Note: Descriptions are shown in the official language in which they were submitted.
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CELL CYCLE PROTEINS AND MITOSIS-ASSOCIATED MOLECULES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of cell cycle
proteins and
mitosis-associated molecules and to the use of these sequences in the
diagnosis, treatment, and
prevention of cell proliferative, developmental, and immune disorders, and in
the assessment of the
effects of exogenous compounds on the expression of nucleic acid and amino
acid sequences of cell
cycle proteins end mitosis-associated molecules.
i0 BACKGROUND OF THE INVENTION
Cell division is the fundamental process by which all living things grow and
reproduce. In
unicellular organisms such as yeast and bacteria, each cell division doubles
the number of organisms.
In multicellular species many rounds of cell division are required to replace
cells lost by wear or by
programmed cell death, and for cell differentiation to produce a new tissue or
organ. Details of the
cell division cycle may vary, but the basic process consists of three
principle events. The first event,
interphase, involves preparations for cell division, replication of the DNA,
and production of essential
proteins. In the second event, mitosis, the iluclear material is divided and
separates to opposite sides
of the cell. The final event, cytokinesis, is division and fission of the cell
cytoplasm. The sequence
and timing of cell cycle transitions is under the control of the cell cycle
regulation system which
controls the process by positive or negative regulatory circuits at various
check points.
Mitosis marks the end of interphase and concludes with the onset of
cytokinesis. There are
four stages in mitosis, occurring in the following order: prophase, metaphase,
anaphase and telophase.
Prophase includes the formation of bi-polar mitotic spindles, composed of
mictrotubules and associated
proteins such as dynein, which originate from polar mitotic centers. During
metaphase, the nuclear
material condenses and develops kinetochore fibers which aid in its physical
attachment to the mitotic
spindles. The ensuing movement of the nuclear material to opposite poles along
the mitotic spindles
occurs during anaphase. Telophase includes the disappearance of the mitotic
spindles and kinetochore
fibers from the nuclear material. Mitosis depends on the interaction of
numerous proteins. For
example, centromere-associated proteins such as CENP-A, -B, and -C, play
structural roles in
kinetochore formation and assembly (Saffery, et al. (2000) Human Mol. Gen. 9:
175-185).
Progression through the cell cycle is governed by the intricate interactions
of protein
complexes. This regulation depends upon the appropriate expression of proteins
which control cell
cycle progression in response to extracellular signals, such as growth factors
and other mitogens, and
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intracellular cues, such as DNA damage or nutrient starvation. Molecules which
directly or indirectly
modulate cell cycle progression fall into several categories, including
cyclins, cyclin-dependent protein
kinases, growth factors and their receptors, second messenger and signal
transduction proteins,
oncogene products, and tumor-suppressor proteins.
The entry and exit of a cell from mitosis is regulated by the synthesis and
destruction of a
family of activating proteins called cyclins. Cyclins act by binding to and
activating a group of cyclin-
dependent protein kinases (Cdks) which then phosphorylate and activate
selected proteins involved in
the mitotic process. Several types of cyclins exist (Ciechanover, A. (1994)
Cell 79:13-21). Cyclins
are characterized by a large region of shared homology that is approximately
180 amino acids in length
and referred to as the "cyclin box" (Chapman, D.L. and Wolgemuth, D.J. (1993)
Development
118:229-40). In addition, cyclins contain a conserved 9 amino acid sequence in
the N-terminal region
of the molecule called the "destruction box." This sequence is believed to be
a recognition code that
triggers ubiquitin-mediated degradation of cyclin B (Hunt, T. (1991) Nature
349:100-101). Cyclin A is
required in higher eukaryotic cells at the Gl/S and the G2/M transitions, and
is involved in the
regulation of cellular DNA replication (Sobczak-Thepot, J. et al. (1993) Exp.
Cell Res. 206:43-48).
Mitotic cyclin, or cyclin B controls entry of the cell into mitosis. G1 cyclin
controls events that drive
the cell out of mitosis. Progression through the G2-M transition is driven by
the activation of mitotic
CDI~ cyclin complexes such as Cdc2-cyclin A, Cdc2-cyclin B 1 and Cdc2-cyclin
B2 complexes
(reviewed in Yang, J. and Kornbluth, S. (1999) Trends in Cell Biology 9:207-
210).
Cyclins are degraded through the ubiquitin conjugation system (UCS), a major
pathway for the
degradation of cellular proteins in eukaroytic cells and in some bacteria. The
UCS mediates the
elimination of abnormal proteins and regulates the half lives of important
regulatory proteins that
control cellular processes such as gene transcription and cell cycle
progression. The UCS is
implicated in the degradation of mitotic cyclin kinases, oncoproteins, tumor
suppressor genes such as
p53, viral proteins, cell surface receptors associated with signal
transduction, transcriptional regulators,
and mutated or damaged proteins (Ciechanover, supra).
The process of ubiquitin conjugation and protein degradation occurs in five
principle steps
(Jentsch, S. (1992) Annu. Rev. Genet. 26:179-207). First ubiquitin (Ub), a
small, heat stable protein is
activated by a ubiquitin-activating enzyme (E1) in an ATP dependent reaction
which binds the C-
terminus of Ub to the thiol group of an internal cysteine residue in El.
Second, activated Ub is
transferred to one of several Ub-conjugating enzymes (E2). Different ubiquitin-
dependent proteolytic
pathways employ structurally similar, but distinct ubiquitin-conjugating
enzymes that are associated
with recognition subunits which direct them to proteins carrying a particular
degradation signal. Third,
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E2 transfers the Ub molecule through its C-terminal glycine to a member of the
ubiquitin-protein lipase
family, E3. Fourth, E3 transfers the Ub molecule to the target protein.
Additional Ub molecules may
be added to the target protein forming a multi-Ub chain structure. Fifth, the
ubiquinated protein is then
recognized and degraded by proteasome, a large, multisubunit proteolytic
enzyme complex, and Ub is
released for re-utilization.
Ub-conjugating enzymes (E2s) are important for substrate specificity in
different UCS
pathways. All E2s have a conserved domain of approximately 16 kDa called the
UBC domain that is
at least 35% identical in all E2s and contains a centrally located cysteine
residue required for ubiquitin-
enzyme thiolester formation (Jentsch, supra). A well conserved proline-rich
element is located N-
l0 terminal to the active cysteine residue. Structural variations beyond this
conserved domain are used to
classify the E2 enzymes. Class I E2s consist almost exclusively of the
conserved UBC domain.
Class II E2s have various unrelated C-terminal extensions that contribute to
substrate specificity and
cellular localization. Class III E2s have unique N-terminal extensions which
are believed to be
involved in enzyme regulation or substrate specificity.
A mitotic cyclin-specific E2 (E2-C) is characterized by the conserved UBC
domain, an N-
terminal extension of 30 amino acids not found in other E2s, and a 7 amino
acid unique sequence
adjacent to this extension. These characteristics together with the high
affinity of E2-C for cyclin
identify it as a new class of E2 (Aristarkhov, A. et al. (1996) Proc. Natl.
Acad. Sci. 93:4294-99).
Ubiquitin-protein ligases (E3s) catalyze the last step in the ubiquitin
conjugation process,
covalent attachment of ubiquitin to the substrate. E3 plays a key role in
determining the specificity of
the process. Only a few E3s have been identified so far. One type of E3
ligases is the HECT
(homologous to E6-AP C-terminus) domain protein family. One member of the
family, E6-AP
(E6-associated protein) is required, along with the human papillomavirus (HP~
E6 oncoprotein, for
the ubiquitination and degradation of p53 (Scheffner et al. et al. (1993) Cell
75:495-505). The
C-terminal domain of the HECT proteins contains the highly conserved ubiquitin-
binding cysteine
residue. The N-terminal region of the various HECT proteins is variable and is
believed to be involved
in specific substrate recognition (Huibregtse, J.M. et al. (1997) Proc. Natl
Acad. Sci. USA
94:3656-3661).
Certain cell proliferation disorders can be identified by changes in the
protein complexes that
normally control progression through the cell cycle. A primary treatment
strategy involves
reestablishing control over cell cycle progression by manipulation of the
proteins involved in cell cycle
regulation (Nigg, E.A. (1995) BioEssays 17:471-4~0).
The discovery of new cell cycle proteins and mitosis-associated molecules and
the
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polynucleotides encoding them satisfies a need in the art by providing new
compositions which are
useful in the diagnosis, prevention, and treatment of cell proliferative,
developmental, and immune
disorders, and in the assessment of the effects of exogenous compounds on the
expression of nucleic
acid and amino acid sequences of cell cycle proteins.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, cell cycle proteins and mitosis-
associated
molecules, referred to collectively as "CCPMAM" and individually as "CCPMAM-
1," "CCPMAM-
2," and "CCPMAM-3." In one aspect, the invention provides an isolated
polypeptide selected from
the group consisting of a) a polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-3, b) a polypeptide comprising a naturally occurring
amino acid sequence
at least 90% identical to an amino acid sequence selected from the group
consisting of SEQ ID N0:1-
3, c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-3, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID N0:1-3. In
one alternative, the
invention provides an isolated polypeptide comprising the amino acid sequence
of SEQ ID NO:1-3,
The invention further provides an isolated polynucleotide encoding a
polypeptide selected from
the group consisting of a) a polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ ID NO:l-3, b) a polypeptide comprising a naturally occurring
amino acid sequence
at least 90% identical to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
3, c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-3, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID N0:1-3. In
one alternative, the
polynucleotide encodes a polypeptide selected from the group consisting of SEQ
ID N0:1-3. In
another alternative, the polynucleotide is selected from the group consisting
of SEQ ID N0:4-6.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID N0:1-3, b) a polypeptide comprising a naturally occurring amino acid
sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO:1-3, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-3, and d) an immunogenic fragment of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-3. In one
alternative, the invention
4
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provides a cell transformed with the recombinant polynucleotide. In another
alternative, the invention
provides a transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ >D NO:1-3, b) a polypeptide comprising a naturally occurring amino acid
sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ m N0:1-3, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m NO:1-3, and d) an immunogenic fragment of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ m NO:l-3. The method
comprises a) culturing
a cell under conditions suitable for expression of the polypeptide, wherein
said cell is transformed with
a recombinant polynucleotide comprising a promoter sequence operably linked to
a polynucleotide
encoding the polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid sequence
selected from the group consisting of SEQ m NO:1-3, b) a polypeptide
comprising a naturally
occurring amino acid sequence at least 90% identical to an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-3, c) a biologically active fragment of a
polypeptide having an amino
acid sequence selected from the group consisting of SEQ m N0:1-3, and d) an
immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
)D N0:1-3.
The invention further provides an isolated polynucleotide selected from the
group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
m N0:4-6, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:4-6, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). In one alternative,
the polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
ID N0:4-6, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ~ N0:4-6, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
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polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) hybridizing the
sample with a probe comprising at least 20 contiguous nucleotides comprising a
sequence
complementary to said target polynucleotide in the sample, and which probe
specifically hybridizes to
said target polynucleotide, under conditions whereby a hybridization complex
is formed between said
probe and said target polynucleotide or fragments thereof, and b) detecting
the presence or absence of
said hybridization complex, and optionally, if present, the amount thereof. In
one alternative, the probe
comprises at least 60 contiguous nucleotides.
The invention fiuther provides a method for detecting a target polynucleotide
in a sample, said
target polynucleotide having a sequence of ,a polynucleotide selected from the
group consisting of a) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:4-6, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:4-6, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) amplifying said
target polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b)
detecting the presence or absence of said amplified target polynucleotide or
fragment thereof, and,
optionally, if present, the amount thereof.
The invention further provides a composition.comprising an effective amount of
a polypeptide
selected from the group consisting of a) a polypeptide comprising an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-3, b) a polypeptide comprising a
naturally occurring amino
acid sequence at least 90% identical to an amino acid sequence selected from
the group consisting of
SEQ ~ NO:1-3, c) a biologically active fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ ID N0:1-3, and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-3, and
a pharmaceutically acceptable excipient. In one embodiment, the composition
comprises an amino
acid sequence selected from the group consisting of SEQ ID N0:1-3. The
invention additionally
provides a method of treating a disease or condition associated with decreased
expression of
functional CCPMAM, comprising administering to a patient in need of such
treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-3, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:l-3, c) a biologically active fragment
of a polypeptide having
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an amino acid sequence selected from the group consisting of SEQ ID NO:l-3,
and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID N0:1-3. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional CCPMAM,
comprising administering to a patient in need of such treatment the
composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
l0 an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1-3, b) a
polypeptide
r
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ ID N0:1-3, c) a
biologically active fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-3, and
d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-3. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a composition comprising an antagonist compound identified
by the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
CCPMAM, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds to
a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ~ NO:1-3, b) a polypeptide
comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-3, c) a biologically active fragment
of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ ~ NO:1-3, and
d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-3. The method comprises a) combining the polypeptide
with at least one
test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
7
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acid sequence selected from the group consisting of SEQ ~ NO:l-3, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-3, c) a biologically active fragment
of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ ID N0:1-3,
and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:l-3. The method comprises a) combining the polypeptide
with at least one
test compound under conditions permissive for the activity of the polypeptide,
b) assessing the activity
of the polypeptide in the presence of the test compound, and c) comparing the
activity of the
polypeptide in the presence of the test compound with the activity of the
polypeptide in the absence of
the test compound, wherein a change in the activity of the polypeptide in the
presence of the test
compound is indicative of a compound that modulates the activity of the
polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of SEQ ID N0:4-6,
the method
' comprising a) exposing a sample comprising the target polynucleotide to a
compound, and b) detecting
altered expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound; b)
hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:4-6, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:4-6,
iii) a polynucleotide
having a sequence complementary to i), iv) a polynucleotide complementary to
the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions
whereby a specific
hybridization complex is formed between said probe and a target polynucleotide
in the biological
sample, said target polynucleotide selected from the group consisting of i) a
polynucleotide comprising
a polynucleotide sequence selected from the group consisting of SEQ ID N0:4-6,
ii) a polynucleotide
comprising a naturally occurring polynucleotide sequence at least 90%
identical to a polynucleotide
sequence selected from the group consisting of SEQ ID N0:4-6, iii) a
polynucleotide complementary
to the polynucleotide of i), iv) a polynucleotide complementary to the
polynucleotide of ii), and v) an
RNA equivalent of i)-iv). Alternatively, the target polynucleotide comprises a
fragment of a
polynucleotide sequence selected from the group consisting of i)-v) above; c)
quantifying the amount
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of hybridization complex; and d) comparing the amount of hybridization complex
in the treated
biological sample with the amount of hybridization complex in an untreated
biological sample, wherein
a difference in the amount of hybridization complex in the treated biological
sample is indicative of
toxicity of the test compound.
BRIEF DESCRLPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability score for the match
between each
polypeptide and its GenBankhomolog is also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold parameters.
DESCRLPTION 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
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forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing the
cell lines, protocols, reagents and vectors which are reported in the
publications and which might be
used in connection with the invention. Nothing herein is to be construed as an
admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"CCPMAM" refers to the amino acid sequences of substantially purified CCPMAM
obtained
from any species, particularly a mammalian species, including bovine, ovine,
porcine, marine, equine,
and human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
CCPMAM. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any
other compound or composition which modulates the activity of CCPMAM either by
directly
interacting with CCPMAM or by acting on components of the biological pathway
in which CCPMAM
participates.
An "allelic variant" is an alternative form of the gene encoding CCPMAM.
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 CCPMAM include those sequences with
deletions, insertions, or substitutions of different nucleotides, resulting in
a polypeptide the same as
CCPMAM or a polypeptide with at least one functional characteristic of CCPMAM.
Included within
this definition are polymorphisms which may or may not be readily detectable
using a particular
oligonucleotide probe of the polynucleotide encoding CCPMAM, and improper or
unexpected
hybridization to allelic variants, with a locus other than the normal
chromosomal locus for the
polynucleotide sequence encoding CCPMAM. The encoded protein may also be
"altered," and may
contain deletions, insertions, or substitutions of amino acid residues which
produce a silent change and
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result in a functionally equivalent CCPMAM. 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
CCPMAM is retained.
For example, negatively charged amino acids may include aspartic acid and
glutamic acid, and
positively charged amino acids may include lysine and arginine. Amino acids
with uncharged polar
side chains having similar hydrophilicity values may include: asparagine and
glutamine; and serine and
threonine. Amino acids with uncharged side chains having similar
hydrophilicity values may include:
leucine, isoleucine, ,and valine; glycine and alanine; and phenylalanine and
tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
l0 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.
25 Amplification is generally carried out using polymerase chain reaction
(PCR) technologies well known
in the art.
The term "antagonist" xefers to a molecule which inhibits or attenuates the
biological activity
of CCPMAM. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates,
small molecules, or any other compound or composition which modulates the
activity of CCPMAM
20 either by directly interacting with CCPMAM or by acting on components of
the biological pathway in
which CCPMAM participates.
The term "antibody" refers to intact immunoglobufin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind CCPMAM polypeptides can be prepared using intact
polypeptides or using
25 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, ox 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 axe chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (I~L,I~. The coupled peptide is
then used to immunize
30 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
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which bind specifically to antigenic determinants (particular regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen used
to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic CCPMAM, 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 CCPMAM or fragments
of CCPMAM
may be employed as hybridization probes. The probes may be stored in freeze-
dried form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts {e.g., NaCI), detergents
(e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to
repeated DNA sequence analysis to resolve uncalled bases, extended using the
XL-PCR kit (Applied
Biosystems, Foster City CA) in the 5' andlor the 3' direction, and
resequenced, or which has been
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assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI) or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended
and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu lle, 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 ar hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
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one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
i0 exon may represent a structural or functional domain of the encoded
protein, new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of CCPMAM or the polynucleotide encoding
CCPMAM
which is identical in sequence to but shorter in length than the parent
sequence. A fragment may
comprise up to the entire length of the defined sequence, minus one
nucleotide/amino acid residue.
For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or
amino acid residues.
A fragment used as a probe, primer, antigen, therapeutic molecule, or for
other purposes, may be at
least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500
contiguous nucleotides or
amino acid residues in length. Fragments may be preferentially selected from
certain regions of a
molecule. For example, a polypeptide fragment may comprise a certain length of
contiguous amino
acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of
a polypeptide as shown
in a certain defined sequence. Clearly these lengths are exemplary, and any
length that is supported
by the specification, including the Sequence Listing, tables, and figures, may
be encompassed by the
present embodiments.
A fragment of SEQ )D N0:4-6 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ )D N0:4-6, for example, as distinct from any other
sequence in the genome
from which the fragment was obtained. A fragment of SEQ ID N0:4-6 is useful,
for example, in
hybridization and amplification technologies and in analogous methods that
distinguish SEQ ID N0:4-6
from related polynucleotide sequences. The precise length of a fragment of SEQ
ID N0:4-6 and the
region of SEQ ID N0:4-6 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:1-3 is encoded by a fragment of SEQ ID N0:4-6. A
fragment of
SEQ ID N0:1-3 comprises a region of unique amino acid sequence that
specifically identifies SEQ ID
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N0:1-3. For example, a fragment of SEQ ID NO:l-3 is useful as an immunogenic
peptide for the
development of antibodies that specifically recognize SEQ ID NO:l-3. The
precise length of a
fragment of SEQ ID NO:1-3 and the region of SEQ ID N0:1-3 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
2o Higgins, D.G. and P.M. Shaip (1989) CABIOS 5:151-153 and in Higgins, D.G.
et al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available from
several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The
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''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 misnzatclz: -2
Open Gap: S and Extension Gap: 2 penalties
Gap x drop-off:' S0
Expect: 10
Word Size: 1l
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ )D 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 ~0, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
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penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported °by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: Il and Extension Gap: 1 penalties
Gap x drop-off 50
Expect: 10
Word Size: 3
Pi.lter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length,
for example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
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conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill in
the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1 % (w/v) SDS, and about 100 p g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (T~ for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions fox nucleic acid hybridization are well known and can be found in
Sambrook, J. et al. (1989)
Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor
Press, Plainview NY;
specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1 % SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used. SSC concentration may
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
sheared and denatured salmon sperm DNA at about 100-200 p g/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
18
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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 CCPMAM
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
CCPMAM which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of CCPMAM. For example,
modulation may cause an increase or a decrease in protein activity, binding
characteristics, or any
other biological, functional, or immunological properties of CCPMAM.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an CCPMAM 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 CCPMAM.
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"Probe" refers to nucleic acid sequences encoding CCPMAM, 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-Tntersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas
South West Medical Center, Dallas TX) is capable of choosing specific primers
from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The
Primer3 primer
selection program (available to the public from the Whitehead 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
CA 02415077 2003-O1-16
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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, s_~ra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for
example, to transform a cell. .
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of
the nitrogenous base thymine are replaced with uracil, and the sugar backbone
is composed of ribose
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instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing
CCPMAM, nucleic acids encoding CCPMAM, or fragments thereof may comprise a
bodily fluid; an
extract from a cell, chromosome, organelle, or membrane isolated from a cell;
a cell; genomic DNA,
RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print;
etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the pxesence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recognized by the
binding molecule. For
example, if an antibody is specific for epitope "A," the presence of a
polypeptide comprising the
epitope A, or the presence of free unlabeled A, in a reaction containing free
labeled A and the
antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at 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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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 91
%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97°~0, at least
98%, or at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The xesulting polypeptides will generally have significant amino acid
identity relative to each
other. A polymorphic variant is a variation in the polynucleotide sequence of
a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
23
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1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human cell cycle proteins and
mitosis-
associated molecules (CCPMAM), the polynucleotides encoding CCPMAM, and the
use of these
compositions for the diagnosis, treatment, or prevention of cell cycle
proteins and mitosis-associated
molecules.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ ID NO:) and an
Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as
shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the
invention. Column 3
shows the GenBank identification number (Genbank ID NO:) of the nearest
GenBank homolog.
Column 4 shows the probability score for the match between each polypeptide
and its GenBank
homolog. Column 5 shows the annotation of the GenBank homolog along with
relevant citations
where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and
2 show the polypeptide sequence identification number (SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention. Column
3 shows the number of amino acid residues in each polypeptide. Column 4 shows
potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the MOTIFS
program of the GCG sequence analysis software package (Genetics Computer
Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structurelfunction analysis and in some
cases, seaxchable
24
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databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are cell cycle proteins and
mitosis-associated
molecules. For example, SEQ )D NO:1 is 36% identical to human cyclin-E binding
protein (GenBank
ID 86630609) as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.) . .
The BLAST probability score is 9.3e-176, which indicates the probability of
obtainin8 the observed
polypeptide sequence alignment by chance. SEQ ID NO:1 also contains a HECT
domain and a
re8ulator of chromosome condensation (RCCl) domain as determined by searchin8
for statistically
significant matches in the hidden Markov model (HMM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN
analyses
provide further corroborative evidence that SEQ ID NO:1 is an ubiquitin-
protein lipase. In an
alternative example, SEQ ID N0:3 is 29% identical to cyclin A (GenBank ID
8984659) as determined
by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score
is 4.0e-1 l, which indicates the probability of obtaining the observed
polypeptide sequence ali8nment by
chance. SEQ ID NO:3 also contains a cyclin domain as determined by searchin8
for statistically
si8nificant matches in the hidden Markov model (HMM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from BLIMPS analysis provides further
corroborative evidence.
that SEQ ID N0:3 is a cyclin. SEQ ID N0:2 was analyzed and annotated in a
similar manner. The
al8orithms and parameters for the analysis of SEQ ID N0:1-3 are described in
Table 7.
As shown in Table 4, the full len8th polynucleotide sequences of the present
invention were
assembled using cDNA sequences or codin8 (exon) sequences derived from 8enomic
DNA, or any
combination of these two types of sequences. Columns 1 and 2 list the
polynucleotide sequence
identification number (Polynucleotide SEQ ID NO:) and the correspondin8 Incyte
polynucleotide
consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide
of the invention.
Column 3 shows the len8th of each polynucleotide sequence in basepairs. Column
4 lists fra8ments of
the polynucleotide sequences which are useful, for example, in hybridization
or amplification
technolo8ies that identify SEQ ID N0:4-6 or that distin8uish between SEQ ID
N0:4-6 and related
polynucleotide sequences. Column 5 shows identification numbers correspondin8
to cDNA
sequences, codin8 sequences (exons) predicted from genomic DNA, and/or
sequence assembla8es
comprised of both cDNA and genomic DNA. These sequences were used to assemble
the full len8th
polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the
nucleotide start (5')
and stop (3') positions of the cDNA and/or 8enomic sequences in column 5
relative to their respective
full length sequences.
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The identification numbers in Column 5 of Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
242354486 is the
identification number of an Incyte cDNA sequence, and SCORNON02 is the cDNA
library from
which it is derived. Incyte cDNAs for which cDNA libraries are not indicated
were derived from
pooled cDNA libraries. Alternatively, the identification numbers in column 5
may refer to GenBank
cDNAs or ESTs which contributed to the assembly of the full length
polynucleotide sequences. In
addition, the identification numbers in column 5 may identify sequences
derived from the ENSEMBL
(The Sanger Centre, Cambridge, UK) database (i.e., those sequences including
the designation
"ENST"). Alternatively, the identification numbers in column 5 may be derived
from the NCBI
RefSeq Nucleotide Sequence Records Database (i.e., those sequences including
the designation
"NM" or "NT") or the NCBI RefSeq Protein Sequence Records (i. e., those
sequences including the
designation "NP"). Alternatively, the identification numbers in column S may
refer to assemblages of
both cDNA and Genscan-predicted exons brought together by an "exon stitching"
algorithm. For
example, FL XXXXXX NI lV2_YYYYY N3 N4 represents a "stitched" sequence in
which XXXXXX is
the identification number of the cluster of sequences to which the algorithm
was applied, and YYYYY is
the number of the prediction generated by the algorithm, and Nl,z.3...~ if
present, represent specific
exons that may have been manually edited during analysis (See Example V).
Alternatively, the
identification numbers in column 5 may refer to assemblages of exons brought
together by an "exon-
stretching" algorithm. For example, FLXXXXXX gAAAAA_~3BBBB_1_Nis the
identification number-
of a "stretched" sequence, with XXXXXX being the Incyte project identification
number, gAA~9AA
being the GenBank identification number of the human genomic sequence to which
the "exon-
stretching" algorithm was applied, gBBBBB being the GenBank identification
number or NCBI RefSeq
identification number of the nearest GenBank protein homolog, and N referring
to specific exons (See
Example V). In instances where a RefSeq sequence was used as a protein homolog
for the "exon-
stretching",algorithm, a RefSeq identifier (denoted by "NM," "NP," or "NT")
may be used in place of
the GenBankidentifier (i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
Prefix ~ Type of analysis andlor examples of programs
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GNN, GFG, Exon prediction from genomic sequences using,
for example,
ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK)
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example ~.
INCY Full length transcript and exon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the exons and
resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in
column 5 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant Incyte
cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library
is the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences
which were used to assemble and confirm the above polynucleotide sequences. .
The tissues and
vectors which were used to construct the cDNA libraries shown in Table 5 are
described in Table 6.
The invention also encompasses CCPMAM variants. A preferred CCPMAM 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 CCPMAM amino acid sequence, and which contains
at least one
functional or structural characteristic of CCPMAM.
The invention also encompasses polynucleotides which encode CCPMAM. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:4-6, which encodes CCPMAM. The
polynucleotide
sequences of SEQ ID N0:4-6, 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
CCPMAM.
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 CCPMAM. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:4-6
which has at least about 70%, or alternatively at least about 85%, or even at
least about 95%
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polynucleotide sequence identity to a nucleic acid sequence selected from the
group consisting of SEQ
1D N0:4-6. Any one of the polynucleotide variants described above can encode
an amino acid
sequence which contains at least one functional or structural characteristic
of CCPMAM.
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 CCPMAM, 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 colon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring CCPMAM, and all such variations
are to be considered
as being specifically disclosed.
Although nucleotide sequences which encode CCPMAM and its variants are
generally
capable of hybridizing to the nucleotide sequence of the naturally occurring
CCPMAM under
appropriately selected conditions of stringency, it may be advantageous to
produce nucleotide
sequences encoding CCPMAM or its derivatives possessing a substantially
different colon usage,
e.g., inclusion of non-naturally occurring colons. Colons may be selected to
increase the rate at
which expression of the peptide occurs in a particular prokaryotic or
eukaryotic host in accordance
with the frequency with which particular colons are utilized by the host.
Other reasons for
substantially altering the nucleotide sequence encoding CCPMAM 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 CCPMAM
and
CCPMAM 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 CCPMAM 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
N0:4-6 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
I~lenow fragment
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of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then earned out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biolo~y and Biotechnology, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding CCPMAM may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent
to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et
al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme digestions and
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.
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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, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode CCPMAM may be cloned in recombinant DNA molecules that direct
expression of
CCPMAM, or fragments or functional equivalents thereof, in appropriate host
cells. Due to the
inherent degeneracy of the genetic code, other DNA sequences which encode
substantially the same
or a functionally equivalent amino acid sequence may be produced and used to
express CCPMAM.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter CCPMAM-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 CCPMAM, 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
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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 CCPMAM 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, CCPMAM itself or a fragment thereof may be synthesized using
chemical methods.
For example, peptide synthesis can be performed using various solution-phase
or solid-phase
techniques. (See, e.g., Creighton, T. (1984) Proteins, Structures and
Molecular Properties, WH
Freeman, New York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science
269:202-204.)
Automated synthesis may be achieved using the ABI 431A peptide synthesizer
(Applied Biosystems).
Additionally, the amino acid sequence of CCPMAM, or any part thereof, may be
altered during direct
synthesis and/or combined with sequences from other proteins, or any part
thereof, to produce a
variant polypeptide or a polypeptide having a sequence of a naturally
occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active CCPMAM, the nucleotide sequences
encoding
CCPMAM 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 CCPMAM. Such elements may vary in their
sirength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding CCPMAM. Such signals include the ATG initiation codon and
adjacent
sequences, e.g. the Kozak sequence. In cases where sequences encoding CCPMAM
and its initiation
codon and upstream regulatory sequences are inserted into the appropriate
expression vector, no
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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 CCPMAM 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 CCPMAM. 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, su ra;
Ausubel, su ra; Van Heeke,
G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et
al. (1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-
1945; Takamatsu, N.
(1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y
(1992) McGraw
Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl.
Acad. Sci. USA
81:3655-3659; and Harrington, 3.J. et al. (1997) Nat. Genet. 15:345-355.)
Expression vectors derived
from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from
vaxious bacterial plasmids, may
be used for delivery of nucleotide sequences to the targeted organ, tissue, or
cell population. (See,
e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et
al. (1993) Proc. Natl.
Acad. Sci. USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature
317(6040):813-815; McGregor,
D.P. et al. (1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia
(1997) Nature
389:239-242.) The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding CCPMAM. For
example, routine
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cloning, subcloning, and propagation of polynucleotide sequences encoding
CCPMAM 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 CCPMAM
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 CCPMAM are
needed, e.g. for the
production of antibodies, vectors which direct high level expression of CCPMAM
may be used. For
example, vectors containing the strong, inducible SP6 or T7 bacteriophage
promoter may be used.
Yeast expression systems may be used for production of CCPMAM. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomvces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et
al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of CCPMAM. Transcription of
sequences
encoding CCPMAM may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV
used alone or in combination with the omega leader sequence from TMV
(Takamatsu, N. (1987)
EMBO J. 6:307-311). Alternatively, plant promoters such as the small subunit
of RUBISCO or heat
shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J.
3:1671-1680; Brogue, R.
et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
Cell Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of
Science and Technolo~y
(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 CCPMAM
may be ligated
into an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential El or E3 region of the viral genome may
be used to obtain
infective virus which expresses CCPMAM 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
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or EB V-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
CCPMAM in cell lines is preferred. For example, sequences encoding CCPMAM 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; fzeo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
lVatl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins,
green fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate f3-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 CCPMAM is inserted within a marker gene sequence,
transformed cells
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containing sequences encoding CCPMAM can be identified by the absence of
marker gene function.
Alternatively, a marker gene can be placed in tandem with a sequence encoding
CCPMAM 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 CCPMAM
and that
express CCPMAM 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 CCPMAM
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 CCPMAM is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. ,et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. 1V; Coligan, J.E. et al. (1997) Current Protocols in 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
CCPMAM include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, the sequences encoding CCPMAM, or any fragments thereof, may be
cloned into a
vector for the production of an mRNA probe. Such vectors are known in the a~-
t, are commercially
available, and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA
polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures
may be conducted
using a variety of commercially available kits, such as those provided by
Amersham Pharmacia
Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter molecules
or labels which
may be used for ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or
chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic
particles, and the like.
Host cells transformed with nucleotide sequences encoding CCPMAM may be
cultured under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
CA 02415077 2003-O1-16
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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 CCPMAM may be designed to contain signal
sequences which direct
secretion of CCPMAM 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 CCPMAM 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 CCPMAM protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of CCPMAM
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 CCPMAM encoding sequence and the
heterologous protein
sequence, so that CCPMAM may be cleaved away from the heterologous moiety
following
purification. Methods for fusion protein expression and purification are
discussed in Ausubel (1995,
supra, ch. 10). A variety of commercially available kits may also be used to
facilitate expression and
purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled CCPMAM may
be
achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ
extract system (Promega).
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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.
CCPMAM of the present invention or fragments thereof may be used to screen for
compounds that specifically bind to CCPMAM. At least one and up to a plurality
of test compounds
may be screened for specific binding to CCPMAM. 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
CCPMAM, e.g., a ligand or fragment thereof, a natural substrate, a structural
or functional mimetic, or
a natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which
CCPMAM 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 CCPMAM,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing CCPMAM or cell membrane fractions which contain CCPMAM
are then
contacted with a test compound and binding, stimulation, or inhibition of
activity of either CCPMAM 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
CCPMAM, either in
solution or affixed to a solid support, and detecting the binding of CCPMAM 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.
CCPMAM of the present invention or fragments thereof may be used to screen for
compounds that modulate the activity of CCPMAM. Such compounds may include
agonists,
antagonists, or partial or inverse agonists. In one embodiment, an assay is
performed under conditions
permissive for CCPMAM activity, wherein CCPMAM is combined with at least one
test compound,
and the activity of CCPMAM in the presence of a test compound is compared with
the activity of
CCPMAM in the absence of the test compound. A change in the activity of CCPMAM
in the
presence of the test compound is indicative of a compound that modulates the
activity of CCPMAM.
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Alternatively, a test compound is combined with an in vitro or cell-free
system comprising CCPMAM
under conditions suitable for CCPMAM activity, and the assay is performed. In
either of these
assays, a test compound which modulates the activity of CCPMAM 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 CCPMAM or their mammalian
homologs
may be "knocked out" in an animal model system using homologous recombination
in embryonic stem
(ES) cells. Such techniques are well known in the art and are useful for the
generation of animal
models of human disease. (See, e.g., U.S. Patent Number 5,175,383 and U.S.
Patent Number
5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cell fine,
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 (March, 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 CCPMAM 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 CCPMAM 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 CCPMAM 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 CCPMAM, e.g., by secreting CCPMAM in its milk,
may also serve
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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 CCPMAM and cell cycle proteins and mitosis-associated molecules. In
addition, the
expression of CCPMAM is closely associated with brain, lung, and reproductive
tumor tissue.
Therefore, CCPMAM appears to play a role in cell proliferative, developmental,
and immune
disorders. In the treatment of disorders associated with increased CCPMAM
expression or activity, it
is desirable to decrease the expression or activity of CCPMAM. In the
treatment of disorders
associated with decreased CCPMAM expression or activity, it is desirable to
increase the expression
or activity of CCPMAM.
Therefore, in one embodiment, CCPMAM 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 CCPMAM. Examples of such disorders include, but are not limited
to, a cell proliferative
disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria, polycythemia
vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular,
cancers of the adrenal
gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal tract,
heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,
prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus; a developmental disorder, such as
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, renal tubular acidosis, anemia, C~shing's syndrome,
achondroplastic dwarfism,
Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wilms'
tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-
Magenis syndrome,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas, hereditary
neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,
hypothyroidism,
hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral
palsy, spina bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, and
sensorineural hearing loss; and an
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immune 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
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic
lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclexosis, 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 extracoiporeal
circulation, viral, bacterial,
fungal, parasitic, protozoal, and helininthic infections, and trauma.
In another embodiment, a vector capable of expressing CCPMAM 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 CCPMAM including, but not limited to, those
described above.
In a further embodiment, a composition comprising a substantially purified
CCPMAM 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 CCPMAM
including, but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of CCPMAM
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of CCPMAM including, but not limited to, those listed above.
In a further embodiment, an antagonist of CCPMAM may be administered to a
subject to
treat or prevent a disorder associated with increased expression or activity
of CCPMAM. Examples
of such disorders include, but are not limited to, those cell proliferative,
developmental, and immune
disorders described above. In one aspect, an antibody which specifically binds
CCPMAM 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 CCPMAM.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding CCPMAM may be administered to a subject to treat or prevent a
disorder associated with
increased expression or activity of CCPMAM including, but not limited to,
those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
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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 CCPMAM may be produced using methods which are generally
known in
the art. In particular, purified CCPMAM may be used to produce antibodies or
to screen libraries of
pharmaceutical agents to identify those which specifically bind CCPMAM.
Antibodies to CCPMAM
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 CCPMAM 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 Calinette-Guerin) and Corynebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
CCPMAM 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 CCPMAM 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 CCPMAM 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.
T_m_m__unol. 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
41
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splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
CCPMAM-specific
single chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may
be generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g.,
Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter,
G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for CCPMAM may also be
generated. For example, such fragments include, but are not limited to, F(ab~2
fragments produced
by pepsin digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide
bridges of the F(ab~2 fragments. Alternatively, Fab expression libraries may
be constructed to allow
rapid and easy identification of monoclonal Fab fragments with the desired
specificity. (See, e.g.,
Huse, W.D. et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
CCPMAM and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering CCPMAM 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 CCPMAM. Affinity is
expressed as an
association constant, I~, which is defined as the molar concentration of
CCPMAM-antibody complex
divided by the molax concentrations of free antigen and free antibody under
equilibrium conditions.
The I~ determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple CCPMAM epitopes, represents the average affinity, or
avidity, of the antibodies
for CCPMAM. The I~ determined for a preparation of monoclonal antibodies,
which are
monospecific for a particular CCPMAM epitope, represents a true measure of
affinity. High-affinity
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antibody preparations with Ka ranging from about 109 to 1012 L/mole are
preferred for use in
immunoassays in which the CCPMAM-antibody complex must withstand rigorous
manipulations.
Low-affinity antibody preparations with Ka ranging from about 106 to 10'
L/mole are preferred for use
in immunopurification and similar procedures which ultimately require
dissociation of CCPMAM,
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 CCPMAM-
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 CCPMAM,
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 CCPMAM. 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 CCPMAM. (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, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997)
Nucleic Acids Res.
43
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25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding CCPMAM may be
used for
somatic or gertriline gene therapy. Gene therapy may be performed to (i)
correct a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SLID)-Xl disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cel175:207-216; Crystal, R.G. et al.
(1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D. (1988)
I5 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 CCPMAM expression or regulation causes
disease, the expression
of CCPMAM 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
CCPMAM are treated by constructing mammalian expression vectors encoding
CCPMAM and
introducing these vectors by mechanical means into CCPMAM-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 CCPMAM include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
CCPMAM may be expressed using (i) a constitutively active promoter, (e.g.,
from cytomegalovirus
44
CA 02415077 2003-O1-16
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(CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ~3-
actin genes), (ii) an
inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and
H. Bujard (1992) Proc.
Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-
1769; Rossi, F.M.V.
and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially
available in the T-REX
plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the
plasmids PVGRXR and
PIND; Invitrogen); the FK506/rapamycin inducible promoter; or the
RU486/mifepristone inducible
promoter (Rossi, F.M.V. and Blau, H.M. supra)), or (iii) a tissue-specific
promoter or the native
promoter of the endogenous gene encoding CCPMAM 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 CCPMAM expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding CCPMAM under the control of an independent promoter or
the retrovirus
long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals,
and (iii) a Rev-
2U responsive element (RRE) along with additional retrovirus cis-acting RNA
sequences and coding
sequences required for efficient vector propagation. Retrovirus vectors (e.g.,
PFB and PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R. et
al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses
a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ T-cells), and the
return of transduced cells to a patient are procedures well known to persons
skilled in the art of gene
therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et
CA 02415077 2003-O1-16
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al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al. (1998)
Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding CCPMAM to cells which have one or more genetic
abnormalities with
respect to the expression of CCPMAM. The construction and packaging of
adenovirus-based vectors
are well known to those with ordinary skill in the art. Replication defective
adenovirus vectors have
proven to be versatile for importing genes encoding immunoregulatory proteins
into intact islets in the
pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially
useful adenoviral vectors
are described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus
vectors for gene
therapy"), hereby incorporated by reference. For adenoviral vectors, see also
Antinozzi, P.A. et al.
(1999) Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature
18:389:239-242,
both incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding CCPMAM to target cells which have one or more genetic
abnormalities with
respect to the expression of CCPMAM. The use of herpes simplex virus (HSV)-
based vectors may
be especially valuable for introducing CCPMAM 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 CCPMAM to target cells. The biology of the
prototypic alphavirus,
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Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
CCPMAM into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
CCPMAM-coding RNAs and the synthesis of high levels of CCPMAM in vector
transduced cells.
While alphavirus infection is typically associated with cell lysis within a
few days, the ability to
establish a persistent infection in hamster normal kidney cells (BHK-21 ) with
a variant of Sindbis virus
(SIN) indicates that the lytic replication of alphaviruses can be altered to
suit the needs of the gene
therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide
host range of
alphaviruses will allow the introduction of CCPMAM into a variety of cell
types. The specific
transduction of a subset of cells in a population may require the sorting of
cells prior to transduction.
The methods of manipulating infectious cDNA clones of alphaviruses, performing
alphavirus cDNA
and RNA transfections, and performing alphavirus infections, are well known to
those with ordinary
skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions -10
and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can,
be achieved using triple helix base-pairing methodology. Triple helix pairing
is useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in
Huber, B.E. and B.I. Carr,
Molecular and Immunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-
177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA
by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding CCPMAM.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
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GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding CCPMAM. 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, 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 CCPMAM.
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 CCPMAM
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding CCPMAM may be therapeutically useful, and in the treatment of
disorders associated with
decreased CCPMAM expression or activity, a compound which specifically
promotes expression of
the polynucleotide encoding CCPMAM may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
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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 andlor structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding CCPMAM 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
CCPMAM 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 CCPMAM. The
amount of
hybridization may be quantified, thus forming the basis for a comparison of
the expression of the
polynucleotide both with and without exposure to one or more test compounds.
Detection of a change
in the expression of a polynucleotide exposed to a test compound indicates
that the test compound is
effective in altering the expression of the polynucleotide. A screen for a
compound effective in
altering expression of a specific polynucleotide can be carried out, for
example, using a
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 (Bruise,
T.W. et al. (1997) U.S.
Patent No. 5,686,242; Bruise, 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 bask
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.
Biotechno1.15:462-466.)
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.
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An additional embodiment of the invention relates to the administration of a
composition which
generally comprises an active ingredient formulated with a pharmaceutically
acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and
proteins. Various
formulations are commonly known and are thoroughly discussed in the latest
edition of Remin on's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of
CCPMAM, antibodies to CCPMAM, and mimetics, agonists, antagonists, or
inhibitors of CCPMAM.
The 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,
l0 sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-
acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and
15 proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the lung
have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton, J:S.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of
administration without
needle injection, and obviates the need for potentially toxic penetration
enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
20 ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising CCPMAM or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
25 macromolecule. Alternatively, CCPMAM 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
30 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.
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A therapeutically effective dose refers to that amount of active ingredient,
for example
CCPMAM or fragments thereof, antibodies of CCPMAM, and agonists, antagonists
or inhibitors of
CCPMAM, 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 LDsolEDso ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDso
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to tlxe
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and response
to therapy. Long-acting compositions may be administered every 3 to 4 days,
every week, or
biweekly depending on the half life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ,ug to 100,000 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 CCPMAM may be used
for the
diagnosis of disorders characterized by expression of CCPMAM, or in assays to
monitor patients
being treated with CCPMAM or agonists, antagonists, or inhibitors of CCPMAM.
Antibodies useful
for diagnostic purposes may be prepared in the same manner as described above
for therapeutics.
Diagnostic assays for CCPMAM include methods which utilize the antibody and a
label to detect
CCPMAM 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 .
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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 CCPMAM, including ELISAs, RIAs, and FACS,
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of CCPMAM
expression. Normal or standard values for CCPMAM expression are established by
combining body
fluids or cell extracts taken from normal mammalian subjects, for example,
human subjects, with
antibodies to CCPMAM under conditions suitable for complex formation. The
amount of standard
complex formation may be quantitated by various methods, such as photometric
means. Quantities of
CCPMAM 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 CCPMAM
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 CCPMAM
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of CCPMAM, and to monitor regulation of CCPMAM levels during
therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding CCPMAM or closely related
molecules may be
used to identify nucleic acid sequences which encode CCPMAM. The specificity
of the probe,
whether it is made from a highly specific region, e.g., the S'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
CCPMAM, allelic variants,
2S or related sequences.
Probes may also be used for the detection of related sequences, and may have
at least SO%
sequence identity to any of the CCPMAM encoding sequences. The hybridization
probes of the
subject invention may be DNA or RNA and may be derived from the sequence of
SEQ ID N0:4-6 or
from genomic sequences including promoters, enhancers, and introns of the
CCPMAM gene.
Means for producing specific hybridization probes for DNAs encoding CCPMAM
include the
cloning of polynucleotide sequences encoding CCPMAM or CCPMAM derivatives into
vectors for
the production of mRNA probes. Such vectors are known in the art, axe
commercially available, and
may be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
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polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding CCPMAM may be used for the diagnosis of
disorders
associated with expression of CCPMAM. Examples of such disorders include, but
are not limited to,
a cell proliferative disorder, such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis,
paroxysmal nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall bladder,
ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis,
prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus;
a developmental disorder,
such as 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, renal tubular acidosis, anemia, Cushing's
syndrome, achondroplastic
dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal
dysgenesis, WAGR syndrome
(Wilins' tumor, aniridia, genitourinary abnormalities, and mental
retardation), Smith-Magens syndrome,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas, hereditary
neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,
hypothyroidism,
hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral
palsy, spina bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, and
sensorineural hearing loss; and an
immune 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
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic
lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
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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. The
polynucleotide sequences
encoding CCPMAM 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 CCPMAM
expression. Such
qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding CCPMAM may be useful
in assays
that detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding CCPMAM 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 CCPMAM in the
sample indicates the presence of the associated disorder. Such assays may also
be used to evaluate
the efficacy of a particular therapeutic treatment regimen in animal studies,
in clinical trials, or to
monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of w
CCPMAM, 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 CCPMAM, under conditions suitable
for hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from normal
subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
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With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding
CCPMAM 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 CCPMAM, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
CCPMAM, 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
i5 encoding CCPMAM 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 CCPMAM are used to amplify
DNA using the
polymerase chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the
secondary and tertiary structures of PCR products in single-stranded form, and
these differences are
detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such as
DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of
individual
overlapping DNA fragments which assemble into a common consensus sequence.
These computer-
based methods filter out sequence variations due to laboratory preparation of
DNA and sequencing
errors using statistical models and automated analyses of DNA sequence
chromatograms. In the
alternative, SNPs may be detected and characterized by mass spectrometry
using, for example, the
high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
Methods which may also be used to quantify the expression of CCPMAM include
radiolabeling or biotinylating nucleotides, coamplification of a control
nucleic acid, and interpolating
CA 02415077 2003-O1-16
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results from standard curves. (See, e.g., Melby, P.C. et al. (1993) J.
Immunol. Methods 159:235-244;
Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed of
quantitation of multiple samples
may be accelerated by running the assay in a high-throughput format where the
oligomer or
polynucleotide of interest is presented in various dilutions and a
spectrophotometric or colorimetric
response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic
profile.
In another embodiment, CCPMAM, fragments of CCPMAM, or antibodies specific for
CCPMAM may be used as elements on a microarray. The microarray may be used to
monitor or
measure protein-protein interactions, drug-target interactions, and gene
expression profiles, as
described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
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or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also. be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a
l0 signature similar to that of a compound with known toxicity, it is likely
to share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain
expression information
from a large number of genes and gene families. Ideally, a genome-wide
measurement of expression
provides the highest quality signature. Even genes whose expression is not
altered by any tested
compounds are important as well, as the levels of expression of these genes
are used to normalize the
rest of the expression data. The normalization procedure is useful for
comparison of expression data
after treatment with different compounds. While the assignment of gene
function to elements of a
toxicant signature aids in interpretation of toxicity mechanisms, knowledge of
gene function is not
necessary for the statistical matching of signatures which Ieads to prediction
of toxicity. (See, for
example, Press Release 00-02 from the National Institute of Environmental
Health Sciences, released
February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.)
Therefore, it is
important and desirable in toxicological screening using toxicant signatures
to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological sample
containing nucleic, acids with the test compound. Nucleic acids that are
expressed in the treated
biological sample are hybridized with one or more probes specific to the
polynucleotides of the present
invention, so that transcript levels corresponding to the polynucleotides of
the present invention may be
quantified. The,transcript levels in the treated biological sample are
compared with levels in an
untreated biological sample. Differences in the transcript levels between the
two samples are
indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern. of protein expression in a particular tissue or cell type. Each
protein component of a proteome
can be subjected individually to further analysis. Proteome expression
patterns, or profiles, are
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analyzed by quantifying the number of expressed proteins and their relative
abundance under given
conditions and at a given time. A profile of a cell's pxoteome may thus be
generated by separating
and analyzing the polypeptides of a particular tissue or cell type. In one
embodiment, the separation is
achieved using two-dimensional gel electrophoresis, in which proteins from a
sample are separated by
isoelectric focusing in the first dimension, and then according to molecular
weight by sodium dodecyl
sulfate slab gel electrophoresis in the second dimension (Steiner and
Anderson, supra). The proteins
are visualized in the gel as discrete and uniquely positioned spots, typically
by staining the geI with an
agent such as Coomassie Blue or silver or fluorescent stains. The optical
density of each protein spot
is generally proportional to the level of the protein in the sample. The
optical densities of equivalently
positioned protein spots from different samples, for example, from biological
samples either treated or
untreated with a test compound or therapeutic agent, are compared to identify
any changes in protein
spot density related to the treatment. The proteins in the spots are partially
sequenced using, for
example, standard methods employing chemical or enzymatic cleavage followed by
mass
spectrometry. The identity of the protein in a spot may be determined by
comparing its partial
sequence, preferably of at least 5 contiguous amino acid residues, to the
polypeptide sequences of the
present invention. In some cases, further sequence data may be obtained for
definitive protein
identification.
A proteomic profile may also be generated using antibodies specific for CCPMAM
to quantify
the levels of CCPMAM expression. In one embodiment, the antibodies are used as
elements on a .
microarray, and protein expression levels are quantified by exposing the
microarray to the sample and
detecting the levels of protein bound to each array element (Lueking, A. et
al. (1999) Anal. Biochem.
270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection
may be performed by
a variety of methods known in the art, for example, by reacting the proteins
in the sample with a thiol-
or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at each
array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances fox some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to rapid
degradation of mRNA, so proteamic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
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sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological sample.
A difference in the amount of protein between the two samples is indicative of
a toxic response to the
test compound in the treated sample. Individual proteins are identified by
sequencing the amino acid
residues of the individual proteins and comparing these partial sequences to
the polypeptides of the
present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared
with the amount in an untreated biological sample. A difference in the amount
of protein between the
two samples is indicative of a toxic response to the test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad.
Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalom D. et
al. (1995) PCT application W095/35505; Holler, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA
94:2150-2155; and Holler, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
Various types of
microarrays are well known and thoroughly described in DNA Microarrays: A
Practical Approach,
M. Schena, ed. (1999) Oxford University Press, London, hereby expressly
incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding CCPMAM
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences may
be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a multi-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial chromosomes
(HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; 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).
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(See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.)
Fluorescent in situ hybridization (FISI~ may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding CCPMAM 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 translocation,
inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, CCPMAM, 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 CCPMAM 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 WO84/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with CCPMAM,
or fragments
thereof, and washed, Bound CCPMAM is then detected by methods well known in
the art. Purified
CCPMAM 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 CCPMAM specifically compete with a test compound
for binding
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CCPMAM. In this manner, antibodies can be used to detect the presence of any
peptide which
shares one or more antigenic determinants with CCPMAM.
In additional embodiments, the nucleotide sequences which encode CCPMAM may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are, therefore,
to be construed as merely illustrative, and not limitative of the remainder of
the disclosure in any way
whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below,
including U.S. Ser No. 60/236,860 and U.S. Ser No. 601220,111, are expressly
incorporated by
reference herein.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some
tissues were
homogenized and lysed in guanidinium isothiocyanate, while others were
homogenized and lysed in
phenol or in a suitable mixture of denaturants, such as TRIZOL (Life
Technologies), a monophasic
solution of phenol and guanidine isothiocyanate. The resulting lysates were
centrifuged over 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,
61.
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units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo Alto
CA), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli cells
including XL1-Blue, XLl-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
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation such
as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200
thermal cycler
(MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
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the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the
techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBankprimate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein
family
databases such as PFAM. (HMM is a probabilistic approach which analyzes
consensus primary
structures of gene families. See, for example, Eddy, S.R. (1996) Curr. Opin.
Struct. Biol. 6:361-365.)
The queries were performed using programs based on BLAST, FASTA, BLIMPS, and
HMMER.
The Incyte cDNA sequences were assembled to produce full length polynucleotide
sequences.
Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched
sequences, or
Genscan-predicted coding sequences (see Examples IV and V) were used to extend
Incyte cDNA
assemblages to full length. Assembly was performed using programs based on
Phred, Phrap, and
Consed, and cDNA assemblages were screened for open reading frames using
programs based on
GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were
translated to derive
the corresponding full length polypeptide sequences. Alternatively, a
polypeptide of the invention may
begin at any of the methionine residues of the full length translated
polypeptide. Full length polypeptide
sequences were subsequently analyzed by querying against databases such as the
GenBank protein
databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and
hidden
Markov model (HMM)-based protein family databases such as PFAM. Full length
polynucleotide
sequences are also analyzed using MACDNASIS PRO software (Hitachi Software
Engineering,
South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and
polypeptide
sequence alignments are generated using default parameters specified by the
CLUSTAL algorithm as
incorporated into the MEGALIGN multisequence alignment program (DNASTAR),
which also
calculates the percent identity between aligned sequences.
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Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and threshold
parameters. The first column of Table 7 shows the tools, programs, and
algorithms used, the second
column provides brief descriptions thereof, the third column presents
appropriate references, all of
which are incorporated by reference herein in their entirety, and the fourth
column presents, where
applicable, the scores, probability values, and other parameters used to
evaluate the strength of a
match between two sequences (the higher the score or the lower the probability
value, the greater the
identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide and
polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:4-6. Fragments from about 20 to about 4000 nucleotides which are useful in
hybridization and
amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative cell cycle proteins and mitosis-associated molecules were initially
identified by
running the Genscan gene identification program against public genomic
sequence databases (e.g.,
gbpri and gbhtg). Genscan is a general-purpose gene identification program
which analyzes genomic
DNA sequences from a variety of organisms (See Burge, C. and S. Marlin (1997)
J. Mol. Biol.
268:78-94, and Burge, C. and S. Marlin (1998) Curr. Opin. Sti~uct. Biol. 8:346-
354). The program
concatenates predicted exons to form an assembled cDNA sequence extending from
a methionine to
a stop codon. The output of Genscan is a FASTA database of polynucleotide and
polypeptide
sequences. The maximum range of sequence for Genscan to analyze at once was
set to 30 kb. To
determine which of these Genscan predicted cDNA sequences encode cell cycle
proteins and mitosis-
associated molecules, the encoded polypeptides were analyzed by querying
against PFAM models for
cell cycle proteins and mitosis-associated molecules. Potential cell cycle
proteins and mitosis-
associated molecules were also identified by homology to Incyte cDNA sequences
that had been
annotated as cell cycle proteins and mitosis-associated molecules. These
selected Genscan-predicted
sequences were then compared by BLAST analysis to the genpept and gbpri public
databases.
Where necessary, the Genscan-predicted sequences were then edited by
comparison to the top
BLAST hit from genpept to correct errors in the sequence predicted by Genscan,
such as extra or
omitted exons. BLAST analysis was also used to find any Incyte cDNA or public
cDNA coverage of
the Genscan-predicted sequences, thus providing evidence for transcription.
When Incyte cDNA
coverage was available, this information was used to correct or confirm the
Genscan predicted
sequence. Full length polynucleotide sequences were obtained by assembling
Genscan-predicted
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Boding sequences with Incyte cDNA sequences and/or public cDNA sequences using
the assembly
process described in Example llT. Alternatively, full length polynucleotide
sequences were derived
entirely from edited or unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Seguences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
IlI were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Seguences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBankprotein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenB ink protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenB ink
protein homolog. The
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GenBank protein homolog, the chimeric protein, or both were used as probes to
search for homologous
genomic sequences from the public human genome databases. Partial DNA
sequences were
therefore "stretched" or extended by the addition of homologous genomic
sequences. The resultant
stretched sequences were examined to determine whether it contained a complete
gene.
VI. Chromosomal Mapping of CCPMAM Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:4-6 were compared with
sequences from the mcyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:4-6 were assembled into clusters of contiguous and overlapping
sequences using
l0 assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances
are based on genetic markers mapped by Genethon which provide boundaries for
radiation hybrid
markers whose sequences were included in each of the clusters. Human genome
maps and other
resources available to the public, such as the NCBI "GeneMap' 99" World Wide
Web site
(http://www.ncbi.nlm.nih.gov/genemapn, can be employed to determine if
previously identified disease
genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel
(1995) supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
search can be modified to determine whether any particular match is
categorized as exact or similar.
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The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identit'~
x minimum { length(Seq. 1 ), length(Seq. 2) }
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding CCPMAM are analyzed with
respect to the
tissue sources from which they were derived. For example, some full length
sequences are
assembled, at least in part, with overlapping Incyte cDNA sequences (see
Example III]. Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following organ/tissue categories: cardiovascular
system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female; genitalia,
male; germ cells; heroic and immune system; liver; musculoskeletal system;
nervous system;
pancreas; respiratory system; sense organs; skin; stomatognathic system;
unclassified/mixed; or
urinary tract. The number of libraries in each category is counted and divided
by the total number of
libraries across all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
by the total number of libraries across all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding CCPMAM. cDNA sequences and cDNA
library/tissue information are found in the LIF'ESEQ GOLD database (Incyte
Genomics, Palo Alto
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CA).
VIII. Extension of CCPMAM Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68 °C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the ait. 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)ZSO4,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE
enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the
following parameters
for primer pair PCI A and PCI B : Step 1: 94 ° C, 3 min; Step 2: 94
° C, 15 sec; Step 3 : 60 ° C, 1 min;
Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step
6: 68°C, 5 min; Step 7: storage
at 4°C. In the alternative, the parameters for primer pair T7 and SI~+
were as follows: Step 1: 94°C;
3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4:
68°C, 2 min; Step 5: Steps 2, 3, and 4
repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C.
'
The concentration of DNA in each well was determined by dispensing 100 ~1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~ 1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~d to 10 ~cl aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison Wl), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
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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 lipase (New England Biolabs, Beverly MA) into
pUC 18 vector
(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to
fill-in restriction
site overhangs, and transfected into competent E. coli cells. Transformed
cells were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37 °C in 384-
well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94 ° C, 3 min; Step 2: 94 ° C, 15 sec; Step
3: 60 ° C, 1 min; Step 4: 72 ° C, 2 min; Step
5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7:
storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with
20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5'regulatory sequences using the above procedure along with
oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:4-6 are employed to screen cDNAs,
genomic
DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about
20 base pairs, is
specifically described, essentially the same procedure is used with larger
nucleotide fragments.
Oligonucleotides are designed using state-of the-art software such as OLIGO
4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ,uCi of
['y-32P] adenosine
triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase
(DuPont NEN, Boston
MA). The labeled oligonucleotides are substantially purified using a SEPHADEX
G-25 superf'me 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
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hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
X. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.), mechanical
microspotting technologies,, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), sera).
Suggested substrates include silicon, silica, glass slides, glass chips, and
silicon wafers. Alternatively, a
procedure analogous to a dot or slot blot may also be used to arrange and link
elements to the surface
of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
A typical array may
be produced using available methods and machines well known to those of
ordinary skill in the art and
may contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science
270:467-470; Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and
J. Hodgson (1998)
Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described
in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pgl~I oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/~l RNase inhibitor, 500 ~M dATP, 500 ~M dGTP, 500 ~M
dTTP, 40 ~M
dCTP, 40 ~M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
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transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of O.SM sodium
hydroxide and
incubated for 20 minutes at 85° C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and resuspended
in 14 ~1 SX SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5 ~ g.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522, incorporated herein by reference. 1 ~l of the array
element DNA, at an average
concentration of 100 ng/~ 1, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINI~ER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2%
SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 ~l of sample mixture consisting of 0.2 ~g
each of Cy3 and
71
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Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of 140
~1 of 5X SSC in a corner of the chamber. The chamber containing the arrays is
incubated for about
6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in
a first wash buffer ( 1X SSC, 0.1 %
SDS), three times for 10 minutes each at 45° C in a second wash buffer
(0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
tluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that location
to be correlated with a weight ratio of hybridizing species of 1:100,000. When
two samples from
different sources (e.g., representing test and control cells), each labeled
with a different fluorophore,
are hybridized to a single array for the purpose of identifying genes that are
differentially expressed,
the calibration is done by labeling samples of the calibrating cDNA with the
two fluorophores and
adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
72
CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping emission
spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
XI. Complementary PolynucIeotides
Sequences complementary to the CCPMAM-encoding sequences, or any parts
thereof, are
used to detect, decrease, or inhibit expression of naturally occurring CCPMAM.
Although use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4..06 software (National Biosciences) and the coding
sequence of CCPMAM.
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 CCPMAM-
encoding transcript.
XII. Expression of CCPMAM
Expression and purification of CCPMAM is achieved using bacterial or virus-
based
expression systems. For expression of CCPMAM 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 CCPMAM upon induction with
isopropyl beta-D-
thiogalactopyranoside (IPTG). Expression of CCPMAM 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 CCPMAM by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera fru~i erda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K. et
73
CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
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, CCPMAM is synthesized as a fusion protein with,
e.g.,
glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-
His, permitting rapid,
single-step, affinity-based purification of recombinant fusion protein from
crude cell lysates. GST, a
26-kilodalton enzyme from Schistosoma japonicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein~activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
CCPMAM 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 CCPMAM obtained by these methods can be used
directly in the
assays shown in Examples XVI and XVII, where applicable.
XIII. Functional Assays
CCPMAM function is assessed by expressing the sequences encoding CCPMAM at
physiologically elevated levels in mammalian cell culture systems. cDNA is
subcloned into a
mammalian expression vector containing a strong promoter that drives high
levels of cDNA
expression. Vectors of choice include PCMV SPORT (Life Technologies) and
PCR3.1 (Invitrogen,
Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ~g of
recombinant vector
are transiently transfected into a human cell line, for example, an
endothelial or hematopoietic cell line,
using either Iiposome formulations or electxoporation. 1-2 ,ug of an
additional plasmid containing
sequences encoding a marker protein are co-transfected. Expression of a marker
protein provides a
means to distinguish transfected cells from nontransfected cells and is a
reliable predictor of cDNA
expression from the recombinant vector. Marker proteins of choice include,
e.g., Green Fluorescent
Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry
(FCM), an
automated, laser optics-based technique, is used to identify transfected cells
expressing GFP or CD64-
GFP and to evaluate the apoptotic state of the cells and other cellular
properties. FCM detects and
quantifies the uptake of fluorescent molecules that diagnose events preceding
or coincident with cell
death. These events include changes in nuclear DNA content as measured by
staining of DNA with
propidium iodide; changes in cell size and granularity as measured by forward
light scatter and 90
degree side light scatter; down-regulation of DNA synthesis as measured by
decrease in
bromodeoxyuridine uptake; alterations in expression of cell surface and
intracellular proteins as
74
CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
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 CCPMAM on gene expression can be assessed using highly
purified
populations of cells transfected with sequences encoding CCPMAM 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 CCPMAM and other genes of interest can be
analyzed by
northern analysis or microarray techniques.
XIV. Production of CCPMAM Specific Antibodies
CCPMAM 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 CCPMAM amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-KL,H complex in complete Freund's adjuvant. Resulting antisera
are tested for
antipeptide and anti-CCPMAM activity by, for example, binding the peptide or
CCPMAM to a
substrate, blocking with 1 °1o BSA, reacting with rabbit antisera,
washing, and reacting with radio-
iodinated goat anti-rabbit IgG.
XV. Purification of Naturally Occurring CCPMAM Using Specific Antibodies
Naturally occurring or recombinant CCPMAM is substantially purified by
immunoaffinity
chromatography using antibodies specific for CCPMAM. An immunoaffinity column
is constructed
by covalently coupling anti-CCPMAM antibody to an activated chromatographic
resin, such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
blocked and washed according to the manufacturer's instructions.
Media containing CCPMAM are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of CCPMAM
(e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/CCPMAM 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 CCPMAM is collected.
XVI. Identification of Molecules Which Interact with CCPMAM
CCPMAM, or biologically active fragments thereof, are labeled with lzsI Bolton-
Hunter
reagent. (See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-
X39.) Candidate
molecules previously arrayed in the wells of a mufti-well plate are incubated
with the labeled
CCPMAM, washed, and any wells with labeled CCPMAM complex are assayed. Data
obtained
using different concentrations of CCPMAM are used to calculate values for the
number, affinity, and
association of CCPMAM with the candidate molecules.
Alternatively, molecules interacting with CCPMAM 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).
CCPMAM may also be used in the PATHCALLING process (CuraGen Corp., New Haven
CT) which employs the yeast two-hybrid system in a high-throughput manner to
determine all
interactions between the proteins encoded by two large libraries of genes
(Nandabalan, K. et al.
(2000) U.S. Patent No. 6,057,101).
XVII. Demonstration of CCPMAM Activity
CCPMAM activity is demonstrated by measuring the induction of cell cycle
progression when
CCPMAM is expressed at physiologically elevated levels in mammalian cell
culture systems.
CCPMAM cDNA is subcloned into a mammalian expression vector containing a
strong promoter that
drives high levels of cDNA expression. Vectors of choice include pCMV SPORT
(Life
Technologies, Gaithersburg, MD) and pCR 3.1 (Invitrogen, Carlsbad, CA), both
of which contain the
cytomegalovirus promoter. 5-10 /.cg of recombinant vector are transiently
transfected into a human
cell line, preferably of endothelial or hematopoietic origin, using either
liposome formulations or
electroporation. 1-2 ~cg of an additional plasmid containing sequences
encoding a marker protein are
co-transfected. Expression of a marker protein provides a means to distinguish
transfected cells from
nontransfected cells and is a reliable predictor of cDNA expression from the
recombinant vector.
Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP)
(Clontech, Palo Alto, CA).
Flow cytometry detects, and quantifies the uptake of fluorescent molecules
that diagnose events
76
CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
preceding or coincident with cell cycle progression or terminal
differentiation. These events include
changes in nuclear DNA content as measured by staining of DNA with propidium
iodide; changes in
cell size and granularity as measured by forward light scatter and 90 degree
side light scatter; up or
down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine
uptake; alterations
in expression of cell surface and intracellular proteins as measured by
reactivity with specific
antibodies; and alterations in plasma membrane composition as measured by the
binding of
fluorescein-conjugated Annexin V protein to the cell surface.
An assay for CCPMAM activity measures cell proliferation as the amount of
newly initiated
DNA synthesis in Swiss mouse 3T3 cells. A plasmid containing polynucleotides
encoding CCPMAM
is transfected into quiescent 3T3 cultured cells using methods well known in
the art. The transiently
txansfected cells are then incubated in the presence of [3H]thymidine, a
radioactive DNA precursor.
Where applicable, varying amounts of CCPMAM ligand are added to the
transfected cells.
Incorporation of [3H]thymidine into acid-precipitable DNA is measured over an
appropriate time
interval, and the amount incorporated is directly proportional to the amount
of newly synthesized DNA
and CCPMAM activity.
Alternatively, CCPMAM activity is measured by the cyclin-ubiquitin ligation
assay
(Aristarkhov, A. et al. (1996) Proc. Natl. Acid. Sci. USA 93:4294-4299). The
reaction contains in a
volume of 10 ~l, 40 mM Tris HCl (pH 7.6), 5 mM Mg C12, 0.5 mM ATP, 10 mM
phosphocreatine, 50
~sg of creative phosphokinase/ml, 1 mg reduced carboxymethylated bovine serum
albumin/ml, 50 ~r.M
ubiquitin, 1 ~cM ubiquitin aldehyde, 1-2 pmol'ZSI-labeled cyclin B, 1 pmol E1,
1 ,uM okadaic acid, l 0
,ug of protein of M-phase fraction 1A (containing active E3-C and essentially
free of E2-C), and
varying amounts of CCPMAM. The reaction is incubated at 18 °C for 60
minutes. Samples are then
separated by electrophoresis on SDS/12% polyacrylamide gel. The amount of lzsl-
cyclin-ubiquitin
formed is quantified by PhosphorImager analysis. The amount of cyclin-
ubiquitin formation is
proportional to the amount of CCPMAM in the reaction.
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the invention.
Although the invention has been described in connection with certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which are obvious
to those skilled in molecular biology or related fields are intended to be
within the scope of the
following claims.
77
CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
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CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
<110> INCYTE GENOMICS, INC.
BAUGHN, Mariah R.
LAL, Preeti
LU, Dyung Aina M.
NGUYEN, Danniel B.
TRIBOULEY, Catherine M.
YAO, Monique G.
YUE, Henry
<120> CELL CYCLE PROTEINS AND MITOSIS-ASSOCIATED MOLECULES
<130> PI-0245 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/220,111; 60/236,860
<151> 2000-07-21; 2000-09-29
<160> 6
<170> PERL Program
<210> 1
<211> 1049
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 1001668CD1
<400> 1
Met Leu Cys Trp Gly Asn Ala Ser Phe Gly Gln Leu Gly Leu Gly
1 5 10 15
Gly Ile Asp Glu Glu Ile Val Leu Glu Pro Arg Lys Ser Asp Phe
20 25 30
Phe Ile Asn Lys Arg Val Arg Asp Val Gly Cys G1y Leu Arg His
35 40 45
Thr Val Phe Val Leu Asp Asp Gly Thr Val Tyr Thr Cys Gly Cys
50 55 60
Asn Asp Leu Gly G1n Leu Gly His Glu Lys Ser Arg Lys Lys Pro
f5 70 75
Glu G1n Val Val Ala Leu Asp A1a Gln Asn Ile Val Ala Val Ser
80 85 90
Cys Gly Glu Ala His Thr Leu Ala Leu Asn Asp Lys Gly Gln Val
95 100 105
Tyr Ala Trp Gly Leu Asp Ser Asp Gly Gln Leu Gly Leu Val Gly
110 115 120
Ser Glu Glu Cys Ile Arg Val Pro Arg Asn Ile Lys Ser Leu Ser
125 130 135
Asp Ile Gln Ile Val Gln Val Ala Cys Gly Tyr Tyr His Ser Leu
140 145 150
A1a Leu Ser Lys Ala Ser Glu Val Phe Cys Trp Gly Gln Asn Lys
155 160 165
Tyr Gly Gln Leu Gly Leu Gly Thr Asp Cys Lys Lys Gln Thr Ser
170 175 180
1/9
CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
Pro Gln Leu Leu Lys Ser Leu Leu Gly Ile Pro Phe Met Gln Val
185 190 195
Ala Ala Gly Gly Ala His Ser Phe Val Leu Thr Leu Ser Gly Ala
200 205 210
Ile Phe Gly Trp Gly Arg Asn Lys Phe Gly Gln Leu Gly Leu Asn
215 220 225
Asp Glu Asn Asp Arg Tyr Val Pro Asn Leu Leu Lys Ser Leu Arg
230 235 240
Ser Gln Lys Ile Val Tyr Ile Cys Cys Gly Glu Asp His Thr Ala
245 250 255
Ala Leu Thr Lys Glu Gly Gly Val Phe Thr Phe Gly Ala Gly Gly
260 265 270
Tyr Gly Gln Leu Gly His Asn Ser Thr Ser His Glu Ile Asn Pro
275 280 285
Arg Lys Val Phe Glu Leu Met Gly Ser Ile Val Thr Glu Ile Ala
290 295 300
Cys Gly Arg Gln His Thr Ser Ala Phe Val Pro Ser Ser Gly Arg
305 310 315
Ile Tyr Ser Phe Gly Leu Gly Gly Asn Gly Gln Leu Gly Thr Gly
320 325 330
Ser Thr Ser Asn Arg Lys Ser Pro Phe Thr Val Lys Gly Asn Trp
335 340 345
Tyr Pro Tyr Asn Gly Gln Cys Leu Pro Asp Ile Asp Ser Glu Glu
350 355 360
Tyr Phe Cys Val Lys Arg Ile Phe Ser Gly Gly Asp Gln Ser Phe
365 370 375
Ser His Tyr Ser Ser Pro Gln Asn Cys Gly Pro Pro Asp Asp Phe
380 385 390
Arg Cys Pro Asn.Pro Thr Lys Gln Ile Trp Thr Val Asn G1u Ala
395 400 405
Leu Ile Gln Lys Trp Leu Ser Tyr Pro Ser Gly Arg Phe Pro Val
410 415 420
Glu Ile Ala Asn Glu Ile Asp Gly Thr Phe Ser Ser Ser Gly Cys
425 430 435
Leu Asn Gly Ser Phe Leu Ala Va1 Ser Asn Asp Asp His Tyr Arg
440 445 450
Thr Gly Thr Arg Phe Ser Gly Val Asp Met Asn Ala Ala Arg Leu
455 460 465
Leu Phe His Lys Leu Ile Gln Pro Asp His Pro Gln Ile Ser Gln
470 475 480
Gln Va1 Ala Ala Ser Leu Glu Lys Asn Leu I1e Pro Lys Leu Thr
485 490 495
Ser Ser Leu Pro Asp Val Glu Ala Leu Arg Phe Tyr Leu Thr Leu
500 505 510
Pro Glu Cys Pro Leu Met Ser Asp Ser Asn Asn Phe Thr Thr 21e
515 520 525
Ala Ile Pro Phe Gly Thr A1a Leu Val Asn Leu Glu Lys Ala Pro
530 535 540
Leu Lys Val Leu Glu Asn Trp Trp Ser Val Leu Glu Pro Pro Leu
545 550 555
Phe Leu Lys I1e Val Glu Leu Phe Lys Glu Val Val Val His Leu
560 565 570
Leu Lys Leu Tyr Lys Ile Gly Ile Pro Pro Ser Glu Arg Arg Ile
575 580 585
Phe Asn Ser Phe Leu His Thr Ala Leu Lys Val Leu Glu Ile Leu
590 595 600
2/9
CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
His Arg Val Asn Glu Lys Met Gly Gln Ile Ile Gln Tyr Asp Lys
605 610 615
Phe Tyr Ile His Glu Val Gln Glu Leu Ile Asp Ile Arg Asn Asp
620 625 630
Tyr Ile Asn Trp Val Gln Gln Gln Ala Tyr Gly Met Leu Ala Asp
635 640 645
Ile Pro Val Thr Ile Cys Thr Tyr Pro Phe Val Phe Asp Ala Gln
650 655 660
Ala Lys Thr Thr Leu Leu G1n Thr Asp Ala Val Leu Gln Met Gln
665 670 675
Met Ala Ile Asp Gln Ala His Arg Gln Asn Val Ser Ser Leu Phe
680 685 690
Leu Pro Val Ile Glu Ser Val Asn Pro Cys Leu Ile Leu Val Val
695 700 705
Arg Arg Glu Asn Ile Val Gly Asp Ala Met Glu Val Leu Arg Lys
710 715 720
Thr Lys Asn Ile Asp Tyr Lys Lys Pro Leu Lys Val Ile Phe Val
725 730 735
Gly Glu Asp Ala Val Asp Ala Gly Gly Val Arg Lys Glu Phe Phe
740 745 750
Leu Leu Tle Met Arg Glu Leu Leu Asp Pro Lys Tyr Gly Met Phe
755 760 765
Arg Tyr Tyr Glu Asp Ser Arg Leu Ile Trp Phe Ser Asp Lys Thr
770 775 780
Phe Glu Asp Ser Asp Leu Phe His Leu Ile Gly Val Ile Cys G1y
785 790 795
Leu Ala I1e Tyr Asn Cys Thr Ile Val Asp Leu His Phe Pro Leu
800 805 810
Ala Leu Tyr Lys Lys Leu Leu Lys Lys Lys Pro Ser Leu Asp Asp
815 820 825
Leu Lys Glu Leu Met Pro Asp Val Gly Arg Ser Met Gln Gln Leu
830 835 840
Leu Asp Tyr Pro Glu Asp Asp Ile Glu Glu Thr Phe Cys Leu Asn
845 850 855
Phe Thr Ile Thr Val Glu Asn Phe Gly Ala Thr G1u Val Lys G1u
860 865 870
Leu Val Leu Asn Gly Ala Asp Thr Ala Val Asn Lys Gln Asn Arg
875 880 885
Gln Glu Phe Val Asp Ala Tyr Val Asp Tyr Ile Phe Asn Lys Ser
890 895 900
Val Ala Ser Leu Phe Asp Ala Phe His Ala G1y Phe His Lys Val
905 910 915
Cys Gly Gly Lys Val Leu Leu Leu Phe Gln Pro Asn Glu Leu Gln
920 925 930
A1a Met Val Ile Gly Asn Thr Asn Tyr Asp Trp Lys Glu Leu Glu
935 940 945
Lys Asn Thr Glu Tyr Lys Gly Glu Tyr Trp Ala Glu His Pro Thr
950 955 960
Ile Lys Ile Phe Trp Glu Val Phe His Glu Leu Pro Leu Glu Lys
965 970 975
Lys Lys Gln Phe Leu Leu Phe ~Leu Thr Gly Ser Asp Arg Ile Pro
980 985 990
Ile Leu Gly Met Lys Ser Leu Lys Leu Val Ile Gln Ser Thr Gly
995 1000 1005
Gly Gly Glu Glu Tyr Leu Pro Val Ser His Thr Cys Phe Asn Leu
1010 1015 1020
3/9
CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
Leu Asp Leu Pro Lys Tyr Thr Glu Lys Glu Thr Leu Arg Ser Lys
1025 1030 1035
Leu Ile Gln Ala Ile Asp His Asn Glu Gly Phe Ser Leu Ile
1040 1045
<210> 2
<211> 375
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 2226248CD1
<400> 2
Met Asn Gly Leu Gly Ile Asp Lys Ile Asn Glu Trp His Ala Gly
1 5 10 15
Glu Ile Ile Lys Leu Ile Ala Asp Tyr Ser Pro Asp Asp Ile Phe
20 25 30
Asn Ala Asp Glu Thr Gly Va1 Phe Phe Gln Leu Leu Pro Gln His
35 40 45
Thr Leu Ala Ala Lys Gly Asp His Cys Arg Gly Gly Lys Lys Ala
50 55 60
Lys Gln Arg Leu Thr Ala Leu Phe Cys Cys Asn Ala Ser Gly Thr
65 70 75
Glu Lys Met Arg Pro Leu Ile Val G1y Arg Ser Ala Ser Pro His
80 85 90
Cys Leu Lys Asn Ile His Ser Leu Pro Cys Asp Tyr Arg A1a Asn
95 100 105
Gln Trp Ala Trp Met Thr Arg Asp Leu Phe Asn Glu Trp Leu Met
110 115 120
Gln Val Asp Ala Arg Met Lys Arg Ala Glu Arg Arg Ile Leu Leu
125 130 135
Leu Ile Asp Asn Cys Ser Ala His Asn Met Leu Pro His Leu Glu
140 145 150
Arg Ile Gln Val Gly Tyr Leu Pro Ser Asn Cys Thr Ala Val Leu
155 160 165
Gln Pro Leu Asn Leu G1y Ile Ile His Thr Met Lys Val Leu Tyr
170 175 180
Gln Ser His Leu Leu Lys Gln Ile Leu Leu Lys Leu Asn Ser Ser
185 190 195
Glu Asp Gln Glu Glu Val Asp Ile Lys G1n Ala Ile Asp Met Ile
200 205 210
Ala Ala Ala Trp Trp Ser Val Lys Pro Ser Thr Val Val Lys Cys
215 220 225
Trp Gln Lys Ala Gly Ile Val Pro Met Glu Phe Ala Glu Cys Asp
230 235 240
Thr Glu Ser Ala Ala Ser Glu Pro Asp Ile Ala Ile Glu Lys Leu
245 250 255
Trp His Thr Val Ala Ile Ala Thr Cys Val Pro Asn Glu Val Asn
260 265 270
Phe Gln Asp Phe Val Thr Ala Asp Asp Asp Leu I1e Ile Ser Gln
275 280 285
Asp Thr Asp Ile Tle Gln Asp Met Val Ala Gly Glu Asn Thr Ser
290 295 300
Glu Ala Gly Ser Glu Asp Glu Gly Glu Val Ser Leu Pro Glu Gln
4/9
CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
305 310 315
Pro Lys Val Thr Ile Thr Glu Ala Ile Ser Ser Val Gln Lys Leu
320 325 330
Arg Gln Phe Leu Ser Thr Cys Val Asp Ile Pro Asp Ala Ile Phe
335 340 345
Gly Gln Leu Asn Gly Ile Asp Glu Tyr Leu Met Lys Arg Va1 Thr
350 355 360
Gln Thr Leu Ile Asp Ser Lys Ile Thr Asp Phe Leu G1n Thr Lys
365 370 375
<210> 3
<211> 351
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 3049930CD1,
<400> 3
Met Ser Arg Arg Phe Phe Val Asp I1e Leu Thr Leu Leu Ser Ser
1 5 10 15
His Cys Gln Leu Cys Pro Ala Ala Arg His Leu Ala Val Tyr Leu
20 25 30
Leu Asp His Phe Met Asp Arg Tyr Asn Val Thr Thr Ser Lys Gln
35 40 45
Leu Tyr Thr Val Ala Val Ser Cys Leu Leu Leu Ala Ser Lys Phe
50 55 60
Glu Asp Arg Glu Asp His Val Pro Lys Leu Glu Gln Ile Asn Ser
65 70 75
Thr Arg Ile Leu Ser Ser Gln Asn Phe Thr Leu Thr Lys Lys Glu
80 85 90
Leu Leu Ser Thr Glu Leu Leu Leu Leu Glu AIa Phe Ser Trp Asn
95 100 105
Leu Cys Leu Arg Thr Pro Ala His Phe Leu Asp Tyr Tyr Leu Leu
110 115 120
Ala Ser Val Ser G1n Lys Asp His His Cys His Thr Trp Pro Thr
125 130 135
Thr Cys Pro Arg Lys Thr Lys Glu Cys Leu Lys Glu Tyr Ala His
140 145 150
Tyr Phe Leu Glu Val Thr Leu Gln Asp His Ile Phe Tyr Lys Phe
155 160 165
Gln Pro Ser Val Val Ala Ala Ala Cys Val G1y Ala Ser Arg Ile
170 175 180
Cys Leu Gln Leu Ser Pro Tyr Trp Thr Arg Asp Leu Gln Arg Ile
185 190 195
Ser Ser Tyr Ser Leu Glu His Leu Ser Thr Cys Ile Glu Ile Leu
200 205 210
Leu Val Val Tyr Asp Asn Val Leu Lys Asp Ala Val Ala Val Lys
215 220 225
Ser Gln Ala Leu Ala Met Val Pro Gly Thr Pro Pro Thr Pro Thr
230 235 240
Gln Val Leu Phe Gln Pro Pro Ala Tyr Pro Ala Leu Gly Gln Pro
245 250 255
Ala Thr Thr Leu Ala Gln Phe Gln Thr Pro Val Gln Asp Leu Cys
5/9
CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
260 265 270
Leu Ala Tyr Arg Asp Ser Leu Gln Ala His Arg Ser Gly Ser Leu
275 280 285
Leu Ser Gly Ser Thr Gly Ser Ser Leu His Thr Pro Tyr Gln Pro
290 295 300
Leu Gln Pro Leu Asp Met Cys Pro Val Pro Val Pro Ala Ser Leu
305 310 315
Ser Met His Met Ala Ile Ala Ala Glu Pro Arg His Cys Leu Ala
320 325 330
Thr Thr Tyr Gly Ser Ser Tyr Phe Ser G1y Ser His Met Phe Pro
335 340 345
Thr Gly Cys Phe Asp Arg
350
<210> 4
<211> 4443
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1001668CB1
<400> 4
cccagtctct tccccaactc tccccacccc tggtgctctc cgccccgctt gcctcccgtt 60
cttccccgcc cgctccatcc gggtcgctga cggctgtctc tggactggac agcagtggcg 120
tccgcttttc tctaaggaac ccgataagaa ctccattttc tgttgatgtg gagacatagg 180
gtacctccaa ctttccgggg tttcagaaga gaatttttta ttgaaactgg aagaccaaaa 240
taattacaag catgttgtgc tggggaaatg catcctttgg gcagctaggt ttgggtggaa 300
ttgatgaaga aattgtacta gagcccagaa aaagtgactt ctttataaat aaaagggtcc 360
gagatgtagg atgtggactc agacatactg tgtttgttct ggatgatgga acagtgtaca 420
catgtggatg taatgatcta ggacagctag gtcatgaaaa atccagaaag aaaccagagc 480
aggttgttgc cctggatgcc caaaatattg tagctgtttc atgtggagaa gctcatacgt 540
tagcgctaaa tgacaaaggc caggtgtatg cttggggtct cgattctgat ggacagcttg 600
gcctggtagg atcagaggaa tgcatcagag tacccagaaa tattaaaagt ttgtcagata 660
tccagattgt acaggttgct tgtggttact atcattcact tgcactttct aaagcaagtg 720
aagtcttctg ttggggacag aataaatatg gccaattggg tttaggtact gactgtaaaa 780
agcaaacttc accgcagctg cttaagtctt tgcttggaat ccctttcatg caagttgcag 840
caggaggagc ccatagtttt gtactcaccc tttctggagc tatctttgga tggggacgca 900
acaagtttgg tcagctaggt cttaatgatg aaaatgatag gtatgttcct aatttactaa 960
agtcactaag atctcagaaa atagtttata tttgttgtgg agaagatcat actgctgctc 1020
taaccaagga aggtggagtg tttacttttg gagctggagg gtatggtcag ttgggccata 1080
attctaccag tcatgaaata aacccaagga aagtttttga acttatggga agcattgtca 1140
ctgagattgc ttgtggacgg cagcacactt ctgcttttgt tccttcatca ggacgaattt 1200
actcttttgg gcttggtggt aatgggcagc tgggaaccgg ttcaacaagc aacaggaaaa 1260
gcccctttac tgtaaaagga aattggtacc cctataatgg gcagtgtcta ccagatattg 1320
attctgaaga atatttctgt gtaaaaagaa ttttctcagg gggagatcaa agcttttcac 1380
attactctag tccccagaac tgtgggccac cagatgactt cagatgtccc aatccgacaa 1440
agcagatctg gacagtgaat gaagctctaa ttcagaaatg gctgagctat ccttctggaa 1500
ggtttcctgt ggagatagcc aatgagatag atggaacgtt ttcttcctct ggttgcctaa 1560
atggaagttt tttagctgtt agcaatgatg atcactatag aacaggtacc agattttcag 1620
gggttgatat gaatgctgct aggcttttat tccacaaact tatacaacct gatcatccgc 1680
agatatctca gcaggtggca gctagtttgg aaaagaatct tattcctaaa ctgactagct 1740
ccttacctga tgttgaagca ttgaggtttt atcttactct accagaatgt cccctgatga 1800
gtgattccaa caatttcaca acaatagcaa ttccctttgg tacagctctt gtgaacctag 1860
aaaaggcacc actgaaagta cttgaaaact ggtggtcagt acttgaacct ccactattcc 1920
6/9
CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
tcaagatagt agaacttttt aaggaagttg tggtacatct tttgaaactc tacaagatcg 1980
gtattccccc ttctgaaaga agaattttca acagttttct tcatactgca ttaaaggttt 2040
tagaaatact acatagggta aatgagaaaa tgggacagat tatacagtat gataaatttt 2100
atatacatga agtacaagaa ttgatagaca taagaaatga ttatatcaac tgggtccaac 2160
agcaggccta tggaatgttg gcagatatcc ctgttacaat ctgtacatat ccatttgtat 2220
ttgatgccca agcaaaaact actctgttac agaccgatgc agtcttacag atgcagatgg 2280
ctattgatca ggcccacagg cagaatgtct cctctctttt tctcccagtg attgaatctg 2340
tgaatccctg cttaattcta gtggtgcgta gagaaaatat tgtaggagat gcaatggaag 2400
tccttaggaa aacaaagaac atagattaca agaagccact caaggttata tttgttggag 2460
aagatgctgt ggatgcagga ggggtgcgca aagaattttt cttgctcatc atgagggaat 2520
tattggatcc taaatacggc atgtttaggt attatgaaga ttccaggctc atttggtttt 2580
ctgataagac atttgaagac agtgatttgt tccatttgat tggtgttatc tgtggcttag 2640
caatttataa ttgtaccatt gtggacctcc attttccttt ggctttatat aagaaactac 2700
tgaaaaagaa gccatccttg gatgatttga aagaactaat gcctgatgtt gggagaagca 2760
tgcaacagtt actggattat ccagaagatg acatagagga aacattttgt cttaatttta 2820
cgatcacagt tgaaaacttt ggtgcaacag aagtgaaaga gctggttcta aatggtgcag 2880
acacagctgt taacaaacaa aatcggcaag agtttgtcga tgcttatgtg gattacatat 2940
tcaataaatc agtggcttcc ttatttgatg cttttcatgc gggctttcat aaggtctgtg 3000
gaggaaaagt ccttctgctc tttcagccta atgaactaca agcaatggtc attggaaata 3060
caaattatga ttggaaggaa ctggaaaaga atacagaata caaaggggaa tattgggcag 3120
aacatcctac gataaaaatt ttttgggaag tatttcacga attaccattg gaaaagaaga 3180
aacagtttct gttatttttg acaggtagtg atcgcattcc tattcttggt atgaagagtc 3240
tgaaactagt catccagtcc acaggaggtg gtgaggagta tctcccagtt tcccatactt 3300
gttttaatct tctggatctt ccaaaatata cagaaaaaga aactctacgc tctaaactga 3360
tccaagctat tgatcacaat gaaggcttca gtttaatata actttggagt tataactatt 3420
cagtttagtg caaaagcatt aaactatttg tgtttttctt gtggtgatga attcagcaag 3480
gtgacagagg tactattata attcttactt gcagaatgtt caatctacga gtgttcatgg 3540
aagccaaaaa atattaaagg aaaatgaaca aactgttaat attattgtac agaaccatgg 3600
attttttttg accatcttct aataaacata gcaagtatta tgaatacatt aaagttttac 3660
taacatgaat tttaagagtt tgcatatttc agaaatgatc tggtgtgagt gcatggaaat 3720
attgcttaat ttttcttcaa tcattgagtg aaaaaccttt aactttggcc tgcaatagtc 3780
atttgattat tttttcattt tgtaaataat gttaagtttt gtaataaaat agttatgttc 3840
tgataccagt acagtttcta tggttgtaat tgaacttgaa cagactttta aaggttaaaa 3900
attatgattt aaatcttact ctgagactct ataaaaagaa aaaaaaggta gcatggtgga 3960
aacactatct tttctttttt gctagaataa gtgtctttgt gcaaacctaa atcacagata 4020
gggaatatac tacataacct gcagttcttt tctttgtgaa tcctgacaca ctgatagatg 4080
ggggattgtc gatcagagaa cttattaata tttagtactg gaagaactct gtctccacag 4140
ttgccagtaa taaaaagaaa cattggctac tatgagcacc aatcactggg ttatagcttt 4200
caaaattatt gatgctgcag tgctttagag ctatttcctt gaatttaaga aacaaatctt 4260
aacagtttta tggtgctgat gcttagttgt ctcatgccat taaattgtaa aagtgagttg 4320
atgcaataca tgtgactttc tgctataatg caaatattta ttttttaaat ttattttaaa 4380
atgccgtgaa aactgtttaa taaagattta ttgttttaat atttaaactt tcaaaaaaaa 4440
aaa 4443
<210> 5
<211> 1387
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2226248CB1
<400> 5
tgcttggcta tgacaatttt caagcaagtg ttgggctggc tgaacagatt tagagatcgc 60
cacggaattg ctttgaaagc agtactgtag agaagatagt gacaggttaa tgaatggtct 120
7/9
CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
aggaatagat aagattaatg agtggcatgc aggggaaatt ataaaactga ttgctgacta 180
cagcccagat gatatcttta atgctgatga gacaggagtg tttttccagt tgcttcccca 240
gcacacactt gctgctaaag gagaccactg tagagggggc aagaaagcaa agcagcggtt 300
gacagcactc ttttgttgca atgcctcggg gactgaaaaa atgagaccat tgattgttgg 360
taggtcagcc agcccacact gcctcaagaa cattcattcc ctcccttgtg attaccgagc 420
caaccagtgg gcttggatga caagggatct gtttaatgag tggctgatgc aagtggatgc 480
caggatgaag agggcggaac gccggatcct cttgctcata gacaactgct ctgctcataa 540
catgcttcca cacttggaaa ggattcaggt tgggtatctg ccctccaact gtactgctgt 600
cctgcagcca ctgaatcttg gcataattca caccatgaaa gtactgtacc agagccacct 660
tctaaaacag atcctcctca agctcaacag cagtgaggat caagaagagg tggacatcaa 720
gcaggccatc gacatgattg ctgcagcgtg gtggtcagtc aagccatcca cagtggtgaa 780
atgttggcag aaggcaggca tcgtccctat ggaatttgca gaatgtgaca cagaatcagc 840
agccagtgaa ccagacattg ccattgaaaa gttgtggcac acagtggcta ttgccacctg 900
tgtcccaaat gaagtaaatt tccaggactt tgttactgca gatgatgatc tcattatctc 960
tcaggacaca gacatcatcc aggacatggt ggctggcgaa aataccagtg aagcaggaag 1020
tgaagatgaa ggggaggtat ctttaccaga gcaaccaaaa gtcaccatca cagaagccat 1080
atcaagtgta cagaaactta gacagttcct ttccacttgt gtagacattc ctgatgccat 1140
ttttggacaa ttaaatggca tagatgaata tttaatgaaa agagtgacac aaacccttat 1200
tgattccaaa attacagatt tcctccaaac aaaataatgc aggaatttat ttcagaaaat 1260
gtagtttaca agaataaaga tttctttaga taggttgttg agccaattta agtaaagcaa 1320
tgttattgtg acaacattcc agtactctga aatagccagg aaacttcttt gaatggaaaa 1380
aaaaaaa 1387
<210> 6
<211> 3023
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3049930CB1
<400> 6
gggacccgag cgcacgcagg ggcgcggcga cggcgggggc tagtcgggct gcggaggcgg 60
ccgtcgggag tgcggagcgc ctcggacgag ggtccaaccg ccggcaggca ccagagggca 120
cggctggctc ggcactggga ggggcccggc gctcgcagcc ccccacgccc agagaggatg 180
cggtgcgccc tgagagccgg gtagcctcgg atagcggcgc tgcgtacgcg atgatggatg 240
agccgccggt tcttcgtgga catcctgacc ctgctgagca gccactgcca gctctgccct 300
gcagcccggc acctggccgt ctacctgctg gaccacttca tggatcgcta caacgtcacc 360
acctccaagc agctctacac cgtggccgtc tcctgcctcc tgcttgcaag taagttcgag 420
gatcgggaag accacgtccc caagttggag caaataaaca gcacgaggat cctgagcagc 480
cagaacttca ccctcaccaa gaaggagctg ctgagcacag agctgctgct cctggaggcc 540
ttcagctgga acctctgcct gcgcacgcct gcccacttcc tggactacta cctcttggcc 600
tccgtcagcc agaaggacca ccactgccac acctggccca ccacctgccc ccgcaagacc 660
aaagagtgcc tcaaggagta tgcccattac ttcctagagg tcaccctgca agatcacata 720
ttctacaaat tccagccttc tgtggtcgct gcggcctgtg ttggggcctc caggatttgc 780
ctgcagcttt ctccctactg gaccagagac ctgcagagga tctcaagcta ttccctggag 840
cacctcagca cgtgtattga aatcctgctg gtagtgtatg acaacgtcct caaggatgcc 900
gtagccgtca agagccaggc cttggcaatg gtgcccggca caccccccac ccccactcaa 960
gtgctgttcc agccaccagc ctacccggcc ctcggccagc cagcgaccac cctggcacag 1020
ttccagaccc ccgtgcagga cctatgcttg gcctatcggg actccttgca ggcccaccgt 1080
tcagggagcc tgctctcggg gagtacaggc tcatccctcc acaccccgta ccaaccgctg 1140
cagcccttgg atatgtgtcc cgtgcccgtc cctgcatccc ttagcatgca tatggccatt 1200
gcagctgagc ccaggcactg cctcgccacc acctatgga'~ gcagctactt cagtgggagc 1260
cacatgttcc ccaccggctg ctttgacaga taggccacct ccagacctca cgaggaagcc 1320
ttggagatgt gggcagagga agaggacact gaagaggaga gctcagccaa gtgaggcagc 1380
8/9
CA 02415077 2003-O1-16
WO 02/08255 PCT/USO1/22805
aggaggccat ccctgaagag ccttggaacg tggagggtct gtgctccttt taaataaaac 1440
tgacccagag caaaacattc aataacatac ctcacccgag agcattcctc tgagaaacgt 1500
ctgccacgtg tggctagggt acaaaaggat ggcttggtgg ccgtcccccc acacaggggc 1560
ccagtgaatc gagaaagact tgataagagg ccaggagagt gggaactgga cacagaccac 1620
tgatctcaag catgtccagt ttttagcatt aaagactttt ctattctttg ctgatggcag 1680
ctacaccgct gaaaaaggag gggcagcctg gcgtgttctc aggacccccg gaggatccca 1740
tattgggtgc ttttctttcc ctggctctat gcagaggggc ctgagttggt gtgtatgcct 1800
gagtgtttgc cttgcgaggc acagtgggtg gctgtcttgc ctttgttttc gaacctaaaa 1860
ccattttcag ccttttagat atgtcatgtg ctgctgcttc ccgaagtggt cttgctttct 1920
gtttcgtaag atgtcttgtt tacacactgt atcagggatt tggtgatact tgaaaattcc 1980
tttggagaaa aaaacaaatt taattgccac actgcctgtc ccacatgagg gctgttaatt 2040
tggaacccaa gtttgaaccc aacttgtgat ggacccgcag gtaaccacag agcttccttt 2100
ttgaaggcat atggttggag agaaccattt tcccagctct cggttccgga agattccacg 2160
tcttggaggc tgtgttcacc actagaactt taataactac ccagggaggg agaagctcgt 2220
tgaaaggaag acaaagattt aaacagttcc cacctctctc caccacatac ttacagggca 2280
tgagtttaac ccagtctagg ctttgagtgt ggctgatagc gaaggatagc aatgtggaaa 2340
atttgcttag tcccacctgt atttggggag tgggatgtac atgggcgttt gataccacca 2400
ttgataggca agcgactggt tggatcaaaa gccagtattt agggatctgc agcgagaggg 2460
ccctcaggaa gactcttgta accatgtgca atatgttttt attctgactc gcagcttgtg 2520
ctcagcatgt tctttggttt agtttggggt tgggggacac attgttcacc cagcagaact 2580
gggaggtgca aaggaccgtg gaagcaattt ttgttttgtt tgaggaatac ctgtcttgga 2640
ttccttagcc ccttgccagt cctggagact gtggcagggg ccgccaggaa ggcagctgtc 2700
tgctgctgag tcagatcgga aggtggtgaa tctttccaga gcagctgaaa atctcagcat 2760
ggagacagta agaaaagaga cggggtgtgg ataagactct gccaccgtgt cacactagca 2820
taggaggctg cacgttcatt tgttgttgtt tttttttcct ttgccaacct ccgttctatt 2880
tatgtgcaag cagtttggat tcaagttctt gtatctgtct gttctgggac ctggggattg 2940
tgagggttcc ctcacagcca gcacgacccc cagaaagagg cgtcccacaa taaacacgtc 3000
acctgctctt gaaaaaaaaa aaa 3023
9/9
