Val Ser Leu Met
155 160 165
Ser His Gln Thr Leu Glu His Met Glu Gly Ala Met Glu Ser Met
170 175 180
Lys Pro Leu Leu Pro Ile Leu His Trp Leu Lys His Pro Gly Asp
185 190 l95
Ser Ile Tyr Lys Asp Val Thr Ser Val His Leu Asp His Leu Arg
200 205 210
Val Tyr Trp Gln Glu Leu Leu Asp Leu His Met Glu Met Gly Leu
215 ~ 220 225
Ser Ile Glu G1u Ala Leu Met Tyr Ser Phe Gln Arg Leu Asp Ser
230 235 240
Asp Ile Ser Leu Glu Ile Gln Ala Pro Leu Glu Asp Glu Val Thr
245 250 255
Arg Asn Leu Ser Leu Gln Val Ala Phe Ser Gly Ala Thr Ala Cys
260 265 270
Met Ala His Val Asp Gly Ile His Leu His Val Ala Asn Ala G1y
275 280 285
Asp Cys Arg Ala Ile Leu Gly Val Gln Glu Asp Asn Gly Met Trp
290 295 300
Ser Cys Leu Pro Leu Thr Arg Asp His Asn A1a Trp Asn Gln Ala
305 310 315
Glu Leu Ser Arg Leu Lys Arg Glu His Pro Glu Ser G1u Asp Arg
320 325 330
Thr I1e Ile Met Glu Asp Arg Leu Leu Gly Val Leu Ile Pro Cys
335 340 345
Arg Ala Phe Gly Asp Val Gln Leu Lys Trp Ser Lys Glu Leu Gln
350 355 360
Arg Ser Ile Leu Glu Arg Gly Phe Asn Thr Glu Ala Leu Asn Ile
365 370 375
Tyr Gln Phe Thr Pro Pro His Tyr Tyr Thr Pro Pro Tyr Leu Thr
380 385 390
Ala Glu Pro Glu Val Thr Tyr His Arg Leu Arg Pro Gln Asp Lys
395 400 405
Phe Leu Val Leu Ala Ser Asp Gly Leu Trp Asp Met Leu Ser Asn
410 415 420
Glu Asp Val Val Arg Leu Val Val Gly His Leu Ala Glu Ala Asp
425 430 435
Trp His Lys Thr Asp Leu Ala Gln Arg Pro Ala Asn Leu Gly Leu
440 445 450
Met Gln Ser Leu Leu Leu Gln Arg Lys Ala Ser Gly Leu His Glu
455 460 465
Ala Asp Gln Asn Ala Ala Thr Arg Leu Ile Arg His Ala Ile Gly
470 475 480
Asn Asn G1u Tyr Gly Glu Met Glu Ala Glu Arg Leu Ala Ala Met
485 490 495
Leu Thr Leu Pro Glu Asp Leu A1a Arg Met Tyr Arg Asp Asp Ile
500 505 510
Thr Val Thr_ Val Val Tyr Phe Asn Ser Glu Ser Ile Gly Ala Tyr
525 520 525
Tyr Lys Gly Gly
3l9
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
<210> 4
<211> 537
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1551235CD1
<400> 4
Met Pro Ala Pro Thr Gln Leu Phe Phe Pro Leu Ile Arg Asn Cys
1 5 10 15
Glu Leu Ser Arg Ile Tyr Gly Thr Ala cys Tyr Cys His His Lys
20 25 30
His Leu Cys Cys Ser Ser Ser Tyr Ile Pro Gln Ser Arg Leu Arg
35 40 45
Tyr Thr Pro His Pro Ala Tyr Ala Thr Phe Cys Arg Pro Lys Glu
50 55 60
Asn Trp Trp Gln Tyr Thr Gln Gly Arg Arg Tyr Ala Ser Thr Pro
65 70 75
Gln Lys Phe Tyr Leu Thr Pro Pro Gln Val Asn Ser Ile Leu Lys
80 85 90
Ala Asn Glu Tyr Ser Phe Lys Val Pro Glu Phe Asp Gly Lys Asn
95 100 105
Val Ser Ser Ile Leu Gly Phe Asp Ser Asn Gln Leu Pro Ala Asn
110 115 120
Ala Pro Ile Glu Asp Arg Arg Ser Ala Ala Thr Cys Leu G1n Thr
125 130 135
Arg Gly Met Leu Leu Gly Val Phe Asp Gly His A1a Gly Cys Ala
140 145 150
Cys Ser Gln Ala Val Ser Glu Arg Leu Phe Tyr Tyr Ile Ala Val
155 160 165
Ser Leu Leu Pro His Glu Thr Leu Leu Glu Ile Glu Asn Ala Val
170 175 180
Glu Ser Gly Arg Ala Leu Leu Pro Ile Leu G1n Trp His Lys His
185 190 195
Pro Asn Asp Tyr Phe Ser Lys Glu Ala Ser Lys Leu Tyr Phe Asn
200 205 210
Ser Leu Arg Thr Tyr Trp Gln Glu Leu Ile Asp Leu Asn Thr Gly
215 220 225
Glu Ser Thr Asp Ile Asp Val Lys Glu Ala Leu Ile Asn Ala Phe
230 235 240
Lys Arg Leu Asp Asn Asp Ile Ser Leu Glu Ala Gln Val Gly Asp
245 250 255
Pro Asn Ser Phe Leu Asn Tyr Leu Val Leu Arg Val Ala Phe Ser
260 265 270
Gly Ala Thr Ala Cys Val Ala His Val Asp Gly Val Asp Leu His
275 280 285
Val Ala Asn Thr Gly Asp Ser Arg Ala Met Leu Gly Val Gln Glu
290 295 300
Glu Asp Gly Ser Trp Ser Ala Val Thr Leu Ser Asn Asp His Asn
305 310 315
Ala Gln Asn Glu Arg Glu Leu Glu Arg Leu Lys Leu Glu His Pro
320 325 330
Lys Ser Glu Ala Lys Ser Val Val Lys Gln Asp Arg Leu Leu Gly
335 340 345
Leu Leu Met Pro Phe Arg Ala Phe Gly Asp Val Lys Phe Lys Trp
350 355 360
Ser Ile Asp Leu Gln Lys Arg Val Ile G1u Ser Gly Pro Asp Gln
365 370 375
Leu Asn Asp Asn Glu Tyr Thr Lys Phe Ile Pro Pro Asn Tyr His
380 385 390
Thr Pro Pro Tyr Leu Thr Ala Glu Pro Glu Val Thr Tyr His Arg
395 400 405
Leu Arg Pro Gln Asp Lys Phe Leu Val Leu Ala Thr Asp Gly Leu
410 415 420
Trp Glu Thr Met His Arg Gln Asp Val Val Arg Ile Val Gly Glu
4/9
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
425 430 435
Tyr Leu Thr Gly Met His His Gln Gln Pro Ile Ala Val Gly Gly
440 445 450
Tyr Lys Val Thr Leu Gly Gln Met His Gly Leu Leu Thr Glu Arg
455 460 465
Arg Thr Lys Met Ser Ser Va1 Phe Glu Asp Gln Asn Ala Ala Thr
470 475 480
His Leu Ile Arg His Ala Val Gly Asn Asn Glu Phe Gly Thr Val
485 490 495
Asp His Glu Arg Leu Ser Lys Met Leu Ser Leu Pro Glu G1u Leu
500 505 510
Ala Arg Met Tyr Arg Asp Asp Ile Thr Ile Ile Val Val Gln Phe
515 520 525
Asn Ser His Val Val Gly Ala Tyr Gln Asn Gln Glu
530 535
<210> 5
<212> 368
<222> PRT
<213> Homo sapiens
<220>
<222> misc_feature
<223> Incyte ID No: 71439661CD1
<400> 5
Met Pro Cys Lys Ser Ala Glu Trp Leu Gln Glu Glu Leu Glu Ala
1 5 ~ 10 15
Arg Gly Gly Ala Ser Leu Leu Leu Leu Asp Cys Arg Pro His Glu
20 25 30
Leu Phe Glu Ser Ser His Ile Glu Thr Ala Ile Asn Leu Ala Ile
35 40 45
Pro Gly Leu Met Leu Arg Arg Leu Arg Lys Gly Asn Leu Pro I1e
50 55 60
Arg Ser Ile IIe Pro Asn His Ala Asp Lys Glu Arg Phe Ala Thr
65 70 75
Arg Cys Lys Ala Ala Thr Val Leu Leu Tyr Asp Glu Ala Thr Ala
80 85 90
Glu Trp Gln Pro Glu Pro Gly Ala Pro Ala Ser Val Leu Gly Leu
95 200 105
Leu Leu Gln Lys Leu Arg Asp Asp Gly Cys Gln Ala Tyr Tyr Leu
110 115 120
Gln Gly Gly Phe Asn Lys Phe Gln Thr Glu Tyr Ser Glu His Cys
125 130 135
G1u Thr Asn Val Asp Ser Ser Ser Ser Pro Ser Ser Ser Pro Pro
140 145 150
Thr Ser Val Leu Gly Leu Gly Gly Leu Arg Ile Ser Ser Asp Cys
155 260 165
Ser Asp Gly Glu Ser Asp Arg Glu Leu Pro Ser Ser Ala Thr Glu
270 175 180
Ser Asp Gly Ser Pro VaI Pro Ser Ser GIn Pro Ala Phe Pro Val
285 190 195
Gln Ile Leu Pro Tyr Leu Tyr Leu Gly Cys Ala Lys Asp Ser Thr
200 205 210
Asn Leu Asp Val Leu Gly Lys Tyr Gly Ile Lys Tyr Ile Leu Asn
215 220 225
Val Thr Pro Asn Leu Pro Asn Ala Phe Glu His Gly Gly Glu Phe
230 235 240
Thr Tyr Lys Gln Ile Pro Ile Ser Asp His Trp Ser Gln Asn Leu
245 250 255
Ser Gln Phe Phe Pro Glu Ala Ile Ser Phe Ile Asp Glu Ala Arg
260 265 270
Ser Lys Lys Cys Gly Val Leu Val His Cys Leu Ala Gly Ile Ser
275 280 285
Arg Ser Val Thr Val Thr Val Ala Tyr Leu Met Gln Lys Met Asn
290 295 300
Leu Ser Leu Asn Asp A1a Tyr Asp Phe Val Lys Arg Lys Lys Ser
5/9
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
305 310 315
Asn Ile Ser Asn Phe Phe Met Gly Leu Leu Asp Phe
Pro Asn Gln
320 325 330
Glu Arg Thr Gly Leu Ser Pro Cys Asn His Ala Ser
Leu Ser Asp
335 340 345
Ser Glu Gln Tyr Phe Thr Pro Thr His Asn Leu Phe
Leu Ser Asn
350 355 360
Pro Leu Asn Leu Glu Thr
Thr Ser
3
65
<210> 6
<211> 2232
<212> DNA
<213> Homo
Sapiens
<220>
<221> misc_feature
<223> Incyte No: 4000156CB1
ID
<400> 6
cctgcctccc ggacacaccg acgctcacgt agtcgcgctt gccacaaccc tgcgggctct 60
ccgatgcggc gagcgagctg gggagggggc ttctccgcgg cccaaaaggg ttctagcctg 120
ttcatctagc cccatgatgg ctgtggacat cgagtacaga tacaactgca tggctccttc 180
cttgcgccaa gagaggtttg cctttaagat ctcaccaaag cccagcaaac cactgaggcc 240
ttgtattcag ctgagcagca agaatgaagc cagtggaatg gtggccccgg ctgtccagga 300
gaagaaggtg aaaaagcggg tgtccttcgc agacaaccag gggctggccc tgacaatggt 360
caaagtgttc tcggaattcg atgacccgct agatatgcca ttcaacatca ccgagctcct 420
agacaacatt gtgagcttga cgacagcaga gagcgagagc tttgttctgg atttttccca 480
gccctctgca gattacttag actttagaaa tcgacttcag gccgaccacg tctgccttga 540
gaactgtgtg ctcaaggaca aggccattgc aggcactgtg aaggttcaga acctcgcatt 600
tgagaagacc gtgaaaataa ggatgacgtt cgacacctgg aagagctaca cagactttcc 660
ttgtcagtac gtgaaggaca cttatgccgg ttcagacagg gacacgttct ccttcgacat 720
cagcttgccc gagaagattc agtcttatga aagaatggag tttgctgtgt actacgagtg 780
caatggacag acgtactggg acagcaacag aggcaagaac tataggatca tccgggctga 840
gttaaaatct acccagggaa tgaccaagcc ccacagtgga ccggatttgg gaatatcctt 900
tgaccagttc ggaagccctc ggtgttccta tggtctgttt ccagagtggc caagttactt 960
aggatatgaa aagctagggc cctactacta gtgactgcag gtgacagggc gtggcggagc 1020
tgccacagac aagcctagct ctgctcactg tgcagtggag atggaaggcc agggaggagc 1080
aacgtggaac ttccatgagg ccccgtttgg gaaaataaaa ggatcctctt cacttctttc 1140
ttaaacagca aatccagcca ggttcagatt acacaaccag tgtctcactc aaaggagcag 1200
tggtggctgg cgcgctttcg cactgtggca gccacgagag tctgtgcacg tctgtgctgg 1260
aaagggtatg gatgggaatc aagcctatgc cagtgctgat gaagctggag gagtctctcc 1320
tctgctctcc actcagatgt gggacatcag tcgccaaaag ccactcagcc ccagccacct 1380
cgcgtgagac cctcactgtt cattgtgttc atctttgggt gctctctgcc agccaggcct 1440
ttcctgcaag ctgctgtgtt tccccgtcca cgtgtatctc tgctgtgaca cactgagctg 1500
acgcacattt ccagtgcagc tgcagaagag aaatgggatt ggctcttgtt ttctgcaagt 1560
tcatgttttg cattttatgt tcttccacaa ttgatctgat gttcaggaaa agataataaa 1620
ggcaaattag ttagtggttg agacaggcat ttcctcctcc cgcttcttga ccccacagat 1680
gtattccagc agagagcaac acaccagtca tcaaaaccca ctggctcctg tgcggtgtca 1740
cagattgcag ggttctgaca aggcaggaca gtcaagagtg gggacacttt cagcttctac 1800
ttttgccttc tagggggagc tttctaagtc cccacattta ccccgagtca ccggaaaaat 1860
ctgatttttc ccccgaaagc tcaatgactt taacgtgctt ggctggtttg tctcattctt 1920
tatgaaagaa ttttggggcc gggcgcggtg gcttatgcct gtaatcccag cactttggga 1980
ggccgaggca ggtggatcac gaggtcagga gatcgagacc atcctggcta acacggtgaa 2040
accctgtctc tactaaaaat acaaaaaaat tagccaggcg tggtggcggg cgcctgtagt 2100
cccagctact tgggaggctg aggcaggaga atggcgggaa cctgggaggc ggacttacag 2160
tgaaccgaga tcacaccact gcattcccgc ctgggcagca gagcgagact ccgtctcaaa 2220
cagaaaaaaa as 2232
<210> 7
<211> 1574
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6704643CB1
6/9
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
<220>
<221> unsure
<222> 38
<223> a, t, c, g, or other
<400> 7
acggccagtg ccaagctaaa attaaccctc actaaagngg aataagcttg cggccgccaa 60
tttttttttt tttttttttt tttttctcca acgaagtctt atattttacc attctgcgtg 120
tattagaaag ctaaatttaa acagcaacaa taagaaatct tccccagtgg atccctcgag 180
cctttcggga aaatgcaggt ttctggctga caggaagaag aaacagcatc caatcggcac 240
ttgtgtctca atgaccaatc aaatttcgcc ttacattgca tcgctgggat aaacggagct 300
ggacgactca gtctcttggt ctgtggctgc tgcggttacc tggatgggcg agcacctctg 360
aggctggctt tgttacctgg gcaataaggg actagcagtt cagccgtttt ctatgcctgc 420
tggatttgtt tgtatttgtt cccagccact gctcatgtaa tgtactccct taaccaggaa 480
attaaagcat tctcccggaa taatctcagg aagcaatgca ccagggtgac aacgctaact 540
ggaaagaaaa ttatagaaac atggaaagat gccagaattc atgttgtgga agaagtagag 600
ccgagcagtg ggggtggttg tggttatgtg caggacctta gctcggacct gcaagttggc 660
gttattaagc catggttgct cctagggtca caagatgctg ctcatgattt ggatacactg 720
aaaaagaata aggtgactca tattcttaat gttgcatatg gagttgaaaa tgctttcctc 780
agtgacttta catataagag catttctata ttggatctgc ctgaaaccaa catcctgtct 840
tattttccag aatgttttga atttattgaa gaagcaaaaa gaaaagatgg agtggttctt 900
gttcattgta atgcaggcgt ttccagggct gctgcaattg taataggttt cctgatgaat 960
tctgaacaaa cctcatttac cagtgctttt tctttggtga aaaatgcaag accttccata 1020
tgtccaaatt ctggcttcat ggagcagctt cgtacatatc aagagggcaa agaaagcaat 1080
aagtgtgaca gaatacagga gaacagttca tgagttgcat tgtagcagac aatggacaac 1140
tgtagtttct gaatgacttc tatagccatc ttttcccttt tttggagagt agactagcaa 1200
aactcccttt tttctcttgc cttttttatg cataaatgga ggtcaatttg attgtcctga 1260
cctactgtat aagtaaattt caaatgtcat tactttctct ttgttattat aatgtgtgat 1320
taaatgcttt tttaaattgc taagggaaaa taaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380
aacaaacaaa cacaacataa acaaagaagg ggggggccac aaaaagagac cccaaaaccg 1440
gaaaaaaatc cgggaaggga acccgggggg aaaccagttc ccaaaaggag gcgcgatata 1500
aaggggggga accaggggca aaaggggccc cggggggaac ccgggatacc cgggacaaaa 1560
tttcccacaa attt 1574
<210> 8
<211> 1924
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6465907CB1
<400> 8
agcgggcggc tgtggcctcg ccagctggtg agaagcgggc gagggtccga ggtttaaaaa 60
tatccttttt tgctgaagga acacatttgc tggtatagtt tcagaatgtc aagtactgtg 120
tcctactgga tcttaaattc tacaaggaac agcattgcca cattgcaagg gggtagacgc 180
ttatactcca ggtatgtctc aaataggaat aaattaaaat ggaggctctt ttcccgggtg 240
ccacccaccc taaacagttc cccatgtggt ggctttactc tgtgcaaagc ctacagacac 300
acatcaacag aggaagatga ttttcacttg caactcagcc ctgagcagat aaatgaagtg 360
cttcgagctg gcgagacaac ccacaagatt cttgaccttg aaagcagagt cccaaattca 420
gtgttgcggt ttgagagcaa ccagctggct gccaattccc cagtggagga ccggcgaggt 480
gtagcctcct gcctgcaaac caatggactg atgtttggca tcttcgatgg acatggtggt 540
catgcatgtg cccaagcagt gagcgagagg ctcttctact atgtggcagt gtccctgatg 600
tcccaccaga ccctggagca catggaggga gctatggaaa gcatgaaacc cttgctgccc 660
atcctgcatt ggctcaagca cccaggggac agtatctaca aggatgtcac atctgtgcat 720
cttgaccacc tccgtgtcta ttggcaggaa ctgcttgatt tgcacatgga aatgggacta 780
agcattgaag aagcattaat gtactccttc cagagactgg attctgacat ctcgctggaa 840
atccaggccc ccctggaaga tgaggtgaca aggaacctgt cactccaggt tgctttctct 900
ggggcaacag cttgcatggc ccatgttgat ggaattcact tgcacgtggc aaatgctggt 960
gactgccgag ccatccttgg tgtccaagag gacaatggca tgtggtcttg tctgcccctt 1020
acacgtgacc acaatgcctg gaaccaggcc gagctgtccc ggctaaagag ggagcaccct 1080
gagtcagagg acaggacgat catcatggag gacaggctac tgggcgtcct catcccctgc 1140
agggcctttg gggatgttca gctgaagtgg agtaaagagt tgcagcgcag cattctggag 1200
aggggcttca ataccgaggc cctcaacatt taccagttca cacccccaca ctactacact 1260
ccaccctacc tgactgctga gcctgaggtc acataccaca ggctgaggcc ccaggataag 1320
ttccttgtgc tggcctcaga tggcctgtgg gacatgctga gcaatgagga cgtggtaagg 1380
7/9
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
ctggtggtgg ggcacctggc tgaggcagat tggcacaaga cagacctggc ccagagaccc 1440
gccaacttgg ggctcatgca gagcctgctg ctgcagagga aagccagcgg gctccacgag 1500
gctgaccaaa atgcagccac gcggctgatc agacatgcca tcgggaacaa tgagtatggg 1560
gagatggagg cagagcggct ggcggcgatg ctgacattgc cagaggactt ggcgaggatg 1620
tacagggatg atatcactgt cactgtggtg tattttaact cagaatcaat cggtgcatat 1680
tacaaggggg gttaagaatc tcccatccta ttgtcaaggt taacataaat gctcttctaa 1740
aatgtttcac ttactcctaa actagctatc caaaccttac tattaaaagc gcaggcagat 1800
ttaatttgct aaatagacta acaggaggaa aaaaacaaac agcctagctt taaaaaacag 1860
tgaaatagca gtgatttcat gtccctgtat gttctgatta agtcttatat gcagagagga 1920
cgtt 1924
<210> 9
<211> 2634
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1551235CB1
<400> 9
gccgggggtg agggctcgcg ctccgggagc tgcacggggc tgcgtggaaa gagcgccgag 60
cggtggcgtc gttgtcgccc cctcctcgtc gggaagaatc gtttggtctc ctgccgtgcc 120
cggttcgtat tccctactcc ctgccacgag ccgccccgtc cgggatcctc cacccgtcca 180
aagttgtgag ggggcgccgg gcgtgctcgc ggatcggcgg ccgcgggcgt gcggagggct 240
ggacgagccc tggagcgcca ggaatcccag tcagaagttc cagcctgcca ctgttctctg 300
atgccatgcc agcaccaact caactgtttt ttcctctcat ccgtaactgt gaactgagca 360
ggatctatgg cactgcatgt tactgccacc acaaacatct ctgttgttcc tcatcgtaca 420
ttcctcagag tcgactgaga tacacacctc atccagcata tgctaccttt tgcaggccaa 480
aggagaactg gtggcagtac acccaaggaa ggagatatgc ttccacacca cagaaatttt 540
acctcacacc tccacaagtc aatagcatcc ttaaagctaa tgaatacagt ttcaaagtgc 600
cagaatttga cggcaaaaat gtcagttcta tccttggatt tgacagcaat cagctgcctg 660
caaatgcacc cattgaggac cggagaagtg cagcaacctg cttgcagacc agagggatgc 720
ttttgggggt ttttgatggc catgcaggtt gtgcttgttc ccaggcagtc agtgaaagac 780
tcttttatta tattgctgtc tctttgttac cccatgagac tttgctagag attgaaaatg 840
cagtggagag cggccgggca ctgetaccca ttctccagtg gcacaagcac cccaatgatt 900
actttagtaa ggaggcatcc aaattgtact ttaacagctt gaggacttac tggcaagagc 960
ttatagacct caacactggt gagtcgactg atattgatgt taaggaggct ctaattaatg 1020
ccttcaagag gcttgataat gacatctcct tggaggcgca agttggtgat cctaattctt 1080
ttctcaacta cctggtgctt cgagtggcat tttctggagc cactgcttgt gtggcccatg 1140
tggatggtgt tgaccttcat gtggccaata ctggcgatag cagagccatg ctgggtgtgc 1200
aggaagagga cggctcatgg tcagcagtca cgctgtctaa tgaccacaat gctcaaaatg 1260
aaagagaact agaacggctg aaattggaac atccaaagag tgaggccaag agtgtcgtga 1320
aacaggatcg gctgcttggc ttgctgatgc catttagggc atttggagat gtaaagttca 1380
aatggagcat tgaccttcaa aagagagtga tagaatctgg cccagaccag ttgaatgaca 1440
atgaatatac caagtttatt cctcctaatt atcacacacc tccttatctc actgctgagc 1500
cagaggtaac ttaccaccga ttaaggccac aggataagtt tctggtgttg gctactgatg 1560
ggttgtggga gactatgcat aggcaggatg tggttaggat tgtgggtgag tacctaactg 1620
gcatgcatca ccaacagcca atagctgttg gtggctacaa ggtgactctg ggacagatgc 1680
atggcctttt aacagaaagg agaaccaaaa tgtcctcggt atttgaggat cagaacgcag 1740
caacccatct cattcgccac gctgtgggca acaacgagtt tgggactgtt gatcatgagc 1800
gcctctctaa aatgcttagt cttcctgaag agcttgctcg aatgtacaga gatgacatta 1860
caatcattgt agttcagttc aattctcatg ttgtaggggc gtatcaaaac caagaatagt 1920
gagtggctct ttcactggca attctcaaat gatacacatt taaagggcag attttttaaa 1980
aagatactac tataataaac atttccagtt ggtcattcta agcatttacc cttttgatac 2040
tctagctagt caggtactcc aaattgactt tgcagcaggg tggcagggtc aggagagtct 2200
ggtcctgcct agctcagatt tcatggcacc tgcacttgaa gcaagtcact tctttatcac 2160
aggtgtcttg aaacattagc ttcttttacc aacctgagaa aattaggatg acctggcaaa 2220
taagatcttg aataggccaa aagcaagtat cttgctgtgt gtagtctctt ggttaaagtg 2280
aagaaacagt actgttcaca cctttcttca ctgagattcc agtgtacatg agaacatata 2340
tttattgcat gattttctag atacacagtc tatgcattat tcatatacat ttattttagc 2400
ctaaagtggt tttcaaatcc agttcttcaa gccataaatg accaagatcc aagcaatctg 2460
aatttgtttt tgtgattatt tgactggaat gcttcttaag tggaataact atactccgtt 2520
atccacccga tttcctaatg taattgaaag attttctatt ttgccacaca cttggagaca 2580
ataagggttt ttagttttat ctactcttct attgaagtta aagaaaaaaa aaaa 2634
<210> 10
8/9
CA 02406264 2002-10-10
WO 01/81590 PCT/USO1/12902
<211> 1696
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 71439661CB1
<400> 10
gcgacggggg caggtgccat gccctgcaag agcgccgagt ggctgcagga ggagctggag 60
gcgcgcggcg gcgcgtcctt gctgctgctc gactgccggc cgcacgagct cttcgagtcg 120
tcgcacatcg agacggccat caacctggcc atcccgggcc tcatgttgcg ccgcctgcgc 180
aagggcaacc tgcccatccg ctccatcatc cccaaccacg ccgacaagga gcgcttcgcc 240
acgcgctgca aggcggccac cgtgctgctc tacgacgagg ccacggccga gtggcagccc 300
gagcccggcg ctcccgcctc cgtgctcggc ctgctcctac agaagctgcg cgacgacggc 360
tgccaggcct actacctcca aggtggtttc aacaagtttc aaacagagta ctctgagcac 420
tgcgagacca acgtggacag ctcttcctcg ccgagcagct cgccacccac ctcagtgctg 480
ggcctggggg gcctgcgcat cagctctgac tgctccgacg gcgagtcgga ccgagagctg 540
cccagcagtg ccaccgagtc agacggcagc cctgtgccat ccagccaacc agccttccct 600
gtccagatcc tgccctacct ctacctcggc tgcgccaagg actccaccaa cctggacgtg 660
ctcggcaagt atggcatcaa gtatatcctc aatgtcacac ccaacctacc caacgccttc 720
gagcacggcg gcgagttcac ctacaagcag atccccatct ctgaccactg gagccagaac 780
ctctcccagt tcttccctga ggccatcagc ttcattgacg aagcccgctc caagaagtgt 840
ggtgtcctgg tgcactgcct ggcaggcatc agccgctcag tgacggtcac tgtggcctat 900
ctgatgcaga agatgaacct gtcactcaac gacgcctacg actttgtcaa gaggaaaaag 960
tccaacatct cgcccaactt caacttcatg gggcagctgc tggactttga gcggacgctg 1020
gggctaagca gcccgtgcga caaccacgcg tcgagtgagc agctctactt ttccacgccc 1080
accaaccaca acctgttccc actcaatacg ctggagtcca cgtgaggcct ggtgcacggg 1140
gggcatggca ccaggcccct gctcggctct ccacagggct aggtgggaga gcccaagccc 1200
gccacctctg gcctgaggaa cccccagatg tcacctgtgc ccagaggccc aggctgatcg 1260
gtgtcggagc gcccctcacc atccttgggg gcagggcccg caggcaaggt ctcccactgc 1320
agggcttgct ggagaggcct cggctcttgg acacgtggct ttgggcgtcc accagggcct 1380
catcctgtcc aggacgctcc tttctgctga cagcccagcc agtttggctg ttttttaaag 1440
acacatccac ggacctgagt ttacttttta cttttggcag gtaaatccaa gctccctgga 1500
gcacaaagag tgtttgagct cttcttgatt tttctttttt tttttttttt taacaaaaag 1560
tgttattttc aggctacatg caacagtgga ttgtataacc cagtatttca tccctttcct 1620
gatcctgcaa gagagagaaa tgttcagttt tcaactttaa tcattgtgaa ttaccttatg 1680
cgattttaag aactgg 1696
9/9
