Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 00/73450 CA 02374222 2001-11-15 pCT~S00/14826
CYTOSKELETON-ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of
cytoskeleton-associated
proteins and to the use of these sequences in the diagnosis, treatment, and
prevention of nervous
system disorders, autoimmune/inflammatory disorders, and cell proliferative
disorders including
cancer.
BACKGROUND OF THE INVENTION
The cytoskeleton, a cytoplasmic system of protein fibers, mediates cell shape,
structure, and
movement. The cytoskeleton supports the cell membrane and forms tracks along
which organelles
and other elements move in the cytosol. The cytoskeleton is a dynamic
structure that allows cells to
adopt various shapes and to carry out directed movements. Major cytoskeletal
fibers are the
microfilaments, the microtubules, and the intermediate filaments. Motor
proteins, including myosin,
dynein, and kinesin, drive movement of or along the fibers. Accessory or
associated proteins modify
the structure or activity of the fibers while cytoskeletal membrane anchors
connect the fibers to the
cell membrane.
Microtubules, cytoskeletal fibers with a diameter of 24 nm, have multiple
roles in the cell.
Bundles of microtubules form cilia and flagella, which are whip-like
extensions of the cell membrane
that are necessary for sweeping materials across an epithelium and for
swimming of sperm,
respectively. Marginal bands of microtubules in red blood cells and platelets
are important for these
cells' pliability. Organelles, membrane vesicles, and proteins are transported
in the cell along tracks
of microtubules. For example, microtubules run through nerve cell axons,
allowing bi-directional
transport of materials and membrane vesicles between the cell body and the
nerve terminal. Failure
to supply the nerve terminal with these vesicles blocks the transmission of
neural signals.
Microtubules are also critical to chromosomal movement during cell division.
Both stable and short-
lived populations of microtubules exist in the cell.
Microtubules are a polymer of GTP-binding tubulin protein subunits. Each
subunit is a
heterodimer of a- and Vii- tubulin, multiple isoforms of which exist. The
hydrolysis of GTP is linked
to the addition of tubulin subunits at the end of a microtubule. The subunits
interact head to tail to
form protofilaments; the protofilaments interact side to side to form a
microtubule. A microtubule is
polarized, one end ringed with a-tubulin and the other with ~i-tubulin, and
the two ends differ in their
rates of assembly. Generally each microtubule is composed of 13 protofilaments
although 11 or 15
protofilament-microtubules are sometimes found. Cilia and flagella contain
doublet microtubules.
A recently described tubulin-related protein, misato, bears structural peptide
motifs like those
WO 00/73450 CA 02374222 2001-11-15 pCT/US00/14826
of a-,(3-, and y-tubulins as well as a myosin heavy chain protein motif. This
unusual protein
performs a critical role during cell division in Drosophila as demonstrated in
mutant organisms
carrying the null allele for this gene locus. Such mutants are unable to
complete embryonic
morphogenesis because of cell cycle defects leading to under-developed
imgainal disks (Miklos, G.L.
et al. (1997) Proc. Natl. Acad. Sci. USA 94:5189-5194).
During cell migration, differentiation, and the cell cycle, the microtubule
cytoskeleton must
rapidly reorganize through assembly and disassembly. Katanin, a heterodimeric
cytoskeleton-
associated protein, reversibly severs and disassembles microtubules to tubulin
dimers. A unique
feature of this protein is its requirment for ATP to sever the tubulin-tubulin
bonds of microtubules
(McNally, F.J. and R.D. Vale (1993) Cell 75:419-429).
Microfilaments, cytoskeletal filaments with a diameter of 7-9 nm, are vital to
cell
locomotion, cell shape, cell adhesion, cell division, and muscle contraction.
Assembly and
disassembly of the microfilaments allow cells to change their morphology.
Microfilaments are the
polymerized form of actin, the most abundant intracellular protein in the
eukaryotic cell. Human
cells contain six isoforms of actin. The three a-actins are found in different
kinds of muscle,
nonmuscle (3-actin and nonmuscle y-actin are found in nonmuscle cells, and
another y-actin is found
in intestinal smooth muscle cells. G-actin, the monomeric form of actin,
polymerizes into polarized,
helical F-actin filaments, accompanied by the hydrolysis of ATP to ADP. Actin
filaments associate
to form bundles and networks, providing a framework to support the plasma
membrane and
determine cell shape. These bundles and networks are connected to the cell
membrane. In muscle
cells, thin filaments containing actin slide past thick filaments containing
the motor protein myosin
during contraction.
Actin-associated proteins have roles in cross-linking, severing, and
stabilizing actin
filaments, and in sequestering actin monomers. Several of the actin-associated
proteins have multiple
functions. Bundles and networks of actin filaments are held together by actin
cross-linking proteins.
These proteins have two actin-binding sites, one for each filament. Short
cross-linking proteins
promote bundle formation while longer, more flexible cross-linking proteins
promote network
formation. Calmodulin-like calcium-binding domains in actin cross-linking
proteins allow calcium
regulation of cross-linking. a-actinin, which is concentrated in actin stress
fibers, provides loose
cross-linking of actin filaments into bundles. Group I actin cross-linking
proteins have unique actin-
binding domains and include the 30 Kd protein, EF-la, fascin, and scrum. Group
II cross-linking
proteins have a 7,000-MW actin-binding domain and include villin and dematin.
Group III cross-
linking proteins have pairs of a 26,000-MW actin-binding domain and include
fimbrin, spectrin,
dystrophin, ABP 120, and filamin.
Severing proteins regulate the length of actin filaments by breaking them into
short pieces or
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by blocking their ends. Severing proteins include gCAP39, severin (fragmin),
gelsolin, and villin.
Capping proteins can cap the ends of actin filaments, but cannot break
filaments. Capping proteins
include CapZ and tropomodulin. The proteins thymosin and profilin sequester
actin monomers in the
cytosol, allowing a pool of unpolymerized actin to exist. The actin-associated
proteins tropomyosin,
troponin, and caldesmon regulate muscle contraction in response to calcium.
Intermediate filaments (IFs) are cytoskeletal fibers with a diameter of 10 nm,
intermediate
between that of microfilaments and microtubules. They serve structural roles
in the cell, reinforcing
cells and organizing cells into tissues. IFs are particularly abundant in
epidermal cells and in
neurons. IFs are extremely stable, and, in contrast to microfilaments and
microtubules, do not
function in cell motility.
Five types of IF proteins are known in mammals. Type I and Type II proteins
are the acidic
and basic keratins, respectively. Heterodimers of the acidic and basic
keratins are the building blocks
of keratin IFs. Keratins are abundant in soft epithelia such as skin and
cornea, hard epithelia such as
nails and hair, and in epithelia that line internal body cavities. Mutations
in keratin genes lead to
epithelial diseases including epidermolysis bullosa simplex, bullous
congenital ichthyosiform
erythroderma (epidermolytic hyperkeratosis), non-epidermolytic and
epidermolytic palmoplantar
keratoderma, ichthyosis bullosa of Siemens, pachyonychia congenita, and white
sponge nevus. Some
of these diseases result in severe skin blistering (Wawersik, M. et al. (1997)
J. Biol. Chem.
272:32557-32565; and Corden, L.D. and W.H. McLean (1996) Exp. Dermatol. 5:297-
307).
IFs have a central a-helical rod region interrupted by short nonhelical linker
segments. The
rod region is bracketed, in most cases, by non-helical head and tail domains.
The rod regions of
intermediate filament proteins associate to form a coiled-coil dimer. A highly
ordered assembly
process leads from the dimers to the IFs. Neither ATP nor GTP is needed for IF
assembly, unlike
that of microfilaments and microtubules.
IF-associated proteins (IFAPs) mediate the interactions of IFs with one
another and with
other cell structures. IFAPs cross-link IFs into a bundle, into a network, or
to the plasma membrane,
and may cross-link IFs to the microfilament and microtubule cytoskeleton.
Microtubules and IFs are
in particular closely associated. IFAPs include BPAG1, plakoglobin,
desmoplakin I, desmoplakin II,
plectin, ankyrin, filaggrin, and lamin B receptor.
Myosins are actin-activated ATPases, found in eukaryotic cells, that couple
hydrolysis of
ATP with motion. Myosin provides the motor function for muscle contraction and
intracellular
movements such as phagocytosis and rearrangement of cell contents during
mitotic cell division
(cytokinesis). The contractile unit of skeletal muscle, termed the sarcomere,
consists of highly
ordered arrays of thin actin-containing filaments and thick myosin-containing
filaments.
Crossbridges form between the thick and thin filaments, and the ATP-dependent
movement of
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myosin heads within the thick filaments pulls the thin filaments, shortening
the sarcomere and thus
the muscle fiber.
Myosins are composed of one or two heavy chains and associated light chains.
Myosin
heavy chains contain an amino-terminal motor or head domain, a neck that is
the site of light-chain
binding, and a carboxy-terminal tail domain. The tail domains may associate to
form an a-helical
coiled coil. Conventional myosins, such as those found in muscle tissue, are
composed of two
myosin heavy-chain subunits, each associated with two light-chain subunits
that bind at the neck
region and play a regulatory role. Unconventional myosins, believed to
function in intracellular
motion, may contain either one or two heavy chains and associated light
chains. There is evidence
for about 25 myosin heavy chain genes in vertebrates, more than half of them
unconventional.
Kinesins are (+) end-directed motor proteins which act on microtubules. The
prototypical
kinesin molecule is involved in the transport of membrane-bound vesicles and
organelles. This
function is particularly important for axonal transport in neurons. Kinesin is
also important in all cell
types for the transport of vesicles from the Golgi complex to the endoplasmic
reticulum. This role is
critical for maintaining the identity and functionality of these secretory
organelles.
Kinesin defines a ubiquitous, conserved family of over 50 proteins that can be
classified into
at least 8 subfamilies based on primary amino acid sequence, domain structure,
velocity of
movement, and cellular function. (Reviewed in Moore, J.D. and S.A. Endow
(1996) Bioessays
18:207-219; and Hoyt, A.M. (1994) Curr. Opin. Cell Biol. 6:63-68.) The
prototypical kinesin
molecule is a heterotetramer comprised of two heavy polypeptide chains (KHCs)
and two light
polypeptide chains (KLCs). The KHC subunits are typically referred to as
"kinesin." KHC is about
1000 amino acids in length, and KLC is about 550 amino acids in length. Two
KHCs dimerize to
form a rod-shaped molecule with three distinct regions of secondary structure.
At one end of the
molecule is a globular motor domain that functions in ATP hydrolysis and
microtubule binding.
Kinesin motor domains are highly conserved and share over 70% identity. Beyond
the motor domain
is an a-helical coiled-coil region which mediates dimerization. At the other
end of the molecule is a
fan-shaped tail that associates with molecular cargo. The tail is formed by
the interaction of the KHC
C-termini with the two KLCs.
A 60 kDa cytoskeletal protein cloned from mouse spermatocytes and termed
meiosis-specific
nuclear structural protein (MNS 1), serves in the organization of the nuclear
or perinuclear
architecture, particularly at the pachytene stage during spermatogenesis. MNS
1 contains long alpha
helical coiled domains that are flanked by non-helical terminal domains. These
domains contribute to
preserve proper nuclear morphology during meotic prophase (Furukawa, K. (1994)
Chromosome
Res. 2:99-113).
The discovery of new cytoskeleton-associated proteins and the polynucleotides
encoding
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them satisfies a need in the art by providing new compositions which are
useful in the diagnoses,
prevention, and treatment of nervous system disorders, autoimmune/inflammatory
disorders, and cell
proliferative disorders including cancer.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, cytoskeleton-associated
proteins, referred to
collectively as "CYAP" and individually as "CYAP-1," "CYAP-2," "CYAP-3," "CYAP-
4," and
"CYAP-5." In one aspect, the invention provides an isolated polypeptide
comprising an amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-5, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-5, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-5, and d) an immunogenic fragment of 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 NO:1-5.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising
an amino acid sequence selected from the group consisting of a) an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-5, b) a naturally occurring amino
acid sequence having at
least 90% sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-5, c) a biologically active fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-5, and d) an immunogenic fragment of an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-5. In one alternative, the
polynucleotide encodes a
polypeptide selected from the group consisting of SEQ ID NO: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 comprising
an amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-5, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-5, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ
)D NO:1-S, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO: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 comprising an
amino acid
CA 02374222 2001-11-15
WO 00/73450 PCT/US00/14826
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-5, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-5, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ
ID NO:l-5, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-5. 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 comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-5, b) a
naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-5, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-5, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-5.
The invention further provides an isolated polynucleotide comprising a
polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:6-10, b) a naturally occurring polynucleotide sequence
having at least 70%
sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID N0:6-
10, c) a polynucleotide sequence complementary to a), d) a polynucleotide
sequence complementary
to b), and e) an RNA equivalent of a)-d). In one alternative, the
polynucleotide comprises at least 60
contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
comprising a polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:6-10, b) a naturally occurring polynucleotide sequence
having at least 70%
sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID N0:6-
10, c) a polynucleotide sequence complementary to a), d) a polynucleotide
sequence complementary
to b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing
the sample with a
probe comprising at least 20 contiguous nucleotides comprising a sequence
complementary to said
target polynucleotide in the sample, and which probe specifically hybridizes
to said target
polynucleotide, under conditions whereby a hybridization complex is formed
between said probe and
said target polynucleotide or fragments thereof, and b) detecting the presence
or absence of said
hybridization complex, and optionally, if present, the amount thereof. In one
alternative, the probe
W~ 00/73450 CA 02374222 2001-11-15 PCT~S00/14826
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 comprising a
polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:6-10, b) a naturally occurring polynucleotide sequence
having at least 70%
sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID N0:6-
10, c) a polynucleotide sequence complementary to a), d) a polynucleotide
sequence complementary
to b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying
said target
polynucleotide or fragment thereof using polymerise 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 pharmaceutical composition comprising an
effective amount
of a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO:1-5, b) a
naturally occurring
amino acid sequence having at least 90% sequence identity to an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-5, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-5, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-5, and a
pharmaceutically
acceptable excipient. In one embodiment, the pharmaceutical composition
comprises an amino acid
sequence selected from the group consisting of SEQ ID NO:1-5. The invention
additionally provides
a method of treating a disease or condition associated with decreased
expression of functional CYAP,
comprising administering to a patient in need of such treatment the
pharmaceutical composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-5, b)
a naturally
occurnng amino acid sequence having at least 90% sequence identity to an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-S, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-5, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO: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
pharmaceutical
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 CYAP, comprising
administering to a
patient in need of such treatment the pharmaceutical composition.
WO 00/73450 CA 02374222 2001-11-15 pCT/US00/14826
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide comprising an amino acid sequence selected from
the group consisting
of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-
5, b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-5, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-5, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO: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 pharmaceutical
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 CYAP, comprising
administering to a patient
in need of such treatment the pharmaceutical composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO:1-5, b) a
naturally occurring
amino acid sequence having at least 90% sequence identity to an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-5, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-5, and d) an immunogenic
fragment of 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 suitable
conditions, and b)
detecting binding of the polypeptide to the test compound, thereby identifying
a compound that
specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-5, b)
a naturally
occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-5, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-5, and d) an
immunogenic
fragment of 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
WO 00/734$0 CA 02374222 2001-11-15 PCT/US00/14826
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.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs),
clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments
used to assemble full-
length sequences encoding CYAP.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of CYAP.
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-
specific
expression patterns of each nucleic acid sequence as determined by northern
analysis; diseases,
disorders, or conditions associated with these tissues; and the vector into
which each cDNA was
cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA clones
encoding CYAP were isolated.
Table S shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
WO 00/73450 CA 02374222 2001-11-15 PCT~S00/14826
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
"CYAP" refers to the amino acid sequences of substantially purified CYAP
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
CYAP. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of CYAP either by
directly interacting with
CYAP or by acting on components of the biological pathway in which CYAP
participates.
An "allelic variant" is an alternative form of the gene encoding CYAP. 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 CYAP include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as CYAP or a
polypeptide with at least one functional characteristic of CYAP. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding CYAP, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
CYAP. 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 CYAP. 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 CYAP 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
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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 CYAP. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of CYAP either by
directly interacting with CYAP or by acting on components of the biological
pathway in which CYAP
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')~, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind CYAP polypeptides can be prepared using intact
polypeptides or using
fragments containing small peptides of interest as the immunizing antigen. The
polypeptide or
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is
then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
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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 CYAP, 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 CYAP or fragments of
CYAP 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
(PE Biosystems,
Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which
has been assembled from
one or more overlapping cDNA, EST, or genomic DNA fragments using a computer
program for
fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison
WI) or Phrap
(University of Washington, Seattle WA). Some sequences have been both extended
and assembled to
produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
S 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, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid 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 sequence can include, for example,
replacement of
hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative
polynucleotide encodes a
polypeptide which retains at least one biological or immunological function of
the natural molecule.
A derivative polypeptide is one modified by glycosylation, pegylation, or any
similar process that
retains at least one biological or immunological function of the polypeptide
from which it was
derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
A "fragment" is a unique portion of CYAP or the polynucleotide encoding CYAP
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
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WO 00/73450 PCT/LJS00/14826
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 NO: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 NO:1-5. The
precise length of a
fragment of SEQ ID NO:1-5 and the region of SEQ ID NO: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 or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
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WO 00/73450 CA 02374222 2001-11-15 pCT/US00/14826
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/612.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: S and Extension Gap: 2 penalties
Gap x drop-off:' S0
Expect: 10
Word Size: 17
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length
supported by the sequences shown herein, in the tables, figures, or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
WO 00/73450 CA 02374222 2001-11-15 pCT~S00/14826
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 (Apr-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 arad Extension Gap: l penalties
Gap x drop-off:' S0
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
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WO 00/73450 CA 02374222 2001-11-15 pCT/US00/14826
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
I % (w/v) SDS, and about 100 ~tg/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 (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.,
1989, Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring
Harbor Press,
Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present
invention include wash conditions of 68°C in the presence of about 0.2
x SSC and about 0.1 % SDS,
for 1 hour. Alternatively, temperatures of about 65°C, 60°C,
55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present
at about 0.1%.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents
include, for instance, sheared and denatured salmon sperm DNA at about 100-200
pg/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
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of CYAP
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 CYAP which is useful in any of the antibody production methods disclosed
herein or known in the
I S 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 CYAP. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of CYAP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an CYAP 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 CYAP.
"Probe" refers to nucleic acid sequences encoding CYAP, 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, for
example Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, 2"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al.,1987, Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al., 1990, PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
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 UK Human Genome
Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
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 nucleic
acids encoding CYAP, or fragments thereof, or CYAP itself, may comprise a
bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a cell; a cell;
genomic DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60°70 free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" refers to the collective pattern of gene expression by a
particular cell
type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or 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.
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants, and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook et al. (1989),
supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
95% or at least 98% or
greater sequence identity over a certain defined length. A variant may be
described as, for example,
an "allelic" (as defined above), "splice," "species," or "polymorphic"
variant. A splice variant may
have significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternative splicing of exons during mRNA processing.
The corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides generally will have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least
98% or greater sequence
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human cytoskeleton-associated
proteins
(CYAP), the polynucleotides encoding CYAP, and the use of these compositions
for the diagnosis,
treatment, or prevention of nervous system disorders, autoimmune/inflammatory
disorders, and cell
proliferative disorders including cancer.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
CYAP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of
the polypeptide
and nucleotide sequences, respectively. Column 3 shows the clone IDs of the
Incyte clones in which
nucleic acids encoding each CYAP were identified, and column 4 shows the cDNA
libraries from
which these clones were isolated. Column 5 shows Incyte clones and their
corresponding cDNA
libraries. Clones for which cDNA libraries are not indicated were derived from
pooled cDNA
libraries. The Incyte clones in column 5 were used to assemble the consensus
nucleotide sequence of
each CYAP and are useful as fragments in hybridization technologies.
IS The columns of Table 2 show various properties of each of the polypeptides
of the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; column 6
shows homologous sequences as identified by BLAST analysis; and column 7 shows
analytical
methods and in some cases, searchable databases to which the analytical
methods were applied. The
methods of column 7 were used to characterize each polypeptide through
sequence homology and
protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding CYAP. The first column of Table
3 lists the
nucleotide SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of
column 1. These
fragments are useful, for example, in hybridization or amplification
technologies to identify SEQ ID
N0:6-10 and to distinguish between SEQ ID N0:6-10 and related polynucleotide
sequences. The
polypeptides encoded by these fragments are useful, for example, as
immunogenic peptides. Column
3 lists tissue categories which express CYAP as a fraction of total tissues
expressing CYAP. Column
4 lists diseases, disorders, or conditions associated with those tissues
expressing CYAP as a fraction
of total tissues expressing CYAP. Column 5 lists the vectors used to subclone
each cDNA library.
Of particular note is the expression of SEQ ID N0:8 in reproductive tissues.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
from which cDNA clones encoding CYAP were isolated. Column 1 references the
nucleotide SEQ
ID NOs, column 2 shows the cDNA libraries from which these clones were
isolated, and column 3
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WO 00/73450 CA 02374222 2001-11-15 pCT/US00/14826
shows the tissue origins and other descriptive information relevant to the
cDNA libraries in column 2.
SEQ ID NO:10 maps to chromosome 16 within the interval from 65.60 to 72.60
centiMorgans.
The invention also encompasses CYAP variants. A preferred CYAP 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 CYAP amino acid sequence, and which contains at least
one functional or
structural characteristic of CYAP.
The invention also encompasses polynucleotides which encode CYAP. 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 CYAP. 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
CYAP. 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 CYAP. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:6-
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 CYAP.
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 CYAP, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring CYAP, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode CYAP and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurnng CYAP under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding CYAP or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
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 CYAP 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 CYAP
and
CYAP 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 CYAP 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; Kimmel, A.R. ( 1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(PE
Biosystems, Foster City CA), 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 (PE Biosystems). Sequencing is then carried out
using either the
ABI 373 or 377 DNA sequencing system (PE 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 Bioloey, John Wiley & Sons,
New York NY, unit
7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnoloey> Wiley VCH, New
York NY, pp.
856-853.)
The nucleic acid sequences encoding CYAP may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
WO UO/7345U CA 02374222 2001-11-15 PCT/US00/14826
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments
adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme
digestions and 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 intron/exon junctions. For all PCR-based methods,
primers may be designed
using commercially available software, such as OLIGO 4.06 Primer Analysis
software (National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the
template at temperatures of
about 68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode CYAP may be cloned in recombinant DNA molecules that direct
expression of CYAP,
or fragments or functional equivalents thereof, in appropriate host cells. Due
to the inherent
degeneracy of the genetic code, other DNA sequences which encode substantially
the same or a
26
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
functionally equivalent amino acid sequence may be produced and used to
express CYAP.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter CYAP-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 CYAP, 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 CYAP 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, CYAP itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solution-phase or
solid-phase techniques.
(See, e.g., Creighton, T. ( 1984) Proteins, Structures and Molecular
Properties, WH Freeman, New
York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.)
Automated synthesis
may be achieved using the ABI 431A peptide synthesizer (PE Biosystems).
Additionally, the amino
acid sequence of CYAP, or any part thereof, may be altered during direct
synthesis and/or combined
with sequences from other proteins, or any part thereof, to produce a variant
polypeptide or a
polypeptide having a sequence of a naturally occurring polypeptide.
27
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier ( 1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active CYAP, the nucleotide sequences
encoding CYAP 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 CYAP. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
CYAP. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding CYAP and its initiation codon and upstream regulatory
sequences are inserted
into the appropriate expression vector, no additional transcriptional or
translational control signals
may be needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted,
exogenous translational control signals including an in-frame ATG initiation
codon should be
provided by the vector. Exogenous translational elements and initiation codons
may be of various
origins, both natural and synthetic. The efficiency of expression may be
enhanced by the inclusion of
enhancers appropriate for the particular host cell system used. (See, e.g.,
Scharf, D. et al. (1994)
Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding CYAP and appropriate transcriptional and
translational
control elements. These methods include in vitro recombinant DNA techniques,
synthetic techniques,
and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. ( 1989)
Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-
17; Ausubel, F.M. et
al. (1995) Current Protocols in Molecular Biolo~v, 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 CYAP. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV,
or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti
or pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Bitter, G.A. et al. (1987) Methods
Enzymol. 153:516-544;
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WO ~~/7345~ CA 02374222 2001-11-15 PCT/US00/14826
Scorer, C.A. et al. (1994) Bio/Technology 12:181-184; 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. 6:307-31 I; Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;
Brogue, R. et al.
(1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105; The
McGraw Hill Yearbook of Science and Technology ( 1992) McGraw Hill, New York
NY, pp.
191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-
3659; and Harrington,
J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from
retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al., (1993)
Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Butler, 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 CYAP. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding CYAP 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 CYAP 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 CYAP are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of CYAP may be used.
For example, vectors
containing the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of CYAP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, su ra; and Scorer, supra.)
Plant systems may also be used for expression of CYAP. Transcription of
sequences
encoding CYAP may be driven viral promoters, e.g., the 35S and 19S promoters
of CaMV used alone
or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
promoters may be used. (See, e.g., Coruzzi, supra; Brogue, supra; and Winter,
sera.) 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 Technology
(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 CYAP
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses CYAP 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 CYAP in cell lines is preferred. For example, sequences encoding CYAP can
be transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media
before being switched to selective media. The purpose of the selectable marker
is to confer resistance
to a selective agent, and its presence allows growth and recovery of cells
which successfully express
the introduced sequences. Resistant clones of stably transformed cells may be
propagated using
tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk- and apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan ( 1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech), 13 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding CYAP is inserted within a marker gene sequence, transformed
cells containing
sequences encoding CYAP can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding CYAP 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 CYAP
and that express
IS CYAP 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 CYAP using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on CYAP is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. ( 1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding CYAP
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding CYAP, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding CYAP may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors
containing polynucleotides which encode CYAP may be designed to contain signal
sequences which
direct secretion of CYAP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding CYAP 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 CYAP protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of CYAP 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 these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the CYAP encoding sequence and the
heterologous protein
sequence, so that CYAP may be cleaved away from the heterologous moiety
following purification.
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
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 CYAP may
be achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
CYAP of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to CYAP. At least one and up to a plurality of test
compounds may be screened
for specific binding to CYAP. 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
CYAP, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, Coligan, J.E. et al. ( 1991 ) Current Protocols
in Immunolo~y 1 (2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which CYAP
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 CYAP,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, DrOSOphila, or
E. coli. Cells expressing CYAP or cell membrane fractions which contain CYAP
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either CYAP 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
CYAP, either in
solution or affixed to a solid support, and detecting the binding of CYAP 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.
CYAP of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of CYAP. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for CYAP
activity, wherein CYAP is combined with at least one test compound, and the
activity of CYAP in the
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
presence of a test compound is compared with the activity of CYAP in the
absence of the test
compound. A change in the activity of CYAP in the presence of the test
compound is indicative of a
compound that modulates the activity of CYAP. Alternatively, a test compound
is combined with an
in vitro or cell-free system comprising CYAP under conditions suitable for
CYAP activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of CYAP
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 CYAP or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem
(ES) cells. Such techniques are well known in the art and are useful for the
generation of animal
models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent
No. 5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi,
M.R. (1989) Science 244:1288-1292). The vector integrates into the
corresponding region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the 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 CYAP may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding CYAP 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 CYAP is injected into animal ES cells, and
the injected sequence
integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the
blastulae are implanted as described above. Transgenic progeny or inbred lines
are studied and
treated with potential pharmaceutical agents to obtain information on
treatment of a human disease.
Alternatively, a mammal inbred to overexpress CYAP, e.g., by secreting CYAP in
its milk, may also
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
serve as a convenient source of that protein (Janne, J. et al. (1998)
Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of CYAP and cytoskeleton-associated proteins. In addition, the
expression of CYAP
is closely associated with cell proliferation, cancer, and inflammation.
Therefore, CYAP appears to
play a role in nervous system disorders, autoimmune/inflammatory disorders,
and cell proliferative
disorders including cancer. In the treatment of disorders associated with
increased CYAP expression
or activity, it is desirable to decrease the expression or activity of CYAP.
In the treatment of
disorders associated with decreased CYAP expression or activity, it is
desirable to increase the
expression or activity of CYAP.
Therefore, in one embodiment, CYAP 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 CYAP. Examples of such disorders include, but are not limited to,
a nervous system
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
disorder of the central
nervous system, cerebral palsy, a neuroskeletal disorder, an autonomic nervous
system disorder, a
cranial nerve disorder, a spinal cord disease, muscular dystrophy and other
neuromuscular disorder, a
peripheral nervous system disorder, dermatomyositis and polymyositis;
inherited, metabolic,
endocrine, and toxic myopathy; myasthenia gravis, periodic paralysis; a mental
disorder including
mood, anxiety, and schizophrenic disorders; seasonal affective disorder (SAD);
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, and Tourette's disorder; an autoimmune/inflammatory disorder such
as acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory
distress syndrome,
allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis, autoimmune
hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-
candidiasis-
ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis,
Crohn's disease, atopic
dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic
lymphopenia with
WO 00/734$0 CA 02374222 2001-11-15 PCT/US00/14826
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; 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 CYAP 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 CYAP including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
CYAP in conjunction with a suitable pharmaceutical carrier may be administered
to a subject to treat
or prevent a disorder associated with decreased expression or activity of CYAP
including, but not
limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of CYAP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of CYAP including, but not limited to, those listed above.
In a further embodiment, an antagonist of CYAP may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of CYAP.
Examples of such
disorders include, but are not limited to, those nervous system disorders,
autoimmune/inflammatory
disorders, and cell proliferative disorders, including cancer, described
above. In one aspect, an
antibody which specifically binds CYAP may be used directly as an antagonist
or indirectly as a
targeting or delivery mechanism for bringing a pharmaceutical agent to cells
or tissues which express
CYAP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding CYAP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of CYAP including, but not limited to, those
described above.
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WO 00/73450 CA 02374222 2001-11-15 PCT/~1500/14g26
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 CYAP may be produced using methods which are generally known
in the
art. In particular, purified CYAP may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind CYAP.
Antibodies to CYAP 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 CYAP or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among
adjuvants used in
humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
CYAP 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 CYAP 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 CYAP 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
37
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. ( 1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. ( 1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
CYAP-specific single
S 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 CYAP may also be
generated.
For example, such fragments include, but are not limited to, F(ab~~ fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab~2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
CYAP and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering CYAP epitopes is generally used, but a
competitive binding assay may
also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for CYAP. Affinity
is expressed as an
association constant, Ka, which is defined as the molar concentration of CYAP-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium conditions.
The Ka determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple CYAP epitopes, represents the average affinity, or
avidity, of the antibodies for
CYAP. The Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular CYAP epitope, represents a true measure of affinity. High-affinity
antibody preparations
with Ka ranging from about 109 to 10''- L/mole are preferred for use in
immunoassays in which the
CYAP-antibody complex must withstand rigorous manipulations. Low-affinity
antibody preparations
with Ka ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
procedures which ultimately require dissociation of CYAP, 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 CYAP-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 CYAP, 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 CYAP. Such technology is well known in the art, and antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding CYAP. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics,
Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. ( 1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. ( 1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, s_unra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. ( 1998) J. Pharm. Sci. 87( 11 ):1308-1315; and Morris, M.C. et al. ( 1997)
Nucleic Acids Res.
25( 14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding CYAP 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
39
WO 00/734$0 CA 02374222 2001-11-15 PCT/US00/14826
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 Somia, N. (1997) Nature
389:239-242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites
(e.g., against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA. 93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trypanosoma cruzi). In the
case where a genetic deficiency in CYAP expression or regulation causes
disease, the expression of
CYAP from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
CYAP are treated by constructing mammalian expression vectors encoding CYAP
and introducing
these vectors by mechanical means into CYAP-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 CYAP 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). CYAP may be
expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV),
Rous sarcoma virus
(RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes), (ii) an
inducible promoter (e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. U.S.A.
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998)
Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the
ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
WO ~0/7345U CA 02374222 2001-11-15 PCT/US00/14826
gene encoding CYAP 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 CYAP expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding CYAP 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. U.S.A. 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987). J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
cells), and the return of transduced cells to a patient are procedures well
known to persons skilled in
the art of gene therapy and have been well documented (Ranga, U. et al. (1997)
J. Virol. 71:7020-
7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 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 CYAP to cells which have one or more genetic
abnormalities with respect
to the expression of CYAP. 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
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
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 CYAP to target cells which have one or more genetic
abnormalities with
respect to the expression of CYAP. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing CYAP to cells of the central nervous
system, for which HSV has a
tropism. The construction and packaging of herpes-based vectors are well known
to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (
1999) Exp. Eye
Res.169:385-395). The construction of a HSV-1 virus vector has also been
disclosed in detail in U.S.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is
hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use
of recombinant
HSV d92 which consists of a genome containing at least one exogenous gene to
be transferred to a
cell under the control of the appropriate promoter for purposes including
human gene therapy. Also
taught by this patent are the construction and use of recombinant HSV strains
deleted for ICP4, ICP27
and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol.
73:519-532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The
manipulation of cloned
herpesvirus sequences, the generation of recombinant virus following the
transfection of multiple
plasmids containing different segments of the large herpesvirus genomes, the
growth and propagation
of herpesvirus, and the infection of cells with herpesvirus are techniques
well known to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding CYAP 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. Biotech. 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
CYAP into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
CYAP-coding RNAs and the synthesis of high levels of CYAP 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)
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
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 CYAP 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
polymerises, 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 ig c Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-
177.) A complementary sequence or antisense molecule may also be designed to
block translation of
mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding CYAP.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for 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 CYAP. Such DNA sequences may be incorporated into a wide
variety of vectors
with suitable RNA polymerise promoters such as T7 or SP6. Alternatively, these
cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines,
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half-
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine,
queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine,
cytidine, guanine, thymine, and uridine which are not as easily recognized by
endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding CYAP. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming
oligonucleotides,
transcription factors and other polypeptide transcriptional regulators, and
non-macromolecular
chemical entities which are capable of interacting with specific
polynucleotide sequences. Effective
compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
polynucleotide expression. Thus, in the treatment of disorders associated with
increased CYAP
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding CYAP may be therapeutically useful, and in the treament of disorders
associated with
decreased CYAP expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding CYAP may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding CYAP 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
CYAP 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 CYAP. The amount of hybridization may be
quantified, thus
forming the basis for a comparison of the expression of the polynucleotide
both with and without
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WO 00/73450 CA 02374222 2001-11-15 pCT/[JS00/14826
exposure to one or more test compounds. Detection of a change in the
expression of a polynucleotide
exposed to a test compound indicates that the test compound is effective in
altering the expression of
the polynucleotide. A screen for a compound effective in altering expression
of a specific
polynucleotide can be carried out, for example, using a Schizosaccharomyces
pombe gene expression
system (Atkins, D. et al. (1999) U.S. Patent No. 5.932,435; Arndt, G.M. et al.
(2000) Nucleic Acids
Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al.
(2000) Biochem. Biophys.
Res. Commun. 268:8-13). A particular embodiment of the present invention
involves screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691 ).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
pharmaceutical
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 Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA).
Such
pharmaceutical compositions may consist of CYAP, antibodies to CYAP, and
mimetics, agonists,
antagonists, or inhibitors of CYAP.
The pharmaceutical compositions utilized in this invention may be administered
by any
number of routes including, but not limited to, oral, intravenous,
intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, pulmonary, transdermal,
subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, or rectal means.
Pharmaceutical 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.
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
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.
Pharmaceutical compositions suitable for use in the invention include
compositions wherein
the active ingredients are contained in an effective amount to achieve the
intended purpose. The
determination of an effective dose is well within the capability of those
skilled in the art.
Specialized forms of pharmaceutical compositions may be prepared for direct
intracellular
delivery of macromolecules comprising CYAP or fragments thereof. For example,
liposome
preparations containing a cell-impermeable macromolecule may promote cell
fusion and intracellular
delivery of the macromolecule. Alternatively, CYAP or a fragment thereof may
be joined to a short
cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus
generated have been
found to transduce into the cells of all tissues, including the brain, in a
mouse model system
(Schwarze, S.R. et al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs,
monkeys, or pigs. An animal model may also be used to determine the
appropriate concentration
range and route of administration. Such information can then be used to
determine useful doses and
routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example CYAP
or fragments thereof, antibodies of CYAP, and agonists, antagonists or
inhibitors of CYAP, 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.
Pharmaceutical compositions
which exhibit large therapeutic indices are preferred. The data obtained from
cell culture assays and
animal studies are used to formulate a range of dosage for human use. The
dosage contained in such
compositions is preferably within a range of circulating concentrations that
includes the EDSO with
little or no toxicity. The dosage varies within this range depending upon the
dosage form employed,
the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
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 pharmaceutical compositions may be
administered every 3 to 4
days, every week, or biweekly depending on the half-life and clearance rate of
the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~cg to 100,000 fig, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind CYAP may be used for
the
diagnosis of disorders characterized by expression of CYAP, or in assays to
monitor patients being
treated with CYAP or agonists, antagonists, or inhibitors of CYAP. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for CYAP include methods which utilize the antibody and a label to detect CYAP
in human body
fluids or in extracts of cells or tissues. The antibodies may be used with or
without modification, and
may be labeled by covalent or non-covalent attachment of a reporter molecule.
A wide variety of
reporter molecules, several of which are described above, are known in the art
and may be used.
A variety of protocols for measuring CYAP, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
CYAP expression. Normal
or standard values for CYAP expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibody to CYAP under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of CYAP
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 CYAP 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 CYAP
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of CYAP, and to monitor regulation of CYAP levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
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WO 00/73450 CA 02374222 2001-11-15 PC'T/US00/14826
sequences, including genomic sequences, encoding CYAP or closely related
molecules may be used
to identify nucleic acid sequences which encode CYAP. 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 CYAP, 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 CYAP 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 CYAP
gene.
Means for producing specific hybridization probes for DNAs encoding CYAP
include the
cloning of polynucleotide sequences encoding CYAP or CYAP 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 3zP or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding CYAP may be used for the diagnosis of
disorders
associated with expression of CYAP. Examples of such disorders include, but
are not limited to, a
nervous system 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
disorder of the central
nervous system, cerebral palsy, a neuroskeletal disorder, an autonomic nervous
system disorder, a
cranial nerve disorder, a spinal cord disease, muscular dystrophy and other
neuromuscular disorder, a
peripheral nervous system disorder, dermatomyositis and polymyositis;
inherited, metabolic,
endocrine, and toxic myopathy; myasthenia gravis, periodic paralysis; a mental
disorder including
mood, anxiety, and schizophrenic disorders; seasonal affective disorder (SAD);
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
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neuralgia, and Tourette's disorder; an autoimmune/inflammatory disorder such
as acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory
distress syndrome,
allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis, autoimmune
hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-
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; 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
CYAP 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 CYAP expression. Such qualitative or
quantitative methods are well
known in the art.
In a particular aspect, the nucleotide sequences encoding CYAP may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding CYAP 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 CYAP 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.
49
W~ 00/73450 CA 02374222 2001-11-15 pCT~S00/14826
In order to provide a basis for the diagnosis of a disorder associated with
expression of
CYAP, 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 CYAP, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from
normal subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding CYAP
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 CYAP, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
CYAP, 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 CYAP 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
CYAP 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
WO 00/73450 CA 02374222 2001-11-15 pC'T/~JS00/14826
differences in the secondary and tertiary structures of PCR products in single-
stranded form, and
these differences are detectable using gel electrophoresis in non-denaturing
gels. In fSCCP, the
oligonucleotide primers are fluorescently labeled, which allows detection of
the amplimers in high-
throughput equipment such as DNA sequencing machines. Additionally, sequence
database analysis
methods, termed in silico SNP (isSNP), are capable of identifying
polymorphisms by comparing the
sequence of individual overlapping DNA fragments which assemble into a common
consensus
sequence. These computer-based methods filter out sequence variations due to
laboratory preparation
of DNA and sequencing errors using statistical models and automated analyses
of DNA sequence
chromatograms. In the alternative, SNPs may be detected and characterized by
mass spectrometry
using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San
Diego CA).
Methods which may also be used to quantify the expression of CYAP 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 in Seilhamer, J.J. et al.,
"Comparative Gene Transcript
Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference. 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, antibodies specific for CYAP, or CYAP or fragments
thereof 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.
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.
51
W~ 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
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 Microarra~s: A Practical Approach,
M. Schena, ed.
( 1999) Oxford University Press, London, hereby expressly incorporated by
reference.
In another embodiment of the invention, nucleic acid sequences encoding CYAP
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a mufti-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. ( 1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
(See, e.g., 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-Ulr:~h, 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 CYAP on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to l 1q22-23,
any sequences mapping to that area may represent associated or regulatory
genes for further
investigation. (See, e.g., Gatti, R.A. et al. ( 1988) Nature 336:577-580.) The
nucleotide sequence of
the instant invention may also be used to detect differences in the
chromosomal location due to
52
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, CYAP, 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 CYAP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. ( 1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with CYAP, or
fragments thereof,
and washed. Bound CYAP is then detected by methods well known in the art.
Purified CYAP 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 CYAP specifically compete with a test compound
for binding CYAP.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with CYAP.
In additional embodiments, the nucleotide sequences which encode CYAP 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 cLrrently 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, in
particular U.S. Ser. No. 60/136,652, are hereby expressly incorporated by
reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
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
53
CA 02374222 2001-11-15
WO 00/73450 PCT/US00/14826
over CsCI cushions or extracted with chloroform. RNA was precipitated from the
lysates with either
isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCR1PT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
pcDNA2.1 plasmid
(Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA).
Recombinant
plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-
BIueMRF, or
SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life
Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP 96
plasmid purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
54
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
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 (PE 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 (PE Biosystems).
Electrophoretic
separation of cDNA sequencing reactions and detection of labeled
polynucleotides were carried out
using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI
PRISM 373
or 377 sequencing system (PE Biosystems) in conjunction with standard ABI
protocols and base
calling software; or other sequence analysis systems known in the art. Reading
frames within the
cDNA sequences were identified using standard methods (reviewed in Ausubel,
1997, supra, unit
7.7). Some of the cDNA sequences were selected for extension using the
techniques disclosed in
Example VI.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilled in the
art. Table 5 summarizes the tools, programs, and algorithms used and provides
applicable
descriptions, references, and threshold parameters. The first column of Table
5 shows the tools,
programs, and algorithms used, the second column provides brief descriptions
thereof, the third
column presents appropriate references, all of which are incorporated by
reference herein in their
entirety, and the fourth column presents, where applicable, the scores,
probability values, and other
parameters used to evaluate the strength of a match between two sequences (the
higher the score, the
greater the homology between two sequences). Sequences were analyzed using
MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software
(DNASTAR). Polynucleotide and polypeptide sequence alignments were generated
using the 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.
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
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 PFAM to acquire
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences
were assembled
into full length polynucleotide sequences using programs based on Phred,
Phrap, and Consed, and
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
amino acid sequences, and these full length sequences were subsequently
analyzed by querying
against databases such as the GenBank databases (described above), SwissProt,
BLOCKS, PRINTS,
DOMO, PRODOM, Prosite, 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, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide
and amino acid 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 were described in The Invention section above.
IV. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel,
1995, su ra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the
computer search can be modified to determine whether any particular match is
categorized as exact or
similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum { length(Seq. I ), length(Seq. 2) }
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
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WO 00/73450 CA 02374222 2001-11-15 pCT/USUO/14826
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.
The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding CYAP occurred. Analysis involved the categorization of
cDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease/condition categories included cancer,
inflammation, trauma,
cell proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
sequence of interest was counted and divided by the total number of libraries
across all categories.
Percentage values of tissue-specific and disease- or condition-specific
expression are reported in
Table 3.
V. Chromosomal Mapping of CYAP Encoding Polynucleotides
The cDNA 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
SEQ ID N0:6-10 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 5). 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.
The genetic map location of SEQ ID NO:10 is described in The Invention as a
range, or
interval, of a human chromosome. 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.
VI. Extension of CYAP Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:6-10 were 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
57
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
primer, to initiate 3' extension of the known fragment. The initial primers
were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4),S04,
and ~i-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 lrl
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 pl 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 ~cl to 10 ~1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose mini-gel to determine which reactions were
successful in extending
the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37°C in
384-well plates in LB/2x carb liquid media.
58
WU 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94'C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, I min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide ( I :2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAVIIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
In like manner, the polynucleotide sequences of SEQ ID N0:6-10 are used to
obtain 5'
regulatory sequences using the procedure above, along with oligonucleotides
designed for such
extension, and an appropriate genomic library.
VII. Labeling and L; se of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:6-10 are employed to screen cDNAs,
genomic DNAs, or mR:'~As. 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-3zPJ adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston 1'L~). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 supe~ne 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. I x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VIII. 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),
59
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
suera). 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, LTV, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinary skill in the art and may contain any appropriate number of
elements. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalom D. et al. (1996) Genome
Res. 6:639-645;
Marshall, A. and J. Hodgson ( 1998) Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof
may comprise the elements of the microarray. Fragments or oligomers suitable
for hybridization can
be selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array 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 VIMLV reverse-transcriptase, 0.05 pg/pl oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/l.~l RNase inhibitor, 500 I,~NI dATP, SOO IdVI
dGTP, 500 E.~M dTTP, 40
L~M dCTP, 401tM 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 (Incvte j. 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 (CLONT'ECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen l 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 pl 5X SSC/0.2% SDS.
Microarrav Preparation
WU 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
pg. 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 pl of the array
element DNA, at an average
concentration of 100 ng/pl, is loaded into the open capillary printing element
by a high-speed robotic
apparatus. The apparatus then deposits about 5 nl 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 pl of sample mixture consisting of 0.2 ftg
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65 °C for 5 minutes and is aliquoted onto the
microarray surface and covered
with an 1.8 cm' coverslip. The anrays 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 ~tl of 5X SSC in a corner of the chamber. The chamber
containing the arrays is
incubated for about 6.5 hours at 60 °C. The arrays are washed for 10
min at 45 °C in a first wash
buffer ( 1X SSC, 0.1 % SDS ), three times for 10 minutes each at 45 °C
in a second wash buffer (O.1X
SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 «' 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
61
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
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 I .8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are
differentially expressed. the calibration is done by labeling samples of the
calibrating cDNA with the
two fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/L~) 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).
IX. Complementary Polynucleotides
Sequences complementary to the CYAP-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring CYAP. 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
62
WO 00/73450 CA 02374222 2001-11-15 PCT/USOU/14826
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of CYAP. 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
CYAP-encoding
transcript.
X. Expression of CYAP
Expression and purification of CYAP is achieved using bacterial or virus-based
expression
systems. For expression of CYAP in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the TS or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express CYAP upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of CYAP 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 CYAP 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 fruQiperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the luaer requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, CYAP is synthesized as a fusion protein with,
e.g., glutathione S-
transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-
kilodalton enzyme from Schistosoma iaponicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
CYAP 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 CYAP obtained by these methods can be used
directly in the assays
shown in Examples XI and XV.
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WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
XI. Demonstration of CYAP Activity
An immuno localization assay for CYAPs as compared with established cell
cytoskeletal
constituents demonstrates CYAP molecules are cytoskeleton-associated proteins.
Using cells in a
variety of stages of differentiation or the cell cycle and immunofluorescent
microscopy with
fluorescently coupled antibodies to intermediate filament components such as
keratin, vimentin or
desmin; microtubule components such as tubulin or microfilament components
such as actin, a
correlation is made between the localization of CYAP-specific fluorescently
coupled antibodies and
each cytoskeleton component. Simultaneous staining of cells for both the known
cytoskeleton
component and CYAP is accomplished through use of differentially excitable
fluorescent species as
the antibody tag. Alternatively, established cytoskeletal components can be
stained directly with
fluorescent dyes such as phalloidin for actin filaments.
Alternatively, an assay for CYAP measures the formation of protein filaments
in vitro. A
solution of CYAP at a concentration greater than the "critical concentration"
for polymer assembly is
applied to carbon-coated grids. Appropriate nucleation sites may be supplied
in the solution. The
grids are negative stained with 0.7~/c (w/v) aqueous uranyl acetate and
examined by electron
microscopy. The appearance of filaments having a diameter of approximately 25
nm (microtubules),
8 nm (actin), or 10 nm (intermediate filaments) is a demonstration of protein
activity.
XII. Functional Assays
CYAP function is assessed by expressing the sequences encoding CYAP at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid
(Invitrogen), both of which
contain the cytomegalovirus promoter. 5-10 ~cg of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ~g of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and ~anularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
64
WO 00/73450 CA 02374222 2001-11-15 pCT~Jg00/14826
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 CYAP on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding CYAP 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 CYAP and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XIII. Production of CYAP Specific Antibodies
CYAP 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 CYAP amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (PE 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-
CYAP activity by, for example, binding the peptide or CYAP to a substrate,
blocking with 1 % BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
XIV. Purification of'.viaturally Occurring CYAP Using Specific Antibodies
Naturally occurring or recombinant CYAP is substantially purified by
immunoaffinity
chromatography using antibodies specific for CYAP. An immunoaffinity column is
constructed by
covalently coupling anti-CYAP 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 CYAP are passed over the immunoaffinity column, and the
column is
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
washed under conditions that allow the preferential absorbance of CYAP (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/CYAP 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 CYAP is collected.
XV. Identification of Molecules Which Interact with CYAP
CYAP, or biologically active fragments thereof, are labeled with ''-SI 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 CYAP, washed,
and any wells with labeled CYAP complex are assayed. Data obtained using
different concentrations
of CYAP are used to calculate values for the number, affinity, and association
of CYAP with the
candidate molecules.
Alternatively, molecules interacting with CYAP 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).
CYAP 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. 6,057,1 O1 ).
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.
66
WO X0/73450 CA 02374222 2001-11-15 PCT/LTS00/14826
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SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
TANG, Y. Tom
YUE, Henry
HILLMAN, Jennifer L.
BAUGHN, Mariah R.
TRAN, Bao
AZIMZAI, Yalda
<120> CYTOSKELETON ASSOCIATED PROTEINS
<130> PF-0707 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/136,652
<151> 99-05-27
<160> 10
<170> PERL Program
<140> To Be Assigned
<141> Herewith
<160> 10
<170> PERL Program
<210> 1
<211> 372
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1446685CD1
<400> 1
Met Ala Thr Ser Pro Gln Lys Ser Pro Ser Val Pro Lys Ser Pro
1 5 10 15
Thr Pro Lys Ser Pro Pro Ser Arg Lys Lys Asp Asp Ser Phe Leu
20 25 30
Gly Lys Leu Gly Gly Thr Leu Ala Arg Arg Lys Lys Ala Lys Glu
35 40 45
Val Ser Glu Leu Gln Glu Glu Gly Met Asn Ala Ile Asn Leu Pro
50 55 60
Leu Ser Pro Ile Pro Phe Glu Leu Asp Pro Glu Asp Thr Met Leu
65 70 75
Glu Glu Asn Glu Val Arg Thr Met Val Asp Pro Asn Ser Arg Ser
80 85 90
Asp Pro Lys Leu Gln Glu Leu Met Lys Val Leu Ile Asp Trp Ile
95 100 105
Asn Asp Val Leu Val Gly Glu Arg Ile Ile Val Lys Asp Leu Ala
110 115 120
Glu Asp Leu Tyr Asp Giy Gln Val Leu Gln Lys Leu Phe Glu Lys
125 130 135
Leu Glu Ser Glu Lys Leu Asn Val Ala Glu Val Thr Gln Ser Glu
140 145 150
Ile Ala Gln Lys Glr. Lys Leu Gln Thr Val Leu Glu Lys Ile Asn
1
WO 00/73450 CA 02374222 2001-11-15 PCT/US~O/14826
155 160 165
Glu Thr Leu Lys Leu Pro Pro Arg Ser Ile Lys Trp Asn Val Asp
170 175 180
Ser Val His Ala Lys Ser Leu Val Ala Ile Leu His Leu Leu Val
185 190 195
Ala Leu Ser Gln Tyr Phe Arg Ala Pro Ile Arg Leu Pro Asp His
200 205 210
Val Ser Ile Gln Val Val Val Val Gln Lys Arg Glu Gly Ile Leu
215 220 225
Gln Ser Arg Gln Ile Gln Glu Glu Ile Thr Gly Asn Thr Glu Ala
230 235 240
Leu Ser Gly Arg His Glu Arg Asp Ala Phe Asp Thr Leu Phe Asp
245 250 255
His Ala Pro Asp Lys Leu Asn Val Val Lys Lys Thr Leu Ile Thr
260 265 270
Phe Val Asn Lys His Leu Asn Lys Leu Asn Leu Glu Val Thr Glu
275 280 285
Leu Glu Thr Gln Phe Ala Asp Gly Val Tyr Leu Val Leu Leu Met
290 295 300
Gly Leu Leu Glu Gly Tyr Phe Val Pro Leu His Ser Phe Phe Leu
305 310 315
Thr Pro Asp Ser Phe Glu Gln Lys Val Leu Asn Val Ser Phe Ala
320 325 330
Phe Glu Leu Met Gln Asp Gly Gly Leu Glu Lys Pro Lys Pro Arg
335 340 345
Pro Glu Asp Ile Val Asn Cys Asp Leu Lys Ser Thr Leu Arg Val
350 355 360
Leu Tyr Asn Leu Phe Thr Lys Tyr Arg Asn Val Glu
365 370
<210> 2
<211> 184
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1553129CD1
<400> 2
Met Ala Ser Pro Ala Ala Ser Ser Val Arg Pro Pro Arg Pro Lys
1 5 10 15
Lys Glu Pro Gln Thr Leu Val Ile Pro Lys Asn Ala Ala Glu Glu
20 25 30
Gln Lys Leu Lys Leu Glu Arg Leu Met Lys Asn Pro Asp Lys Ala
35 40 45
Val Pro Ile Pro Glu Lys Met Ser Glu Trp Ala Pro Arg Pro Pro
50 55 60
Pro Glu Phe Val Arg Asp Val Met Gly Ser Ser Ala Gly Ala Gly
65 70 75
Ser Gly Glu Phe His Val Tyr Arg His Leu Arg Arg Arg Glu Tyr
80 85 90
Gln Arg Gln Asp Tyr Met Asp Ala Met Ala Glu Lys Gln Lys Leu
95 100 105
Asp Ala Glu Phe Gln Lys Arg Leu Glu Lys Asn Lys Ile Ala Ala
110 115 120
Glu Glu Gln Thr Ala Lys Arg Arg Lys Lys Arg Gln Lys Leu Lys
125 130 135
Glu Lys Lys Leu Leu Ala Lys Lys Met Lys Leu Glu Gln Lys Lys
140 145 150
Gln Glu Gly Pro Gly Gln Pro Lys Glu Gln Gly Ser Ser Ser Ser
2
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
155 160 165
Ala Glu Ala Ser Gly Thr Glu Glu Glu Glu Glu Val Pro Ser Phe
170 175 180
Thr Met Gly Arg
<210> 3
<211> 281
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2572333CD1
<400> 3
Met Ile Asp Glu Ile Val Arg Lys Ile Tyr Glu Glu Asp Gln Leu
1 5 10 15
Glu Lys Gln Gln Lys Leu Glu Lys Met Asn Ala Met Arg Arg Tyr
20 25 30
Ile Glu Glu Phe Gln Lys Glu Gln Ala Leu Trp Arg Lys Lys Lys
35 40 45
Arg Glu Glu Met Glu Glu Glu Asn Arg Lys Ile Ile Glu Phe Ala
50 55 60
Asn Met Gln Gln Gln Arg Glu Glu Asp Arg Met Ala Lys Val Gln
65 70 75
Glu Asn Glu Glu Lys Arg Leu Gln Leu Gln Asn Ala Leu Thr Gln
80 85 90
Lys Leu Glu Glu Met Leu Arg Gln Arg Glu Asp Leu Glu Gln Val
95 100 105
Arg Gln Glu Leu Tyr Gln Glu Glu Gln Ala Glu Ile Tyr Lys Ser
110 115 120
Lys Leu Lys Glu Glu Ala Glu Lys Lys Leu Arg Lys Gln Lys Glu
125 130 135
Met Lys Gln Asp Phe Glu Glu Gln Met Ala Leu Lys Glu Leu Val
140 145 150
Leu Gln Ala Ala Lys Glu Glu Glu Glu Asn Phe Arg Lys Thr Met
155 160 165
Leu Ala Lys Phe Ala Glu Asp Asp Arg Ile Glu Leu Met Asn Ala
170 175 180
Gln Lys Gln Arg Met Lys Gln Leu Glu His Arg Arg Ala Val Glu
185 190 195
Lys Leu Ile Glu Glu Arg Arg Gln Gln Phe Leu Ala Asp Lys Gln
200 205 210
Arg Glu Leu Glu Glu Trp Gln Leu Gln Gln Arg Arg Gln Gly Phe
215 220 225
Ile Asn Ala Ile Ile Glu Glu Glu Arg Leu Lys Leu Leu Lys Glu
230 235 240
His Ala Thr Asn Leu Leu Gly Tyr Leu Pro Lys Gly Val Phe Lys
245 250 255
Lys Glu Asp Asp Ile Asp Leu Leu Gly Glu Glu Phe Arg Lys Val
260 265 270
Tyr Gln Gln Arg Ser Glu Ile Cys Glu Glu Lys
275 280
<210> 4
<211> 557
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3616028CD1
3
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
<400> 4
Met Ala Gly Gly Ala Arg Glu Val Leu Thr Leu Gln Leu Gly His
1 5 10 15
Phe Ala Gly Phe Val Gly Ala His Trp Trp Asn Gln Gln Asp Ala
20 25 30
Ala Leu Gly Arg Ala Thr Asp Ser Lys Glu Pro Pro Gly Glu Leu
35 40 45
Cys Pro Asp Val Leu Tyr Arg Thr Gly Arg Thr Leu His Gly Gln
50 55 60
Glu Thr Tyr Thr Pro Arg Leu Ile Leu Met Asp Leu .Lys Gly Ser
65 70 75
Leu Ser Ser Leu Lys Glu Glu Gly Gly Leu Tyr Arg Asp Lys Gln
80 85 90
Leu Asp Ala Ala Ile Ala Trp Gln Gly Lys Leu Thr Thr His Lys
95 100 105
Glu Glu Leu Tyr Pro Lys Asn Pro Tyr Leu Gln Asp Phe Leu Ser
110 115 120
Ala Glu Gly Val Leu Ser Ser Asp Gly Val Trp Arg Val Lys Ser
125 130 135
Ile Pro Asn Gly Lys Gly Ser Ser Pro Leu Pro Thr Ala Thr Thr
140 145 150
Pro Lys Pro Leu Ile Pro Thr Glu Ala Ser Ile Arg Val Trp Ser
155 160 165
Asp Phe Leu Arg Val His Leu His Pro Arg Ser Ile Cys Met Ile
170 175 180
Gln Lys Tyr Asn His Asp Gly Glu Ala Gly Arg Leu Glu Ala Phe
185 190 195
Gly Gln Gly Glu Ser Val Leu Lys Glu Pro Lys Tyr Gln Glu Glu
200 205 210
Leu Glu Asp Arg Leu His Phe Tyr Val Glu Glu Cys Asp Tyr Leu
215 220 225
Gln Gly Phe Gln Ile Leu Cys Asp Leu His Asp Gly Phe Ser Gly
230 235 240
Val Gly Ala Lys Ala Ala Glu Leu Leu Gln Asp Glu Tyr Ser Gly
245 250 255
Arg Gly Ile Ile Thr Trp Gly Leu Leu Pro Gly Pro Tyr His Arg
260 265 270
Gly Glu Ala Gln Arg Asn Ile Tyr Arg Leu Leu Asn Thr Ala Phe
275 280 285
Gly Leu Val His Leu Thr Ala His Ser Ser Leu Val Cys Pro Leu
290 295 300
Ser Leu Gly Gly Ser Leu Gly Leu Arg Pro Glu Pro Pro Val Ser
305 310 315
Phe Pro Tyr Leu His Tyr Asp Ala Thr Leu Pro Phe His Cys Ser
320 325 330
Ala Ile Leu Ala Thr Ala Leu Asp Thr Val Thr Val Pro Tyr Arg
335 340 345
Leu Cys Ser Ser Pro Val Ser Met Val His Leu Ala Asp Met Leu
350 355 360
Ser Phe Cys Gly Lys Lys Val Val Thr Ala Gly Ala Ile Ile Pro
365 370 375
Phe Pro Leu Ala Pro Gly Gln Ser Leu Pro Asp Ser Leu Met Gln
380 385 390
Phe Gly Gly Ala Thr Pro Trp Thr Pro Leu Ser Ala Cys Gly Glu
395 400 405
Pro Ser Gly Thr Arg Cys Phe Ala Gln Ser Val Val Leu Arg Gly
410 415 420
Ile Asp Arg Ala Cys His Thr Ser His Arg Leu Met Val Val Leu
425 430 435
Ala Leu Phe Trp Gln Pro Ala His Pro Arg Asp Thr Ser Thr Leu
440 445 450
4
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
Cys Pro Ser Cys Met Tyr His Trp Gly Arg Asn Leu Gly Ser Val
455 460 465
Phe Thr Thr Thr Ala Ala Trp Ser His Glu Phe Phe Pro Ser Ala
470 475 480
Ala Asp Ser Leu Gln Gly Gly Ser Ser Leu Pro Pro Pro Leu Leu
485 490 495
Lys Leu Gln Ser Thr Gly Tyr Gly Ser Gly Trp Phe Pro Gln Gly
500 505 510
Ser Ser Gly Glu His Pro Ser Val Trp Gly Thr Val Phe Leu Phe
515 520 525
Val Pro Ala Pro Asp Pro Gly Ser Leu Gly Gln Arg Pro His Gln
530 535 540
Thr Arg Leu Ala Ala Leu Gly Gln Leu His Gly Cys Trp Ser Gly
545 550 555
Ala Arg
<210> 5
<211> 334
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5422507CD1
<400> 5
Met Ser Trp Ile Lys Glu Gly Glu Leu Ser Leu Trp Glu Arg Phe
1 5 10 15
Cys Ala Asn Ile Ile Lys Ala Gly Pro Met Pro Lys His Ile Ala
20 25 30
Phe Ile Met Asp Gly Asn Arg Arg Tyr Ala Lys Lys Cys Gln Val
35 40 45
Glu Arg Gln Glu Gly His Ser Gln Gly Phe Asn Lys Leu Ala Glu
50 55 60
Thr Leu Arg Trp Cys Leu Asn Leu Gly Ile Leu Glu Val Thr Val
65 70 75
Tyr Ala Phe Ser Ile Glu Asn Phe Lys Arg Ser Lys Ser Glu Val
80 85 90
Asp Gly Leu Met Asp Leu Ala Arg Gln Lys Phe Ser Arg Leu Met
95 100 105
Glu Glu Lys Glu Lys Leu Gln Lys His Gly Val Cys Ile Arg Val
110 115 120
Leu Gly Asp Leu His Leu Leu Pro Leu Asp Leu Gln Glu Leu Ile
125 130 135
Ala Gln Ala Val Gln Ala Thr Lys Asn Tyr Asn Lys Cys Phe Leu
140 145 150
Asn Val Cys Phe Ala Tyr Thr Ser Arg His Glu Ile Ser Asn Ala
155 160 165
Val Arg Glu Met Ala Trp Gly Val Glu Gln Gly Leu Leu Asp Pro
170 175 180
Ser Asp Ile Ser Glu Ser Leu Leu Asp Lys Cys Leu Tyr Thr Asn
185 190 195
Arg Ser Pro His Pro Asp Ile Leu Ile Arg Thr Ser Gly Glu Val
200 205 210
Arg Leu Ser Asp Phe Leu Leu Trp Gln Thr Ser His Ser Cys Leu
215 220 225
Val Phe Gln Pro Val Leu Trp Pro Glu Tyr Thr Phe Trp Asn Leu
230 235 240
Phe Glu Ala Ile Leu Gln Phe Gln Met Asn His Ser Val Leu Gln
245 250 255
Gln Lys Ala Arg Asp Met Tyr Ala Glu Glu Arg Lys Arg Gln Gln
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
260 265 270
Leu Glu Arg Asp Gln Ala Thr Val Thr Glu Gln Leu Leu Arg Glu
275 280 285
Gly Leu Gln Ala Ser Gly Asp Ala Gln Leu Arg Arg Thr Arg Leu
290 295 300
His Lys Leu Ser Ala Arg Arg Glu Glu Arg Val Gln Gly Phe Leu
305 310 315
Gln Ala Leu Glu Leu Lys Arg Ala Asp Trp Leu Ala Arg Leu Gly
320 325 330
Thr Ala Ser Ala
<210> 6
<211> 1826
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1446685CB1
<400> 6
gaaagccgca gcctcagtcc cgccgccgcc cgctgcgtcc gcccagcgcc agctccgcgt 60
cccgaccggc ccgcggcagc ctgcgccgcg ccatggccac ctccccgcag aagtcgcctt 120
ctgtccccaa gtctcccact cccaagtcgc ccccgtcccg caagaaagat gattccttct 180
tggggaaact cggagggacc ctggcccgga ggaagaaagc caaggaggtg tccgagctgc 240
aggaggaggg aatgaacgcc atcaacctgc ccctcagccc aattcccttt gagctggacc 300
ccgaggacac gatgctggag gagaatgagg tgcgaacaat ggtggatcca aactcacgca 360
gtgaccccaa gcttcaagaa ctgatgaagg tattaattga ctggattaat gatgtgttgg 420
ttggagaaag aatcattgtg aaagacctag ctgaagattt gtatgatgga caagtcctgc 480
agaagctttt cgagaaactg gagagtgaga agctaaatgt ggctgaggtc acccagtcag 540
agattgctca gaagcaaaaa ctgcagactg tcctggagaa gatcaatgaa accctgaaac 600
ttcctcccag gagcatcaag tggaatgtgg attctgttca tgccaagagc ctggtggcca 660
tcttacacct gctcgttgct ctgtctcagt atttccgcgc accaattcga ctcccagacc 720
atgtttccat ccaagtggtt gtggtccaga aacgagaagg aatcctccag tctcggcaaa 780
tccaagagga aataactggt aacacagagg ctctttccgg gaggcatgaa cgtgatgcct 840
ttgacacctt gttcgaccat gccccagaca agctgaatgt ggtgaaaaag acactcatca 900
ctttcgtgaa caagcacctg aataaactga acctggaggt cacagaactg gaaacccagt 960
ttgcagatgg ggtgtacctg gtgctgctca tggggctcct ggagggctac tttgtgcccc 1020
tgcacagctt cttcctgacc ccggacagct ttgaacagaa ggtcttgaat gtctcctttg 1080
cctttgagct catgcaagat ggagggttgg aaaagccaaa accgcggcca gaagacatag 1140
tcaactgtga cctgaaatct acactacgag tgttgtacaa cctcttcacc aagtaccgta 1200
acgtggagtg aggggctgcc ctgggcccac cactgcccaa gagttcttgc tgttggcgta 1260
ctggaccctc ctccgaactg ccttaccctg cttattcctg tctcttgcac tgtgctctcc 1320
cacaagtcca gctgcaaccc agagatagtg gaaactgaaa ttaggaagga aatcatcaat 1380
aactcagtgg gctgacccat ccctcccagg cgctggggac caacctagca atgaaggttg 1440
ggaaggttgt tcccttcccg gtgccaggtc cagatttccc tccatgattt gggaaccagg 1500
ttaggcaaaa gagtccccac aagatgaaaa taaagatcct agttaccatt caaaggatgc 1560
taactgtgtg tcaggcccca cactaagtgc tctgctctga tatactcaag gccattaatc 1620
ttcaggactc ccattgacgt aggtgtttca ttcccctttt acagatgagg aaactaaggc 1680
ttggaggtta aatgacttgc cagaagttgg aatttttttc ctctttgaac ataacctctc 1740
ccttctccct aaaggtaacc actattctga gtccaatcat caaggttttg cttttctttt 1800
tagctaagta tgcattcctc aatagt 1826
<210> 7
<211> 959
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1553129CB1
6
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
<400> 7
ctcgagccgc tcgagccgcg ccgccgctcg cttgtgaaac tggaaggctg ccatggctag 60
cccagccgcc tcctcggtgc gaccaccgag gcccaagaaa gagccgcaga cgctcgtcat 120
ccccaagaat gcggcggagg agcagaagct caagctggag cggctcatga agaacccgga 180
caaagcagtt ccaattccag agaaaatgag tgaatgggca cctcgacctc ccccagaatt 240
tgtccgagat gtcatgggtt caagtgctgg ggccggcagt ggagagttcc acgtgtacag 300
acatctgcgc cggagagaat atcagcgaca ggactacatg gatgccatgg ctgagaagca 360
aaaattggat gcagagtttc agaaaagact ggaaaagaat aaaattgctg cagaggagca 420
gaccgcaaag cgccggaaga agcgccagaa gttaaaagag aagaaattac tggcaaagaa 480
gatgaaactt gaacagaaga aacaagaagg acccggtcag cccaaggagc aggggtccag 540
cagctctgcg gaggcatctg gaacagagga ggaggaggaa gtgcccagtt tcaccatggg 600
gcgatgacaa tgtttgccac agcctctgcc tggaacctgg ctcgtgctgt gaccagaagg 660
gaaaggcggc tgtttggctc tttctccccc gcaaggacct gctgacccgc tggatggaga 720
gcaaaggaga cccctcccga gccgctcaca gtcctgtatt tggcaggttt gggagcctga 780
ggggccatct ccctgacact cagaggcact gccttgcaga caccatccgt gctcctggta 840
aagggggaca gagagcctca ccttgccaca tatttgaaca gtgatgagtt tggggctggt 900
ttctgggaag ggaacgttta tttagtaaag agcagaacac ccttgaaaaa aaaaaaaaa 959
<210> 8
<211> 1372
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2572333CB1
<400> 8
tcatgattga tgaaattgtt aggaagatct atgaagaaga tcagttggaa aaacaacaaa 60
agttagaaaa aatgaatgca atgcgaaggt atatagaaga gtttcagaaa gagcaggctc 120
tctggagaaa aaagaaacgt gaggagatgg aagaagaaaa cagaaaaatc atagagtttg 180
ctaacatgca gcagcaaaga gaagaagatc ggatggcaaa agttcaagaa aatgaggaga 240
aaaggctaca gcttcagaat gcgttgacac agaaattaga agaaatgctg cggcaacgtg 300
aagatttgga acaagtgcga caagaattat accaggaaga acaagctgaa atatataaga 360
gcaagctaaa agaagaagca gaaaagaaat tgagaaagca aaaagagatg aagcaagatt 420
ttgaagaaca aatggccttg aaggaattag tgctacaggc tgcaaaagag gaagaggaga 480
actttagaaa aactatgcta gctaaatttg ctgaggatga tcgaatagaa ttaatgaatg 540
ctcagaaaca aagaatgaag cagctggaac acaggagggc tgtggaaaaa cttattgaag 600
agcgtcgcca acaattcctt gcagacaaac aacgtgaact agaagagtgg cagttgcagc 660
aaaggcggca aggatttatt aatgcaatta ttgaagaaga aaggctaaaa cttcttaaag 720
agcatgctac aaacttacta ggctatctcc ctaaaggagt atttaaaaaa gaggatgata 780
ttgatctgct tggtgaagag ttcaggaaag tatatcaaca aaggagtgaa atttgtgaag 840
agaaatgata tcatcaaaat tgggtaaagc atagattttt tgtatgttac cactagatgt 900
cagcataact tttgttttac agttcagtgg cattaggtat ccattgtctg tttggatttt 960
gtaaatcatc actgaatttc ataacttgta aacaattatc agatacaaat taattttaat 1020
caagctgtta tttttgtact gataatttca aaatccgatt tctacaacac tacagagcac 1080
tgtttgcatc cccatcctca agacagtata tttaccttga ctaatacaga actctacccc 1140
aaagtgaact gccttctgtc tgtgtttacg aactttactt acttgattta gccagggaaa 1200
taaatatttg gaattttctt ttaaaaaaaa aaaaaaaggg cggccgctcg cccgacatct 1260
gtggccctgg caccaagaag gttcatgtca tcttcaacta caagggcaag aacgtgctga 1320
tcaacaagga catccgttgc aaggatgatg agtttacaca cctgtacaca ct 1372
<210> 9
<211> 1867
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3616028CB1
WU 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
<400> 9
aatcggctag gagcagcgag cggcgcggct gaggcgcggc ggccccgtgg agcagcgcag 60
tatggcgggc ggggcccggg aggtgctcac actgcagttg ggacattttg ccggtttcgt 120
gggcgcgcac tggtggaacc agcaggatgc tgcgctgggc cgagcgaccg attccaagga 180
gcccccggga gagctgtgcc ccgacgtcct gtatcgtacg ggccggacgc tgcacggcca 240
ggagacctac acgccgcgac tcatcctcat ggatctgaag ggtagtttga gctccctaaa 300
agaggaaggt ggactctaca gggacaaaca gttggatgct gcaatagcat ggcaggggaa 360
gctcaccaca cacaaagagg aactctatcc caagaaccct tatctccaag actttctgag 420
tgcagaggga gtgctgagta gtgatggtgt ctggagggtc aaatccattc ccaatggcaa 480
aggttcctca ccactcccca ccgctacaac tccaaaacca cttatcccta cagaggccag 540
catcagggtc tggtcagact tcctcagagt ccatctccat ccccggagca tctgtatgat 600
tcagaagtac aaccacgatg gggaagcagg tcggctggag gcttttggcc aaggggaaag 660
tgtcctaaag gaacccaagt accaggaaga gctggaggac aggctgcatt tctacgtgga 720
ggaatgtgac tacttgcagg gcttccagat cctgtgtgac ctgcacgatg gcttctctgg 780
ggtaggcgcg aaggcggcag agctgctaca agatgaatat tcagggcggg gaataataac 840
ctggggcctg ctacctggtc cctaccatcg tggggaggcc cagagaaaca tctatcgtct 900
attaaacaca gcttttggtc tcgtgcacct gactgctcac agctctcttg tctgcccctt 960
gtccttgggt gggagcctgg gcctgcgacc cgagccacct gtcagcttcc cttacctgca 1020
ttatgatgcc actctgccct tccactgcag tgccatcctg gctacagccc tggacacagt 1080
cactgttcct tatcgcctgt gttcctctcc agtttccatg gttcatctgg ctgacatgct 1140
gagcttctgt gggaaaaagg tggtgacagc aggagcaatc atccctttcc ccttggctcc 1200
aggccagtcc cttcctgatt ccctgatgca gtttggagga gccaccccat ggaccccact 1260
gtctgcatgt ggggagcctt ctggaacacg ttgctttgcc cagtcagtgg tgctgagggg 1320
tatagacaga gcatgccaca caagccacag actaatggtg gttttggctt tgttctggca 1380
gccagctcac cccagggaca cctccaccct ctgcccttca tgcatgtacc actggggaag 1440
aaatcttggc tcagtattta caacaacagc agcctggagt catgagttct tcccatctgc 1500
tgctgactcc ctgcagggtg gctcctcctt acccccacct cttctcaagc tgcagtccac 1560
cgggtatggt tctggatggt tcccccaagg gagcagtgga gagcatccca gtgtttgggg 1620
cactgtgttc ctcttcgtcc ctgcaccaga ccctggaagc cttggccaga gacctcacca 1680
aactcgactt gcggcgctgg gccagcttca tggatgctgg agtggagcac gatgacgtag 1740
cagagctgct gcaggagcta caaagcctgg cccagtgcta ccagggtggt gacagcctcg 1800
tggactaaag ttcccagtgt gggagaaagg agctagtttg caataaaaac agctggatgc 1860
1867
aggaggg
<210> 10
<211> 1429
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5422507CB1
<400> 10
ggtgactggg cggggagcag ctgcgggaga agcaaaggga cgactgaggg aataatcagg 60
agaccactga ggcgtgaagt actaggcgtg cgataactga aggaattacc tggctggtgt 120
ttgcttgttc tggagtgatc ttctgactgg aaaagaacta tgtcatggat caaggaagga 180
gagctgtcac tttgggagcg gttctgtgcc aacatcataa aggcaggccc aatgccgaaa 240
cacattgcat tcataatgga cgggaaccgt cgctatgcca agaagtgcca ggtggagcgg 300
caggaaggcc actcacaggg cttcaacaag ctagctgaga ctctgcggtg gtgtttgaac 360
ctgggcatcc tagaggtgac agtctacgca ttcagcattg agaacttcaa acgctccaag 420
agtgaggtag acgggcttat ggatctggcc cggcagaagt tcagccgctt gatggaagaa 480
aaggagaaac tgcagaagca tggggtgtgt atccgggtcc tgggcgatct gcacttgttg 540
cccttggatc tccaggagct gattgcacaa gctgtacagg ccacgaagaa ctacaacaag 600
tgtttcctga atgtctgttt tgcatacaca tcccgtcatg agatcagcaa tgctgtgaga 660
gagatggcct ggggggtgga gcaaggcctg ttggatccca gtgatatctc tgagtctctg 720
cttgataagt gcctctatac caaccgctct cctcatcctg acatcttgat acggacttct 780
ggagaagtgc ggctgagtga cttcttgcta tggcagacct ctcactcctg cctggtgttc 840
caacccgttc tgtggccaga gtatacattt tggaacctct tcgaggccat cctgcagttc 900
cagatgaacc atagcgtgct tcagcagaag gcccgagaca tgtatgcaga ggagcggaag 960
aggcagcagc tggagaggga ccaggctaca gtgacagagc agctgctgcg agaggggctc 1020
WO 00/73450 CA 02374222 2001-11-15 PCT/US00/14826
caagccagtg gggacgccca gctccgaagg acacgcttgc acaaactctc ggccagacgg 1080
gaagagcgag tccaaggctt cctgcaggcc ttggaactca agcgagctga ctggctggcc 1140
cgtctgggca ctgcatcagc ctgaatgagg ctggccacct gccactttgc cctgccctct 1200
gcctccaggg ctccactccc cttccttttc ttggtgaaag gcacctcctt tcctgataat 1260
gaatggtgtt ccctttgctt ggctggggag ccccccaggc caggtttgct ggccatagat 1320
acctttgggc tgcctgggac aggctcctga ggaggattga gggtgaaagt ctcccacgag 1380
tacactaaac ctaggtctgg tcaccaatag ggtttggaga gctaaggtc 1429
