Note: Descriptions are shown in the official language in which they were submitted.
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PROTEINS ASSOCIATED WITH CELL GROWTH, DIFFERENTIATION, AND DEATH
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of proteins
associated with
cell growth, differentiation, and death and to the use of these sequences in
the diagnosis, treatment,
and prevention of cell proliferative disorders including cancer, developmental
disorders, neurological
disorders, reproductive disorders, and autoimmune/inflammatory disorders, and
in the assessment of
the effects of exogenous compounds on the expression of nucleic acid and amino
acid sequences of
proteins associated with cell growth, differentiation, and death.
BACKGROUND OF THE INVENTION
Human growth and development requires the spatial and temporal regulation of
cell
differentiation, cell proliferation, and apoptosis. These processes
coordinately control reproduction,
aging, embryogenesis, morphogenesis, organogenesis, and tissue repair and
maintenance. At the
cellular level, growth and development is governed by the cell's decision to
enter into or exit from
the cell division cycle and by the cell's commitment to a terminally
differentiated state. These
decisions are made by the cell in response to extracellular signals and other
environmental cues it
receives. The following discussion focuses on the molecular mechanisms of cell
division,
embryogenesis, cell differentiation and proliferation, and apoptosis, as well
as disease states such as
cancer which can result from disruption of these mechanisms.
Cell Cycle
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. 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
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.
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,
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and production of essential proteins. In the second event, mitosis, the
nuclear 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 microtubules
and associated proteins such as dynein, which originate from polax 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, R. et
al. (2000) Human Mol.
Gen. 9: 175-185).
During the M phase of eukaryotic cell cycling, structural rearrangements occur
ensuring
appropriate distribution of cellulax components between daughter cells.
Breakdown of interphase
structures into smaller subunits is common. The nuclear envelope breaks into
vesicles, and nuclear
lamins axe disassembled. Subsequent phosphorylation of these lamins occurs and
is maintained until
telophase, at which time the nuclear lamina structure is reformed. cDNAs
responsible for encoding
M phase phosphorylation (MPPs) are components of U3 small nucleolax
ribonucleoprotein (snoRNP),
and relocalize to the nucleolus once mitosis is complete (Westendorf, J.M. et
al. (1998) J. Biol.
Chem. 9:437-449). U3 snoRNPs are essential mediators of RNA processing events.
Proteins involved in the regulation of cellular processes such as mitosis
include the Ser/Thr-
protein phosphatases type 1 (PP-1). PP-is act by dephosphorylation of key
proteins involved in the
metaphase-anaphase transition. The gene PP1R7 encodes the regulatory
polypeptide sds22, having at
least six splice variants (Ceulemans, H. et al. (1999) Eur. J. Biochem. 262:36-
42). Sds22 modulates
the activity of the catalytic subunit of PP-ls, and enhances the PP-1-
dependent dephosphorylation of
mitotic substrates.
Cell cycle regulatory proteins play an important role in cell proliferation
and cancer. For
example, failures in the proper execution and timing of cell cycle events can
lead to chromosome
segregation defects resulting in aneuploidy or polyploidy. This genomic
instability is characteristic of
transformed cells (Luca, F.C. and Winey, M. (1998) Mol. Biol. Cell. 9:29-46).
A recently identified
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protein, mMOB 1, is the mammalian homolog of yeast MOB 1, an essential yeast
gene required for
completion of mitosis and maintenance of ploidy. The mammalian mMOB 1 is a
member of protein
complexes including protein phosphatase 2A (PP2A), and its phosphorylation
appears to be regulated
by PP2A (Moreno, C.S. et al. (2001) J. Biol. Chem. 276:24253-24260). PP2A has
been implicated in
the development of human cancers, including lung and colon cancers and
leukemias.
Cell cycle regulation involves numerous proteins interacting in a sequential
manner. The
eukaryotic cell cycle consists of several highly controlled events whose
precise order ensures
successful DNA replication and cell division. Cells maintain the order of
these events by making
later events dependent on the successful completion of earlier events. This
dependency is enforced
by cellular mechanisms called checkpoints. Examples of additional cell cycle
regulatory proteins
include the histone deacetylases (HDACs). HDACs are involved in cell cycle
regulation, and
modulate chromatin structure. Human HDAC1 has been found to interact in vitro
with the human
Hus1 gene product, whose Schizosaccharofnyces pombe homolog has been
implicated in GZ/M
checkpoint control (Cai, R.L. et al. (2000) J. Biol. Chem. 275:27909-27916).
DNA damage (G~) and DNA replication (S-phase) checkpoints arrest eukaryotic
cells at the
GZ/M transition. This arrest provides time for DNA repair or DNA replication
to occur before entry
into mitosis. Thus, the GZ/M checkpoint ensures that mitosis only occurs upon
completion of DNA
replication and in the absence of chromosomal damage. The Husl gene of
Schizosaccharomyces
pombe is a cell cycle checkpoint gene, as are the rad family of genes (e.g.,
radl and rad9) (Volkmer,
E. and Karnitz, L.M. (1999) J. Biol. Chem. 274:567-570; Kostrub C.F. et al.
(1998) EMBO J.
17:2055-2066). These genes are involved in the mitotic checkpoint, and are
induced by either DNA
l
damage or blockage of replication. Induction of DNA damage or replication
block leads to loss of
function of the Hus 1 gene and subsequent cell death. Human homologs have been
identified for most
of the rad genes, including ATM and ATR, the human homologs of rad3p.
Mutations in the ATM
gene are correlated with the severe congenital disease ataxia-telagiectasia
(Savitsky, K. et al. (1995)
Science 268:1749-1753). The human Husl protein has been shown to act in a
complex with radl
protein which interacts with rad9, making them central components of a DNA
damage-responsive
protein complex of human cells (Volkmer, E. and Karnitz, L.M. (1999) J. Biol.
Chem. 274:567-570).
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. 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
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believed to be a recognition code that triggers ubiquitin-mediated degradation
of cyclin B (Hunt, T.
(1991) Nature 349:100-101). Several types of cyclins exist (Ciechanover, A.
(1994) Cell 79:13-21).
Progression through Gl and S phase is driven by the G1 cyclins and their
catalytic subunits, including
Cdk2-cyclin A, Cdk2-cyclin E, Cdk4-cyclin D and Cdk6-cyclin D. Progression
through the G2-M
transition is driven by the activation of mitotic CDK-cyclin complexes such as
Cdc2-cyclin A,
Cdc2-cyclin B1 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 (El) in an ATP dependent
reaction which binds the C-
terminus of Ub to the thiol group of an internal cysteine residue in E1.
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, E2 transfers the Ub molecule through its C-terminal glycine to
a member of the
ubiquitin-protein ligase family, E3. Fourth, E3 transfers the Ub molecule to
the target protein.
Additional Ub molecules may be added to the target protein forming a mufti-Ub
chain structure.
Fifth, the ubiquinated protein is then recognized and degraded by the
proteasome, a large,
multisubunit proteolytic enzyme complex, and Ub is released for re-
utilization.
Prior to activation, Ub is usually.expressed as a fusion protein composed of
an N-terminal
ubiquitin and a C-terminal extension protein (CEP) or as a polyubiquitin
protein with Ub monomers
attached head to tail. CEPS have characteristics of a variety of regulatory
proteins; most are highly
basic, contain up to 30% lysine and arginine residues, and have nucleic acid-
binding domains
(Monia, B.P. et al. (1989) J. Biol. Chem. 264:4093-4103). The fusion protein
is an important
intermediate which appears to mediate co-regulation of the cell's
translational and protein
degradation activities, as well as localization of the inactive enzyme to
specific cellular sites. Once
delivered, C-terminal hydrolases cleave the fusion protein to release a
functional Ub (Monia et al.,
supra).
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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-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) Proe. 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 (HPV)
E6 oncoprotein, for
the ubiquitination and degradation of p53 (Scheffner et al. (1993) Cell 75:495-
505). The C-terminal
domain of 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). The
SCF (Skpl-Cdc53lCullin-F box receptor) family of proteins comprise another
group of ubiquitin
ligases (Deshaies, R. (1999) Annu. Rev. Dev. Biol. 15:435-467). Multiple
proteins are recruited into
the SCF complex, including Skpl, cullin, and an F box domain containing
protein. The F box protein
binds the substrate for the ubiquitination reaction and may play roles in
determining substrate
specificity and orienting the substrate for reaction. Skp1 interacts with both
the F box protein and
cullin and may be involved in positioning the F box protein and cullin in the
complex for transfer of
ubiquitin from the E2 enzyme to the protein substrate. Substrates of SCF
ligases include proteins
involved in regulation of CDK activity, activation of transcription, signal
transduction, assembly of
kinetochores, and DNA replication.
Sgtl was identified in a screen for genes in yeast that suppress defects in
kinetochore
function caused by mutations in Skpl (Kitagawa, K. et al. (1999) Mol. Cell
4:21-33). Sgtl interacts
with Skpl and associates with SCF ubiquitin ligase. Defects in Sgtl cause
arrest of cells at either Gl
or G2 stages of the cell cycle. A yeast Sgtl null mutant can be rescued by
human Sgtl, an indication
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of the conservation of Sgtl function across species. Sgtl is required for
assembly of kinetochore
complexes in yeast.
Abnormal activities of the UCS are implicated in a number of diseases and
disorders. These
include, e.g., cachexia (Llovera, M. et al. (1995) Int. J. Cancer 61: 138-
141), degradation of the
tumor-suppressor protein, p53 (Ciechanover, supra), and neurodegeneration such
as observed in
Alzheimer's disease (Gregori, L. et al. (1994) Biochem. Biophys. Res. Commun.
203: 1731-1738).
Since ubiquitin conjugation is a rate-limiting step in antigen presentation,
the ubiquitin degradation
pathway may also have a critical role in the immune response (Grant E.P. et
al. (1995) J. lmmunol.
155: 3750-3758).
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 17:471-480).
Embryogenesis
Mammalian embryogenesis is a process which encompasses the first few weeks of
development following conception. During this period, embryogenesis proceeds
from a single
fertilized egg to the formation of the three embryonic tissues, then to an
embryo which has most of its
internal organs and all of its external features.
The normal course of mammalian embryogenesis depends on the correct temporal
and spatial
regulation of a large number of genes and tissues. These regulation processes
have been intensely
studied in mouse. An essential process that is still poorly understood is the
activation of the
embryonic genome after fertilization. As mouse oocytes grow, they accumulate
transcripts that are
either translated directly into proteins or stored for later activation by
regulated polyadenylation.
During subsequent meiotic maturation and ovulation, the maternal genome is
transcriptionally inert,
and most maternal transcripts are deadenylated and/or degraded prior to, or
together with, the
activation of the zygotic genes at the two-cell stage (Stutz, A. et al. (
1998) Genes Dev. 12:2535-
2548). The maternal to embryonic transition involves the degradation of
oocyte, but not zygotic
transcripts, the activation of the embryonic genome, and the induction of cell
cycle progression to
accommodate early development.
MATER (Maternal Antigen That Embryos Require) was initially identified as a
target of
antibodies from mice with ovarian immunity (Tong, Z-B., and Nelson, L.M.
(1999) Endocrinology
140:3720-3726). Expression of the gene encoding MATER is restricted to the
oocyte, making it one
of a limited number of known maternal-effect genes in mammals (Tong, Z-B., et
al. (2000) Mamm.
Genome 11:281-287). The MATER protein is required for embryonic development
beyond two cells,
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based upon preliminary results from mice in which this gene has been
inactivated. The 1111-amino
acid MATER protein contains a hydrophilic repeat region in the amino terminus,
and a region
containing 14 leucine-rich repeats in the carboxyl terminus. These repeats
resemble the sequence
found in porcine ribonuclease inhibitor that is critical for protein-protein
interactions.
The degradation of maternal transcripts during meiotic maturation and
ovulation may involve
the activation of a ribonuclease just prior to ovulation. Thus the function of
MATER may be to bind
to the maternal ribonuclease and prevent degradation of zygotic transcripts
(Tong (2000) supra). In
addition to its role in oocyte development and embryogenesis, MATER may also
be relevant to the
pathogenesis of ovarian immunity, as it is a target of autoantibodies in mice
with autoimmune
oophoritis (Tong (1999) supra).
The maternal mRNA D7 is a moderately abundant transcript in Xeno~us laevis
whose
expression is highest in, and perhaps restricted to, oogenesis and early
embryogenesis. The
D7 protein is absent from oocytes and first begins to accumulate during oocyte
maturation. Its, levels
are highest during the first day of embryonic development and then they
decrease. The loss of D7
protein affects the maturation process itself, significantly delaying the time
course of germinal vesicle
breakdown. Thus, D7 is a newly described protein involved in oocyte maturation
(Smith R.C., et al.
(1988) Genes Dev. 2(10):1296-306.)
Many other genes are involved in subsequent stages of embryogenesis. After
fertilization, the
oocyte is guided by fimbria at the distal end of each fallopian tube into and
through the fallopian tube
and thence into the uterus. Changes in the uterine endometrium prepare the
tissue to support the
implantation and embryonic development of a fertilized ovum. Several stages of
division have
occurred before the dividing ovum, now a blastocyst with about 100 cells,
enters the uterus. Upon
reaching the uterus, the developing blastocyst usually remains in the uterine
cavity an additional two
to four days before implanting in the endometrium, the inner lining of the
uterus. Implantation results
from the action of trophoblast cells that develop over the surface of the
blastocyst. These cells
secrete proteolytic enzymes that digest and liquefy the cells of the
endometrium. The invasive
process is reviewed in Fisher and Damsky (1993; Semin Cell Biol 4:183-188) and
Graham and Lala
(1992; Biochem Cell Biol 70:867-874). Once implantation has taken place, the
trophoblast and other
sublying cells proliferate rapidly, forming the placenta and the various
membranes of pregnancy.
(See Guyton, A.C. (1991) Textbook of Medical Physiology, 8"' ed. W.B. Saunders
Company,
Philadelphia pp. 915-919.)
The placenta has an essential role in protecting and nourishing the developing
fetus. In most
species the syncytiotrophoblast layer is present on the outside of the
placenta at the fetal-maternal
interface. This is a continuous structure, one cell deep, formed by the fusion
of the constituent
trophoblast cells. The syncytiotrophoblast cells play important roles in
maternal-fetal exchange, in
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tissue remodeling during fetal development, and in protecting the developing
fetus from the maternal
immune response (Stoye, J.P. and Coffin, J.M. (2000) Nature 403:715-717).
A gene called syncytin is the envelope gene of a human endogenous defective
provirus.
Syncytin is expressed in high levels in placenta, and more weakly in testis,
but is not detected in any
other tissues (Mi, S. et al. (2000) Nature 403:785-789). Syncytin expression
in the placenta is
restricted to the syncytiotrophoblasts. Since retroviral env proteins are
often involved in promoting
cell fusion events, it was thought that syncytin might be involved in
regulating the fusion of
trophoblast cells into the syncytiotrophoblast layer. Experiments demonstrated
that syncytin can
mediate cell fusion in vitro, and that anti-syncytin antibodies can inhibit
the fusion of placental
cytotrophoblasts (Mi, supra). In addition, a conserved immunosuppressive
domain present in
retroviral envelope proteins, and found in syncytin at amino acid residues 373-
397, might be involved
in preventing maternal immune responses against the developing embryo.
Syncytin may also be involved in regulating trophoblast invasiveness by
inducing trophoblast
fusion and terminal differentiation (Mi, supra). Insufficient trophoblast
infiltration of the uterine wall
is associated with placental disorders such as preeclampsia, or pregnancy
induced hypertension, while
uncontrolled trophoblast invasion is observed in choriocarcinoma and other
gestational trophoblastic
diseases. Thus syncytin function may be involved in these diseases.
Cell Differentiation
Multicellular organisms are comprised~of diverse cell types that differ
dramatically both in
structure and function, despite the fact that each cell is like the others in
its hereditary endowment.
Cell differentiation is the process by which cells come to differ in their
structure and physiological
function. The cells of a multicellular organism all arise from mitotic
divisions of a single-celled
zygote. The zygote is totipotent, meaning that it has the ability to give rise
to every type of cell in the
adult body. During development the cellular descendants of the zygote lose
their totipotency and
become determined. Once its prospective fate is achieved, a cell is said to
have differentiated. All
descendants of this cell will be of the same type.
Human growth and development requires the spatial and temporal regulation of
cell
differentiation, along with cell proliferation and regulated cell death. These
processes coordinate to
control reproduction, aging, embryogenesis, morphogenesis, organogenesis, and
tissue repair and
maintenance. The processes involved in cell differentiation are also relevant
to disease states such as
cancer, in which case the factors regulating normal cell differentiation have
been altered, allowing the
cancerous cells to proliferate in an anaplastic, or undifferentiated, state.
The mechanisms of differentiation involve cell-specific regulation of
transcription and
translation, so that different genes are selectively expressed at different
times in different cells.
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Genetic experiments using the fruit fly Drosophila melano ag ster have
identified regulated cascades of
transcription factors which control pattern formation during development and
differentiation. These
include the homeotic genes, which encode transcription factors containing
homeobox motifs. The
products of homeotic genes determine how the insect's imaginal discs develop
from masses of
undifferentiated cells to specific segments containing complex organs. Many
genes found to be
involved in cell differentiation and development in Drosophila have homologs
in mammals. Some
human genes have equivalent developmental roles to their Drosophila homologs.
The human
homolog of the Drosophila eyes absent gene (eya) underlies branchio-oto-renal
syndrome, a
developmental disorder affecting the ears and kidneys (Abdelhak, S. et al.
(1997) Nat. Genet. 15:157-
164). The Drosophila slit gene encodes a secreted leucine-rich repeat
containing protein expressed
by the midline glial cells and required for normal neural development.
At the cellular level, growth and development are governed by the cell's
decision to enter
into or exit from the cell cycle and by the cell's commitment to a terminally
differentiated state.
Differential gene expression within cells is triggered in response to
extracellular signals and other
environmental cues. Such signals include growth factors and other mitogens
such as retinoic acid;
cell-cell and cell-matrix contacts; and environmental factors such as
nutritional signals, toxic
substances, and heat shock. Candidate genes that may play a role in
differentiation can be identified
by altered expression patterns upon induction of cell differentiation in
vitro.
The final step in cell differentiation results in a specialization that is
characterized by the
production of particular proteins, such as contractile proteins in muscle
cells, serum proteins in liver
cells and globins in red blood cell precursors. The expression of these
specialized proteins depends at
least in part on cell-specific transcription factors. For example, the homobox-
containing transcription
factor PAX-6 is essential for early eye determination, specification of ocular
tissues, and normal eye
development in vertebrates.
In the ease of epidermal differentiation, the induction of differentiation-
specific genes occurs
either together with or following growth arrest and is believed to be linked
to the molecular events
that control irreversible growth arrest. Irreversible growth arrest is an
early event which occurs when
cells transit from the basal to the innermost suprabasal layer of the skin and
begin expressing
squamous-specific genes. These genes include those involved in the formation
of the cross-linked
envelope, such as transglutaminase I and III, involucrin, loricin, and small
proline-rich repeat (SPRR)
proteins. The SPRR proteins are 8-10 kDa in molecular mass, rich in proline,
glutamine, and
cysteine, and contain similar repeating sequence elements. The SPRR proteins
may be structural
proteins with a strong secondary structure or metal-binding proteins such as
metallothioneins.
(Jetten, A. M. and Harvat, B. L. (1997) J. Dermatol. 24:711-725; PRINTS Entry
PR00021 PRORICH
Small proline-rich protein signature.)
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The Wnt gene family of secreted signaling molecules is highly conserved
throughout
eukaryotic cells. Members of the Wnt family are involved in regulating
chondrocyte differentiation
within the cartilage template. Wnt-5a, Wnt-5b and Wnt-4 genes are expressed in
chondrogenic
regions of the chicken limb, Wnt-5a being expressed in the perichondrium
(mesenchymal cells
immediately surrounding the early cartilage template). Wnt-5a misexpression
delays the maturation
of chondrocytes and the onset of bone collar formation in chicken limb
(Hartmann, C. and Tabin, C.J.
(2000) Development 127:3141-3159).
Glypicans are a family of cell surface heparan sulfate proteoglycans that play
an important
role in cellular growth control and differentiation. Cerebroglycan, a heparan
sulfate proteoglycan
expressed in the nervous system, is involved with the motile behavior of
developing neurons (Stipp,
C.S. et al. (1994) J. Cell Biol. 124:149-160).
Notch plays an active role in the differentiation of glial cells, and
influences the length and
organization of neuronal processes (for a review, see Frisen, J. and Lendahl,
U. (2001) Bioessays
23:3-7). The Notch receptor signaling pathway is important for morphogenesis
and development of
many organs and tissues in multicellular species. Drosophila fringe proteins
modulate the activation
of the Notch signal transduction pathway at the dorsal-ventral boundary of the
wing imaginal disc.
Mammalian fringe-related family members participate in boundary determination
during
segmentation (Johnston, S.H. et al. (1997) Development 124:2245-2254).
Recently a number of proteins have been found to contain a conserved cysteine-
rich domain
of about 60 amino-acid residues called the LIM domain (for Lin-11 Isl-1 Mec-3)
(Freyd G. et al.
(1990) Nature 344:876-879; Baltz R. et al. (1992) Plant Cell 4:1465-1466). In
the LIM-domain, there
are seven conserved cysteine residues and a histidine. The LIM domain binds
two zinc ions
(Michelsen J.W. et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:4404-4408). LIM
does not bind DNA,
rather it seems to act as an interface for protein-protein interaction.
Apoptosis
Apoptosis is the genetically controlled process by which unneeded or defective
cells undergo
programmed cell death. Selective elimination of cells is as important for
morphogenesis and tissue
remodeling as is cell proliferation and differentiation. Lack of apoptosis may
result in hyperplasia
and other disorders associated with increased cell proliferation. Apoptosis is
also a critical
component of the immune response. Immune cells such as cytotoxic T-cells and
natural killer cells
prevent the spread of disease by inducing apoptosis in tumor cells and virus-
infected cells. In
addition, immune cells that fail to distinguish self molecules from foreign
molecules must be
eliminated by apoptosis to avoid an autoimmune response.
Apoptotic cells undergo distinct morphological changes. Hallmarks of apoptosis
include cell
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shrinkage, nuclear and cytoplasmic condensation, and alterations in plasma
membrane topology.
Biochemically, apoptotic cells are characterized by increased intracellular
calcium concentration,
fragmentation of chromosomal DNA, and expression of novel cell surface
components.
The molecular mechanisms of apoptosis are highly conserved, and many of the
key protein
regulators and effectors of apoptosis have been identified. Apoptosis
generally proceeds in response
to a signal which is transduced intracellularly and results in altered
patterns of gene expression and
protein activity. Signaling molecules such as hormones and cytokines are known
both to stimulate
and to inhibit apoptosis through interactions with cell surface receptors.
Transcription factors also
play an important role in the onset of apoptosis. A number of downstream
effector molecules,
especially proteases, have been implicated in the degradation of cellular
components and the
proteolytic activation of other apoptotic effectors.
The Bcl-2 family of proteins, as well as other cytoplasmic proteins, are key
regulators of
apoptosis. There are at least 15 Bcl-2 family members within 3 subfamilies.
These proteins have
been identified in mammalian cells and in viruses, and each possesses at least
one of four Bcl-2
homology domains (BHl to BH4), which are highly conserved. Bcl-2 family
proteins contain the
BH1 and BH2 domains, which are found in members of the pro-survival subfamily,
while those
proteins which are most similar to Bcl-2 have all four conserved domains,
enabling inhibition of
apoptosis following encounters with a variety of cytotoxic challenges. Members
of the pro-survival
subfamily include Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1 in mammals; NF-13
(chicken); CED-9
(Caenorhabditis ele~ans); and viral proteins BHRF1, LMWS-HL, ORF16, KS-Bcl-2,
and ElB-19K.
The BH3 domain is essential for the function of pro-apoptosis subfamily
proteins. The two pro-
apoptosis subfamilies, Bax and BH3, include Bax, Bak, and Bok (also called
Mtd); and Bik, Blk, Hrk,
BNIP3, BimL, Bad, Bid, and Egl-1 (C. ele_~ans); respectively. Members of the
Bax subfamily contain
the BHl, BH2, and BH3 domains, and resemble Bcl-2 rather closely. In contrast,
members of the
BH3 subfamily have only the 9-16 residue BH3 domain, being otherwise unrelated
to any known
protein, and only Bik and Blk share sequence similarity. The proteins of the
two pro-apoptosis
subfamilies may be the antagonists of pro-survival subfamily proteins. This is
illustrated in C.
elegans where Egl-1, which is required for apoptosis, binds to and acts via
CED-9 (for review, see
Adams, J.M. and Cory, S. (1998) Science 281:1322-1326).
Heterodimerization between pro-apoptosis and anti-apoptosis subfamily proteins
seems to
have a titrating effect on the functions of these protein subfamilies, which
suggests that relative
concentrations of the members of each subfamily may act to regulate apoptosis.
Heterodimerization
is not required for a pro-survival protein; however, it is essential in the
BH3 subfamily, and less so in
the Bax subfamily.
The Bcl-2 protein has 2 isoforms, alpha and beta, which are formed by
alternative splicing. It
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forms homodimers and heterodimers with Bax and Bak proteins and the Bcl-X
isoform Bcl-xs.
Heterodimerization with Bax requires intact BHl and BH2 domains, and is
necessary for pro-survival
activity. The BH4 domain seems to be involved in pro-survival activity as
well. Bcl-2 is located
within the inner and outer mitochondria) membranes, as well as within the
nuclear envelope and
endoplasmic reticulum, and is expressed in a variety of tissues. Its
involvement in follicular
lymphoma (type II chronic lymphatic leukemia) is seen in a chromosomal
translocation T(14;18)
(q32;q21) and involves immunoglobulin gene regions.
The Bcl-x protein is a dominant regulator of apoptotic cell death. Alternative
splicing results
in three isoforms, Bcl-xB, a long isoform, and a short isoform. The long
isoform exhibits cell death
repressor activity, while the short isofonn promotes apoptosis. Bcl-xL forms
heterodimers with Bax
and Bak, although heterodimerization with Bax does not seem to be necessary
for pro-survival (anti-
apoptosis) activity. Bcl-xS forms heterodimers with Bcl-2. Bcl-x is found in
mitochondria)
membranes and the perinuclear envelope. Bcl-xS is expressed at high levels in
developing
lymphocytes and other cells undergoing a high rate of turnover. Bcl-xL is
found in adult brain and in
other tissues' long-lived post-mitotic cells. As with Bcl-2, the BHl, BH2, and
BH4 domains are
involved in pro-survival activity.
The Bcl-w protein is found within the cytoplasm of almost all myeloid cell
lines and in
numerous tissues, with the highest levels of expression in brain, colon, and
salivary gland. This
protein is expressed in low levels in testis, liver, heart, stomach, skeletal
muscle, and placenta, and a
few lymphoid cell lines. Bcl-w contains the BHI, BH2, and BH4 domains, all of
which are needed
for its cell survival promotion activity. Although mice in which Bcl-w gene
function was disrupted
by homologous recombination were viable, healthy, and normal in appearance,
and adult females had
normal reproductive function, the adult males were infertile. In these males,
the initial, prepuberty
stage of spermatogenesis was largely unaffected and the testes developed
normally. However, the
seminiferous tubules were disorganized, contained numerous apoptotic cells,
and were incapable of
producing mature sperm. This mouse model may be applicable to some cases of
human male sterility
and suggests that alteration of programmed cell death in the testes may be
useful in modulating
fertility (Print, C.G. et al. (1998) Proc. Nat). Acad. Sci. USA 95:12424-
12431).
Studies in rat ischemic brain found Bcl-w to be overexpressed relative to its
normal low
constitutive level of expression in nonischemic brain. Furthermore, in vitro
studies to examine the
mechanism of action of Bcl-w revealed that isolated rat brain mitochondria
were unable to respond to
an addition of recombinant Bax or high concentrations of calcium when Bcl-w
was also present. The
normal response would be the release of cytochrome c from the mitochondria.
Additionally,
recombinant Bcl-w protein was found to inhibit calcium-induced loss of
mitochondria)
transmembrane potential, which is indicative of permeability transition.
Together these findings
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suggest that Bcl-w may be a neuro-protectant against ischemic neuronal death
and may achieve this
protection via the mitochondria) death-regulatory pathway (Yan, C. et al.
(2000) J. Cereb. Blood Flow
Metab. 20:620-630).
The bfl-1 gene is an additional member of the Bcl-2 family, and is also a
suppressor of
apoptosis. The Bfl-1 protein has 175 amino acids, and contains the BHl, BH2,
and BH3 conserved
domains found in Bcl-2 family members. It also contains a Gln-rich NH2-
terminal region and lacks
an NH domain 1, unlike other Bcl-2 family members. The mouse Al protein shares
high sequence
homology with Bfl-1 and has the 3 conserved domains found in Bfl-1. Apoptosis
induced by the p53
tumor suppressor protein is suppressed by Bfl-1, similar to the action of Bcl-
2, Bcl-xL, and EBV-
BHRFl (D'Sa-Eipper, C. et al. (1996) Cancer Res. 56:3879-3882). Bfl-1 is found
intracellularly,
with the highest expression in the hematopoietic compartment, i.e. blood,
spleen, and bone marrow;
moderate expression in lung, small intestine, and testis; and minimal
expression in other tissues. It is
also found in vascular smooth muscle cells and hematopoietic malignancies. A
correlation has been
noted between the expression level of bfl-1 and the development of stomach
cancer, suggesting that
the Bfl-1 protein is involved in the development of stomach cancer, either in
the promotion of
cancerous cell survival or in cancer (Choi, S.S. et al. (1995) Oncogene
11:1693-1698).
Cancers are characterized by continuous or uncontrolled cell proliferation.
Some cancers are
associated with suppression of normal apoptotic cell death. Strategies for
treatment may involve
either reestablishing control over cell cycle progression, or selectively
stimulating apoptosis in
cancerous cells (Nigg, E.A. (1995) BioEssays 17:471-480). Immunological
defenses against cancer
include induction of apoptosis in mutant cells by tumor suppressors, and the
recognition of tumor
antigens by T lymphocytes. Response to mitogenic stresses is frequently
controlled at the level of
transcription and is coordinated by various transcription factors. For
example, the Rel/NF-kappa B
family of vertebrate transcription factors plays a pivotal role in
inflammatory and immune responses
to radiation. The NF-kappa B family includes p50, p52, ReIA, ReIB, cRel, and
other DNA-binding
proteins. The p52 protein induces apoptosis, upregulates the transcription
factor c-Jun, and activates
c-Jun N-terminal kinase 1 (JNKl) (Sun, L. et al. (1998) Gene 208:157-166).
Most NF-kappa B
proteins form DNA-binding homodimers or heterodimers. Dimerization of many
transcription factors
is mediated by a conserved sequence known as the bZIP domain, characterized by
a basic region
followed by a leucine zipper.
The Fas/Apo-1 receptor (FAS) is a member of the tumor necrosis factor (TNF)
receptor
family. Upon binding its ligand (Fas ligand), the membrane-spanning FAS
induces apoptosis by
recruiting several cytoplasmic proteins that transmit the death signal. One
such protein, termed FAS-
associated protein factor 1 (FAFl), was isolated from mice, and it was
demonstrated that expression
of FAF1 in L cells potentiated FAS-induced apoptosis (Chu, K. et al. (1995)
Proc. Nat). Acad. Sci.
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USA 92:11894-11898). Subsequently, FAS-associated factors have been isolated
from numerous
other species, including fruit fly and quail (Frohlich, T. et al. (1998) J.
Cell Sci. 111:2353-2363).
Another cytoplasmic protein that functions in the transmittal of the death
signal from Fas is the Fas-
associated death domain protein, also known as FADD. FADD transmits the death
signal in both
FAS-mediated and TNF receptor-mediated apoptotic pathways by activating
caspase-8 (Bang, S. et al.
(2000) J. Biol. Chem. 275:36217-36222).
Fragmentation of chromosomal DNA is one of the hallmarks of apoptosis. DNA
fragmentation factor (DFF) is a protein composed of two subunits, a 40-kDa
caspase-activated
nuclease termed DFF40/CAD, and its 45-kDa inhibitor DFF45/ICAD. Two mouse
homologs of
DFF45/ICAD, termed C>DE-A and CIDE-B, have recently been described (Inohara,
N. et al. (1998)
EMBO J. 17:2526-2533). CIDE-A and CIDE-B expression in mammalian cells
activated apoptosis,
while expression of CIDE-A alone induced DNA fragmentation. In addition, FAS-
mediated
apoptosis was enhanced by CIDE-A and CIDE-B, further implicating these
proteins as effectors that
mediate apoptosis.
Transcription factors play an important role in the onset of apoptosis. A
number of
downstream effector molecules, particularly proteases such as the cysteine
proteases called caspases,
are involved in the initiation and execution phases of apoptosis. The
activation of the caspases results
from the competitive action of the pro-survival and pro-apoptosis Bcl-2-
related proteins (Print, C.G. .
et al. (1998) Proc. Natl. Acad. Sci. USA 95:12424-12431). A pro-apoptotic
signal can activate
initiator caspases that trigger a proteolytic caspase cascade, leading to the
hydrolysis of target
proteins and the classic apoptotic death of the cell. Two active site
residues, a cysteine and a
histidine, have been implicated in the catalytic mechanism. Caspases are among
the most specific
endopeptidases, cleaving after aspartate residues.
Caspases are synthesized as inactive zymogens consisting of one large (p20)
and one small
(p10) subunit separated by a small spacer region, and a variable N-terminal
prodomain. This
prodomain interacts with cofactors that can positively or negatively affect
apoptosis. An activating
signal causes autoproteolytic cleavage of a specific aspartate residue (D297
in the caspase-1
numbering convention) and removal of the spacer and prodomain, leaving a
p10/p20 heterodimer.
Two of these heterodimers interact via their small subunits to form the
catalytically active tetramer.
The long prodomains of some caspase family members have been shown to promote
dimerization and
auto-processing of procaspases. Some caspases contain a "death effector
domain" in their prodomain
by which they can be recruited into self activating complexes with other
caspases and FADD protein-
associated death receptors or the TNF receptor complex. In addition, two
dimers from different
caspase family members can associate, changing the substrate specificity of
the resultant tetramer.
Tumor necrosis factor (TNF) and related cytokines induce apoptosis in lymphoid
cells.
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(Reviewed in Nagata, S. (1997) Cell 88:355-365.) Binding of TNF to its
receptor triggers a signal
transduction pathway that results in the activation of a proteolytic caspase
cascade. One such
caspase, ICE (Interleukin-1(3 converting enzyme), is a cysteine protease
comprised of two large and
two small subunits generated by ICE auto-cleavage (Dinarello, C. A. (1994)
FASEB J. 8:1314-1325).
ICE is expressed primarily in monocytes. ICE processes the cytokine precursor,
interleukin-1(3, into
its active form, which plays a central role in acute and chronic inflammation,
bone resorption,
myelogenous leukemia, and other pathological processes. ICE and related
caspases cause apoptosis
when overexpressed in transfected cell lines.
A caspase recruitment domain (CARD) is found within the prodomain of several
apical
caspases and is conserved in several apoptosis regulatory molecules such as
Apaf 2, RAIDD, and
cellular inhibitors of apoptosis proteins (IAPs) (Hofmann, K. et al. (1997)
Trends Biochem. Sci.
22:155-157). The regulatory role of CARD in apoptosis may be to allow proteins
such as Apaf 1 to
associate with caspase-9 (Li, P. et al. ( 1997) Cell 91:479-489). A human cDNA
encoding an
apoptosis repressor with a CARD (ARC) which is expressed in both skeletal and
cardiac muscle has
been identified and characterized. ARC functions as an inhibitor of apoptosis
and interacts
selectively with caspases (I~oseki, T. et al. (1998) Proc. Natl. Acad. Sci.
USA 95:5156-5160). All of
these interactions have clear effects on the control of apoptosis (reviewed in
Chan S.L. and M.P.
Mattson (1999) J. Neurosci. Res. 58:167-190; Salveson, G.S. and V.M. Dixit
(1999) Proc. Natl. Acad.
Sci. USA 96:10964-10967).
ES18 was identified as a potential regulator of apoptosis in mouse T-cells
(Park, E.J. et al.
(1999) Nuc. Acid. Res. 27:1524-1530). ES18 is 428 amino acids in length,
contains an N-terminal
proline-rich region, an acidic glutamic acid-rich domain, and a putative LXXLL
nuclear receptor
binding motif. The protein is preferentially expressed in lymph nodes and
thymus. The level of ES18
expression increases in T-cell thymoma 549.1 in response to treatment with
dexamethasone,
staurosporine, or C2-ceramide, which induce apoptosis. ES 18 may play a role
in stimulating
apoptotic cell death in T-cells.
The rat ventral prostate (RVP) is a model system for the study of hormone-
regulated
apoptosis. RVP epithelial cells undergo apoptosis in response to androgen
deprivation. Messenger
RNA (mRNA) transcripts that are up-regulated in the apoptotic RVP have been
identified (Briehl, M.
M. and Miesfeld, R. L. (1991) Mol. Endocrinol. 5:1381-1388). One such
transcript encodes RVP.1,
the precise role of which in apoptosis has not been determined. The human
homolog of RVP.1,
hRVPl, is 89% identical to the rat protein (I~atahira, J. et al. (1997) J.
Biol. Chem. 272:26652-
26658). hRVPl is 220 amino acids in length and contains four transmembrane
domains. hRVPl is
highly expressed in the lung, intestine, and liver. Interestingly, hRVP1
functions as a low affinity
receptor for the Clostridium perfrin~ens enterotoxin, a causative agent of
diarrhea in humans and
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other animals.
Cytokine-mediated apoptosis plays an important role in hematopoiesis and the
immune
response. Myeloid cells, which are the stem cell progenitors of macrophages,
neutrophils,
erythrocytes, and other blood cells, proliferate in response to specific
cytokines such as
granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin-3
(IL-3). When
deprived of GM-CSF or IL-3, myeloid cells undergo apoptosis. The murine
requiem (req) gene
encodes a putative transcription factor required for this apoptotic response
in the myeloid cell Brie
FDCP-1 (Gabig, T. G. et al. (1994) J. Biol. Chem. 269:29515-29519). The Req
protein is 371 amino
acids in length and contains a nuclear localization signal, a single Kruppel-
type zinc finger, an acidic
domain, and a cluster of four unique zinc-forger motifs enriched in cysteine
and histidine residues
involved in metal binding. Expression of req is not myeloid- or apoptosis-
specific, suggesting that
additional factors regulate Req activity in myeloid cell apoptosis.
Dysregulation of apoptosis has recently been recognized as a significant
factor in the
pathogenesis of many human diseases. For example, excessive cell survival
caused by decreased
apoptosis can contribute to disorders related to cell proliferation and the
immune response. Such
disorders include cancer, autoimmune diseases, viral infections, and
inflammation. In contrast,
excessive cell death caused by increased apoptosis can lead to degenerative
and immunodeficiency
disorders such as AIDS, neurodegenerative diseases, and myelodysplastic
syndromes. (Thompson,
C.B. (1995) Science 267:1456-1462.)
Impaired regulation of apoptosis is also associated with loss of neurons in
Alzheimer's
disease. Alzheimer's disease is a progressive neurodegenerative disorder that
is characterized by the
formation of senile plaques and neurofibrillary tangles containing amyloid
beta peptide. These
plaques are found in limbic and association cortices of the brain, including
hippocampus, temporal
cortices, cingulate cortex, amygdala, nucleus basalis and locus caeruleus. B-
amyloid peptide
participates in signaling pathways that induce apoptosis and lead to the death
of neurons (Kajkowski,
C. et al. (2001) J. Biol. Chem. 276:18748-18756). Early in Alzheimer's
pathology, physiological
changes are visible in the cingulate cortex (Minoshima, S. et al. ( 1997)
Annals of Neurology 42:85-
94). In subjects with advanced Alzheimer's disease, accumulating plaques
damage the neuronal
architecture in limbic areas and eventually cripple the memory process.
Cancer
Cancer remains a major public health cancer, and current preventative measures
and
treatments do not match the needs of most patients. Cancers are characterized
by continuous or
uncontrolled cell proliferation. Some cancers are associated with suppression
of normal apoptotic
cell death. Understanding of the neoplastic process can be aided by the
identification of molecular
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markers of prognostic and diagnostic importance. Cancers are associated with
oncoproteins which
are capable of transforming normal cells into malignant cells. Some
oncoproteins are mutant
'isoforms of the normal protein while others are abnormally expressed with
respect to location or level
of expression. Normal cell proliferation begins with binding of a growth
factor to its receptor on the
cell membrane, resulting in activation of a signal system that induces and
activates nuclear regulatory
factors to initiate DNA transcription, subsequently leading to cell division.
Classes of oncoproteins
known to affect the cell cycle controls include growth factors, growth factor
receptors, intracellular
signal transducers, nuclear transcription factors, and cell-cycle control
proteins. Several types of
cancer-specific genetic markers, such as tumor antigens and tumor suppressors,
have also been
identified.
Onco_enes
Oncoproteins are encoded by genes, called oncogenes, that are derived from
genes that
normally control cell growth and development. Many oncogenes have been
identified and
characterized. These include growth factors such as sis, receptors such as
erbA, erbB, zzeu, and ros,
intracellular receptors such as src, yes, fps, abl, and znet, protein-
serine/threonine kinases such as nzos
and raf, nuclear transcription factors such as jun, fos, myc, N-myc, nzyb,
ski, and rel, cell cycle control
proteins such as RB and p53, mutated tumor-suppressor genes such as zzzdzn2,
Cipl, p16, and cyclin
D, ras, set, cazz, sec, and gag R10.
Viral oncogenes are integrated into the human genome after infection of human
cells by
certain viruses. Examples of viral oncogenes include v-src, v-abl, and v-fps.
Transformation of
normal genes to oncogenes may also occur by chromosomal translocation. The
Philadelphia
chromosome, characteristic of chronic myeloid leukemia and a subset of acute
lymphoblastic
leukemias, results from a reciprocal translocation between chromosomes 9 and
22 that moves a
truncated portion of the proto-oncogene c-abl to the breakpoint cluster region
(bcr) on chromosome
22. The hybrid c-abl-bcr gene encodes a chimeric protein that has tyrosine
kinase activity. In chronic
myeloid leukemia, the chimeric protein has a molecular weight of 210 kd,
whereas in acute leukemias
a more active 180 kd tyrosine kinase is formed (Robbins, S.L. et al. (1994)
Pathologic Basis of
Disease, W.B. Saunders Co., Philadelphia PA).
The Ras superfamily of small GTPases is involved in the regulation of a wide
range of
cellular signaling pathways. Ras family proteins are membrane-associated
proteins acting as
molecular switches that bind GTP and GDP, hydrolyzing GTP to GDP. The GTPase-
activating
protein of Ras (RasGAP) is activated by the GTPase-activating family of
proteins (GAPs). A central
conserved GAP-related domain, and a C-terminal pleckstrin homology (PIE domain
are characteristic
of the GAP1 subfamily of RasGAP proteins (Allen, M. et al., (1998) Gene 218:17-
25). In the active
GTP-bound state Ras family proteins interact with a variety of cellular
targets to activate downstream
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signaling pathways. For example, members of the Ras subfamily are essential in
transducing signals
from receptor tyrosine kinases (RTKs) to a series of serine/threonine kinases
which control cell
growth and differentiation. Activated Ras genes were initially found in human
cancers and
subsequent studies confirmed that Ras function is critical in the
determination of whether cells
continue to grow or become terminally differentiated. Stimulation of cell
surface receptors activates
Ras which, in turn, activates cytoplasmic kinases. The kinases translocate to
the nucleus and activate
key transcription factors that control gene expression and protein synthesis
(Barbacid, M. (1987)
Annu. Rev. Biochem. 56:779-827, Treisman, R. (1994) Curr. Opin. Genet. Dev.
4:96-98). Mutant
Ras proteins, which bind but can not hydrolyze GTP, are permanently activated,
and cause continuous
cell proliferation or cancer.
Activation of Ras family proteins is catalyzed by guanine nucleotide exchange
factors (GEFs)
which catalyze the dissociation of bound GDP and subsequent binding of GTP. A
recently
discovered RalGEF-like protein, RGL3, interacts with both Ras and the related
protein Rit.
Constitutively active Rit, like Ras, can induce oncogenic transformation,
although since Rit fails to
interact with most known Ras effector proteins, novel cellular targets may be
involved in Rit
transforming activity. RGL3 interacts with both Ras and Rit, and thus may act
as a downstream
effector for these proteins (Shao, H. and Andres, D.A. (2000) J. Biol. Chem.
275:26914-26924).
Tumor antigens
Tumor antigens are cell surface molecules that are differentially expressed in
tumor cells
relative to non-tumor tissues. Tumor antigens make tumor cells immunologically
distinct from
normal cells and are potential diagnostics for human cancers. Several
monoclonal antibodies have
been identified which react specifically with cancerous cells such as T-cell
acute lymphoblastic
leukemia and neuroblastoma (Minegishi et al. (1989) Leukemia Res. 13:43-51;
Takagi et al. (1995)
Int. J. Cancer 61:706-715). In addition, the discovery of high level
expression of the HER2 gene in
breast tumors has led to the development of therapeutic treatments (Liu et al.
(1992) Oncogene 7:
1027-1032; Kern (1993) Am. J. Respir. Cell Mol. Biol. 9:448-454). Tumor
antigens axe found on the
cell surface and have been characterized either as membrane proteins or
glycoproteins. For example,
MAGE genes encode a family of tumor antigens recognized on melanoma cell
surfaces by autologous
cytolytic T lymphocytes. Among the 12 human MAGE genes isolated, half are
differentially
expressed in tumors of various histological types (De Plaen et al. (1994)
Immunogenetics 40:360-
369). None of the 12, MAGE genes, however, is expressed in healthy tissues
except testis and
placenta.
TA1, a tumor-associated gene, was identified and cloned based on its increased
expression in
rat hepatoma cells compared to normal rat liver (Sang, J. et al. (1995) Cancer
Res. 55:1152-1159).
The deduced amino acid sequence encodes an integral membrane protein which
contains multiple
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transmembrane domains. TA1 exhibits an oncofetal expression pattern in liver.
Transcripts for TAl
are present in rat fetal liver and hepatoma, but they are not present in
normal adult rat liver. In
normal adult rat, TA1 is expressed at moderate-to-high levels in testes and
brain, and at low levels in
ovary, spleen, mammary gland, and uterus. TA1 expression is most abundant in
placenta, which
suggests a developmental role for the molecule (Sang et al., supra).
The E16 gene cloned from human peripheral blood lymphocytes encodes a 241
amino acid
integral membrane protein with multiple predicted transmembrane domains
(Gaugitsch, H.W. et al.
(1992) J. Biol. Chem. 267:11267-73 ). E16 gene expression is closely linked to
cellular activation
and division. In myeloid and lymphoid cells, E16 transcripts are rapidly
induced and rapidly
degraded after stimulation. This pattern of expression resembles the kinetics
seen for proto-oncogenes
and lymphokines in the T cell system (Gaugitsch et al., supra). E16 expression
was not detected in
normal (non-cancerous) human tissues such as adult brain, lung, liver, colon,
esophagus, stomach, or
kidney, nor in four-month fetal brain, lung, liver, or kidney (Wolf, D.A. et
al. (1996) Cancer Res.
56:5012-5022; Gaugitsch et al., supra). E16 was detected in every cell line
tested (Gaugitsch et al.,
supra). Its presence in rapidly dividing cell lines and its absence in human
tissues with low
proliferative potential suggest a direct involvement of E16 protein in the
cell division process
(Gaugitsch et al., supra).
The proteins encoded by the rat TAl and human E16 genes share 95% amino acid
sequence
identity (Wolf et al., supra). Nucleotide probes and antibodies specific for
homologous regions of
TA1 and E16 were prepared in order to detect TA1/E16 expression in various
human cancers. With
these probes, elevated levels of TA1/E16 expression were detected in colonic,
gastric, and breast
adenocarcinomas, and in lymphoma. Although E16 was originally described by
Gaugitsch et al.
(supra) as a lymphocyte activation marker, no significant levels of TA1/E16
message was detected in
tissues from patients with active ulerative colitis and Crohn's disease (Wolf
et al., supra).
The TA1 and E16 proteins show significant homology to a putative amino acid
permease
from the helminth Schistosoma mansoni (GenBank 407047; unpublished). These
sequence
similarities suggest a potential role for TAl and E16 proteins in amino acid
or nutrient uptake which
may be up-regulated in tumor cells (Wolf et al., supra).
Tumor sup ressors
Tumor suppressor genes are generally defined as genetic elements whose loss or
inactivation
contributes to the deregulation of cell proliferation and the pathogenesis and
progression of cancer.
Tumor suppressor genes normally function to control or inhibit cell growth in
response to stress and
to limit the proliferative life span of the cell. Several tumor suppressor
genes have been identified
including the genes encoding the retinoblastoma (Rb) protein, p53, and the
breast cancer 1 and 2
proteins (BRCA1 and BRCA2). Mutations in these genes are associated with
acquired and inherited
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genetic predisposition to the development of certain cancers.
The role of p53 in the pathogenesis of cancer has been extensively studied.
(Reviewed in
Aggarwal, M. L. et al. (1998) J. Biol. Chem. 273:1-4; Levine, A. (1997) Cell
88:323-331.) About
50% of all human cancers contain mutations in the p53 gene. These mutations
result in either the
absence of functional p53 or, more commonly, a defective form of p53 which is
overexpressed. p53
is a transcription factor that contains a central core domain required for DNA
binding. Most cancer-
associated mutations in p53 localize to this domain. In normal proliferating
cells, p53 is expressed at
low levels and is rapidly degraded. p53 expression and activity is induced in
response to DNA
damage, abortive mitosis, and other stressful stimuli. In these instances, p53
induces apoptosis or
arrests cell growth until the stress is removed. Downstream effectors of p53
activity include
apoptosis-specific proteins and cell cycle regulatory proteins, including Rb,
oncogene products,
cyclins, and cell cycle-dependent kinases.
A novel gene, INGI , encoding a potential tumor suppressor protein has been
cloned.
(Garkavtsev, I. et al. (1996) Nat. Genet. 14:415-420.) Overexpression of ING1
in normal and
transformed cell lines inhibits their growth in vitro. Furthermore, expression
of antisense INGI
promotes neoplastic transformation of cultured cells, as demonstrated by their
ability to grow in soft
agar and to induce tumors when injected into immunodeficient mice. p33, the
protein encoded by
INGI , localizes to the nucleus and has similarity to retinoblastoma binding
protein 2 (RbBP2) and to
zinc finger motifs. Decreased expression of p33 is observed in some breast
cancer cell linen and a
truncated form of p33 is expressed at high levels in a neuroblastoma cell
line. Truncated p33 results
from genomic rearrangement at the ING1 locus. Moreover, levels of INGl RNA and
protein are
increased about 10-fold in senescent cells, which are ageing, non-
proliferative cells, compared to the
levels expressed in young, proliferating cells. (Garkavtsev, I. and Riabowol,
K. (1997) Mol. Cell
Biol. 17:2014-2019.) These observations indicate that p33 normally functions
to inhibit cell growth
and limit cellular life span.
Recent studies show that p33 cooperates with p53 in the negative regulation of
cell
proliferation. (Garkavtsev, I. et al. (1998) Nature 391:295-298.) The
functions of p53 and p33 are
interdependent, and p33 directly modulates p53-dependent transcriptional
activation. A direct
physical association between p33 and p53 has been demonstrated by co-
immunoprecipitation,
indicating that p33 may influence the activity of p53 in cell cycle control,
ageing, and apoptosis.
The metastasis-suppressor gene KAIl (CD82) has been reported to be related to
the tumor
suppressor gene p53. KAIl is involved in the progression of human prostatic
cancer and possibly
lung and breast cancers when expression is decreased. KAI1 encodes a member of
a structurally
distinct family of leukocyte surface glycoproteins. The family is known as
either the tetraspan
transmembrane protein family or transmembrane 4 superfamily (TM4SF) as the
members of this
CA 02441495 2003-08-06
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family span the plasma membrane four times. The family is composed of integral
membrane proteins
having a N-terminal membrane-anchoring domain which functions as both a
membrane anchor and a
translocation signal during protein biosynthesis. The N-terminal membrane-
anchoring domain is not
cleaved during biosynthesis. TM4SF proteins have three additional
transmembrane regions, seven or
more conserved cysteine residues, are similar in size (218 to 284 residues),
and all have a large
extracellular hydrophilic domain with three potential N-glycosylation sites.
The promoter region
contains many putative binding motifs for various transcription factors,
including five AP2 sites and
nine SpI sites. Gene structure comparisons of KAIl and seven other members of
the TM4SF indicate
that the splicing sites relative to the different structural domains of the
predicted proteins are
conserved. This suggests that these genes are related evolutionarily and arose
through gene
duplication and divergent evolution (Levy, S. et al. (1991) J. Biol. Chem.
266:14597-14602; Dong,
J.T. et al. (1995) Science 268:884-886; Dong, J:T. et al., (1997) Genomics
41:25-32).
The Leucine-rich gene-Glioma Inactivated (LGI1) protein shares homology with a
number of
transmembrane and extracellular proteins which function as receptors and
adhesion proteins. LGI1 is
encoded by an LLR (leucine-rich, repeat-containing) gene and maps to 10q24.
LGIl has four LLRs
which are flanked by cysteine-rich regions and one transmembrane domain
(Somerville, R.P., et al.
(2000) Mamm. Genome 11:622-627). LGI1 expression is seen predominantly in
neural tissues,
especially brain. The loss of tumor suppresser activity is seen in the
inactivation of the LGI1 protein
which occurs during the transition from low to high-grade tumors in malignant
gliomas. The
reduction of LGI1 expression in low grade brain tumors and its significant
reduction or absence of
expression in malignant gliomas suggests that it could be used for diagnosis
of glial tumor
progression (Chernova, O.B., et al. (1998) Oncogene 17:2873-2881).
The ST13 tumor suppresser was identified in a screen for factors related to
colorectal
carcinomas by subtractive hybridization between cDNA of normal mucosal tissues
and mRNA of
colorectal carcinoma tissues (Cao, J. et al. (1997) J. Cancer Res. Clin.
Oncol. 123:447-451). ST13 is
down-regulated in human colorectal carcinomas.
Mutations in the von Hippel-Lindau (VHL) tumor suppresser gene are associated
with retinal
and central nervous system hemangioblastomas, clear cell renal carcinomas, and
pheochromocytornas
(Hoffman, M. et al. (2001) Hum. Mol. Genet. 10:1019-1027; Kamada, M. (2001)
Cancer Res.
61:4184-4189). Tumor progression is linked to defects or inactivation of the
VHL gene. VHL
regulates the expression of transforming growth factor-a, the GLUT-1 glucose
transporter and
vascular endothelial growth~factor. The VHL protein associates with elongin B,
elongin C, Cult and
Rbxl to form a complex that regulates the transcriptional activator hypoxia-
inducible factor (HIF).
HIF induces genes involved in angiogenesis such as vascular endothelial growth
factor and platelet-
derived growth factor B. Loss of control of HIF caused by defects in VHL
results in the excessive
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production of angiogenic peptides. VHL may play roles in inhibition of
angiogenesis, cell cycle
control, fibronectin matrix assembly, cell adhesion, and proteolysis.
Mutations in tumor suppressor genes are a common feature of many cancers and
often appear
to affect a critical step in the pathogenesis and progression of tumors.
Accordingly, Chang, F. et al.
(1995; J. Clin. Oncol. 13: 1009-1022) suggest that it may be possible to use
either the gene or an
antibody to the expressed protein 1) to screen patients at increased risk for
cancer, 2) to aid in
diagnosis made by traditional methods, and 3) to assess the prognosis of
individual cancer patients.
In addition, Hamada, K et al. (1996; Cancer Res. 56:3047-3054) are
investigating the introduction of
p53 into cervical cancer cells via an adenoviral vector as an experimental
therapy for cervical cancer.
The PR-domain genes were recently recognized as playing a role in human
tumorigenesis.
PR-domain genes normally produce two protein products: the PR-plus product,
which contains the PR
domain, and the PR-minus product which lacks this domain. In cancer cells, PR-
plus is disrupted or
overexpressed, while PR-minus is present or overexpressed. The imbalance in
the amount of these
two proteins appears to be an important cause of malignancy (Jiang, G.L. and
Huang, S. (2000)
Histol. Histopathol. 15:109-117).
Many neoplastic disorders in humans can be attributed to inappropriate gene
transcription.
Malignant cell growth may result from either excessive expression of tumor
promoting genes or
insufficient expression of tumor suppressor genes (Cleary, M.L. (1992) Cancer
Surv. 15:89-104).
Chromosomal translocations may also produce chimeric loci which fuse the
coding sequence of one
gene with the regulatory regions of a second unrelated gene. An important
class of transcriptional
regulators are the zinc forger proteins. The zinc finger motif, which binds
zinc ions, generally
contains tandem repeats of about 30 amino acids consisting of periodically
spaced cysteine and
histidine residues. Examples of this sequence pattern include the C2H2-type,
C4-type, and C3HC4-
type zinc forgers, and the PHD domain (Lewin, supra; Aasland, R., et al.
(1995) Trends Biochem. Sci.
20:56-59). One clinically relevant zinc-finger protein is WTl, a tumor-
suppressor protein that is
inactivated in children with Wilm's tumor. The oncogene bcl-6, which plays an
important role in
large-cell lymphoma, is also a zinc-forger protein (Papavassiliou, A.G. (1995)
N. Engl. J. Med.
332:45-47).
Tumor responsive proteins
Cancers, also called neoplasias, are characterized by continuous and
uncontrolled cell
proliferation. They can be divided into three categories: carcinomas,
sarcomas, and leukemias.
Carcinomas are malignant growths of soft epithelial cells that may infiltrate
surrounding tissues and
give rise to metastatic tumors. Sarcomas may be of epithelial origin or arise
from connective tissue.
Leukemias are progressive malignancies of blood-forming tissue characterized
by proliferation of
leukocytes and their precursors, and may be classified as myelogenous
(granulocyte- or monocyte-
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derived) or lymphocytic (lymphocyte-derived). Tumorigenesis refers to the
progression of a tumor's
growth from its inception. Malignant cells may be quite similar to normal
cells within the tissue of
origin or may be undifferentiated (anaplastic). Tumor cells may possess few
nuclei or one large
polymorphic nucleus. Anaplastic cells may grow in a disorganized mass that is
poorly vascularized
and as a result contains large areas of ischemic necrosis. Differentiated
neoplastic cells may secrete
the same proteins as the tissue of origin. Cancers grow, infiltrate, invade,
and destroy the
surrounding tissue through direct seeding of body cavities or surfaces,
through lymphatic spread, or
through hematogenous spread. Cancer remains a major public health concern and
current
preventative measures and treatments do not match the needs of most patients.
Understanding of the
neoplastic process of tumorigenesis can be aided by the identification of
molecular markers of
prognostic and diagnostic importance.
Current forms of cancer treatment include the use of immunosuppressive drugs
(Morisaki, T.
et al. (2000) Anticancer Res. 20: 3363-3373; Geoerger, B. et al. (2001) Cancer
Res. 61: 1527-1532).
The identification of proteins involved in cell signaling, and specifically
proteins that act as receptors
for immunosuppressant drugs, may facilitate the development of anti-tumor
agents. For example,
immunophilins are a family of conserved proteins found in both prokaryotes and
eukaryotes that bind
to immunosuppressive drugs with varying degrees of specificity. One such group
of irnmunophilic
proteins is the peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) family
(PPIase, rotamase). These
enzymes, first isolated from porcine kidney cortex, accelerate protein folding
by catalyzing the cis-
trans isomerization of proline imidic peptide bonds in oligopeptides (Fischer,
G. and Schmid, F.X.
(1990) Biochemistry 29: 2205-2212). Included within the immunophilin family
are the cyclophilins
(e.g., peptidyl-prolyl isomerase A or PPIA) and FK-binding protein (e.g.,
FKBP) subfamilies.
Cyclophilins are multifunctional receptor proteins which participate in signal
transduction activities,
including those mediated by cyclosporin (or cyclosporine). The PPIase domain
of each family is
highly conserved between species. Although structurally distinct, these
multifunctional receptor
proteins are involved in numerous signal transduction pathways, and have been
implicated in folding
and trafficking events.
The immunophilin protein cyclophilin binds to the immunosuppressant drug
cyclosporin A.
FKBP, another immunophilin, binds to FK506 (or rapamycin). Rapamycin is an
immunosuppressant
agent that arrests cells in the G, phase of growth, inducing apoptosis. Like
cyclophilin, this macrolide
antibiotic (produced by Streptomyces tsukubaensis) acts by binding to
ubiquitous, predominantly
cytosolic immunophilin receptors. These immunophilin/immunosuppressant
complexes (e.g.,
cyclophilin A/cyclosporin A (CypA/CsA) and FKBP12/FK506) achieve their
therapeutic results
through inhibition of the phosphatase calcineurin, a calcium/calmodulin-
dependent protein kinase that
participates in T-cell activation (Hamilton, G.S. and Steiner, J.P. (1998) J.
Med. Chem. 41: 5119-
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5143). The marine fkbp5l gene is abundantly expressed in immunological
tissues, including the
thymus and T lymphocytes (Baughman, G. et al. (1995) Molec. Cell. Biol. 15:
4395-4402).
FKBPI2lrapamycin-directed immunosuppression occurs through binding to TOR
(yeast) or FRAP
(FKBP12-rapamycin-associated protein, in mammalian cells), the kinase target
of rapamycin essential
for maintaining normal cellular growth patterns. Dysfunctional TOR signaling
has been linked to
various human disorders including cancer (Metcalfe, S.M. et al. (1997)
Oncogene 15: 1635-1642;
Emami, S. et al. (2001) FASEB J. 15: 351-361), and autoimmunity (Damoiseaux,
J.G. et al. (1996)
Transplantation 62: 994-1001).
Several cyclophilin isozymes have been identified, including cyclophilin B,
cyclophilin C,
mitochondrial matrix cyclophilin, bacterial cytosolic and periplasmic PPIases,
and natural-killer cell
cyclophilin-related protein possessing a cyclophilin-type PPIase domain, a
putative tumor-recognition
complex involved in the function of natural killer (NK) cells. These cells
participate in the innate
cellular immune response by lysing virally-infected cells or transformed
cells. NK cells specifically
target cells that have lost their expression of major histocompatibility
complex (MHC) class I genes
(common during tumorigenesis), endowing them with the potential for
attenuating tumor growth. A
150-kDa molecule has been identified on the surface of human NK cells that
possesses a domain
which is highly homologous to cyclophilin/peptidyl-prolyl cis-trans isomerase.
This cyclophilin-type
protein may be a component of a putative tumor-recognition complex, a NK tumor
recognition
sequence (NK-TR) (Anderson, S.K. et al. (1993) Proc. Natl. Acad. Sci. USA 90:
542-546). The
NKTR tumor recognition sequence mediates recognition between tumor cells and
large granular
lymphocytes (LGLs), a subpopulation of white blood cells (comprised of
activated cytotoxic T cells
and natural killer cells) capable of destroying tumor targets. The protein
product of the NKTR gene
presents on the surface of LGLs and facilitates binding to tumor targets. More
recently, a mouse Nktr
gene and promoter region have been located on chromosome 9. The gene encodes a
NK-cell-specific
150-kDa protein (NK-TR) that is homologous to cyclophilin and other tumor-
responsive proteins
(Simons-Evelyn, M. et al. (1997) Genomics 40: 94-100).
Other proteins that interact with tumorigenic tissue include cytokines such as
tumor necrosis
factor (TNF). The TNF family of cytokitnes are produced by lymphocytes and
macrophages, and can
cause the lysis of transformed (tumor) endothelial cells. Endothelial protein
1 (Edpl) has been
identified as a human gene activated transcriptionally by TNF-alpha in
endothelial cells, and a TNF-
alpha inducible Edpl gene has been identified in the mouse (Swift, S. et al.
(1998) Biochim. Biophys.
Acta 1442: 394-398).
Expression profiling
Array technology can provide a simple way to explore the expression of a
single polymorphic
gene or the expression profile of a large number of related or unrelated
genes. When the expression
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of a single gene is examined, arrays are employed to detect the expression of
a specific gene or its
variants. When an expression profile is examined, arrays provide a platform
for identifying genes
that are tissue specific, are affected by a substance being tested in a
toxicology assay, are part of a
signaling cascade, carry out housekeeping functions, or are specifically
related to a particular genetic
predisposition, condition, disease, or disorder.
The discovery of new proteins associated with cell growth, differentiation,
and death, and the
polynucleotides encoding them, satisfies a need in the art by providing new
compositions which are
useful in the diagnosis, prevention, and treatment of cell proliferative
disorders including cancer,
developmental disorders, neurological disorders, reproductive disorders, and
autoimmune/inflammatory disorders, and in the assessment of the effects of
exogenous compounds on
the expression of nucleic acid and amino acid sequences of proteins associated
with cell growth,
differentiation, and death.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, proteins associated with cell
growth,
differentiation, and death, referred to collectively as "CGDD" and
individually as "CGDD-1,"
"CGDD-2," "CGDD-3," "CGDD-4," "CGDD-5," "CGDD-6," "CGDD-7," "CGDD-8," "CGDD-
9,"
"CGDD-10," "CGDD-1 l," and "CGDD-12." .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 N0:1-12, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ m NO:1-12, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ )D
NO:1-12, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-12. In one alternative, the invention provides an
isolated polypeptide
comprising the amino acid sequence of SEQ ID NO:1-12.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected
from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-12, b) a polypeptide comprising a naturally
occurring amino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
)D NO:1-12, c) a biologically active fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ )D NO:1-12, and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ~ID NO: l-12.
In one alternative, the polynucleotide encodes a polypeptide selected from the
group consisting of
SEQ ID NO:1-12. In another alternative, the polynucleotide is selected from
the group consisting of
CA 02441495 2003-08-06
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SEQ m N0:13-24.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-12, 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:1-12, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-12. In one
alternative, the
invention provides a cell transformed with the recombinant polynucleotide. In
another alternative, the
invention provides a transgenic organism comprising the recombinant
polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ m NO:1-12, b) a polypeptide comprising a naturally occurring amino acid
sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ m NO:1-12, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m NO:1-12, and.d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ~ NO:1-12. 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:1-12, b) a polypeptide
comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ >D NO:1-12, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ 1D
NO:1-12, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-12.
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 ~ N0:13-24, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ )D
N0:13-24, 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
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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 )D N0:13-24, 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 N0:13-24, c) a polynucleotide complementary to the polynucleotide of
a), d) a
polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). The
method comprises a) hybridizing the sample with a probe comprising at least 20
contiguous
nucleotides comprising a sequence complementary to said target polynucleotide
in the sample, and
which probe specifically hybridizes to said target polynucleotide, under
conditions whereby a
hybridization complex is formed between said probe and said target
polynucleotide or fragments
thereof, and b) detecting the presence or absence of said hybridization
complex, and optionally, if
present, the amount thereof. In one alternative, the probe comprises at least
60 contiguous
nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ ID N0:13-24, 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
N0:13-24, 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 N0:1-12, 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:1-12, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ )D
NO:1-12, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m NO:1-12, and a pharmaceutically acceptable excipient. In
one embodiment, the
composition comprises an amino acid sequence selected from the group
consisting of SEQ m NO:1-
12. The invention additionally provides a method of treating a disease or
condition associated with
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decreased expression of functional CGDD, 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 ff~ NO: l-12, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ )D NO:1-12, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ >D
NO:1-12, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ )D NO:1-12. 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 expxession of
functional CGDD, comprising
administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ 117 NO:1-12, 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:1-12, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ )D NO:1-12,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ >D NO:1-12. 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
CGDD, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: l-12, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ )D NO:1-12, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ JD
NO:1-12, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
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consisting of SEQ ID NO:1-12. The method comprises a) combining the
polypeptide with at least
one test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-12, 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:1-12, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
N0:1-12, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-12. 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 N0:13-24,
the method
comprising. a) exposing a sample comprising the target polynucleotide to a
compound, b) detecting
altered expression of the target polynucleotide, and c) comparing the
expression of the target
polynucleotide in the presence of varying amounts of the compound and in the
absence of the
compound.
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 N0:13-24, ii) a
polynucleotide comprising a naturally occurnng polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:13-24,
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
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N0:13-24, 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 N0:13-24,
iii) a polynucleotide complementary to the polynucleatide of i), iv) a
polynucleotide complementary
to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
Alternatively, the target
polynucleotide comprises a fragment of a polynucleotide sequence selected from
the group consisting
of i)-v) above; c) quantifying the amount of hybridization complex; and d)
comparing the amount of
hybridization complex in the treated biological sample with the amount of
hybridization complex in
an untreated biological sample, wherein a difference in the amount of
hybridization complex in the
treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences ofthe presentinvention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability scores for the
matches between each
polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for .
analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to,be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
will be limited only by the appended claims.
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It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection with the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"CGDD" refers to the amino acid sequences of substantially purified CGDD
obtained~from
any species, particularly a mammalian species, including bovine, ovine,
porcine, murine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
CGDD. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of CGDD either by
directly interacting with
CGDD or by acting on components of the biological pathway in which CGDD
participates.
An "allelic variant" is an alternative form of the gene encoding CGDD. 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 CGDD include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as CGDD or a
polypeptide with at least one functional characteristic of CGDD. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding CGDD, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
CGDD. The encoded protein may also be "altered," and may contain deletions,
insertions, or
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substitutions of amino acid residues which produce a silent change and result
in a functionally
equivalent CGDD. 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 CGDD is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid
sequence to the complete native amino acid sequence associated with the
recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of CGDD. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of CGDD either by
directly interacting with CGDD or by acting on components of the biological
pathway in which
CGDD participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')Z, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind CGDD polypeptides can be prepared using intact
polypeptides or using
fragments containing small peptides of interest as the immunizing antigen. The
polypeptide or
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLI~. The coupled peptide is
then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
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on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an in vitro evolutionary
process (e.g., SELEX
(Systematic Evolution of Ligands by Exponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects fox target-specific aptamer sequences from large
combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may
include
deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other
nucleotide-like molecules.
The nucleotide components of an aptamer may have modified sugar groups (e.g.,
the 2'-OH group of a
ribonucleotide may be replaced by 2'-F or 2'-NHZ), which may improve a desired
property, e.g.,
resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
e.g., a high molecular weight carrier to slow clearance of the aptamer from
the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e:g., by
photo-activation of a
cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-
13.)
The term "intramer" refers to an aptarrier which is expressed in vivo. For
example, a vaccinia
virus-based RNA expression system has been used to express specific RNA
aptamers at high levels in
the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA
96:3606-3610).
The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or
other left-
handed nucleotide derivatives or nucleotide-like molecules. Aptamers
containing left-handed
nucleotides are resistant to degradation by naturally occurring enzymes, which
normally act on
substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a 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 CGDD, or of
any oligopeptide
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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 CGDD or fragments of
CGDD may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCl), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or 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
AIa GIy, 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 Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe . His, Met, Leu, Trp, Tyr
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Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which
retains at least one biological or immunological function of the natural
molecule. A derivative
polypeptide is one modified by glycosylation, pegylation, or any similar
process that retains at least
one biological or immunological function of the polypeptide from which it was
derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
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
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 CGDD or the polynucleotide encoding CGDD
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
CA 02441495 2003-08-06
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defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:13-24 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:13-24, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ >D N0:13-24 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:13-24 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:13-24 and the region of SEQ ID N0:13-24 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:1-12 is encoded by a fragment of SEQ ID N0:13-24. A
fragment
of SEQ ID N0:1-12 comprises a region of unique amino acid sequence that
specifically identifies
SEQ 1D NO:1-12. For example, a fragment of SEQ ID NO:1-12 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID N0:1-12.
The precise length of
a fragment of SEQ ID NO:1-12 and the region of SEQ ID NO:1-12 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
codan (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 "°lo identity," as applied to
polynucleotide sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
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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.nebi.nlm.nih.govBLAST/. 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/gorflbl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSZIM62
Reward for match: 1
Penalty far mismatch: -2
Operz Gap: 5 arzd Exterzsiorz Gap: 2 penalties
Gap x drop-off.' S0
Expect: l0
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length
supported by the sequences shown herein, in the tables, figures, or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
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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=l, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off: 50 .
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
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
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complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1 % (w/v) SDS, and about 100 ~.g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.
(1989) Molecular Cloning: A Laboratory Manual, 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
~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
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to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of CGDD
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 CGDD 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 CGDD. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of CGDD.
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 CGDD may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in
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the art. These processes may occur synthetically or biochemically. Biochemical
modifications will
vary by cell type depending on the enzymatic milieu of CGDD.
"Probe" refers to nucleic acid sequences encoding CGDD, 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, 2nd
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel,F.M. et al. (1987) Current
Protocols in Molecular
Biolo , Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
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
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selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may also be obtained from their respective sources and modified to
meet the user's specific
needs.) The PrimeGen program (available to the public from the UK Human Genome
Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription, '
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
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The term "sample" is used in its broadest sense. A sample suspected of
containing CGDD,
nucleic acids encoding CGDD, or fragments thereof may comprise a bodily fluid;
an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at 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" or "expression profile" refers to the collective pattern
of gene
expression by a particular cell type or tissue under given conditions at a
given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or
viral infection, electroporation, heat shock, lipofection, and particle
bombardment. The term
"transformed cells" includes stably transformed cells in which the inserted
DNA is capable of
replication either as an autonomously replicating plasmid or as part of the
host chromosome, as well
as transiently transformed cells which express the inserted DNA or RNA for
limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
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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%, 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 resulting polypeptides will generally have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 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.
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THE INVENTION
The invention is based on the discovery of new human proteins associated with
cell growth,
differentiation, and death (CGDD), the polynucleotides encoding CGDD, and the
use of these
compositions for the diagnosis, treatment, or prevention of cell proliferative
disorders including
cancer, developmental disorders, neurological disorders, reproductive
disorders, and
autoimmune/inflammatory disorders.
Table 1 summarizes the nomenclature~for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide 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 ~ NO:) of the nearest GenBank
homolog.
Column 4 shows the probability scores for the matches between each polypeptide
and its homolog(s).
Column 5 shows the annotation of the GenBank homolog(s) along with relevant
citations where
applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1
I 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 structure/function
analysis and in some cases,
searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are proteins associated
with cell growth,
differentiation, and death. For example, SEQ ID N0:3 is 45°lo
identical, from residue M1 to residue
I454, to rat RING finger protein terf (GenBank ID g5114353) as determined by
the Basic Local
Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is
2.2e-102, which
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indicates the probability of obtaining the observed polypeptide sequence
alignment by chance. SEQ
ID N0:3 also contains SPRY, zinc forger (C3HC4 type; RING finger), B-box zinc
finger domains as
determined by searching for statistically significant matches in the hidden
Markov model (HMM)-
based PFAM database of conserved protein family domains. (See Table 3.) Data
from BLIMPS and
PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:3
is a RING forger
protein.
In another example, SEQ )D N0:5 is 59% identical, from residue E14 to residue
S1159, to
human nGAP (GenBank )D g4105589) as determined by the Basic Local Alignment
Search Tool
(BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates
the probability of
obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:5
also contains a
GTPase-activator protein for Ras-like GTPase as determined by searching for
statistically significant
matches in the hidden Markov model (HMM)-based PFAM database of conserved
protein family
domains. (See Table 3.) Data from PROFILESCAN analysis provide further
corroborative evidence
that SEQ ID N0:5 is a Ras-specific GTPase-activating protein.
In another example, SEQ ID N0:7 is 82% identical, from residue M1 to residue
8579, to
Rattus norve i~ cus cerebroglycan (GenBank ID g440127) as determined by the
Basic Local Alignment
Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.4e-260,
which indicates the
probability of obtaining the observed polypeptide sequence alignment by
chance. SEQ ID N0:7 also
contains a glypican domain as determined by searching for statistically
significant matches in the
hidden Markov model (HMM)-based PFAM database of conserved protein family
domains. (See
Table 3.) Data from BLIMPS and MOTIFS analyses provide further corroborative
evidence that SEQ
ID N0:7 is a glypican.
For example, SEQ ID N0:9 is 99% identical, from residue M1 to residue D448, to
the human
TRPM-2 gene product (GenBank ID g339973) as determined by the Basic Local
Alignment Search
Tool (BLAST). (See Table 2.) The BLAST probability score is 3.9e-244, which
indicates the
probability of obtaining the observed polypeptide sequence alignment by
chance. SEQ ~ N0:9 also
contains a clusterin domain as determined by searching 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 )D N0:9 is a clusterin. SEQ ID NO:1-2, SEQ ID N0:4, SEQ ID
N0:6, SEQ ID
N0:8 and SEQ ID NO:10-12 were analyzed and annotated in a similar manner. The
algorithms and
parameters for the analysis of SEQ ID NO:1-12 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Column 1 lists the polynucleotide
sequence
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identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte
polynucleotide
consensus sequence number (Incyte ID) for each polynucleotide of the
invention, and the length of
each polynucleotide sequence in basepairs. Column 2 shows the nucleotide start
(5') and stop (3')
positions of the cDNA and/or genomic sequences used to assemble the full
length polynucleotide
sequences of the invention, and of fragments of the polynucleotide sequences
which are useful, for
example, in hybridization or amplification technologies that identify SEQ ID
N0:13-24 or that
distinguish between SEQ ID NO:13-24 and related polynucleotide sequences.
The polynucleotide fragments described in Column 2 of Table 4 may refer
specifically, for
example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from
pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2
may refer to GenBank
cDNAs or ESTs which contributed to the assembly of the full length
polynucleotide sequences. In
addition, the polynucleotide fragments described in column 2 may identify
sequences derived from
the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e.~ those sequences
including the
designation "ENST"). Alternatively, the polynucleotide fragments described in
column 2 may be
derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those
sequences
including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence
Records (i.e., those
sequences including the designation "NP"). Alternatively, the polynucleotide
fragments described in
column 2 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together by
an "exon stitching" algorithm. For example, a polynucleotide sequence
identified as
FL XXXXXX_Nl Nz 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 NI,2, j..., if
present, represent specific
exons that may have been manually edited during analysis (See Example V).
Alternatively, the
polynucleotide fragments in column 2 may refer to assemblages of exons brought
together by an
"exon-stretching" algorithm. For example, a polynucleotide sequence identified
as
FLXXXXXX gAAAAA_gBBBBB_1 1V is a "stretched" sequence, with XXXXXX being the
Incyte
project identification number, gAAAAA being the GenBank identification number
of the human
genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB
being the
GenBank identification number or NCBI RefSeq identification number of the
nearest GenBank
protein homolog, and N referring to specific exons (See Example V). In
instances where a RefSeq
sequence was used as a protein homolog for the "exon-stretching" algorithm, a
RefSeq identifier
(denoted by "NM," "NP," or "NT") may be used in place of the GenBank
identifier (i.e., gBBBBB).
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
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methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs
GNN, GFG,Exon prediction from genomic sequences using,
for example,
ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK).
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example V).
INCY Full length transcript and exon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the exons and
resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in Table
4 was obtained to confirm the final consensus polynucleotide sequence, but the
relevant Incyte cDNA
identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
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 CGDD variants. A preferred CGDD 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 CGDD amino acid sequence, and which contains at least
one functional or
structural characteristic of CGDD.
The invention also encompasses polynucleotides which encode CGDD. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:13-24, which encodes CGDD. The
polynucleotide
sequences of SEQ ID N0:13-24, 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
CGDD. 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 CGDD. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ m
N0:13-24 which has at least about 70%, or alternatively at least about 85%, or
even at least about
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95% polynucleotide sequence identity to a nucleic acid sequence selected from
the group consisting
of SEQ ID N0:13-24. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of CGDD.
In addition, or in the alternative, a polynucleotide variant of the invention
is a splice variant
of a polynucleotide sequence encoding CGDD. A splice variant may have portions
which have
significant sequence identity to the polynucleotide sequence encoding CGDD,
but will generally have
a greater or lesser number of polynucleotides due to additions or deletions of
blocks of sequence
arising from alternate splicing of exons during mRNA processing. A splice
variant may have less
than about 70%, or alternatively less than about GO%, or alternatively less
than about 50%
polynucleotide sequence identity to the polynucleotide sequence encoding CGDD
over its entire
length; however, portions of the splice variant will have at least about 70%,
or alternatively at least
about 85%, or alternatively at least about 95%, or alternatively 100%
polynucleotide sequence
identity to portions of the polynucleotide sequence encoding CGDD. For
example, a polynucleotide
comprising a sequence of SEQ ID N0:23 is a splice variant of a polynucleotide
comprising a
sequence of SEQ ID N0:17 and a polynucleotide comprising a sequence of SEQ ID
N0:24 is a splice
variant of a polynucleotide comprising a sequence of SEQ ID N0:21. Any one of
the splice variants
described above can encode an amino acid sequence which contains at least one
functional or
structural characteristic of CGDD. '
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 CGDD, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring CGDD, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode CGDD and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring CGDD under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding CGDD or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding CGDD and its derivatives without altering the encoded amino
acid sequences
include the production of RNA transcripts having more desirable properties,
such as a greater
49
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half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode CGDD
and
CGDD 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 CGDD 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
N0:13-24 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M, and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
( 1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biol~y and Biotechnology, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding CGDD 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
CA 02441495 2003-08-06
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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 intronlexon junctions. For all PCR-based methods,
primers may be designed
using commercially available software, such as OLIGO 4.06 primer analysis
software (National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the
template at temperatures of
about 68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera fox 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 CGDD may be cloned in recombinant DNA molecules that direct
expression of CGDD,
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 CGDD.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter CGDD-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
51
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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 Clam CA; described in U.S. Patent
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of CGDD, such as its biological or enzymatic
activity or its ability
to bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding CGDD 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, CGDD 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
rnay be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the
amino acid sequence of CGDD, or any part thereof, may be altered during direct
synthesis andlor
combined with sequences from other proteins, or any part thereof, to produce a
vaxiant 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 CGDD, the nucleotide sequences
encoding CGDD or
52
CA 02441495 2003-08-06
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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 CGDD. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
CGDD. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding CGDD and its initiation codon and upstream regulatory
sequences are inserted
into the appropriate expression vector, no additional transcriptional or
translational control signals
may be needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted,
exogenous translational control signals including an in-frame ATG initiation
codon should be
provided by the vector. Exogenous translational elements and initiation codons
may be of various
origins, both natural and synthetic. The efficiency of expression may be
enhanced by the inclusion of
enhancers appropriate for the particular host cell system used. (See, e.g.,
Scharf, D. et al. (1994)
Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding CGDD and appropriate transcriptional and
translational
control elements. These methods include in vitro recombinant DNA techniques,
synthetic techniques,
and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989)
Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-
17; Ausubel, F.M. et
al. (1995) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York
NY, ch. 9, 13, and
16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding CGDD. 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, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw
Hill, New
York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and
Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
53
CA 02441495 2003-08-06
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delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993)
Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Butler, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.)
The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding CGDD. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding CGDD 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 CGDD 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 CGDD are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of CGDD 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 CGDD. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia asp
toris. 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 CGDD. Transcription of
sequences
encoding CGDD may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of
Science and 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 CGDD
may be ligated into
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CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
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 CGDD in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of CGDD in cell lines is preferred. For example, sequences encoding CGDD can
be transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media
before being switched to selective media. The purpose of the selectable marker
is to confer resistance
to a selective agent, and its presence allows growth and recovery of cells
which successfully express
the introduced sequences. Resistant clones of stably transformed cells may be
propagated using
tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk- and apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate l3-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.)
CA 02441495 2003-08-06
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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 CGDD is inserted within a marker gene sequence, transformed
cells containing
sequences encoding CGDD can be identified by the absence of marker gene
function. Alternatively,
a marker gene can be placed in tandem with a sequence encoding CGDD 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 CGDD
and that express
CGDD 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 CGDD 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 CGDD is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Irnmunolo~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 CGDD
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding CGDD, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
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 CGDD may be cultured
under
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conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors
containing polynucleotides which encode CGDD may be designed to contain signal
sequences which
direct secretion of CGDD 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 CGDD 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 CGDD protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of CGDD 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-rnyc, 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-fnyc, and hemagglutinin (HA)
enable immunoaffmity
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 CGDD encoding sequence and the
heterologous protein
sequence, so that CGDD 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 CGDD may
be achieved
in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
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CA 02441495 2003-08-06
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T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
CGDD of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to CGDD. At least one and up to a plurality of test
compounds may be screened
for specific binding to CGDD. 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
CGDD, 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 CGDD
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 CGDD,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or
E. coli. Cells expressing CGDD or cell membrane fractions which contain CGDD
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either CGDD 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
CGDD, either in
solution or affixed to a solid support, and detecting the binding of CGDD 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.
CGDD of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of CGDD. Such compounds may include agonists,
antagonists, or partial
or inverse agonists. In one embodiment, an assay is performed under conditions
permissive for
CGDD activity, wherein CGDD is combined with at least one test compound, and
the activity of
CGDD in the presence of a test compound is compared with the activity of CGDD
in the absence of
the test compound. A change in the activity of CGDD in the presence of the
test compound is
indicative of a compound that modulates the activity of CGDD. Alternatively, a
test compound is
combined with an in vitro or cell-free system comprising CGDD under conditions
suitable for CGDD
activity, and the assay is performed. In either of these assays, a test
compound which modulates the
activity of CGDD may do so indirectly and need not come in direct contact with
the test compound.
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At least one and up to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding CGDD or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem
(ES) cells. Such techniques are well known in the art and are useful for the
generation of animal
models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent
No. 5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding
region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding CGDD 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 CGDD 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 CGDD 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 CGDD, e.g., by secreting CGDD in
its milk, may also
serve as a convenient source of that protein (Janne, J. et al. (1998)
Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of CGDD and proteins associated with cell growth,
differentiation, and death. In
addition, examples of tissues expressing CGDD can be found in Table 6.
Therefore, CGDD appears
to play a role in cell proliferative disorders including cancer, developmental
disorders, neurological
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disorders, reproductive disorders, and autoimmune/inflammatory disorders. In
the treatment of
disorders associated with increased CGDD expression or activity, it is
desirable to decrease the
expression or activity of CGDD. In the treatment of disorders associated with
decreased CGDD
expression or activity, it is desirable to increase the expression or activity
of CGDD.
Therefore, in one embodiment, CGDD 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 CGDD. 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
renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism,
Duchenne and Becker
muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor,
aniridia,
genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome,
myelodysplastic
syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas,
hereditary neuropathies such
as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,
hydrocephalus, seizure
disorders such as Syndenham's chorea and cerebral palsy, spina bifida,
anencephaly,
craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing
loss; a neurological
disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral
neoplasms, Alzheimer's
disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease
and other extrapyramidal
disorders, amyotrophic lateral sclerosis and other motor neuron disorders,
progressive neural
muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis
and other demyelinating
diseases, bacterial and viral meningitis, brain abscess, subdural empyema,
epidural abscess,
suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral
central nervous system
disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-
Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system including Down syndrome, cerebral palsy, neuroskeletal
disorders, autonomic
nervous system disorders, cranial nerve disorders, spinal cord diseases,
muscular dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis,
periodic paralysis, mental
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disorders including mood, anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD),
akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,
dystonias, paranoid psychoses,
postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy,
corticobasal degeneration,
and familial frontotemporal dementia; a reproductive disorder such as a
disorder of prolactin
production, infertility, including tubal disease, ovulatory defects,
endometriosis, a disruption of the
estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome,
ovarian
hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid,
autoimmune
disorders, ectopic pregnancy, teratogenesis; cancer of the breast, fibrocystic
breast disease,
galactorrhea; a disruption of spermatogenesis, abnormal sperm physiology,
cancer of the testis, cancer
of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's
disease, impotence, carcinoma of
the male breast, gynecomastia, hypergonadotropic and hypogonadotropic
hypogonadism,
pseudohermaphroditism, azoospermia, premature ovarian failure, acrosin
deficiency, delayed puperty,
retrograde ejaculation and anejaculation, haemangioblastomas,
cystsphaeochromocytomas,
paraganglioma, cystadenomas of the epididymis, and endolymphatic sac tumors;
and an
autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(A)DS), Addison's
disease, adult respiratorydistress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema,
episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema
nodosum, atrophic
gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis,
psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's
syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and
extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic
infections, and trauma.
In another embodiment, a vector capable of expressing CGDD 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 CGDD including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
CGDD 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 CGDD including,
but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of CGDD
may be
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administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of CGDD including, but not limited to, those listed above.
In a further embodiment, an antagonist of CGDD may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of CGDD.
Examples of such
disorders include, but are not limited to, those cell proliferative disorders
including cancer,
developmental disorders, neurological disorders, reproductive disorders, and
autoimmunelinflammatory disorders described above. In one aspect, an antibody
which specifically
binds CGDD 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 CGDD.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding CGDD may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of CGDD including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of CGDD may be produced using methods which are generally known
in the
art. In particular, purified CGDD may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind CGDD.
Antibodies to CGDD 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. Single chain
antibodies (e.g., from
camels or llamas) may be potent enzyme inhibitors and may have advantages in
the design of peptide
mimetics, and in the development of immuno-adsorbents and biosensors
(Muyldennans, S. (2001) J.
Biotechnol. 74:277-302).
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, camels,
dromedaries, llamas, humans, and others may be immunized by injection with
CGDD 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 axe
not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface
active substances such
as lysolecithin, platonic polyols, polyanions, peptides, oil emulsions, KLH,
and dinitrophenol.
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Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
CGDD 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 CGDD 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 CGDD 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., I~ohler, G. et al. (1975) Nature 256:495-497; I~ozbor,
D. et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
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
CGDD-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 nay 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 CGDD may also be
generated.
For example, such fragments include, but are not limited to, F(ab')z fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by 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
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polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
CGDD and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering CGDD 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 CGDD. Affinity
is expressed as an
association constant, Ka, which is defined as the molar concentration of CGDD-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium conditions.
The Ka determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple CGDD epitopes, represents the average affinity, or
avidity, of the antibodies for
CGDD. The Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular CGDD epitope, represents a true measure of affinity. High-affinity
antibody preparations
with Ka ranging from about 109 to 10'Z L/mole are preferred for use in
immunoassays in which the
CGDD-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
innnunopurification and similar
procedures which ultimately require dissociation of CGDD, preferably in active
form, from the
antibody (Catty, D. (1988) Antibodies. Volume I: A Practical Approach, lRL
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 CGDD-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, s. u~ra,, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding CGDD, 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 CGDD. 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 CGDD. (See, e.g., Agrawal, S., ed. (1996) Antisense Thera et~ utics,
Humana Press Inc.,
Totawa NJ.)
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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.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding CGDD may be
used for .
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SC)D)-X1 disease
characterized by X
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) 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) 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 CGDD expression or regulation causes
disease, the expression of
CGDD 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
CGDD are treated by constructing mammalian expression vectors encoding CGDD
and introducing
these vectors by mechanical means into CGDD-deficient cells. Mechanical
transfer technologies for
use with cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii)
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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 CGDD 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).
CGDD may
be expressed using (i) a constitutively active promoter, (e.g., from
cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes),
(ii) an inducible
promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard
(1992) Proc. Natl.
Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769;
Rossi, F.M.V. and
H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially. available
in the T-REX plasmid
(Invitrogen)); the ecdysone-inducible promoter (available in the plasmids
PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486lmifepristone
inducible promoter
(Rossi, F.M.V. and H.M. Blau, supra)), or (iii) a tissue-specific promoter or
the native promoter of the
endogenous gene encoding CGDD 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 CGDD expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding CGDD under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
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A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell.lines producing high transducing efficiency
retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
cells), and the return of transduced cells to a patient are procedures well
known to persons skilled in
the art of gene therapy and have been well documented (Ranga, U. et al. (1997)
J. Virol. 71:7020-
7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)
Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding CGDD to cells which have one or more genetic
abnormalities with respect
to the expression of CGDD. 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 No. 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 CGDD to target cells which have one or more genetic
abnormalities with
respect to the expression of CGDD. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing CGDD to cells of the central nervous
system, for which HSV has
a tropism. The construction and packaging of herpes-based vectors are well
known to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of
recombinant HSV d92 which
consists of a genome containing at least one exogenous gene to be transferred
to a cell under the
control of the appropriate promoter for purposes including human gene therapy.
Also taught by 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
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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 CGDD to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and I~.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
CGDD into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
CGDD-coding RNAs and the synthesis of high levels of CGDD 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 CGDD 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 Immunologic Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-
177.) A complementary sequence or antisense molecule may also be designed to
block translation of
mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
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engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding CGDD.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding CGDD. Such DNA sequences may be incorporated into a wide
variety of vectors
with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these
cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines,
cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half-
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine,
queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine,
cytidine, guanine, thymine, and uridine which are not as easily recognized by
endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding CGDD.
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
a
CGDD expression or activity, a compound which specifically inhibits expression
of the
polynucleotide encoding CGDD may be therapeutically useful, and in the
treatment of disorders
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associated with decreased CGDD expression or activity, a compound which
specifically promotes
expression of the polynucleotide encoding CGDD may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurnng 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 CGDD 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
CGDD 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 CGDD. 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 (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues axe available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
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monkeys.
An additional embodiment of the invention relates to the administration of a
composition
which generally comprises an active ingredient formulated with a
pharmaceutically acceptable
excipient. Excipients may include, for example, sugars, starches, celluloses,
gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the
latest edition of
Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
compositions may
consist of CGDD, antibodies to CGDD, and mimetics, agonists, antagonists, or
inhibitors of CGDD.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary,.
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of
fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g. larger peptides
and proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the
lung have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton,
J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage
of administration
without needle injection, and obviates the need for potentially toxic
penetration enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising CGDD or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of
the macromolecule. Alternatively, CGDD or a fragment thereof may be joined to
a short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs,
monkeys, or pigs. An animal model may also be used to determine the
appropriate concentration
range and route of administration. Such information can then be used to
determine useful doses and
routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
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CGDD or fragments thereof, antibodies of CGDD, and agonists, antagonists or
inhibitors of CGDD,
which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity
may be determined
by standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting compositions may be administered every 3 to 4
days, every week,
or biweekly depending on the half life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1,ug to 100,000 ,ug, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind CGDD may be used for
the
diagnosis of disorders characterized by expression of CGDD, or in assays to
monitor patients.being
treated with CGDD or agonists, antagonists, or inhibitors of CGDD. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for CGDD include methods which utilize the antibody and a label to detect CGDD
in human body
fluids or in extracts of cells or tissues. The antibbdies may be used with or
without modification, and
may be labeled by covalent or non-covalent attachment of a reporter molecule.
A wide variety of
reporter molecules, several of which are described above, are known in the art
and may be used.
A variety of protocols for measuring CGDD, including ELISAs, RIAs, and FAGS,
are lrnown
in the art and provide a basis for diagnosing altered or abnormal levels of
CGDD expression. Normal
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or standard values for CGDD expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibodies to CGDD
under conditions suitable for complex formation. The amount of standard
complex formation may be
quantitated by various methods, such as photometric means. Quantities of CGDD
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 CGDD 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 CGDD
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of CGDD, and to monitor regulation of CGDD levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding CGDD or closely related
molecules may be used
to identify nucleic acid sequences which encode CGDD. The specificity of the
probe, whether it is
made from a highly specific region, e.g., the 5'regulatory region, or from a
less specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding CGDD, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50°l0
sequence identity to any of the CGDD encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ m
N0:13-24 or from
genomic sequences including promoters, enhancers, and introns of the CGDD
gene.
Means for producing specific hybridization probes for DNAs encoding CGDD
include the
cloning of polynucleotide sequences encoding CGDD or CGDD derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding CGDD may be used for the diagnosis of
disorders
associated with expression of CGDD. 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
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adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas, parathyroid,
penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and
uterus; a developmental
disorder such as renal tubular acidosis, anemia, Cushing's syndrome,
achondroplastic dwarfism,
Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wilms'
tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-
Magenis syndrome,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas, hereditary
neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,
hypothyroidism,
hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral
palsy, spina bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, and
sensorineural hearing loss; a
neurological disorder such as epilepsy, ischemic cerebrovascular disease,
stroke, cerebral neoplasms,
Alzheimer's disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and other
extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron
disorders, progressive
neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple
sclerosis and other
demyelinating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis,
viral central nervous
system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-
Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system including Down syndrome, cerebral palsy, neuroskeletal
disorders, autonomic
nervous system disorders, cranial nerve disorders, spinal cord diseases,
muscular dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic rnyopathies, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD),
akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,
dystonias, paranoid psychoses,
postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy,
corticobasal degeneration,
and familial frontotemporal dementia; a reproductive disorder such as a
disorder of prolactin
production, infertility, including tubal disease, ovulatory defects,
endometriosis, a disruption of the
estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome,
ovarian
hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid,
autoimmune
disorders, ectopic pregnancy, teratogenesis; cancer of the breast, fibrocystic
breast disease,
galactorrhea; a disruption of spermatogenesis, abnormal sperm physiology,
cancer of the testis, cancer
of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's
disease, impotence, carcinoma of
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the male breast, gynecomastia, hypergonadotropic and hypogonadotropic
hypogonadism,
pseudohermaphroditism, azoospermia, premature ovarian failure, acrosin
deficiency, delayed puperty,
retrograde ejaculation and anejaculation, haemangioblastomas,
cystsphaeochromocytomas,
paraganglioma, cystadenomas of the epididymis, and endolymphatic sac tumors;
and an
autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS), Addison's
disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema,
episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema
nodosum, atrophic
gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis,
psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's
syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and
extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic
infections, and trauma. The
polynucleotide sequences encoding CGDD 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 CGDD
expression. Such qualitative or quantitative methods are well known in the
art.
In a particular aspect, the nucleotide sequences encoding CGDD may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding CGDD 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 CGDD 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
CGDD, 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 CGDD, under conditions suitable for
hybridization or
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amplification. Standard hybridization may be quantified by comparing the
values obtained from
normal subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding CGDD
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 CGDD, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
CGDD, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding CGDD 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
CGDD 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
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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).
SNPs may be used to study the genetic basis of human disease. For example, at
least 16
common SNPs have been associated with non-insulin-dependent diabetes mellitus.
SNPs are also
useful for examining differences in disease outcomes in monogenic disorders,
such as cystic fibrosis,
sickle cell anemia, or chronic granulomatous disease. For example, variants in
the mannose-binding
lectin, MBL2, have been shown to be correlated with deleterious pulmonary
outcomes in cystic
fibrosis. SNPs also have utility in pharmacogenomics, the identification of
genetic variants that
influence a patient's response to a drug, such as life-threatening toxicity.
For example, a variation in
N-acetyl transferase is associated with a high incidence of peripheral
neuropathy in response to the
anti-tuberculosis drug isoniazid, while a variation in the core promoter of
the ALOX5 gene results in
diminished clinical response to treatment with an anti-asthma drug that
targets the 5-lipoxygenase
pathway. Analysis of the distribution of SNPs in different populations is
useful for investigating
genetic drift, mutation, recombination, and selection, as well as for tracing
the origins of populations
and their migrations. (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y. and Z. Gu
(1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr. Opin.
Neurobiol. 11:637-641.)
Methods which may also be used to quantify the expression of CGDD include
radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives
rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
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to develop a pharmacogenomic pxofile 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, CGDD, fragments of CGDD, or antibodies specific for
CGDD 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 No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines,
biopsies, or other biological samples. The transcript image may thus reflect
gene expression in vivo,
as in the case of a tissue or biopsy sample, or in vitro, as in the case of a
cell line.
Transcript images which profile the expression of the polynucleotides of the
present
invention may also be used in conjunction with in vitro model systems and
preclinical evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share
those toxic properties. These fingerprints or signatures are most useful and
refined when they contain
expression information from a large number of genes and gene families.
Ideally, a genome-wide
measurement of expression provides the highest quality signature. Even genes
whose expression is
not altered by any tested compounds are important as well, as the levels of
expression of these genes
are used to normalize the rest of the expression data. The normalization
procedure is useful for
comparison of expression data after treatment with different compounds. While
the assignment of
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gene function to elements of a toxicant signature aids in interpretation of
toxicity mechanisms,
knowledge of gene function is not necessary for the statistical matching of
signatures which leads to
prediction of toxicity. (See, for example, Press Release 00-02, from the
National Institute of
Environmental Health Sciences, released February 29, 2000, available at
http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a
proteome can be subjected individually to further analysis. Proteome
expression patterns, or profiles,
are analyzed by quantifying the number of expressed proteins and their
relative abundance under
given conditions and at a given time. A profile of a cell's proteome may thus
be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample are
separated by isoelectric focusing in the first dimension, and then according
to molecular weight by
sodium dodecyl sulfate slab gel electrophoresis in the second dimension
(Steiner and Anderson,
supra). The proteins are visualized in the gel as discrete and uniquely
positioned spots, typically by
staining the gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical
density of each protein spot is generally proportional to the level of the
protein in the sample. The
optical densities of equivalently positioned protein spots from different
samples, for example, from
biological samples either treated or untreated with a test compound or
therapeutic agent, are
compared to identify any changes in protein spot density related to the
treatment. The proteins in the
spots are partially sequenced using, for example, standard methods employing
chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein in a spot
may be deternnined 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 CGDD
to quantify
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the levels of CGDD 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 arilount of
fluorescence bound at
each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to
rapid degradation of mRNA, so proteomic profiling may be more reliable and
informative in such
cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological
sample. A difference in the amount of protein between the two samples is
indicative of a toxic
response to the test compound in the treated sample. Individual proteins are
identified by sequencing
the amino acid residues of the individual proteins and comparing these partial
sequences to the
polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are
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 W095135505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are
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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 CGDD
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences may
be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a mufti-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial
chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial
chromosomes
(BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See,
e.g., Harrington, J.J.
et al. (1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134;
and Trask, B.J.
(1991) Trends Genet. 7:149-154.) Once mapped, the nucleic acid sequences of
the invention may be
used to develop genetic linkage maps, for example, which correlate the
inheritance of a disease state
with the inheritance of a particular chromosome region or restriction fragment
length polymorphism
(RFLP). (See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl.
Acad. Sci. USA 83:7353-
7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding CGDD 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, CGDD, its catalytic or immunogenic
fragments, or
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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 CGDD and the agent being tested may be measured.
Another technique for drug screening provides fox high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds axe
synthesized on a solid substrate. The test compounds are reacted with CGDD, or
fragments thereof,
and washed. Bound CGDD is then detected by methods well known in the art.
Purified CGDD 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 CGDD specifically compete with a test compound
for binding CGDD.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with CGDD.
In additional embodiments, the nucleotide sequences which encode CGDD 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, in
particular U.S. Ser. No. 60/268,111, U.S. Ser. No. 60/271,175, U.S. Ser. No.
60/274,552, and U.S.
Ser. No. 60/274,503, 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). Some tissues were homogenized and lysed in
guanidinium
isothiocyanate, while others were homogenized and lysed in phenol or in a
suitable mixture of
denaturants, such as TRIZOL (Life Technologies), a monophasic solution of
phenol and guanidine
isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or
extracted with
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chloroform. RNA was precipitated from the lysates with either isopropanol or
sodium acetate and
ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid
(Invitrogen),
PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE
(Incyte
Genomics), or pINCY (Incyte Genomics), or derivatives thereof. Recombinant
plasmids were
transformed into competent E. coli cells including XLl-Blue, XL1-BlueMRF, or
SOLR from
Stratagene or DHSa, DH10B, or ElectroMAX DHlOB 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 carned out in a single reaction mixture. Samples were
processed and stored in
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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
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 GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sa
iens,
Rattus norve,i~ cus, Mus musculus, Caenorhabditis elegy, Saccharomyces
cerevisiae,
Schizosaccharom,~pombe, and Candida albicans (Incyte Genomics, Palo Alto CA);
hidden
Markov model (HMM)-based protein family databases such as PFAM; and HMM-based
protein
domain databases such as SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci.
USA 95:5857-5864;
Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244). (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 eDNA 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
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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, the PROTEOME databases,
BLOCKS,
PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family
databases
such as PFAM; and HMM-based protein domain databases such as SMART. Full
length
polynucleotide sequences are also analyzed using MACDNASIS PRO software
(Hitachi Software
Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide
and polypeptide sequence alignments are generated using default parameters
specified by the
CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment
program
(DNASTAR), which also calculates the percent identity between aligned
sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and
threshold parameters. The first column of Table 7 shows the tools, programs,
and algorithms used,
the second column provides brief descriptions thereof, the third column
presents appropriate
references, all of which are incorporated by reference herein in their
entirety, and the fourth column
presents, where applicable, the scores, probability values, and other
parameters used to evaluate the
strength of a match between two sequences (the higher the score or the lower
the probability value,
the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide
and polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ
ID N0:13-24. Fragments from about 20 to about 4000 nucleotides which are
useful in hybridization
and amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative proteins associated with cell growth, differentiation, and death were
initially
identified by running the Genscan gene identification program against public
genomic sequence
databases (e.g., gbpri and gbhtg). Genscan is a general-purpose gene
identification program which
analyzes genomic DNA sequences from a variety of organisms (See Burge, C. and
S. Karlin (1997) J.
Mol. Biol. 268:78-94, and Burge, C. and S. Karlin (1998) Curr. Opin. Struct.
Biol. 8:346-354). The
program concatenates predicted exons to form an assembled cDNA sequence
extending from a
methionine to a stop codon. The output of Genscan is a FASTA database of
polynucleotide and
polypeptide sequences. The maximum range of sequence for Genscan to analyze at
once was set to
30 kb. To determine which of these Genscan predicted cDNA sequences encode
proteins associated
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with cell growth, differentiation, and death, the encoded polypeptides were
analyzed by querying
against PFAM models fox proteins associated with cell growth, differentiation,
and death. Potential
proteins associated with cell growth, differentiation, and death were also
identified by homology to
Incyte cDNA sequences that had been annotated as proteins associated with cell
growth,
differentiation, and death. These selected Genscan-predicted sequences were
then compared by
BLAST analysis to the genpept and gbpri public databases. Where necessary, the
Genscan-predicted
sequences were then edited by comparison to the top BLAST hit from genpept to
correct errors in the
sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis
was also used to
find any Incyte cDNA or public cDNA coverage of the Genscan-predicted
sequences, thus providing
evidence for transcription. When Incyte cDNA coverage was available, this
information was used to
correct or confirm the Genscan predicted sequence. Full length polynucleotide
sequences were
obtained by assembling Genscan-predicted coding sequences with Incyte cDNA
sequences and/or
public cDNA sequences using the assembly process described in Example III.
Alternatively, full
length polynucleotide sequences were derived entirely from edited or unedited
Genscan-predicted
codingsequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Se uences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
i
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
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with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" SecLuences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for
homologous genomic sequences from the public human genome databases. Partial
DNA sequences
were therefore "stretched" or extended by the addition of homologous genomic
sequences. The
resultant stretched sequences were examined to determine whether it contained
a complete gene.
VI. Chromosomal Mapping of CGDD Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:13-24 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ~ N0:13-24 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM
distances are based on genetic markers mapped by Genethon which provide
boundaries for radiation
hybrid markers whose sequences were included in each of the clusters. Human
genome maps and
other resources available to the public, such as the NCBI "GeneMap'99" World
Wide Web site
(http:l/www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
In this manner, SEQ ID NO:15 was mapped to chromosome 1 within the interval
from
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242.50 to 258.70 centiMorgans. SEQ ID N0:20 was mapped to chromosome 7 within
the interval
from 180.8 centiMorgans to the q-terminus.
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. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding CGDD 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 )II). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following organltissue categories: cardiovascular
system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female;
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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 CGDD. cDNA sequences and cDNA
library/tissue
information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto
CA).
VIII. Extension of CGDD Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional.or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NHø)zS04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
l: 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 ~,l
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ,ul 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
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concentration of DNA. A 5 ,u1 to 10 /.d aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37°C in
384-well plates in LB/2x curb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise
(Amersham Phannacia Biotech) and Pfu DNA polymerise (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified 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. Identification of Single Nucleotide Polymorphisms in CGDD Encoding
Polynucleotides
Common DNA sequence variants known as single nucleotide polymorphisms (SNPs)
were
identified in SEQ ID N0:13-24 using the L1FESEQ database (Incyte Genomics).
Sequences from the
same gene were clustered together and assembled as described in Example III,
allowing the
identification of all sequence variants in the gene. An algorithm consisting
of a series of filters was
used to distinguish SNPs from other sequence variants. Preliminary filters
removed the majority of
basecall errors by requiring a minimum Phred quality score of 15, and removed
sequence alignment
errors and errors resulting from improper trimming of vector sequences,
chimeras, and splice
variants. An automated procedure of advanced chromosome analysis analysed the
original
chromatogram files in the vicinity of the putative SNP. Clone error filters
used statistically generated
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algorithms to identify errors introduced during laboratory processing, such as
those caused by reverse
transcriptase, polymerase, or somatic mutation. Clustering error filters used
statistically generated
algorithms to identify errors resulting from clustering of close homologs ox
pseudogenes, or due to
contamination by non-human sequences. A final set of filters removed
duplicates and SNPs found in
immunoglobulins or T-cell receptors.
Certain SNPs were selected for further characterization by mass spectrometry
using the high
throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at
the SNP sites in
four different human populations. The Caucasian population comprised 92
individuals (46 male, 46
female), including 83 from Utah, four French, three Venezualan, and two Amish
individuals. The
African population comprised 194 individuals (97 male, 97 female), all African
Americans. The
Hispanic population comprised 324 individuals (162 male, 162 female), all
Mexican Hispanic. The
Asian population comprised 126 individuals (64 male, 62 female) with a
reported parental breakdown
of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
Allele
frequencies were first analyzed in the Caucasian population; in some cases
those SNPs Which showed
no allelic variance in this population wexe not further tested in the other
three populations.
X. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ )D N0:13-24 are employed to screen
cDNAs,
genoxnic 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
~,~ 32P~ adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at zoom temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
XI. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
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photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena ( 1999),
supra). Suggested substrates include silicon, silica, glass slides, glass
chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to
arrange and link
elements to the surface of a substrate using thermal, UV, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinary skill in the art and may 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 guanidiniurn thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/p,l oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/~1 RNase inhibitor, 500 ,uM dATP, 500 ~M dGTP,
500 ~tM dTTP, 40
p.M dCTP, 40 ~.M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium
hydroxide and
incubated for 20 minutes at 85° C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive 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 nnl of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
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then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 ~.l 5X SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
~,g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water,
and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides
are cured in a
110°C oven.
Array elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~.1 of the array
element DNA, at an average
concentration of 100 ng/~.1, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER LTV-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 ,u1 of sample mixture consisting of 0.2 ~,g
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered
with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just
slightly larger than a microscope slide. The chamber is kept at 100% humidity
internally by the
addition of 140 ~,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
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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
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are
differentially expressed, the calibration is done by labeling samples of the
calibrating cDNA with the
two fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each
spot is centered in each element of the grid. The fluorescence signal within
each element is then
integrated to obtain a numerical value corresponding to the average intensity
of the signal. The
software used for signal analysis is the GEMTOOLS gene expression analysis
program (Incyte).
XII. Complementary Polynucleotides
Sequences complementary to the CGDD-encoding sequences, or any parts thereof,
are used to
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detect, decrease, or inhibit expression of naturally occurring CGDD. 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 CGDD. 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
CGDD-encoding
transcript.
XIII. Expression of CGDD
Expression and purification of CGDD is achieved using bacterial or virus-based
expression
systems. For expression of CGDD 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 T5 or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express CGDD upon induction with isopropyl beta-
D-
thiogalactopyranoside (Il'TG). Expression of CGDD in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autog~raphica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding CGDD 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~iperda (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 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, CGDD 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
CGDD at specifically engineered sites. FLAG, an ~-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
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resins (QIAGEN). Methods for protein expression and purification are discussed
in Ausubel (1995,
supra, ch. 10 and 16). Purified CGDD obtained by these methods can be used
directly in the assays
shown in Examples XVII, and XVIll where applicable.
XIV. Functional Assays
CGDD function is assessed by expressing the sequences encoding CGDD 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 ,ug of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ,ug of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow CytometrX, Oxford, New York NY.
The influence of CGDD on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding CGDD 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 CGDD and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XV. Production of CGDD Specific Antibodies
CGDD substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
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Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using
standard protocols.
Alternatively, the CGDD 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, sera, 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
Ki,H (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide and anti-CGDD activity by, for example, binding the peptide or
CGDD to a substrate,
blocking with 1 °lo BSA, reacting with rabbit antisera, washing, and
reacting with radio-iodinated goat
anti-rabbit IgG.
XVI. Purification of Naturally Occurring CGDD Using Specific Antibodies
Naturally occurring or recombinant CGDD is substantially purified by
immunoaffmity
chromatography using antibodies specific for CGDD. An immunoaffmity column is
constructed by
covalently coupling anti-CGDD antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing CGDD are passed over the immunoaffmity column, and the column
is
washed under conditions that allow the preferential absorbance of CGDD (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/CGDD 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 CGDD is collected.
XVII. Identification of Molecules Which Interact with CGDD
CGDD, or biologically active fragments thereof, are labeled with'zsI Bolton-
Hunter reagent.
(See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a mufti-well plate are incubated with the
labeled CGDD, washed,
and any wells with labeled CGDD complex are assayed. Data obtained using
different concentrations
of CGDD are used to calculate values for the number, affinity, and association
of CGDD with the
candidate molecules.
Alternatively, molecules interacting with CGDD are analyzed using the yeast
two-hybrid
system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or
using commercially
97
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available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
CGDD may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
XVIII. Demonstration of CGDD Activity
CGDD activity is demonstrated by measuring the induction of termanal
differentiation,
apoptosis or cell cycle progression when CGDD is expressed 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, Gaithersburg, MD) and PCR 3.1 (Invitrogen, Carlsbad,
CA), both of
which contain the cytomegalovirus promoter. 5-10 ,ug 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 ~g of an additional plasmid
containing sequences
encoding a marker protein are co-transfected. Expression of a marker protein
provides a means to
distinguish transfected cells from nontransfected cells and is a reliable
predictor of cDNA expression
from the recombinant vector. Marker proteins of choice include, e.g., Green
Fluorescent Protein
(GFP) (Clontech, Palo Alto, CA), CD64, or a GD64-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 their physiological state. FCM detects and quantifies
the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
cycle progression, cell
death 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. Methods in flow cytometry are discussed in Ormerod, M. G.
(1994) Flow
Cytometry, Oxford, New York, NY.
Alternatively, an in vitro assay for CGDD activity measures the transformation
of normal
human fibroblast cells overexpressing antisense CGDD RNA (Garkavtsev, I. and
Riabowol, K.
(1997) Mol. Cell Biol. 17:2014-2019). cDNA encoding CGDD is subcloned into the
pLNCX
retroviral vector to enable expression of antisense CGDD RNA. The resulting
construct is transfected
into the ecotropic BOSC23 virus-packaging cell line. Virus contained in the
BOSC23 culture
supernatant is used to infect the amphotropic CAK8 virus-packaging cell line.
Virus contained in the
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CAK8 culture supernatant is used to infect normal human fibroblast (Hs68)
cells. Infected cells are
assessed for the following quantifiable properties characteristic of
transformed cells: growth in
culture to high density associated with loss of contact inhibition, growth in
suspension or in soft agar,
formation of colonies or foci, lowered serum requirements, and ability to
induce tumors when
injected into immunodeficient mice. The activity of CGDD is proportional to
the extent of
transformation of Hs68 cells.
Alternatively, CGDD can be expressed in a mammalian cell line by transforming
the cells
with a eukaryotic expression vector encoding CGDD. Eukaryotic expression
vectors are
commercially available, and the techniques to introduce them into cells are
well known to those
skilled in the art. To assay the cellular localization of CGDD, cells are
fractionated as described by
Jiang H. P. et al. (1992; Proc. Natl. Acad. Sci. 89: 7856-7860). Briefly,
cells pelleted by low-speed
centrifugation are resuspended in buffer (10 mM TRIS-HCI, pH 7.4/ 10 mM NaCI/
3 mM MgCh/ 5
mM EDTA with 10 uglml aprotinin, 10 ug/ml leupeptin, 10 ugJml pepstatin A, 0.2
mM
phenylmethylsulfonyl fluoride) and homogenized. The homogenate is centrifuged
at 600 x g for 5
minutes. The particulate and cytosol fractions are separated by
ultracentrifugation of the supernatant
at 100,000 x g for 60 minutes. The nuclear fraction is obtained by
resuspending the 600 x g pellet in
sucrose solution (0.25 M sucrose/ 10 mM TRIS-HCI, pH 7.412 mM MgClz) and
recentrifuged at 600
x g. Equal amounts of protein from each fraction are applied to an SDS/10%
polyacrylamide gel and
blotted onto membranes. Western blot analysis is performed using CGDD anti-
serum. The
localization of CGDD is assessed by the intensity of the corresponding band in
the nuclear fraction
relative to the intensity in the other fractions. Alternatively, the presence
of CGDD in cellular
fractions is examined by fluorescence microscopy using a fluorescent antibody
specific for CGDD.
Alternatively, CGDD activity may be demonstrated as the ability to interact
with its
associated Ras superfamily protein, in an in vitro binding assay. The
candidate Ras superfamily
proteins are expressed as fusion proteins with glutathione S-transferase
(GST), and purified by
affinity chromatography on glutathione-Sepharose. The Ras superfamily proteins
are loaded with
GDP by incubating 20 mM Tris buffer, pH 8.0, containing 100 mM NaCI, 2 mM
EDTA, 5 mM
MgCl2, 0.2 mM DTT, 100 ~,M AMP-PNP and 10 ~,M GDP at 30°C for 20
minutes. CGDD is
expressed as a FLAG fusion protein in a baculovirus system. Extracts of these
baculovirus cells
containing CGDD-FLAG fusion proteins are precleared with GST beads, then
incubated with GST-
Ras superfamily fusion proteins. The complexes formed are precipitated by
glutathione-Sepharose
and separated by SDS-polyacrylamide gel electrophoresis. The separated
proteins are blotted onto
nitrocellulose membranes and probed with commercially available anti-FLAG
antibodies. CGDD
activity is proportional to the amount of CGDD-FLAG fusion protein detected in
the complex.
Alternatively, as demonstrated by Li and Cohen (Li, L. and S.N. Cohen (1995)
Cell 85:319-
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329), the ability of CGDD to suppress tumorigenesis can be measured by
designing an antisense
sequence to the 5' end of the gene and transfecting NIH 3T3 cells with a
vector transcribing this
sequence. The suppression of the endogenous gene will allow transformed
fibroblasts to produce
clumps of cells capable of forming metastatic tumors when introduced into nude
mice.
Alternatively, an assay for CGDD activity measures the effect of injected CGDD
on the
degradation of maternal transcripts. Procedures for oocyte collection from
Swiss albino mice,
injection, and culture are as described in Stutz (supra). A decrease in the
degradation of maternal
RNAs as compared to control oocytes is indicative of CGDD activity. In the
alternative, CGDD
activity is measured as the ability of purified CGDD to bind to RNAse as
measured by the assays
described in Example XVII.
Alternatively, an assay for CGDD activity measures syncytium formation in COS
cells
transfected with an CGDD expression plasmid, using the two-component fusion
assay described in
Mi (supra). This assay takes advantage of the fact that human interleukin 12
(IL-12) is a heterodimer
comprising subunits with molecular weights of 35 kD (p35) and 40 kD (p40). COS
cells transfected
with expression plasmids carrying the gene for p35 are mixed with COS cells
cotransfected with
expression plasmids carrying the genes for p40 and CGDD. The level of IL-12
activity in the
resulting conditioned medium corresponds to the activity of CGDD in this
assay. Syncytium
formation may also be measured by light microscopy (Mi et al. supra).
An alternative assay for CGDD activity measures cell proliferation as the
amount of newly
initiated DNA synthesis in Swiss mouse 3T3 cells. A plasmid containing
polynucleotides encoding
CGDD is transfected into quiescent 3T3 cultured cells using methods well known
in the art. The
transiently transfected cells are then incubated in the presence of
[3H]thymidine or a radioactive DNA
precursor such as [oc3zP]ATP. Where applicable, varying amounts of CGDD 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 CGDD activity.
Alternatively, CGDD activity is measured by the cyclin-ubiquitin ligation
assay (Townsley,
F.M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2362-2367). The reaction
contains in a volume of
10 ,u1, 40 mM Tris.HCl (pH 7.6), 5 mM Mg Clz, 0.5 mM ATP, 10 mM
phosphocreatine, 50 ,ug of
creatine phosphokinase/ml, 1 mg reduced carboxymethylated bovine serum
albumin/ml, 50 ,uM
ubiquitin, 1 ,uM ubiquitin aldehyde, 1-2 pmol'z5I-labeled cyclin B, 1 pmol E1,
1,uM okadaic acid, 10
,ug of protein of M-phase fraction 1A (containing active E3-C and essentially
free of E2-C), and
varying amounts of CGDD. The reaction is incubated at 18 °C for 60
minutes. Samples are then
separated by electrophoresis on an SDS polyacrylamide gel. The amount of'z5I-
cyclin-ubiquitin
formed is quantified by PHOSPHORIMAGER analysis. The amount of cyclin-
ubiquitin formation is
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proportional to the activity of CGDD in the reaction.
Alternatively, an assay for CGDD activity uses radiolabeled nucleotides, such
as [a3zP]ATP,
to measure either the incorporation of radiolabel into DNA during DNA
synthesis, or fragmentation
of DNA that accompanies apoptosis. Mammalian cells are transfected with
plasmid containing
cDNA encoding CGDD by methods well known in the art. Cells are then incubated
with radiolabeled
nucleotide for various lengths of time. Chromosomal DNA is collected, and
radioactivity is detected
using a scintillation counter. Incorporation of radiolabel into chromosomal
DNA is proportional to
the degree of stimulation of the cell cycle. To determine if CGDD promotes
apoptosis, chromosomal
DNA is collected as above, and analyzed using polyacrylamide gel
electrophoresis, by methods well
known in the art. Fragmentation of DNA is quantified by comparison to
untransfected control cells,
and is proportional to the apoptotic activity of CGDD.
Alternatively, cyclophilin activity of CGDD is measured using a chymotrypsin-
coupled assay
to measure the rate of cis to trans interconversion (Fischer, G., Bang, H.,
and Mech, C. (1984)
Biomed. Biochim. Acta 43: 1101-1111). The chymotrypsin is used to estimate the
trans-substrate
cleavage activity at Xaa-Pro peptide bonds, wherein the rate constant for the
cis to trans isomerization
can be obtained by measuring the rate constant of the substrate hydrolysis at
the slow phase. Samples
are incubated in the presence or absence of the immunosuppressant drugs CsA or
FK506, reactions
initiated by addition of chymotrypsin, and the fluorescent reaction measured.
The enzymatic rate
constant is calculated from the equation kapP = kHZO + ke~, wherein first
order kinetics are displayed,
and where one unit of PPIase activity is defined as kenZ (S ~).
Alternatively, cyclophilin activity of CGDD is monitored by a quantitative
immunoassay that
measures its affinity for stereospecific binding to the immunosuppressant drug
cyclosporin
(Quesniaux, V.F. et al. (1987) Eur. J. Irnmunol. 17: 1359-1365). In this
assay, the cyclophilin-
cyclosporin complex is coated on a solid phase, with binding detected using
anti-cyclophilin rabbit
antiserum enhanced by an antiglobulin-enzyme conjugate.
Alternatively, activity of CGDD is monitored by a binding assay developed to
measure the
non-covalent binding between FKBPs and immunosuppressant drugs in the gas
phase using
electrospray ionization mass spectrometry (Trepanier, D.J., et al. (1999)
Ther. Drug Monit. 21: 274-
280). In electrospray ionization, ions are generated by creating a fine spray
of highly charged
droplets in the presence of a strong electric field; as the droplet decreases
in size, the charge density
on the surface increases. Ions are electrostatically directed into a mass
analyzer, where ions of
opposite charge are generated in spatially separate sources and then swept
into capillary inlets where
the flows are merged and where reactions occur. By comparing the charge states
of bound versus
unbound CGDD/immunosuppressive drug complexes, relative binding affinities can
be established
and correlated with in vitro binding~and immunosuppressive activity.
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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.
102
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
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Table 5
PolynucleotideIncyte ProjectRepresentative Library
SEQ ID:
ID NO:
13 1567742CB1 NGANNOTOl
14 7485501CB1 SPLNNOT04
15 3089944CB SKINBITO1
1
16 5284076CB TESTNON04
1
17 2899903CB BRABDIE02
1
18 7491355CB PROSTUT09
1
19 3333288CB BRAIFER06
1
20 7488313CB COLNNOT01
1
21 6013113CB BRATNOT05
1
22 7488573CB OVARDIRO1
1
23 7506027CB1 BRABDIE02
24 7503618CB1 CARGDITOl
125
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<110> INCYTE GENOMICS, INC.
YUE, Henry
YAO, Monique G.
ISON, Craig H.
LU, Yan
WARREN, Bridget A.
ELLIOTT, Vicki S.
BAUGHN, Mariah R.
DING, Li
XU, Yuming
GIETZEN, Kimberly J.
TANG, Tom Y.
LAL, Preeti
DUGGAN, Brendan M.
BURFORD, Neil
LU, Dyung Aina M.
RICHARDSON, Thomas W.
TRAN, Uyen K.
KHARE, Reena
WALIA, Narinder K.
<120> PROTEINS ASSOCIATED WITH CELL GROWTH, DIFFERENTIATION, AND DEATH
<130> PF-0903 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/268,111; 60/271,175; 60/274,503; 60/274,552
<151> 2001-02-09; 2001-02-23; 2001-03-08; 2001-03-09
<160> 24
<170> PERL Program
<210> 1
<211> 977
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1567742CD1
<400> 1
Met Ala Ser Ser His Ser Ser Ser Pro Val Pro Gln Gly Ser Ser
1 5 10 15
Ser Asp Val Phe Phe Lys Ile Glu Val Asp Pro Ser Lys His Ile
20 25 30
Arg Pro Val Pro Ser Leu Pro Asp Val Cys Pro Lys Glu Pro Thr
35 40 45
Gly Asp Ser His Ser Leu Tyr Val A1a Pro Ser Leu Val Thr Asp
50 ~ 55 60
Gln His Arg Trp Thr Val Tyr His Ser Lys Val Asn Leu Pro Ala
65 70 75
Ala Leu Asn Asp Pro Arg Leu Ala Lys Arg Glu Ser Asp Phe Phe
80 85 90
1/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
Thr Lys Thr Trp Gly Leu Asp Phe Val Asp Thr Glu Val Ile Pro
95 100 105
Ser Phe Tyr Leu Pro Gln Ile Ser Lys Glu His Phe Thr Val Tyr
110 115 120
Gln Gln Glu Ile Ser Gln Arg Glu Lys Ile His Glu Arg Cys Lys
125 130 135
Asn Ile Cys Pro Pro Lys Asp Thr Phe Glu Arg Thr Leu Leu His
140 145 150
Thr His Asp Lys Ser Arg Thr Asp Leu Glu Gln Val Pro Lys Ile
155 160 165
Phe Met Lys Pro Asp Phe Ala Leu Asp Asp Ser Leu Thr Phe Asn
170 175 180
Ser Val Leu Pro Trp Ser His Phe Asn Thr Ala Gly Gly Lys Gly
185 190 195
Asn Arg Asp Ala Ala Ser Ser Lys Leu Leu Gln Glu Lys Leu Ser
200 205 210
His Tyr Leu Asp Ile Val Glu Val Asn Ile Ala His Gln Ile Ser
215 220 225
Leu Arg Ser Glu Ala Phe Phe His Ala Met Thr Ser Gln His Glu
230 235 240
Leu Gln Asp Tyr Leu Arg Lys Thr Ser Gln A1a Val Lys Met Leu
245 250 255
Arg Asp Lys Ile Ala Gln Ile Asp Lys Val Met Cys Glu Gly Ser
260 265 270
Leu His Ile Leu Arg Leu Ala Leu Thr Arg Asn Asn Cys Val Lys
275 280 285
Val Tyr Asn Lys Leu Lys Leu Met Ala Thr Val His Gln Thr Gln
290 295 300
Pro Thr Val Gln Val Leu Leu Ser Thr Ser Glu Phe Val Gly Ala
305 310 315
Leu Asp Leu Ile Ala Thr Thr Gln G1u Val Leu Gln Gln Glu Leu
320 325 330
Gln Gly Ile His Ser Phe Arg His Leu Gly Ser Gln Leu Cys Glu
335 340 345
Leu G1u Lys Leu Ile Asp Lys Met Met Ile Ala Glu Phe Ser Thr
350 355 360
Tyr Ser His Ser Asp Leu Asn Arg Pro Leu Glu Asp Asp Cys G1n
365 370 375
Val Leu Glu Glu Glu Arg Leu Ile Ser Leu Val Phe Gly Leu Leu
380 385 390
Lys Gln Arg Lys Leu Asn Phe Leu Glu Ile Tyr Gly Glu Lys Met
395 400 ~ 405
Val Ile Thr Ala Lys Asn Ile Ile Lys Gln Cys Val I1e Asn Lys
410 415 420
Val Ser Gln Thr Glu Glu Ile Asp Thr Asp Val Val Val Lys Leu
425 430 435
Ala Asp G1n Met Arg Met Leu Asn Phe Pro Gln Trp Phe Asp Leu
440 445 450
Leu Lys Asp Ile Phe Ser Lys Phe Thr Ile Phe Leu Gln Arg Val
455 460 465
Lys Ala Thr Leu Asn Ile Ile His Ser Va1 Val Leu Ser Val Leu
470 475 480
Asp Lys Asn Gln Arg Thr Arg Glu Leu Glu G1u Ile Ser Gln Gln
485 490 495
Lys Asn Ala Ala Lys Asp Asn Ser Leu Asp Thr Glu Val Ala Tyr
500 505 510
2/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
Leu Ile His Glu Gly Met Phe Ile Ser Asp Ala Phe Gly Glu Gly
515 520 525
Glu Leu Thr Pro Ile Ala Val Asp Thr Thr Ser Gln Arg Asn Ala
530 535 540
Ser Pro Asn Ser Glu Pro Cys Ser Ser Asp Ser Val Ser Glu Pro
545 550 555
Glu Cys Thr Thr Asp Ser Ser Ser Ser Lys Glu His Thr Ser Ser
560 565 570
Ser Ala Ile Pro Gly Gly Val Asp Ile Met Val Ser Glu Asp Met
575 580 585
Lys Leu Thr Asp Ser Glu Leu Gly Lys Leu Ala Asn Asn Ile Gln
590 595 600
Glu Leu Leu Tyr Ser Ala Ser Asp Ile Cys His Asp Arg Ala Val
605 610 615
Lys Phe Leu Met Ser Arg Ala Lys Asp Gly Phe Leu Glu Lys Leu
620 625 630
Asn Ser Met Glu Phe Ile Thr Leu Ser Arg Leu Met Glu Thr Phe
635 640 645
Ile Leu Asp Thr Glu Gln Ile Cys Gly Arg Lys Ser Thr Ser Leu
650 655 660
Leu Gly Ala Leu Gln Ser Gln Ala Ile Lys Phe Val Asn Arg Phe
665 670 675
His Glu Glu Arg Lys Thr Lys Leu Ser Leu Leu Leu Asp Asn Glu
680 685 690
Arg Trp Lys Gln Ala Asp Val Pro Ala Glu Phe Gln Asp Leu Val
695 700 705
Asp Ser Leu Ser Asp Gly Lys Ile Ala Leu Pro Glu Lys Lys Ser
710 715 720
Gly Ala Thr Glu Glu Arg Lys Pro Ala Glu Val Leu Ile Val Glu
725 730 735
Gly Gln Gln Tyr Ala Val Val Gly Thr Val Leu Leu Leu Ile Arg
740 745 750
Ile Ile Leu Glu Tyr Cys Gln Cys Val Asp Asn Ile Pro Ser Val
755 760 765
Thr Thr Asp Met Leu Thr Arg Leu Ser Asp Leu Leu Lys Tyr Phe
770 775 780
Asn Ser Arg Ser Cys Gln Leu Val Leu Gly Ala Gly Ala Leu Gln
785 790 795
Val Val Gly Leu Lys Thr Ile Thr Thr Lys Asn Leu Ala Leu Ser
800 805 810
Ser Arg Cys Leu Gln Leu Ile Val His Tyr Ile Pro Val Ile Arg
815 820 825
Ala His Phe Glu Ala Arg Leu Pro Pro Lys Gln Tyr Ser Met Leu
830 835 840
Arg His Phe Asp His Ile Thr Lys Asp Tyr His Asp His Ile Ala
845 850 855
Glu Ile Ser Ala Lys Leu Val Ala Ile Met Asp Ser Leu Phe Asp
860 865 870
Lys Leu Leu Ser Lys Tyr Glu Val Lys Ala Pro Val Pro Ser Ala
875 880 885
Cys Phe Arg Asn Ile Cys Lys Gln Met Thr Lys Met His Glu Ala
890 895 900
Ile Phe Asp Leu Leu Pro G1u Glu G1n Thr Gln Met Leu Phe Leu
905 910 915
Arg Ile Asn Ala Ser Tyr Lys Leu His Leu Lys Lys Gln Leu Ser
920 925 930
3/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
His Leu Asn Va1 Ile Asn Asp Gly Gly Pro Gln Asn Gly Leu Val
935 940 945
Thr Ala Asp Val Ala Phe Tyr Thr Gly Asn Leu Gln Ala Leu Lys
950 955 960
Gly Leu Lys Asp Leu Asp Leu Asn Met Ala Glu Ile Trp Glu Gln
965 970 975
Lys Arg
<210> 2
<211> 109
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7485501CD1
<400> 2
Met Ser Tyr Lys Pro Thr Thr Pro Ala Pro Ser Ser Thr Pro Gly
1 5 10 15
Phe Ser Thr Pro Gly Pro Gly Thr Pro Val Pro Thr Gly Ser Val
20 25 30
Pro Ser Pro Ser Gly Ser Gly Pro Gly Ala Thr Ala Pro Cys Arg
35 40 45
Pro Leu Phe Lys Asp Phe Gly Pro Pro Thr Val Gly Cys Val Gln
50 55 60
Ala Met Lys Pro Pro Gly Ala Gln Gly Ser Gln Ser Thr Tyr Thr
65 70 75
Glu Leu Leu Leu Val Thr Gly Glu Met Gly Lys Gly Ile Arg Pro
80 85 90
Thr Tyr A1a Gly Ser Lys Ser Ala Ala Glu Arg Leu Lys Arg Gly
95 100 105
Ile Ile His Pro
<210> 3
<211> 468
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3089944CD1
<400> 3
Met Ala Ala Pro Asp Leu Ser Thr Asn Leu Gln Glu G1u Ala Thr
1 5 10 15
Cys Ala Ile Cys Leu Asp Tyr Phe Thr Asp Pro Val Met Thr Asp
20 25 30
Cys Gly His Asn Phe Cys Arg Glu Cys Ile Arg Arg Cys Trp Gly
35 40 45
Gln Pro Glu G1y Pro Tyr Ala Cys Pro Glu Cys Arg Glu Leu Ser
50 55 60
Pro Gln Arg Asn Leu Arg Pro Asn Arg Pro Leu Ala Lys Met Ala
65 70 75
4/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
Glu Met Ala Arg Arg Leu His Pro Pro Ser Pro Val Pro Gln Gly
80 85 90
Val Cys Pro Ala His Arg Glu Pro Leu Ala Ala Phe Cys Gly Asp
95 100 105
Glu Leu Arg Leu Leu Cys Ala Ala Cys Glu Arg Ser Gly Glu His
110 115 120
Trp Ala His Arg Val Arg Pro Leu Gln Asp Ala Ala Glu Asp Leu
125 230 135
Lys Ala Lys Leu Glu Lys Ser Leu Glu His Leu Arg Lys Gln Met
140 145 150
Gln Asp Ala Leu Leu Phe Gln Ala Gln Ala Asp Glu Thr Cys Val
155 160 165
Leu Trp Gln Lys Met Val Glu Ser Gln Arg Gln Asn Val Leu Gly
170 175 180
Glu Phe Glu Arg Leu Arg Arg Leu Leu Ala Glu Glu Glu G1n Gln
185 190 195
Leu Leu Gln Arg Leu Glu Glu Glu Glu Leu Glu Val Leu Pro Arg
200 205 2l0
Leu Arg Glu Gly Ala Ala His Leu Gly Gln Gln Ser Ala His Leu
215 220 225
Ala Glu Leu Ile Ala G1u Leu Glu G1y Arg Cys Gln Leu Pro Ala
230 235 240
Leu Gly Leu Leu Gln Asp Ile Lys Asp Ala Leu Arg Arg Val Gln
245 250 255
Asp Val Lys Leu Gln Pro Pro Glu Val Val Pro Met Glu Leu Arg
260 265 270
Thr Val Cys Arg Val Pro Gly Leu Val Glu Thr Leu Arg Arg Phe
275 280 285
Arg Gly Asp Val Thr Leu Asp Pro Asp Thr Ala Asn Pro GIu Leu
290 295 300
Ile Leu Ser Glu Asp Arg Arg Ser Val Gln Arg Gly Asp Leu Arg
305 310 315
Gln Ala Leu Pro Asp Ser Pro Glu Arg Phe Asp Pro Gly Pro Cys
320 325 330
Val Leu Gly Gln Glu Arg Phe Thr Ser Gly Arg His Tyr Trp Glu
335 340 345
Val Glu Val Gly Asp Arg Thr Ser Trp Ala Leu Gly Val Cys Arg
350 355 360
Glu Asn Val Asn Arg Lys Glu Lys Gly Glu Leu Ser Ala Gly Asn
365 370 375
Gly Phe Trp 21e Leu Val Phe Leu Gly Ser Tyr Tyr Asn Ser Ser
380 385 390
Glu Arg Ala Leu Ala Pro Leu Arg Asp Pro Pro Arg Arg Val Gly
395 400 405
Ile Phe Leu Asp Tyr Glu Ala Gly His Leu Ser Phe Tyr Ser Ala
410 415 420
Thr Asp Gly Ser Leu Leu Phe Ile Phe Pro Glu Ile Pro Phe Ser
425 430 435
Gly Thr Leu Arg Pro Leu Phe Ser Pro Leu Ser Ser Ser Pro Thr
440 445 450
Pro Met Thr Ile Cys Arg Pro Lys Gly Gly Ser Gly Asp Thr Leu
455 460 ' 465
Ala Pro G1n
<210> 4
5/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
<211> 158
<212> PRT
<213> Homo Sapiens
<220>
<221> mist feature
<223> Incyte ID No: 5284076CD1
<400> 4
Met Ala Leu Glu Val Leu Met Leu Leu Ala Val Leu Ile Trp Thr
1 5 10 15
Gly Ala Glu Asn Leu His Val Lys Ile Ser Cys Ser Leu Asp Trp
20 25 30
Leu Met Val Ser Val Ile Pro Val Ala Glu Ser Arg Asn Leu Tyr
35 40 45
Ile Phe Ala Asp G1u Leu His Leu Gly Met Gly Cys Pro Ala Asn
50 55 60
Arg Ile His Thr Tyr Val Tyr Glu Phe Ile Tyr Leu Val Arg Asp
65 70 75
Cys Gly Ile Arg Thr Arg Val Va1 Ser Glu Glu Thr Leu Leu Phe
80 85 90
Gln Thr Glu Leu Tyr Phe Thr Pro Arg Asn Ile Asp His Asp Pro
95 100 105
Gln Glu Ile His Leu Glu Cys Ser Thr Ser Arg Lys Ser Val Trp
110 115 120
Leu Thr Pro Val Ser Thr Glu Asn Glu Ile Lys Leu Asp Pro Ser
125 130 135
Pro Phe Ile Ala Asp Phe Gln Thr Thr Ala Glu Glu Leu Gly Leu
140 145 150
Leu Ser Ser Ser Pro Asn Leu Leu
155
<210> 5
<211> 1161
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2899903CD1
<400> 5
Met Glu Pro Asp Ser Leu Leu Asp Gln Asp Asp Ser Tyr G1u Ser
1 5 10 15
Pro Gln Glu Arg Pro Gly Ser Arg Arg Ser Leu Pro G1y Ser Leu
20 25 30
Ser Glu Lys Ser Pro Ser Met Glu Pro Ser Ala Ala Thr Pro Phe
35 40 45
Arg Val Thr Gly Phe Leu Ser Arg Arg Leu Lys G1y Ser Ile Lys
50 55 . 60
Arg Thr Lys Ser Gln Pro Lys Leu Asp Arg Asn His Ser Phe Arg
65 70 75
His Ile Leu Pro Gly Phe Arg Ser Ala Ala Ala Ala Ala Ala Asp
80 85 90
Asn Glu Arg Ser His Leu Met Pro Arg Leu Lys Glu Ser Arg Ser
95 100 105
6/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
His Glu Ser Leu Leu Ser Pro Ser Ser Ala Val Glu Ala Leu Asp
110 115 120
Leu Ser Met Glu Glu Glu Val Val Ile Lys Pro Val His Ser Ser
125 130 135
Ile Leu Gly Gln Asp Tyr Cys Phe Glu Val Thr Thr Ser Ser Gly
140 145 150
Ser Lys Cys Phe Ser Cys Arg Ser Ala Ala Glu Arg Asp Lys Trp
155 160 165
Met Glu Asn Leu Arg Arg Ala Val His Pro Asn Lys Asp Asn Ser
170 175 180
Arg Arg Val Glu His Ile Leu Lys Leu Trp Val Ile Glu Ala Lys
185 190 195
Asp Leu Pro Ala Lys Lys Lys Tyr Leu Cys Glu Leu Cys Leu Asp
200 205 210
Asp Val Leu Tyr Ala Arg Thr Thr Gly Lys Leu Lys Thr Asp Asn
215 220 225
Val Phe Trp Gly Glu His Phe Glu Phe His Asn Leu Pro Pro Leu
230 235 240
Arg Thr Val Thr Val His Leu Tyr Arg G1u Thr Asp Lys Lys Lys
245 250 255
Lys Lys Glu Arg Asn Ser Tyr Leu Gly Leu Val Ser Leu Pro Ala
260 265 270
Ala Ser Val Ala Gly Arg Gln Phe Val Glu Lys Trp Tyr Pro Val
275 280 285
Val Thr Pro Asn Pro Lys Gly Gly Lys Gly Pro Gly Pro Met Ile
290 295 300
Arg Ile Lys Ala Arg Tyr Gln Thr Ile Thr Ile Leu Pro Met Glu
305 310 315
Met Tyr Lys Glu Phe Ala Glu His Ile Thr Asn His Tyr Leu Gly
320 325 330
Leu Cys Ala Ala Leu Glu Pro Ile Leu Ser Ala Lys Thr Lys Glu
335 340 345
Glu Met Ala Ser Ala Leu Val His Ile Leu Gln Ser Thr Gly Lys
350 355 360
Val Lys Asp Phe Leu Thr Asp Leu Met Met Ser Glu Val Asp Arg
365 370 375
Cys Gly Asp Asn Glu His Leu Ile Phe Arg Glu Asn Thr Leu Ala
380 385 390
Thr Lys Ala Ile Glu Glu Tyr Leu Lys Leu Val Gly Gln Lys Tyr
395 400 405
Leu Gln Asp Ala Leu Gly Glu Phe Ile Lys Ala Leu Tyr Glu Ser
410 415 420
Asp Glu Asn Cys Glu Val Asp Pro Ser Lys Cys Ser Ala Ala Asp
425 430 435
Leu Pro Glu His Gln Gly Asn Leu Lys Met Cys Cys Glu Leu Ala
440 445 450
Phe Cys Lys Ile Ile Asn Ser Tyr Cys Val Phe Pro Arg Glu Leu
455 460 465
Lys Glu Val Phe Ala Ser Trp Arg Gln Glu Cys Ser Ser Arg Gly
470 475 480
Arg Pro Asp Ile Ser Glu Arg Leu Ile Ser Ala Ser Leu Phe Leu
485 490 495
Arg Phe Leu Cys Pro Ala Ile Met Ser Pro Ser Leu Phe Asn Leu
500 505 510
Leu Gln Glu Tyr Pro Asp Asp Arg Thr Ala Arg Thr Leu Thr Leu
515 520 525
7/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
Ile Ala Lys Val Thr Gln Asn Leu Ala Asn Phe Ala Lys Phe Gly
530 535 540
Ser Lys Glu Glu Tyr Met Ser Phe Met Asn Gln Phe Leu Glu His
545 550 555
Glu Trp Thr Asn Met Gln Arg Phe Leu Leu Glu Ile Se'r Asn Pro
560 565 570
Glu Thr Leu Ser Asn Thr Ala Gly Phe Glu Gly Tyr Ile Asp Leu
575 580 585
Gly Arg Glu Leu Ser Ser Leu His Ser Leu Leu Trp Glu Ala Val
590 595 600
Ser Gln Leu Glu Gln Ser Ile Va1 Ser Lys Leu Gly Pro Leu Pro
605 610 615
Arg Ile Leu Arg Asp Val His Thr Ala Leu Ser Thr Pro Gly Ser
620 625 630
Gly Gln Leu Pro Gly Thr Asn Asp Leu Ala Ser Thr Pro Gly Ser
635 640 645
Gly Ser Ser Ser 21e Ser Ala Gly Leu Gln Lys Met Val Ile Glu
650 655 660
Asn Asp Leu Ser Gly Leu Ile Asp Phe Thr Arg Leu Pro Ser Pro
665 670 675
Thr Pro Glu Asn Lys Asp Leu Phe Phe Val Thr Arg Ser Ser Gly
680 685 690
Va1 Gln Pro Ser Pro Ala Arg Sex Ser Ser Tyr Ser Glu Ala Asn
695 700 705
Glu Pro Asp Leu Gln Met Ala Asn G1y Gly Lys Ser Leu Ser Met
710 715 720
Val Asp Leu Gln Asp Ala Arg Thr Leu Asp Gly Glu Ala Gly Sex
725 730 735
Pro A1a Gly Pro Asp Val Leu Pro Thr Asp Gly Gln Ala Ala Ala
740 745 750
Ala Gln Leu Val Ala Gly Trp Pro Ala Arg Ala Thr Pro Val Asn
755 760 765
Leu Ala Gly Leu Ala Thr Val Arg Arg Ala Gly Gln Thr Pro Thr
770 775 780
Thr Pro Gly Thr Ser Glu Gly Ala Pro Gly Arg Pro Gln Leu Leu
785 790 795
Ala Pro Leu Ser Phe Gln Asn Pro Va1 Tyr Gln Met Ala Ala Gly
800 805 810
Leu Pro Leu Ser Pro Arg Gly Leu Gly Asp Ser G1y Ser Glu Gly
815 820 825
His Ser Ser Leu Ser Ser His Ser Asn Ser Glu Glu Leu Ala Ala
830 835 840
Ala Ala Lys Leu Gly Ser Phe Ser Thr Ala Ala Glu Glu Leu Ala
845 850 855
Arg Arg Pro Gly Glu Leu Ala Arg Arg Gln Met Ser Leu Thr Glu
860 865 870
Lys Gly Gly Gln Pro Thr Val Pro Arg Gln Asn Ser Ala Gly Pro
875 880 885
Gln Arg Arg Ile Asp Gln Pro Pro Pro Pro Pro Pro Pro Pro Pro
890 895 900
Pro Ala Pro Arg Gly Arg Thr Pro Pro Asn Leu Leu Ser Thr Leu
905 910 915
Gln Tyr Pro Arg Pro Ser Ser Gly Thr Leu Ala Ser Ala Ser Pro
920 925 930
Asp Trp Val Gly Pro Ser Thr Arg Leu Arg Gln Gln Ser Ser Ser
935 940 945
8/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
Ser Lys Gly Asp Ser Pro Glu Leu Lys Pro Arg Ala Val His Lys
950 955 960
Gln Gly Pro Ser Pro Val Ser Pro Asn Ala Leu Asp Arg Thr Ala
965 970 975
A1a Trp Leu Leu Thr Met Asn Ala Gln Leu Leu Glu Asp Glu Gly
980 985 990
Leu Gly Pro Asp Pro Pro His Arg Asp Arg Leu Arg Ser Lys Asp
995 1000 1005
Glu Leu Ser Gln Ala Glu Lys Asp Leu Ala Val Leu Gln Asp Lys
1010 1015 1020
Leu Arg Ile Ser Thr Lys Lys Leu Glu Glu Tyr Glu Thr Leu Phe
1025 1030 1035
Lys Cys Gln G1u Glu Thr Thr Gln Lys Leu Val Leu Glu Tyr Gln
1040 1045 1050
Ala Arg Leu Glu Glu Gly Glu Glu Arg Leu Arg Arg Gln G1n Glu
1055 1060 1065
Asp Lys Asp Ile G1n Met Lys Gly Ile Ile Ser Arg Leu Met Ser
1070 1075 1080
Val G1u Glu Glu Leu Lys Lys Asp His Ala Glu Met Gln Ala Ala
1085 1090 1095
Val Asp Ser Lys Gln Lys I1e Ile Asp Ala Gln Glu Lys Arg Ile
1100 1105 1110
Ala Ser Leu Asp Ala Ala Asn Ala Arg Leu Met Ser Ala Leu Thr
1115 1120 1125
Gln Leu Lys Glu Arg Tyr Ser Met Gln Ala Arg Asn Gly Ile Ser
1130 1135 1140
Pro Thr Asn Pro Thr Lys Leu Gln Ile Thr Glu Asn Gly Glu Phe
1145 1150 1155
Arg Asn Ser Ser Asn Cys
1160
<210> 6
<211> 331
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7491355CD1
<400> 6
Met Ser Arg Ala Arg Gly Ala Leu Cys Arg Ala Cys Leu Ala Leu
1 5 10 15
Ala Ala Ala Leu Ala Ala Leu Leu Leu Leu Pro Leu Pro Leu Pro
20 25 30
Arg Ala Pro Ala Pro Ala Arg Thr Pro Ala Pro Ala Pro Arg Ala
35 40 45
Pro Pro Ser Arg Pro Ala Ala Pro Ser Leu Arg Pro Asp Asp Va1
50 55 60
Phe I1e Ala Val Lys Thr Thr Arg Lys Asn His Gly Pro Arg Leu
65 70 75
Leu Leu Leu Leu Arg Thr Trp Ile Ser Arg Ala Arg Gln Gln Thr
80 85 90
Phe Ile Phe Thr Asp Gly Asp Asp Pro Glu Leu Glu Leu Gln Gly
95 100 105
Gly Asp Arg Val I1e Asn Thr Asn Cys Ser Ala Val Arg Thr Arg
9/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
120 115 220
G1n Ala Leu Cys Cys Lys Met Ser Val Glu Tyr Asp Lys Phe Ile
125 130 135
Glu Ser Gly Arg Lys Trp Phe Cys His Val Asp Asp Asp Asn Tyr
140 145 150
Val Asn Ala Arg Ser Leu Leu His Leu Leu Ser Ser Phe Ser Pro
155 160 165
Ser Gln Asp Val Tyr Leu Gly Arg Pro Ser Leu Asp His Pro Ile
170 175 180
Glu Ala Thr Glu Arg Val Gln Gly Gly Arg Thr Val Thr Thr Val
185 190 195
Lys Phe Trp Phe Ala Thr Gly Gly Ala Gly Phe Cys Leu Ser Arg
200 205 210
Gly Leu Ala Leu Lys Met Ser Pro Trp Ala Ser Leu Gly Ser Phe
215 220 225
Met Ser Thr Ala Glu Gln Val Arg Leu Pro Asp Asp Cys Thr Val
230 235 240
Gly Tyr Ile Val Glu Gly Leu Leu Gly Ala Arg Leu Leu His Ser
245 250 255
Pro Leu Phe His Ser His Leu Glu Asn Leu Gln Arg Leu Pro Pro
260 265 270
Asp Thr Leu Leu Gln Gln Val Thr Leu Ser His Gly Gly Pro Glu
275 280 285
Asn Pro Gln Asn Val Va1 Asn Val Ala Gly Gly Phe Ser Leu His
290 295 300
Gln Asp Pro Thr Arg Phe Lys~Ser Ile His Cys Leu Leu Tyr Pro
305 310 315
Asp Thr Asp Trp Cys Pro Arg Gln Lys Gln Gly Ala Pro Thr Ser
320 325 330
Arg
<210> 7
<212> 579
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3333288CD1
<400> 7
Met Ser Ala Leu Arg Pro Leu Leu Leu Leu Leu Leu Pro Leu Cys
1 5 10 25
Pro Gly Pro Gly Pro Gly Pro Gly Ser Glu Ala Lys Val Thr Arg
20 25 30
Ser Cys Ala Glu Thr Arg Gln Val Leu Gly Ala Arg Gly Tyr Ser
35 40 45
Leu Asn Leu Ile Pro Pro Ala Leu Ile Ser Gly Glu His Leu Arg
50 55 60
Val Cys Pro G1n Glu Tyr Thr Cys Cys Ser Ser Glu Thr Glu Gln
65 70 75
Arg Leu Ile Arg Glu Thr Glu Ala Thr Phe Arg Gly Leu Val Glu
80 85 90
Asp Ser Gly Ser Phe Leu Val His Thr Leu Ala Ala Arg His Arg
95 100 105
10/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
Lys Phe Asp Glu Phe Phe Leu Glu Met Leu Ser Val Ala Gln His
110 115 120
Ser Leu Thr Gln Leu Phe Ser His Ser Tyr Gly Arg Leu Tyr Ala
125 130 135
Gln His Ala Leu Ile Phe Asn Gly Leu Phe Ser Arg Leu Arg Asp
140 145 150
Phe Tyr Gly Glu Ser Gly G1u Gly Leu Asp Asp Thr Leu Ala Asp
155 160 165
Phe Trp Ala Gln Leu Leu Glu Arg Val Phe Pro Leu Leu His Pro
170 175 180
Gln Tyr Ser Phe Pro Pro Asp Tyr Leu Leu Cys Leu Ser Arg Leu
185 190 195
Ala Ser Ser Thr Asp Gly Ser Leu Gln Pro,Phe Gly Asp Ser Pro
200 205 210
Arg Arg Leu Arg Leu Gln Ile Thr Arg Thr Leu Val Ala Ala Arg
215 220 225
Ala Phe Val G1n Gly Leu Glu Thr Gly Arg Asn Val Val Ser Glu
230 235 240
Ala Leu Lys Val Pro Val Ser Glu Gly Cys Ser Gln Ala Leu Met
245 250 255
Arg Leu Ile Gly Cys Pro Leu Cys Arg Gly Val Pro Ser Leu Met
260 265 270
Pro Cys Gln Gly Phe Cys Leu Asn Val Val Arg Gly Cys Leu Ser
275 280 285
Ser Arg Gly Leu Glu Pro Asp Trp Gly Asn Tyr Leu Asp Gly Leu
290 295 300
Leu Ile Leu Ala Asp Lys Leu Gln G1y Pro Phe Ser Phe Glu Leu
305 310 315
Thr Ala Glu Ser Ile Gly Val Lys Ile Ser Glu Gly Leu Met Tyr
320 325 330
Leu Gln Glu Asn Ser Ala Lys Va1 Ser Ala Gln Val Phe Gln Glu
335 340 345
Cys Gly Pro Pro Asp Pro Val Pro Ala Arg Asn Arg Arg Ala Pro
350 355 360
Pro Pro Arg Glu Glu Ala Gly Arg Leu Trp Ser Met Val Thr Glu
365 370 375
Glu Glu Arg Pro Thr Thr Ala A1a Gly Thr Asn Leu His Arg Leu
380 385 390
Val Trp Glu Leu Arg Glu Arg Leu Ala Arg Met Arg Gly Phe Trp
395 400 405
Ala Arg Leu Ser Leu Thr Val Cys Gly Asp Ser Arg Met Ala Ala
410 415 420
Asp Ala Ser Leu G1u Ala Ala Pro Cys Trp Thr Gly Ala Gly Arg
425 430 435
Gly Arg Tyr Leu Pro Pro Val Val Gly Gly Ser Pro Ala Glu Gln
440 445 450
Val Asn Asn Pro Glu Leu Lys Val Asp Ala Ser Gly Pro Asp Val
455 460 465
Pro Thr Arg Arg Arg Arg Leu Gln Leu Arg Ala Ala Thr Ala Arg
470 475 480
Met Lys Thr Ala Ala Leu Gly His Asp Leu Asp Gly Gln Asp Ala
485 490 495
Asp Glu Asp Ala Ser G1y Ser Gly Gly Gly Gln Gln Tyr Ala Asp
500 505 510
Asp Trp Met Ala G1y Ala Val Ala Pro Pro Ala Arg Pro Pro Arg
515 520 525
11/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
Pro Pro Tyr Pro Pro Arg Arg Asp Gly Ser Gly Gly Lys Gly Gly
530 535 540
Gly Gly Ser Ala Arg Tyr Asn Gln Gly Arg Ser Arg Ser Gly Gly
545 550 555
Ala Ser Ile Gly Phe His Thr Gln Thr Ile Leu Ile Leu Ser Leu
560 565 570
Ser Ala Leu Ala Leu Leu Gly Pro Arg
575
<210> 8
<211> 490
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7488313CD1
<400> 8
Met Glu Gly Gln Asp Glu Val Ser Ala Arg Glu Gln His Phe His
1 5 10 15
Ser Gln Val Arg Glu Ser Thr Ile Cys Phe Leu Leu Phe Ala Ile
20 25 30
Leu Tyr Val Val Ser Tyr Phe Ile Ile Thr Arg Tyr Lys Arg Lys
35 40 45
Ser Asp Glu Gln Glu Asp Glu Asp Ala Ile Val Asn Arg Ile Ser
50 55 60
Leu Phe Leu Ser Thr Phe Thr Leu Ala Val Ser Ala Gly Ala Val
65 70 75
Leu Leu Leu Pro Phe Ser Ile Ile Ser Asn Glu Ile Leu Leu Ser
80 85 90
Phe Pro Gln Asn Tyr Tyr Ile Gln Trp Leu Asn G1y Ser Leu Ile
95 100 105
His Gly Leu Trp Asn Leu A1a Ser Leu Phe Ser Asn Leu Cys Leu
110 115 120
Phe Val Leu Met Pro Phe Ala Phe Phe Phe Leu G1u Ser Glu Gly
125 130 135
Phe Ala Gly Leu Lys Lys Gly Ile Arg Ala Arg I1e Leu Glu Thr
140 145 150
Leu Val Met Leu Leu Leu Leu Ala Leu Leu Ile Leu Gly Ile Val
l55 160 165
Trp Va1 Ala Ser Ala Leu Ile Asp Asn Asp Ala Ala Ser Met Glu
170 175 180
Ser Leu Tyr Asp Leu Trp Glu Phe Tyr Leu Pro Tyr Leu Tyr Ser
185 190 195
Cys Ile Ser Leu Met Gly Cys Leu Leu Leu Leu Leu Cys Thr Pro
200 205 210
Val Gly Leu Ser Arg Met Phe Thr Val Met Gly Gln Leu Leu Val
215 220 225
Lys Pro Thr Ile Leu Glu Asp Leu Asp Glu G1n Ile Tyr Ile Ile
230 235 240
Thr Leu Glu Glu Glu Ala Leu Gln Arg Arg Leu Asn Gly Leu Ser
245 250 255
Ser Ser Val Glu Tyr Asn Ile Met Glu Leu Glu Gln Glu Leu Glu
260 265 270
Asn Val Lys Thr Leu Lys Thr Lys Leu G1u Arg Arg Lys Lys Ala
12/41
CA 02441495 2003-08-06
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275 280 285
Ser Ala Trp Glu Arg Asn Leu Val Tyr Pro Ala Val Met Val Leu
290 295 300
Leu Leu Ile Glu Thr Ser Ile Ser Val Leu Leu Val Ala Cys Asn
305 310 315
Ile Leu Cys Leu Leu Val Asp Glu Thr Ala Met Pro Lys Gly Thr
320 325 330
Arg Gly Pro Gly Ile Gly Asn Ala Ser Leu Ser Thr Phe Gly Phe
335 340 345
Val Gly Ala Ala Leu Glu Tle Ile Leu Ile Phe Tyr Leu Met Val
350 355 360
Ser Ser Va1 Val Gly Phe Tyr Ser Leu Arg Phe Phe Gly Asn Phe
365 370 375
Thr Pro Lys Lys Asp Asp Thr Thr Met Thr Lys Ile Ile Gly Asn
380 385 390
Cys Val Ser Ile Leu Val Leu Ser Ser Ala Leu Pro Val Met Ser
395 400 405
Arg Thr Leu Gly Ile Thr Arg Phe Asp Leu Leu Gly Asp Phe Gly
410 415 420
Arg Phe Asn Trp Leu Gly Asn Phe Tyr Ile Val Leu Ser Tyr Asn
425 430 435
Leu Leu Phe Ala Ile Val Thr Thr Leu Cys Leu Val Arg Lys Phe
440 445 450
Thr Ser Ala Val Arg Glu Glu Leu Phe Lys Ala Leu Gly Leu His
455 460 465
Lys Leu His Leu Pro Asn Thr Ser Arg Asp Ser Glu Thr Ala Lys
470 475 480
Pro Ser Val Asn Gly His Gln Lys Ala Leu
485 490
<210> 9
<211> 544
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6013113CD1
<400> 9
Met Met Lys Thr Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp
1 5 10 15
Glu Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu
20 25 30
Leu Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu
35 40 45
Ile Gln Asn Ala Val Asn Gly Val Lys G1n Ile Lys Thr Leu Ile
50 55 60
Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu
65 70 75
Glu Ala Lys Lys Lys Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu
80 85 90
Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr
95 100 105
Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr
110 115 120
13/41
CA 02441495 2003-08-06
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Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu
125 130 135
Val Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe
140 145 150
Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn
155 160 165
Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln Asp His Phe
170 175 180
Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe
185 190 195
Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser
200 205 210
Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile
215 220 225
Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe
230 235 240
His Ala Met Phe Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln
245 250 255
Gln Ala Met Asp Ile His Phe His Ser Pro Ala Phe Gln His Pro
260 265 270
Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr Val Cys
275 280 285
Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp
290 295 300
G1n'Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp Cys Ser Thr
305 310 315
Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser
320 325 330
Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu
335 340 345
Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu
350 355 360
Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu
365 370 375
Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala
380 385 390
Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu Val
395 400 405
Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro
410 415 420
Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala
425 430 435
Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Asp Ser Leu
440 445 450
Leu Lys Leu Leu Ser Arg Arg Ala Thr Trp Ala Glu Leu Arg Gly
455 460 465
Pro Gly Ala Leu Leu Glu Leu Leu Ala Val Arg Arg Lys Val Ala
470 475 480
Gly Phe Cys Asp Glu Lys Arg Glu Glu Glu Lys Gly Lys G1u Gln
485 490 495
Arg Gly Cys Val Cys Asp Ala Gln Glu Lys Ala Glu Val Ala Val
500 505 510
Lys Leu Leu Arg Asp Glu G1y Gly Arg Ala Leu Cys Asn Cys Gln
515 520 525
Ser Thr Asp Met Gln Gln Gly Pro Phe Leu Ile Val Thr Val Ser
530 535 540
14/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
Gln Arg Arg Gln
<210> 10
<211> 2758
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7488573CD1
<400> 10
Met Asp Val Lys Glu Arg Lys Pro Tyr Arg Ser Leu Thr Arg Arg
1 5 10 15
Arg Asp Ala Glu Arg Arg Tyr Thr Ser Ser Ser Ala Asp Ser G1u
20 25 30
Glu Gly Lys Ala Pro Gln Lys Ser Tyr Ser Ser Ser Glu Thr Leu
35 40 45
Lys Ala Tyr Asp Gln Asp Ala Arg Leu Ala Tyr Gly Ser Arg Val
50 55 60
Lys Asp Ile Val Pro Gln Glu Ala Glu Glu Phe Cys Arg Thr Gly
65 70 75
Ala Asn Phe Thr Leu Arg Glu Leu Gly Leu Glu Glu Val Thr Pro
80 85 90
Pro His Gly Thr Leu Tyr Arg Thr Asp Ile Gly Leu Pro His Cys
95 100 105
Gly Tyr Ser Met Gly Ala Gly Ser Asp Ala Asp Met Glu Ala Asp
110 115 120
Thr Val Leu Ser Pro Glu His Pro Val Arg Leu Trp G1y Arg Ser
125 130 135
Thr Arg Ser Gly Arg Ser Ser Cys Leu Ser Ser Arg A1a Asn Ser
140 145 150
Asn Leu Thr Leu Thr Asp Thr Glu His G1u Asn Thr Glu Thr Pro
155 160 165
Gly G1y Leu Gln Asn His Ala Arg Leu Arg Thr Pro Pro Pro Pro
170 175 180
Leu Ser His Ala His Thr Pro Asn Gln His His A1a A1a Ser Ile
185 190 195
Asn Sex Leu Asn Arg Gly Asn Phe Thr Pro Arg Ser Asn Pro Ser
200 205 210
Pro Ala Pro Thr Asp His Ser Leu Ser Gly Glu Pro Pro Ala Gly
215 220 225
Gly Ala Gln Glu Pro Ala His Ala Gln Glu Asn Trp Leu Leu Asn
230 235 240
Ser Asn Ile Pro Leu Glu Thr Arg Asn Leu.Gly Lys Gln Pro Phe
245 250 255
Leu Gly Thr Leu Gln Asp Asn Leu Ile Glu Met Asp Ile Leu Gly
260 265 270
Ala Ser Arg His Asp Gly Ala Tyr Ser Asp G1y His Phe Leu Phe
275 280 285
Lys Pro Gly Gly Thr Ser Pro Leu Phe Cys Thr Thr Ser Pro Gly
290 295 300
Tyr Pro Leu Thr Ser Ser Thr Val Tyr Ser Pro Pro Pro Arg Pro
305 310 315
Leu Pro Arg Ser Thr Phe Ala Arg Pro Ala Phe Asn Leu Lys Lys
15/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
320 325 330
Pro Ser Lys Tyr Cys Asn Trp Lys Cys Ala Ala Leu Ser Ala Ile
335 340 345
Val Ile Ser Ala Thr Leu Val I1e Leu Leu Ala Tyr Phe Val Gly
350 355 360
Lys His Leu Phe Asn Trp His Leu Gln Pro Met Glu Gly Gln Met
365 370 375
Tyr Glu Ile Thr Glu Asp Thr Ala Ser Ser Trp Pro Val Pro Thr
380 385 390
Asp Val Ser Leu Tyr Pro Ser Gly Gly Thr Gly Leu Glu Thr Pro
395 400 405
Asp Arg Lys Gly Lys Gly Thr Thr Glu Gly Lys Pro Ser Ser Phe
410 415 420
Phe Pro Glu Asp Ser Phe Ile Asp Ser Gly Glu Ile Asp Val Gly
425 430 435
Arg Arg Ala Ser Gln Lys Ile Pro Pro Gly Thr Phe Trp Arg Ser
440 445 450
G1n Val Phe Ile Asp His Pro Val His Leu Lys Phe Asn Val Ser
455 460 465
Leu Gly Lys Ala Ala Leu Val Gly I1e Tyr Gly Arg Lys Gly Leu
470 475 480
Pro Pro Ser His Thr Gln Phe Asp Phe Val Glu Leu Leu Asp Gly
485 490 495
Arg Arg Leu Leu Thr Gln Glu Ala Arg Ser Leu Glu Gly Thr Pro
500 505 510
Arg Gln Ser Arg Gly Thr Val Pro Pro Ser Ser His Glu Thr Gly
515 520 525
Phe I1e Gln Tyr Leu Asp Ser Gly Ile Trp His Leu Ala Phe Tyr
530 535 540
Asn Asp Gly Lys Glu Ser Glu Val Val Ser Phe Leu Thr Thr Ala
545 550 555
Ile Glu Ser Val Asp Asn Cys Pro Ser Asn Cys Tyr Gly Asn Gly
560 565 570
Asp Cys Ile Ser Gly Thr Cys His Cys Phe Leu Gly Phe Leu Gly
575 580 585
Pro Asp Cys Gly Arg Ala Ser Cys Pro Val Leu Cys Ser Gly Asn
590 595 600
Gly Gln Tyr Met Lys Gly Arg Cys Leu Cys His Ser Gly Trp Lys
605 610 615
Gly,Ala Glu Cys Asp Val Pro Thr Asn Gln Cys Ile Asp Val Ala
620 625 630
Cys Ser Asn His Gly Thr Cys Ile Met Gly Thr Cys Ile Cys Asn
635 640 645
Pro Gly Tyr Lys Gly Glu Ser Cys Glu Glu Val Asp Cys Met Asp
650 655 660
Pro Thr Cys Ser Gly Arg Gly Val Cys Val Arg Gly Glu Cys His
665 670 675
Cys Ser Val Gly Trp Gly Gly Thr Asn Cys Glu Thr Pro Arg Ala
680 685 690
Thr Cys Leu Asp Gln Cys Ser Gly His Gly Thr Phe Leu Pro Asp
695 700 705
Thr Gly Leu Cys Ser Cys Asp Pro Ser Trp Thr Gly His Asp Cys
710 715 720
Ser Ile Glu Ile Cys Ala Ala Asp Cys Gly Gly His Gly Val Cys
725 730 735
Val Gly Gly Thr Cys Arg Cys Glu Asp Gly Trp Met Gly Ala Ala
16!41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
740 745 750
Cys Asp Gln Arg Ala Cys His Pro Arg Cys Ala Glu His Gly Thr
755 760 765
Cys Arg Asp Gly Lys Cys Glu Cys Ser Pro Gly Trp Asn Gly Glu
770 775 780
His Cys Thr Ile Ala His Tyr Leu Asp Arg Val Val Lys Glu Gly
785 790 795
Cys Pro Gly Leu Cys Asn Gly Asn Gly Arg Cys Thr Leu Asp Leu
800 805 810
Asn Gly Trp His Cys Val Cys Gln Leu Gly Trp Arg Gly Ala Gly
815 820 825
Cys Asp Thr Ser Met Glu Thr Ala Cys Gly Asp Ser Lys Asp Asn
830 835 840
Asp Gly Asp Gly Leu Val Asp Cys Met Asp Pro Asp Cys Cys Leu
845 850 855
Gln Pro Leu Cys His Ile Asn Pro Leu Cys Leu Gly Ser Pro Asn
860 865 870
Pro Leu Asp Ile Tle Gln Glu Thr Gln Val Pro Val Ser Gln Gln
875 880 885
Asn Leu His Ser Phe Tyr Asp Arg Ile Lys Phe Leu Va1 Gly Arg
890 895 900
Asp Ser Thr His Ile Ile Pro Gly Glu Asn Pro Phe Asp Gly Gly
905 910 915
His Ala Cys Val Ile Arg Gly G1n Val Met Thr Ser Asp Gly Thr
920 925 930
Pro Leu Val G1y Val Asn Ile Ser Phe Val Asn Asn Pro Leu Phe
935 940 945
Gly Tyr Thr Ile Ser Arg Gln Asp Gly Ser Phe Asp Leu Val Thr
950 955 960
Asn Gly Gly Ile Ser Ile Ile Leu Arg Phe Glu Arg Ala Pro Phe
965 970 975
Ile Thr Gln Glu His Thr Leu Trp Leu Pro Trp Asp Arg Phe Phe
980 985 990
Val Met Glu Thr Tle Ile Met Arg His Glu Glu Asn Glu Ile Pro
995 1000 1005
Ser Cys Asp Leu Ser Asn Phe Ala Arg Pro Asn Pro Val Val Ser
1010 1015 1020
Pro Ser Pro Leu Thr Ser Phe Ala Ser Ser Cys Ala Glu Lys Gly
1025 1030 1035
Pro Ile Val Pro Glu Ile Gln Ala Leu Gln Glu Glu 21e Ser Ile
1040 1045 1050
Ser Gly Cys Lys Met Arg Leu~Ser Tyr Leu Ser Ser Arg Thr Pro
1055 1060 1065
Gly Tyr Lys Ser Val Leu Arg Ile Ser Leu Thr His Pro Thr Ile
1070 1075 1080
Pro Phe Asn Leu Met Lys Val His Leu Met Val Ala Val Glu Gly
1085 1090 1095
Arg Leu Phe Arg Lys Trp Phe Ala Ala Ala Pro Asp Leu Ser Tyr
1100 1105 1110
Tyr Phe Ile Trp Asp Lys Thr Asp Val Tyr Asn Gln Lys Val Phe
1115 1120 1125
Gly Leu Ser Glu Ala Phe Val Ser Val Gly Tyr Glu Tyr Glu Ser
1130 1135 1140
Cys Pro Asp Leu Ile Leu Trp Glu Lys Arg Thr Thr Val Leu Gln
1145 1150 1155
Gly Tyr Glu Ile Asp Ala Ser Lys Leu Gly Gly Trp Ser Leu Asp
17/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
2260 1165 1170
Lys His His Ala Leu Asn Ile Gln Ser Gly Ile Leu His Lys Gly
1175 1180 1185
Asn Gly Glu Asn Gln Phe Val Ser Gln Gln Pro Pro Val Ile Gly
1190 1195 2200
Ser Ile Met Gly Asn Gly Arg Arg Arg Ser Ile Ser Cys Pro Ser
1205 1210 1215
Cys Asn Gly Leu Ala Asp Gly Asn Lys Leu Leu Ala Pro Val Ala
1220 1225 1230
Leu Thr Cys Gly Ser Asp Gly Ser Leu Tyr Val Gly Asp Phe Asn
1235 1240 1245
Tyr Ile Arg Arg Ile Phe Pro Ser Gly Asn Val Thr Asn Ile Leu
1250 1255 1260
Glu Leu Ser His Ser Pro Ala His Lys Tyr Tyr Leu Ala Thr Asp
1265 1270 1275
Pro Met Ser Gly Ala Val Phe Leu Ser Asp Ser Asn Ser Arg Arg
1280 1285 1290
Val Phe Lys Ile Lys Ser Thr Val Val Val Lys Asp Leu Val Lys
1295 1300 1305
Asn Ser Glu Val Val Ala Gly Thr Gly Asp Gln Cys Leu Pro Phe
1310 1315 1320
Asp Asp Thr Arg Cys Gly Asp Gly Gly Lys Ala Thr Glu Ala Thr
1325 2330 1335
Leu Thr Asn Pro Arg Gly Ile Thr Val Asp Lys Phe Gly Leu Ile
1340 1345 1350
Tyr Phe Val Asp Gly Thr Met Ile Arg Arg Ile Asp Gln Asn Gly
2355 1360 1365
Ile Ile Ser Thr Leu Leu Gly Ser Asn Asp Leu Thr Ser Ala Arg
1370 1375 1380
Pro Leu Ser Cys Asp Ser Val Met Asp Ile Ser Gln Val His Leu
1385 1390 1395
Glu Trp Pro Thr Asp Leu Ala Ile Asn Pro Met Asp Asn Ser Leu
1400 1405 1410
Tyr Val Leu Asp Asn Asn Val Val Leu Gln Ile Ser Glu Asn His
1415 1420 1425
Gln Va1 Arg Ile Val Ala Gly Arg Pro Met His Cys Gln Val Pro
1430 1435 1440
Gly Ile Asp His Phe Leu Leu Ser Lys Val Ala Ile His Ala Thr
1445 1450 1455
Leu Glu Ser Ala Thr Ala Leu Ala Val Ser His Asn Gly Val Leu
1460 1465 1470
Tyr Ile Ala Glu Thr Asp Glu Lys Lys Ile Asn Arg I1e Arg Gln
1475 1480 1485
Val Thr Thr Ser Gly Glu Ile Ser Leu Val Ala Gly Ala Pro Ser
1490 1495 1500
Gly Cys Asp Cys Lys Asn Asp Ala Asn Cys Asp Cys Phe Ser Gly
1505 1510 1515
Asp Asp Gly Tyr Ala Lys Asp Ala Lys Leu Asn Thr Pro Ser Ser
1520 ~ 1525 1530
Leu Ala Va1 Cys Val Asp Gly Glu Leu Tyr Val Ala Asp Leu Gly
1535 1540 1545
Asn Ile Arg Ile Arg Phe Ile Arg Lys Asn Lys Pro Phe Leu Asn
1550 1555 1560
Thr Gln Asn Met Tyr Glu Leu Ser Ser Pro Ile Asp Gln Glu Leu
1565 1570 1575
Tyr Leu Phe Asp Thr Thr Gly Lys His Leu Tyr Thr Gln Ser Leu
18/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
1580 1585 1590
Pro Thr Gly Asp Tyr Leu Tyr Asn Phe Thr Tyr Thr Gly Asp Gly
1595 1600 . 1605
Asp Ile Thr Leu Ile Thr Asp Asn Asn Gly Asn Met Val Asn Val
1610 1615 1620
Arg Arg Asp Ser Thr Gly Met Pro Leu Trp Leu Val Val Pro Asp
1625 1630 1635
Gly Gln Val Tyr Trp Val Thr Met Gly Thr Asn Ser Ala Leu Lys
1640 ' 1645 1650
Ser Val Thr Thr Gln Gly His Glu Leu Ala Met Met Thr Tyr His
1655 1660 1665
Gly Asn Ser Gly Leu Leu Ala Thr Lys Ser Asn G1u Asn Gly Trp
1670 1675 1680
Thr Thr Phe Tyr Glu Tyr Asp Ser Phe Gly Arg Leu Thr Asn Val
1685 1690 1695
Thr Phe Pro Thr Gly Gln Val Ser Ser Phe Arg Ser Asp Thr Asp
1700 1705 1710
Ser Ser Val His Val Gln Val Glu Thr Ser Ser Lys Asp Asp Val
1715 1720 1725
Thr Ile Thr Thr Asn Leu Ser Ala Ser Gly Ala Phe Tyr Thr Leu
1730 1735 1740
Leu Gln Asp Gln Val Arg Asn Ser Tyr Tyr Ile Gly Ala Asp Gly
1745 1750 1755
Ser Leu Arg Leu Leu Leu Ala Asn Gly Met Glu Val Ala Leu Gln
1760 1765 1770
Thr Glu Pro His Leu Leu Ala Gly Thr Val Asn Pro Thr Val Gly
1775 1780 1785
Lys Arg Asn Val Thr Leu Pro Ile Asp Asn Gly Leu Asn Leu Val
1790 1795 1800
Glu Trp Arg G1n Arg Lys Glu Gln Ala Arg Gly Gln Val Thr Val
1805 1810 1815
Phe Gly Arg Arg Leu Arg Val His Asn Arg Asn Leu Leu Ser Leu
1820 1825 1830
Asp Phe Asp Arg Val Thr Arg Thr Glu Lys Ile Tyr Asp Asp His
1835 1840 1845
Arg Lys Phe Thr Leu Arg Ile Leu Tyr Asp Gln Ala Gly Arg Pro
1850 1855 1860
Ser Leu Trp Ser Pro Ser Ser Arg Leu Asn G1y Val Asn Val Thr
1865 1870 1875
Tyr Ser Pro Gly Gly Tyr Ile Ala Gly Ile Gln Arg G1y Ile Met
1880 1885 1890
Ser Glu Arg Met Glu Tyr Asp Gln Ala Gly Arg Ile Thr Ser Arg
1895 1900 1905
Ile Phe Ala Asp G1y Lys Thr Trp Ser Tyr Thr Tyr Leu Glu Lys
1910 1915 1920
Ser Met Val Leu Leu Leu His Ser Gln Arg Gln Tyr 21e Phe Glu
1925 1930 1935
Phe Asp Lys Asn Asp Arg Leu Ser Ser Val Thr Met Pro Asn Val
1940 1945 1950
Ala Arg Gln Thr Leu Glu Thr Ile Arg Ser Val Gly Tyr Tyr Arg
1955 1960 1965
Asn Ile Tyr Gln Pro Pro Glu Gly Asn Ala Ser Va1 Ile Gln Asp
1970 1975 1980
Phe Thr Glu Asp Gly His Leu Leu His Thr Phe Tyr Leu Gly Thr
1985 1990 1995
Gly Arg Arg Val Ile Tyr Lys Tyr Gly Lys Leu Ser Lys Leu Ala
19/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
2000 2005 2010
Glu Thr Leu Tyr Asp Thr Thr Lys Val Ser Phe Thr Tyr Asp G1u
2015 2020 2025
Thr Ala Gly Met Leu Lys Thr Ile Asn Leu Gln Asn Glu Gly Phe
2030 2035 2040
Thr Cys Thr Ile Arg Tyr Arg Gln Ile Gly Pro Leu Ile Asp Arg
2045 2050 2055
Gln Ile Phe Arg Phe Thr Glu Glu Gly Met Val Asn Ala Arg Phe
2060 2065 2070
Asp Tyr Asn Tyr Asp Asn Ser Phe Arg Val Thr Ser Met Gln Ala
2075 2080 2085
Val Ile Asn Glu Thr Pro Leu Pro Ile Asp Leu Tyr Arg Tyr Asp
2090 2095 2100
Asp Val Ser Gly Lys Thr Glu Gln Phe Gly Lys Phe Gly Val Ile
2105 2110 2115
Tyr Tyr Asp Ile Asn Gln Ile Ile Thr Thr Ala Val Met Thr His
2120 2125 2130
Thr Lys His Phe Asp Ala Tyr Gly Arg Met Lys Glu Val Gln Tyr
2135 2140 2145
Glu Ile Phe Arg Ser Leu Met Tyr Trp Met Thr Val Gln Tyr Asp
2150 2155' 2160
Asn Met Gly Arg Val Val Lys Lys Glu Leu Lys Val Gly Pro Tyr
2165 2170 2175
Ala Asn Thr Thr Arg Tyr Ser Tyr Glu Tyr Asp Ala Asp Gly Gln
2180 2185 2190
Leu Gl.n Thr Val Ser Ile Asn Asp Lys Pro Leu Trp Arg Tyr Ser
2195 2200 2205
Tyr Asp Leu Asn Gly Asn Leu His Leu Leu Ser Pro Gly Asn Ser
2210 2215 2220
Ala Arg Leu Thr Pro Leu Arg Tyr Asp Ile Arg Asp Arg Ile Thr
2225 2230 2235
Arg Leu Gly Asp Val Gln Tyr Lys Met Asp Glu Asp Gly Phe Leu
2240 2245 2250
Arg Gln Arg Gly Gly Asp Ile Phe Glu Tyr Asn Ser Ala Gly Leu
2255 2260 2265
Leu Ile Lys Ala Tyr Asn Arg Ala Gly Ser Trp Ser Val Arg Tyr
2270 2275 2280
Arg Tyr Asp Gly Leu Gly Arg Arg Val Ser Ser Lys Ser Ser His
2285 2290 2295
Ser His His Leu Gln Phe Phe Tyr Ala Asp Leu Thr Asn Pro Thr
2300 2305 2310
Lys Val Thr His Leu Tyr Asn His Ser Ser Ser Glu Ile Thr Ser
2315 2320 2325
Leu Tyr Tyr Asp Leu Gln G1y His Leu Phe Ala Met Glu Leu Ser
2330 2335 2340
Ser Gly Asp Glu Phe Tyr Ile Ala Cys Asp Asn Ile Gly Thr Pro
2345 2350 2355
Leu A1a Val Phe Ser Gly Thr Gly Leu Met Ile Lys Gln Ile Leu
2360 2365 2370
Tyr Thr Ala Tyr Gly Glu Ile Tyr Met Asp Thr Asn Pro Asn Phe
2375 2380 2385
Gln Ile Ile Ile Gly Tyr His Gly Gly Leu Tyr Asp Pro Leu Thr
2390 2395 2400
Lys Leu Val His Met Gly Arg Arg Asp Tyr Asp Val Leu Ala Gly
2405 2410 2415
Arg Trp Thr Ser Pro Asp His Glu Leu Trp Lys His Leu Ser Ser
20/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
2420 2425 2430
Ser Asn Val Met Pro Phe Asn Leu Tyr Met Phe Lys Asn Asn Asn
2435 2440 2445
Pro Ile Ser Asn Ser Gln Asp Ile Lys Cys Phe Met Thr Asp Val
2450 2455 2460
Asn Ser Trp Leu Leu Thr Phe Gly Phe Gln Leu His Asn Val Ile
2465 2470 2475
Pro Gly Tyr Pro Lys Pro Asp Met Asp Ala Met Glu Pro Ser Tyr
2480 2485 2490
Glu Leu Ile His Thr Gln Met Lys Thr Gln Glu Trp Asp Asn Ser
2495 2500 2505
Lys Ser Ile Leu Gly Val Gln Cys Glu Val Gln Lys Gln Leu Lys
2510 2525 2520
Ala Phe Val Thr Leu Glu Arg Phe Asp Gln Leu Tyr Gly Ser Thr
2525 2530 2535
Ile Thr Ser Cys Gln Gln Ala Pro Lys Thr Lys Lys Phe Ala Ser
2540 2545 2550
Ser G1y Ser Val Phe Gly Lys Gly Val Lys Phe Ala Leu Lys Asp
2555 2560 2565
Gly Arg Val Thr Thr Asp Ile Ile Ser Val Ala Asn Glu Asp Gly
2570 2575 2580
Arg Arg Val Ala Ala Ile Leu Asn His Ala His Tyr Leu G1u Asn
2585 2590 2595
Leu His Phe Thr Ile Asp Gly Val Asp Thr His Tyr Phe Val Lys
2600 2605 2610
Pro Gly Pro Ser Glu Gly Asp Leu Ala Ile Leu Gly Leu Ser Gly
2615 2620 2625
Gly Arg Arg Thr Leu G1u Asn Gly Val Asn Val Thr~Val Ser Gln
2630 2635 2640
Ile Asn Thr Va1 Leu Asn Gly Arg Thr Arg Arg Tyr Thr Asp Ile
2645 2650 2655
Gln Leu Gln Tyr Gly Ala Leu Cys Leu Asn Thr Arg Tyr Gly Thr
2660 2665 2670
Thr Leu Asp Glu Glu Lys Ala Arg Val Leu Glu Leu Ala Arg Gln
2675 2680 2685
Arg Ala Va1 Arg Gln Ala Trp Ala Arg Glu Gln Gln Arg Leu Arg
2690 2695 2700
Glu Gly Glu Glu Gly Leu Arg Ala Trp Thr Glu Gly Glu Lys Gln
2705 2710 2715
Gln Val Leu Ser Thr Gly Arg Val Gln Gly Tyr Asp Gly Phe Phe
2720 2725 2730
Val Ile Ser Val Glu Gln Tyr Pro Glu Leu Ser Asp Ser Ala Asn
2735 2740 2745
Asn Ile His Phe Met Arg G1n Ser Glu Met Gly Arg Arg
2750 2755
<210> 11
<211> 1139
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7506027CD1
<400> 11
21/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
Met G1u Pro Asp Ser Leu Leu Asp G1n Asp Asp Ser Tyr Glu Ser
1 5 10 15
Pro Gln Glu Arg Pro Gly Ser Arg Arg Ser Leu Pro Gly Ser Leu
20 25 30
Ser Glu Lys Ser Pro Ser Met Glu Pro Ser Ala Ala Thr Pro Phe
35 40 45
Arg Val Thr Gly Phe Leu Ser Arg Arg Leu Lys Gly Ser I1e Lys
50 55 60
Arg Thr Lys Ser Gln Pro Lys Leu Asp Arg Asn His Ser Phe Arg
65 70 75
His Ile Leu Pro Gly Phe Arg Ser A1a Ala Ala Ala Ala Ala Asp
80 85 90
Asn Glu Arg Ser His Leu Met Pro Arg Leu Lys Glu Ser Arg Ser
95 100 105
His Glu Ser Leu Leu Ser Pro Ser Ser Ala Val Glu Ala Leu Asp
110 115 120
Leu Ser Met Glu Glu Glu Val Val Ile Lys Pro Val His Ser Ser
125 130 135
Ile Leu Gly Gln Asp Tyr Cys Phe G1u Val Thr Thr Ser Ser Gly
140 145 150
Ser Lys Cys Phe Ser Cys Arg Ser Ala Ala Glu Arg Asp Lys Trp
155 160 165
Met Glu Asn Leu Arg Arg Ala Val His Pro Asn Lys Asp Asn Ser
170 175 180
Arg Arg Val Glu His Ile Leu Lys Leu Trp Val Ile Glu Ala Lys
185 190 195
Asp Leu Pro Ala Lys Lys Lys Tyr Leu Cys Glu Leu Cys Leu Asp
200 205 210
Asp Val Leu Tyr Ala Arg Thr Thr Gly Lys Leu Lys Thr Asp Asn
215 220 225
Val Phe Trp Gly Glu His Phe Glu Phe His Asn Leu Pro Pro Leu
230 235 240
Arg Thr Val Thr Val His Leu Tyr Arg Glu Thr Asp Lys Lys Lys
245 250 255
Lys Lys Glu Arg Asn Ser Tyr Leu Gly Leu Val Ser Leu Pro Ala
260 265 270
Ala Ser Val Ala Gly Arg Gln Phe Val G1u Lys Trp Tyr Pro,Val
275 280 285
Val Thr Pro Asn Pro Lys Gly Gly Lys G1y Pro Gly Pro Met Ile
290 295 300
Arg Ile Lys Ala Arg Tyr Gln Thr I1e Thr Ile Leu Pro Met Glu
305 310 315
Met Tyr Lys Glu Phe Ala Glu His I1e Thr Asn His Tyr Leu Gly
320 325 330
Leu Cys Ala Ala Leu Glu Pro I1e Leu Ser Ala Lys Thr Lys G1u
335 340 345
Glu Met Ala Ser Ala Leu Val His I1e Leu Gln Ser Thr Gly Lys
350 355 360
Val Lys Asp Phe Leu Thr Asp Leu Met Met Ser Glu Val Asp Arg
365 370 375
Cys Gly Asp Asn Glu His Leu Ile Phe Arg Glu Asn Thr Leu Ala
380 385 390
Thr Lys Ala Ile G1u G1u Tyr Leu Lys Leu Val Gly Gln Lys Tyr
395 400 405
Leu Gln Asp Ala Leu Gly Glu Phe Ile Lys Ala Leu Tyr Glu Ser
410 415 420
22/41
CA 02441495 2003-08-06
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Asp Glu Asn Cys Glu Val Asp Pro Ser Lys Cys Ser Ala Ala Asp
425 430 435
Leu Pro Glu His Gln Gly Asn Leu Lys Met Cys Cys Glu Leu Ala
440 445 450
Phe Cys Lys Ile Ile Asn Ser Tyr Cys Val Phe Pro Arg Glu Leu
455 460 465
Lys Glu Val Phe Ala Ser Trp Arg Gln Glu Cys Ser Ser Arg Gly
470 475 480
Arg Pro Asp I1e Ser Glu Arg Leu Ile Ser Ala Ser Leu Phe Leu
485 490 495
Arg Phe Leu Cys Pro Ala Ile Met Ser Pro Ser Leu Phe Asn Leu
500 505 510
Leu Gln Glu Tyr Pro Asp Asp Arg Thr Ala Arg Thr Leu Thr Leu
515 520 525
Ile Ala Lys Val Thr Gln Asn Leu Ala Asn Phe Ala Lys Phe Gly
530 535 540
Ser Lys Glu Glu Tyr Met Ser Phe Met Asn Gln Phe Leu Glu His
545 550 555
Glu Trp Thr Asn Met Gln Arg Phe Leu Leu Glu Ile Ser Asn Pro
560 565 570
Glu Thr Leu Ser Asn Thr Ala Gly Phe Glu Gly Tyr Ile Asp Leu
575 580 585
Gly Arg Glu Leu Ser Ser Leu,His Ser Leu Leu Trp Glu Ala Val
590 595 600
Ser Gln Leu Glu Gln Ser Ile Val Ser Lys Leu Gly Pro Leu Pro
605 610 615
Arg Ile Leu Arg Asp Val His Thr Ala Leu Ser Thr Pro Gly Ser
620 625 630
Gly Gln Leu Pro Gly Thr Asn Asp Leu Ala Ser Thr Pro Gly Ser
635 640 645
Gly Ser Ser Ser Ile Ser Ala Gly Leu Gln Lys Met Val Tle Glu
650 655 660
Asn Asp Leu Ser Gly Ser Ser Gly Val Gln Pro Ser Pro Ala Arg
665 670 675
Ser Ser Ser Tyr Ser G1u Ala Asn Glu Pro Asp Leu Gln Met Ala
680 685 690
Asn Gly Gly Lys Ser Leu Ser Met Val Asp Leu Gln Asp Ala Arg
695 700 705
Thr.Leu Asp Gly Glu Ala Gly Ser Pro Ala G1y Pro Asp Val Leu
710 715 720
Pro Thr Asp Gly Gln Ala Ala A1a Ala Gln Leu Val Ala Gly Trp
725 730 735
Pro A1a Arg Ala Thr Pro Val Asn Leu Ala Gly Leu Ala Thr Val
740 745 750
Arg Arg Ala Gly Gln Thr Pro Thr Thr Pro G1y Thr Ser Glu Gly
755 760 765
Ala Pro Gly Arg Pro Gln Leu Leu Ala Pro Leu Ser Phe Gln Asn
770 775 780
Pro Val Tyr Gln Met Ala Ala Gly Leu Pro Leu Ser Pro Arg Gly
785 790 795
Leu Gly Asp Ser Gly Ser Glu Gly His Ser Ser Leu Ser Ser His
800 805 810
Ser Asn Ser Glu Glu Leu Ala Ala Ala Ala Lys Leu Gly Ser Phe
815 820 825
Ser Thr Ala Ala Glu Glu Leu Ala Arg Arg Pro Gly Glu Leu Ala
830 835 840
23/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
Arg Arg Gln Met Ser Leu Thr Glu Lys Gly Gly Gln Pro Thr Val
845 850 855
Pro Arg Gln Asn Ser Ala G1y Pro Gln Arg Arg Ile Asp Gln Pro
860 865 870
Pro Pro Pro Pro Pro Pro Pro Pro Pro Ala Pro Arg Gly Arg Thr
875 880 885
Pro Pro Asn Leu Leu Ser Thr Leu Gln Tyr Pro Arg Pro Ser Ser
890 895 900
Gly Thr Leu Ala Ser Ala Ser Pro Asp Trp Val Gly Pro Ser Thr
905 910 915
Arg Leu Arg Gln Gln Ser Sex Ser Ser Lys Gly Asp Ser Pro Glu
920 925 930
Leu Lys Pro Arg Ala Va1 His Lys Gln Gly Pro Ser Pro Val Ser
935 940 945
Pro Asn Ala Leu Asp Arg Thr Ala Ala Trp Leu Leu Thr Met Asn
950 955 960
Ala Gln Leu Leu Glu Asp Glu Gly Leu Gly Pro Asp Pro Pro His
965 970 975
Arg Asp Arg Leu Arg Ser Lys Asp Glu Leu Ser Gln Ala Glu Lys
980 985 990
Asp Leu Ala Val Leu Gln Asp Lys Leu Arg Ile Ser Thr Lys Lys
995 1000 1005
Leu Glu Glu Tyr Glu Thr Leu Phe Lys Cys Gln Glu Glu Thr Thr
1010 1015 1020
Gln Lys Leu Val Leu Glu Tyr Gln Ala Arg Leu Glu Glu Gly Glu
1025 1030 1035
Glu Arg Leu Arg Arg Gln Gln Glu Asp Lys Asp Ile Gln Met Lys
1040 1045 1050
Gly Ile Ile Ser Arg Leu Met Ser Val Glu Glu Glu Leu Lys Lys
1055 1060 1065
Asp His Ala Glu Met Gln Ala Ala Val Asp Ser Lys Gln Lys Ile
1070 1075 1080
Ile Asp Ala G1n Glu Lys Arg Ile Ala Ser Leu Asp Ala Ala Asn
1085 1090 1095
Ala Arg Leu Met Ser Ala Leu Thr Gln Leu Lys Glu Arg Tyr Ser
1100 1105 1110
Met Gln Ala Arg Asn Gly Ile Ser Pro Thr Asn Pro Thr Lys Leu
1115 1120 1125
Gln Ile Thr G1u Asn Gly Glu Phe Arg Asn Ser Ser Asn Cys
1130 1135
<210> 12
<211> 503
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503618CD1
<400> 12
Met Met Lys Thr Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp
1 5 10 15
Glu Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu
20 25 30
Leu G1n Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu
24141
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
35 40 45
Ile Gln Asn Ala Val Asn Gly Va1 Lys Gln Ile Lys Thr Leu Ile
50 .55 60
Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu
65 70 75
Glu Ala Lys Lys L°ys Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu
80 85 90
Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr
95 100 105
Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr
110 115 120
Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu
125 130 135
Val Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe
140 145 150
Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn
155 160 165
Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln Asp His Phe
170 175 180
Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Ser Pro Tyr Glu
185 190 195
Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu Glu Met Ile
200 205 210
His Glu Ala G1n Gln Ala Met Asp Ile His Phe His Ser Pro Ala
215 220 225
Phe Gln His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp
230 235 240
Arg Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu
245 250 255
Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val
260 265 . 270
Asp Cys Ser Thr Asn Asn Pro Ser G1n A1a Lys Leu Arg Arg Glu
275 280 285
Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr
290 295 300
Asn Glu Leu Leu Lys Ser Tyr G1n Trp Lys Met Leu Asn Thr Ser
305 310 315
Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg
320 325 330
Leu Ala Asn Leu Thr Gln G1y Glu Asp Gln Tyr Tyr Leu Arg Val
335 340 345
Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly
350 355 360
Val Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr
365 370 375
Val Thr Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met
380 385 390
Glu Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys His
395 400 405
Arg Asp Ser Leu Leu Lys Leu Leu Ser Arg Arg Ala Thr Trp A1a
410 415 420
Glu Leu Arg Gly Pro Gly Ala Leu Leu Glu Leu Leu Ala Val Arg
425 430 435
Arg Lys Val Ala Gly Phe Cys Asp Glu Lys Arg Glu Glu G1u Lys
440 445 450
Gly Lys Glu Gln Arg Gly Cys Val Cys Asp Ala Gln Glu Lys Ala
25/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
455 460 465
Glu Val Ala Val Lys Leu Leu Arg Asp Glu Gly Gly Arg Ala Leu
470 475 480
Cys Asn Cys Gln Ser Thr Asp Met Gln Gln Gly Pro Phe Leu Tle
485 490 495
Val Thr Val Ser Gln Arg Arg Gln
500
<210> 13
<211> 3971
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1567742CB1
<400> 13
gtgggctggg ggctgcggcg gctccggcgc tgtctccccg cacccgaccg ggcgagccgg 60
ctgggccggc ggggtgaggg aaagcagtgg agtcgggagc agaagcgcta gaggcagtgg 120
tcgtggcgcg gcggcggcgg ctcccctgga ggccggggat gtgggagagg cggtggcagc 180
agcggggagg cggctgctgc tggacccggg ggaaactgct ggctgacagg acacccggga 240
gagacgtgag ggagcctgcg tgccacctct cacccctgag tgaagctggg ctcgagaggt 300
cggccctgtg ctccccgggc cgactggcca gcgggcgcgg ggcgggggcg ggaacccggg 360
ctcgggcccg gccgggcgcc gggcggcggc ggccgtggag cagcagcctc ggtgcgacgt 420
ggagggctgg aggcggcggc gatgcactag gcctcgctca gggcggctgc cccgggaccc 480
gcagttgagt ggtgatttta tgcaatggct tcaagccaca gttcttcacc agtgcctcaa 540
ggaagcagca gtgatg'tttt ctttaaaata gaggtagatc cgtcaaaaca cattcgacct 600
gtgccatcac tgccagatgt gtgtcccaag gaacccacag gtgattcaca tagtttatat 660
gttgccccat ctctagttac agatcaacat agatggactg tatatcattc caaagtaaat 720
ctcccagcag cattaaacga tcctagatta gcaaaaagag aatctgactt cttcacaaaa 780
acatggggat tggactttgt ggacactgaa gtcatacctt cattctacct cccacagatc 840
agcaaggaac attttacagt atatcaacag gaaatctctc agagagagaa gattcatgag 900
agatgcaaga atatttgtcc tcctaaagat accttcgaaa ggactctttt acatactcat 960
gataaatcca ggacagatct ggagcaagta cctaagattt ttatgaaacc agattttgcc 1020
ttggatgatt ccttaacttt taattcagtt ttaccatggt ctcattttaa tactgctggt 1080
ggaaaaggaa atcgtgatgc agcttcctca aagttgcttc aagaaaagct gagccattat 1140
ctggatattg tggaagtaaa cattgctcac cagatctctc tacgttcaga agcatttttt 1200
catgcaatga cctctcaaca cgagttgcag gactacctca ggaaaacttc ccaggctgta 1260
aaaatgcttc gagataaaat tgcacagatt gataaagtaa tgtgtgaagg atcactccac 1320
attttaagac tggcacttac cagaaataat tgtgttaaag tatacaataa gctgaagtta 1380
atggccactg tacaccagac tcagcctaca gtacaggtgt tattatctac ttctgaattt 1440
gttggagcat tggacttaat agcaacaaca caagaggttc tacagcagga acttcagggc 1500
attcacagtt tccggcattt gggatcacag ctttgtgaat tagaaaaact gatagataaa 1560
atgatgattg cagaattttc tacttattct cacagtgact taaatagacc actggaagat 1620
gactgtcaag ttttagaaga ggaaagacta atatctcttg tatttggact tttaaaacaa 1680
agaaagctta attttttaga aatctatggt gaaaaaatgg ttattacagc aaagaatatc 1740
attaaacagt gtgtgattaa taaagtttca caaacagaag aaatagacac agatgttgtt 1800
gtgaagcttg cagatcagat gagaatgttg aattttcccc agtggtttga tctgctcaag 1860
gatattttct ctaagtttac aattttccta cagagagtga aggcaacatt aaatatcatt 1920
cacagtgttg ttctctcagt tcttgacaaa aaccaaagga ctagagaatt ggaagagatt 1980
tcacaacaga agaatgctgc aaaagataat tcactggaca cagaggtggc ttatttaatc 2040
catgaaggca tgtttataag tgatgcattc ggtgagggtg agctaacacc tatagcagtt 2100
gacactacct ctcaaagaaa tgcatctcca aatagtgagc cctgcagcag tgattctgta 2160
tccgagccag aatgtactac tgattcttca tccagcaaag agcacacatc atcatctgct 2220
attccaggag gtgtggatat tatggtcagt gaagatatga aattaactga ctcagagcta 2280
26/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
ggaaagctgg caaataatat ccaggaatta ttatatagtg cctcagatat atgccatgat 2340
cgagctgtca aatttctcat gtcaagagca aaggatggtt ttcttgagaa gctaaattcc 2400
atggaattca taacactttc tagattaatg gaaacattca ttttagacac cgaacagatc 2460
tgtggaagaa aaagcacgtc attacttgga gcacttcaga gccaagctat taagtttgta 2520
aataggtttc atgaagagag aaaaaccaag ctcagcctcc tcttagacaa tgagcgctgg 2580
aagcaagcag atgttcctgc agaatttcag gatcttgttg attctctgtc agatgggaag 2640
attgctttac ctgaaaaaaa atcaggagcc acagaagaaa ggaaaccagc tgaagttctt 2700
attgtcgagg gacaacagta tgcagttgtt ggaaccgtat tgctgttaat aagaattatc 2760
cttgaatatt gccagtgtgt ggataacatc ccatctgtta ctactgacat gcttactcgt 2820
ctgtcagatt tattgaagta cttcaattca agaagttgcc agttagttct tggagctggt 2880
gcactgcaag ttgttggact aaaaacgata actacaaaaa atttggctct ttcttcacga 2940
tgtttgcagt taattgtgca ctacattcct gtgatccggg ctcattttga agctcgacta 3000
ccacctaagc aatatagcat gcttaggcat tttgatcata tcactaagga ctaccatgat 3060
cacatagctg aaatatcagc taagcttgta gcgataatgg atagcttatt tgacaagctg 3120
ttatctaagt atgaagtgaa ggctcctgtt ccttctgcct gtttcaggaa tatttgtaag 3180
caaatgacaa aaatgcacga agctatattt gatctccttc cagaagaaca aacacagatg 3240
ttatttttaa gaattaatgc aagttataaa ctccacttga aaaagcagtt atctcactta 3300
aatgtgataa atgatggagg acctcaaaat gggttggtca cagcagatgt agctttttac 3360
actggaaatc ttcaagcctt aaaaggcctt aaagatttgg acctaaatat ggccgaaatt 3420
tgggagcaga agaggtgatg tcatcctgga aaactgggta gttcatctga ccatgggatg 3480
tgtttgttat gaagaaaatc tggatgcctg tgattcgaga attgaacctg aaacccaaag 3540
tgaactgggg tgggggaagg gaaaaaggaa agtatcaagt gttgggaaac tggattcagt 3600
gggatctaca aggaatgtca tttttgtgca tcctacagtg aggagtaact gatcaggtgt 3660
ctataacatt tttcattctc tctggaaaca gactcaggtt tctttggacc aaatccaaaa 3720
gaacacatag ctgtaacaca gctgtagttg.actagaatgc tctgtatact ttatattaaa 3780
aaatgctttg catttcttcc agtgcaatga aattcatatg gtgtcccacc ttatttaatg 3840
atggtacaat ttaaaatctt agtcaacttc tgtagaaagt tttctctatg aaagtaaagc 3900
tgtttgaaaa attattattt ttttacagat ctttctataa aaaataaaca tcttttgatt 3960
gcttggaaaa a
3971
<210> 14
<211> 410
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7485501CB1
<400> 14
cccaggagtt ggggatgtcc tacaaaccta ccacccctgc ccccagcagc acccccggct 60
tcagcacccc tgggccaggc actccggtcc ctacaggaag cgtcccgtcg ccgtcgggct 120
cagggccggg agccactgcc ccttgcagac cgctgtttaa agactttgga ccacctacgg 180
tcggttgtgt gcaggccatg aaaccacctg gtgcccaggg ctcccagagc acctacacgg 240
aactgctgtt ggtcacaggg gagatgggca aagggatccg gcccacctat gctggcagca 300
agagcgccgc ggagcgcctg aagagaggta tcatccatcc ctagtcagag tgcctggtag 360
agacagagcg gaacgcccac acttaacagg aagctcctag gcctctgtgt 410
<210> 15
<211> 2597
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3089944CB1
27/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
<400> 15
gcgccccgtg agcccgagca cccgggagtc ccgagcctcg cgccccggag tgcccgagcc 60
tgcgccgccg cacccggata ccccgcgtcc ccgcgagctg ccgaggccgc ccgccgccgc 120
cccgcggaca gtaccgcctt cctcccctct gtccgcgcca tggccgcccc cgacctgtcc 180
accaacctcc aggaggaggc cacctgcgcc atctgcctcg actacttcac ggatccggtg 240
atgaccgact gcggccacaa cttctgccgc gagtgcatcc ggcgctgctg gggccagccc 300
gagggcccgt acgcgtgccc cgagtgccgc gagctgtccc cgcagaggaa cctgcggccc 360
aaccgcccgc ttgctaagat ggccgagatg gcgcggcgcc tgcacccgcc gtcgccggtc 420
ccgcagggcg tgtgccccgc gcaccgcgag ccactggccg ccttctgtgg cgacgagctg 480
cgcctcctgt gtgcggcctg cgagcgctct ggggagcact gggcgcaccg cgtgcggccg 540
ctgcaggacg cggccgaaga cctcaaggcg aagctggaga agtcactgga gcatctccgg 600
aagcagatgc aggatgcgtt gctgttccaa gcccaggcgg atgagacctg cgtcttgtgg 660
cagaagatgg tggagagcca gcggcagaac gtgctgggtg agttcgagcg tcttcgccgt 720
ttgctggcag aggaggagca gcagctgctg cagaggctgg aggaggagga gctggaggtg 780
ctgccccggc tgcgggaggg cgcagcccac ctaggccagc agagcgccca cctagctgag 840
ctcatcgccg agctcgaggg ccgctgccag ctgcctgctc tggggctgct gcaggacatc 900
aaggacgccc tgcgcagggt ccaggatgtg aagctgcagc ccccagaagt tgtgcctatg 960
gagctgagga ccgtgtgcag ggtcccggga ctggtagaga cactgcggag gtttcgaggg 1020
gacgtgacct tggacccgga caccgccaac cctgagctga tcctgtctga agacaggcgg 1080
agcgtgcagc ggggggacct acggcaggcc ctgccggaca gcccagagcg ctttgacccc 1140
ggcccctgcg tgctgggcca ggagcgcttc acctcaggcc gcCactactg ggaggtggag 1200
gttggggacc gcaccagctg ggccctgggg gtgtgcaggg agaacgtgaa caggaaggag 1260
aagggcgagc tgtccgcggg caacggcttc tggatcctgg tcttcctggg gagctattac 1320
aattcctcgg aacgggcctt ggctccactc cgggacccac ccaggcgcgt ggggatcttt 1380
ctggactacg aggctggaca tctctctttc tacagtgcca ccgatgggtc actgctattc 1440
atctttcccg agatcccctt ctcggggacg ctgcggcccc tcttctcacc cctgtccagc 1500
agcccgaccc cgatgactat ctgccggccg aaaggtgggt ccggggacac cctggctccc 1560
cagtgactcg ggccctcctg gaggagtcct gttgcctctc ctgcccctcc aggccactga 1620
gtgttttggc cacttggagg acctgggagg agggagtgtg tcctttgagc aagaggagga 1680
actcctggtg cctttctgag cctgcgtggg agaaccccaa ttctagcact ccaggaaact 1740
gtgggagagt gtggggcagg ctccgtcctc cctgggagac ccctccagcc accgggtgcc 1800
acttaatgcc aacagccctt accaaagctg ggagccccat tgccccggca gctctggcct 1860
gtggttccag aagctgagaa aactccactg gggcttgcag aatccagggt tcacctaagc 1920
tgCaCagttC CtgCagCttt gCCagCCCCC tgaaagtctt gtgtacccca cctctgaaga 1980
tgctggggga ggcagctggg atgggagcca gCCCCatgCC tgtctgtgac cccacagtgg 2040
gtgagagccc gtcacagtcc tgggtgtggc tgctctggaa gaattaggag gcagccataa 2100
taagagtctt cagagagatg atgggagggg ccagtgagga caggaacaga gagtagatgt 2160
cctataataa aggggcttct gggaggtgcc tgggcacaga tgtctgttca gcaggtgtgt 2220
gggcctagag gagagagcag agcccagaaa tgtcttttgc aggcccacgt tctgacttga 2280
agctttcgtg ggcatgttgc cattgggttt tgcccttgca aaggcttcct aggtctccag 2340
tggcccctca ggacccaggg tcccagctgc tgcttgggga tgtgcactgc tggccgccgg 2400
ccttgcagtc tctctaccct ggggaggaac agtggcttct cagagcctgg ggcatacaga 2460
agaaggcagg agttgatttt tgtgttgggt ttggggtttc tttgtcctca aggtactgtt 2520
ctgtttctct ttacccctct gctttattta ttgtaagcat tcccacgtta aataaacttt 2580
ggctgttgtc tacaaaa 2597
<210> 16
<211> 1480
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5284076CB1
<400> 16
28/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
ctggaggtct gctcagacga aggtctccat ggcgttagaa gtcttgatgc tcctcgctgt 60
cttgatttgg accggtgctg agaacctcca tgtgaaaata agttgctctc tggactggtt 120
gatggtctca gttatcccag ttgcagaaag cagaaatctg tatatatttg cggatgaatt 180
acatctggga atgggctgcc ctgcaaatcg gatacataca tatgtatatg agtttatata 240
tcttgttcgt gattgtggca tcaggacaag ggtagtttct gaggaaactc tcctttttca 300
aaccgagctg tactttaccc caaggaatat agatcatgac cctcaggaaa tccatttgga 360
gtgttccacc tctaggaaat cagtgtggct tacaccagtt tctactgaga atgaaataaa 420
attggatcct agtcctttta ttgctgactt tcagacaaca gcagaagagt taggattatt 480
atcttctagt ccaaacttgc tctgagctaa aggagaaatg gaaacttgaa gctggtgtta 540
tgtattttgc aggaaaacag tttcattttt tcatagcaaa aatatagttg gtgtatatct 600
ctccttaagt ctctggtttc taaaaaccct acttcagtaa aggtcctgat tagttgatta 660
gtgaatgtgt atttctaaat atttgtattc agtaggggta tggctgatta atttaacatt 720
aactattagg taattcatat tatacattta agttctttct gttctgtgta gaagattcag 780
aaatatgtct tcaaagacaa tgacttgatc taattgataa gaacctccaa taaatatgtt 840
ctaatatttt tcaggaagaa taaagaatag agagagacat ataaatgtgc aagaggcaaa 900
actttgagca tagtgtaaaa tttaacatat taactctcac gaaaggcaaa atccttttat 960
gtgcagatac tttaattcat gtagattttc ctattaatca gtaaagttga atcctaacaa 1020
taatgccatg tgacaaccta tttagattat tccagaatta aattcaattt attttctaga 1080
gctcaagtaa ccactacctt aactgaaatt tgatgttagg tttcccttgt tcctccgaat 1140
ggttcttcca cactcaaaat aattgaatgg ttgagttggt taagcaaaga gttatcctgc 1200
cacctaagag cattcattaa atgattattt attaccacct actttatact atcttccttt 1260
ctttaaacat ggagtctaaa tatgtaatat atcaaaaaat acttctgatt tggtagattt 1320
cttatatcaa gggtgagaat tgaactgtgc cattggctat tcaatagctt attgaatgta 1380
tgttttggat gccacatcct cctggaagca aattttgcca agatactgtt tattattatt 1440
tttaattaaa gtgatactat ccattttca aaaaaaaaaa 1480
<210> 17
<211> 6877
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2899903CB1
<400> 17
gtctcagcct cacctcttag cttttccatc tgcacagccg ggccagatcc ccgcagccag 60
catcacgggc agccaggcca accgtcccgg cgtcttccta ttttagacat ctcgctgcct 120
cagtcccttc taatgtttcc agccaggctg cggggggagg aaaaagaggt tactgctact 180
ttaaatgtac tgtatgaagg cgagggctgg aaaggggcct gcttgcagga atacccagtc 240
atctagttgg aaaagccgcc agatggaata caaaaggagg aacccagacg ctcatggaga 300
cagcctcggt tcataaatca ggtggggcca ggggctgggg gcccacacgc catggagccc 360
gactcccttc tggaccaaga cgactcctac gagtcgcctc aagaaaggcc gggctctcgg 420
cgcagcctgc ctggcagcct ttccgagaag agccccagca tggagccctc ggccgccacg 480
ccgttccggg tcacgggctt cctcagccgc cgcctcaagg gctccatcaa gcgcaccaag 540
agccagccca agctggaccg caaccacagc ttccgccaca tcctgccggg gttccggagc 600
gccgccgccg ccgccgcgga caatgagagg tcccatctga tgccgaggct gaaggagtct 660
cgctcccacg agtccctgct cagccccagc agtgcggtgg aggcgctgga cctcagcatg 720
gaggaagagg tggtcatcaa gcccgtgcac agcagcatcc ttggccagga ctactgcttc 780
gaggtgacga cgtcatcagg aagcaagtgc ttttcctgcc ggtctgcagc tgagcgggat 840
aagtggatgg agaacctccg gcgagcggtg catcccaaca aggacaacag ccggcgtgtg 900
gagcacatcc tgaagctgtg ggtgatcgag gccaaggacc tgccagccaa gaagaagtac 960
ctgtgcgagc tgtgcctgga cgatgtgctc tatgcccgca ccacgggcaa gctcaagacg 1020
gacaatgttt tctggggcga gcacttcgag ttccacaact tgccgcctct gcgcacggtc 1080
actgtccacc tgtaccggga gaccgacaag aagaagaaga aggagcgcaa cagttacctg 1140
ggcctggtga gcctacctgc tgcctcggtg gccgggcggc agttcgtgga gaagtggtac 1200
29/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
ccggtggtga cgcccaaccc caagggcggc aagggccctg gacccatgat ccgcatcaag 1260
gcgcgctacc aaaccatcac catcctgccc atggagatgt acaaagagtt cgctgagcac 1320
atcaccaacc actacctggg gctgtgtgca gccctcgagc ccatcctcag tgccaagacc 1380
aaggaggaga tggcatctgc cctggtgcac atcctgcaga gcacgggcaa ggtgaaggac 1440
ttcctgacag acctgatgat gtcagaggtg gaccgctgcg gggacaacga gcacctcatc 1500
ttccgggaga acacactggc caccaaggcc attgaggagt acctcaagct agtgggccag 1560
aagtacctgc aggacgccct aggtgagttc atcaaagcgc tgtatgagtc agatgagaac 1620
tgcgaagtgg atcccagcaa gtgctcggcc gctgacctcc cagagcacca gggcaacctc 1680
aagatgtgct gcgagctggc cttctgcaag atcatcaact cctactgtgt cttcccacgg 1740
gagttgaaag aggtgtttgc ctcgtggagg caggagtgca gcagtcgcgg ccgcccggac 1800
atcagtgagc ggctcatcag cgcctccctc ttcctgcgct tcctctgccc agccatcatg 1860
tcgccctcac tcttcaacct gctgcaggag taccctgatg accgcactgc ccgcaccctc 1920
accctcatcg ccaaggtcac ccagaacctg gccaactttg ccaaatttgg cagcaaggag 1980
gaatacatgt ccttcatgaa ccagttccta gagcatgagt ggaccaacat gcagcgcttc 2040
ctgctggaga tctccaaccc cgagaccctc tccaatacag ccggcttcga gggctacatc 2100
gacctgggcc gcgagctctc cagcctgcac tcactgctct gggaggccgt cagccagctg 2160
gagcagagca tagtatccaa actgggaccc ctgcctcgga tcctgaggga cgtccacaca 2220
gcactgagca ccccaggtag cgggcagctc ccagggacca atgacctggc ctccacaccg 2280
ggctctggca gcagcagcat ctcagctggg ctgcagaaga tggtgattga gaacgatctt 2340
tccggtctga tagatttcac ccggttaccg tctccaaccc ccgaaaacaa ggacttgttt 2400
tttgtcacaa ggtcctccgg ggtccagccc tcacctgccc gcagctcgag ttactcggaa 2460
gccaacgagc ctgatcttca gatggccaac ggtggcaaga gcctctccat ggtggacctc 2520
caggacgccc gcacgctgga tggggaggca ggctccccgg cgggccccga cgtcctcccc 2580
acagatgggc aggccgctgc agctcagctg gtggccgggt ggccggcccg ggcaacccca 2640
gtgaacctgg cagggctggc cacggtgcgg cgggcaggcc agacaccaac cacaccaggc 2700
acctccgagg gcgcgccagg ccggccccag ctgttggcac cgctctcctt ccagaaccct 2760
gtgtaccaga tggcggctgg cctgccgctg tcaccccgtg gccttggcga ctcaggctct 2820
gagggccaca gctccctgag ctcacacagc aacagcgagg agttggcggc tgctgccaag 2880
ctgggaagtt tcagcactgc cgcggaggag ctggctcggc ggcccggtga gctggcacgg 2940
cgacagatgt cactgactga aaaaggcggg cagcccacgg tgccacggca gaacagtgct 3000
ggcccccaga ggaggatcga ccagcctccg cccccacccc cgccgccacc tcctgccccc 3060
cgcggccgga cgccccccaa cctgctgagc accctgcagt acccaagacc ctcaagcgga 3120
accctggcgt cggcctcacc tgattgggtg ggccccagta cccgcctgag gcagcagtcc 3180
tcttcctcca agggggacag cccagaactg aagccacggg cagtgcacaa gcagggccct 3240
tcacctgtga gccccaatgc cctggaccgc acagccgctt ggctcttgac catgaacgcg 3300
cagttgttag aagacgaggg cctgggccca gacccccccc acagggatag gctaaggagt 3360
aaggacgagc tcagccaagc agaaaaggac ctggcggtgc tgcaggacaa gctgcgaatc 3420
tccaccaaga agctggagga gtatgagacc ctgttcaagt gccaggagga gacgacgcag 3480
aagctggtgc tggagtacca ggcacggctg gaggagggcg aggagcggct gcggcggcag 3540
caggaggaca aggacatcca gatgaagggc atcatcagca ggttgatgtc cgtggaggaa 3600
gaactgaaga aggaccacgc agagatgcaa gcggctgtgg actccaaaca gaagatcatt 3660
gatgcccagg agaagcgcat tgcctcgttg gatgccgcca atgcccgcct catgagtgcc 3720
ctgacccagc tgaaagagag gtacagcatg caagcccgta acggcatctc ccccaccaac 3780
cccaccaaat tgcagattac tgagaacggc ~gagttcagaa acagcagcaa ttgttaacct 3840
gcctgaggag ggaggaagct acccaaggag agggggacta tggtggccaa gggcagggtc 3900
tcggcctggg gaggcaccca cggttgcagc cccagcgcgg gtgtcaggag gccgagcctc 3960
ccctccctgc cgctgtccag ggggcggccg cagagggagc caccagagac tgaagcagcg 4020
tgaggcgagg tcgccagccg ctccctgtgg ggtgcgggca gaagagactg cacgctgggg 4080
agtggggaca gcctgatggg gcagggggcc tgccaaaaat atgtctgttg gttcctgaat 4140
gtggtgtgtc cttgtcctcc tggatctggc cgagtgcatg tgtcccccca cacctgtgcc 4200
agggaggggg cttcctggag gggggattca agggctaggg gcctacacct gtggcttccc 4260
ctcgcctcct tggggggccc gggactccct ggcagccagg ccctgtcatg tgggacctgg 4320
cacttggcag atcagttggc aggcaggaag ataggaggac acagagcagg aggtcagtgt 4380
CCCCtgCCtg tCtCCatCCg aagcacctgc cactgcatgc agcctgttgg gaccttcctg 4440
gctgtgagga actgaggatt cctacccacc caccccctct gaacctgtcc ccagagcacc 4500
acctgctacc ttcttccctg ccttagttgt attgccagat agacccagtg agggccatgg 4560
30/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
ctttttcttg tgagctcttg tccctgtggg gaggacccac agcttcccac acctcccaca 4620
caggcccagg ctgatgctct agggctccca gaagccagag atctgggcgg atctggccag 4680
atggctctga gcactgtatc tgccttctcc tggggcccag cacacccagg gcacagtggt 4740
cctgtaggga gtgccacctg gtgctcaccc tgaaagaaaa ggtgatcctt cctctgagtg 4800
atggtttaaa aaaagattct aacgcctgca ggccctgaga aggtggataa ctgtgatttt 4860
ttttcctttc acagtatgca ttagaaacaa aagcccgctt gctcgcttgc tggaacacag 4920
gggcctttta agttgagcgt gcgcactgca tgggaaatag cggccctgga ggatgttaga 4980
cttgctccct ctccaagaca gcagcagcct gcacctgccc cgtgtgtgtg gccggcctcc 5040
tcctcaccct tcccggcccc cggccaagga cccaggcgct gcatacaggg gaggggcgca 5100
ccccacagct ggggccggtt ttcctcagct ctaggctgtt ctgtagctta tctgcccctc 5160
ccccactttc aagacagatg agcaggagct tgggtctctc tcggcccctg tctgttccca 5220
gcccctgcag attctgagca aaggccctgg gtaagaaggg tgggagtggg gcctttgcca 5280
gcagagccag ggcagggcga gctgcaggaa tcacccctct gcccctgcag ctggaatgtg 5340
ccacagaggc cccacctgaa gggtggatgt gctggagggg tggcccagag ccatactgcg 5400
tccaccctga gctcggggac aggtgacagt ggctgctctg ggaaggggct tttagatgta 5460
acctacaatt cagttaggct agagacagat gctggtggag gaagggctgg gccaccaggg 5520
atcacagacc acaggaagat gggaggtgga agcagaggcc ctgcccccac cccttcctgt 5580
ctcactcttc tgtcttgtcc ccacccatgc gccttcgtgc ctgagaccag ggtggccaca 5640
caggcagggc ctggctccag tctcatcctc ccattgccca gtgagccctg ctcttctctc 5700
cccagccccc tcccaccgct gcctcgtaga gtgacctcgg acagagcccc cctagcaata 5760
cagggaggct cccggggcct ggacaggcgg gctcggaggc tacccgctgt ggccggtgcc 5820
agctgccctt gcagggtggg tgagctctca ggccgagagc cttatttacc tagtgcaaaa 5880
actgtaaaag tgtacagact cttcacagat ttttatctta attgcaagtc tgccgatttt 5940
gtaaatgttc ttggtgtttg actgtaatgt aactatctca cctaatggtt gtacatatcc 6000
tttggtcctg gtgctgccga gggctggccg ggactgctgc tctcccaagg gttttatttt 6060
atttctgaat ctagagaaca gtattgggca ggaggaaaag gcttggtgtc tgcggggggt 6120
gtcttccctg cctgtggcat ttgtgtgttg gctttgcagc tgctgtctga gtagtggcca 6180
ctggggtgcc ttcactgggc cagtcaacgg ggggctcctg cccaggccac agagaacctg 6240
agttcccggg agctgggccc tgcctgcagc cagggctggg gttgccagag gccctggagg 6300
gaaggacagt ccctgctggg gaagaacagc cccggggccc cctggtcacc gagactcagc 6360
ctctgctgga gaaagccacg CCCtCCCtgC tagcacagag gcctgactga cttttttgct 6420
taacttccat gttctgggtg atggaaactg ccaaacctcc tgtcagtgag gactctttcc 6480
gactgcccag aaagtggggg tggaggaccg aggctacagc tccacacgcc ccggtccccc 6540
agagcatctg ccccaggtac acctccccct gcgccccgca cgactgcggg agccagactg 6600
tccagggaaa cagcctctct cttttctaca cactcagcca caaagccccc cagctcccac 6660
accgcgtccc agctcccctc ttttgtaagt atgtgaaaag gaaaaaatgc aaacgttgga 6720
gtttgggctg gagctcctcc ctccagctgc gacttttaac tatgtaataa tgtacagagg 6780
aagctgttgg tgttctaaga ctctgtgtgg ctgtgcaatt tctgtacatt tgcaattaga 6840
aatattaaag atttatttag ctattttaaa aaaaaaa 6877
<210> 18
<211> 1290
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte TD No: 7491355CB1
<400> 18
atgagccgcg cgcgtggggc gctgtgccgg gcctgcctcg cgctggccgc ggccctggcc 60
gcgctgctgt tactgccgct gccgctgccc cgcgcgcccg ccccggcccg gacccccgcc 120
CCggCCCCJC gcgcgocccc gtcccggccc gctgccccca gcctgcggcc tgacgacgtc 180
ttcatcgccg tcaagaccac ccggaagaac cacgggccgc gcctgctgct gctgctgcgc 240
acctggatct cccgggcccg ccagcagacg tttatcttca ccgacgggga cgaccctgag 300
ctcgagctcc agggcggcga ccgtgtcatc aacaccaact gctcggcggt gcgcactcgt 360
31/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
caggccctct gctgcaagat gtccgtggag tatgacaagt tcattgagtc cgggcgcaag 420
tggttttgcc acgtggatga tgacaattat gtgaacgcaa ggagcctcct gcacctgctc 480
tccagcttct cacccagcca ggacgtctac ctggggcggc ccagcctgga ccaccccatt 540
gaggccaccg agagggtcca gggtggcaga actgtgacca cggtcaagtt ctggtttgct 600
actggtgggg ccgggttctg cctcagcaga ggccttgccc tcaagatgag cccatgggcc 660
agcctgggca gcttcatgag cacagctgag caggtgcggc tgccggatga ctgcacagtt 720
ggctacatcg tggaggggct cctgggcgcc cgcctgctgc acagccccct cttccactct 780
cacctggaga acctgcagag gctgccgccc gacaccctgc tccagcaggt taccttgagc 840
catgggggtc ctgagaaccc acagaacgtg gtgaacgtgg ctggaggctt cagcctgcat 900
caagacccca cacggtttaa gtctatccat tgtcttctgt acccagacac ggactggtgt 960
cccaggcaga aacagggcgc cccgacctct cggtgacacc aaccaccccg acccagggct 1020
gcctggctct gtcccaggcg cggggaacca gagcccccta tgggctcagt ggggctccct 1080
caggtgccac ggccacacca gtgagatgca ggcacctggc agaccctctg gctagcctgc 1140
agccccccct ctcccagccc ctggtgggtg cggtgatggg tgttttggga gaacgaagac 1200
agccaggctg atggccaggg ccgcagtggc cctccccccg acccagcccc aaggttgatc 1260
tcacgggaac aggcttccac cccagcactc 1290
<210> 19
<211> 2133
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3333288CB1
<400> 19
ctcggcagat gccgcctggt ccagctatcg tgctcggtat tcagttttcc ggagcagcgc 60
tctttctctg gcccgcggag cggtcccgcg gccgagtacc ggattcccga gtttgggagg 120
ctctgctttc ctccttagga cccactttgc cgtcctgggg tggctgcagt tatgtccgcg 180
ctgcgacctc.tcctgcttct gctgctgcct ctgtgtcccg gtcctggtcc cggacccggg 240
agcgaggcaa aggtcacccg gagttgtgca gagacccggc,aggtgctggg ggcccgggga 300
tatagcttaa acctaatccc tcccgccctg atctcaggtg agcacctccg ggtctgtccc 360
caggagtaca cctgctgttc cagtgagaca gagcagaggc tgatcaggga gactgaggcc 420
accttccgag gcctggtgga ggacagcggc tcctttctgg ttcacacact ggctgccagg 480
cacagaaaat ttgatgagtt ttttctggag atgctctcag tagcccagca ctctctgacc 540
cagctcttct CCCaCtCCta CggCCCJCCtg tatgCCCagC aCgCCCtCat attcaatggc 600
ctgttctctc ggctgcgaga cttctatggg gaatctggtg aggggttgga tgacaccctg 660
gcggatttct gggcacagct cctggagaga gtgttcccgc tgctgcaccc acagtacagc 720
ttcccccctg actacctgct ctgcctctca cgcttggcct catctaccga tggctctctg 780
cagccctttg gggactcacc ccgccgcctc cgcctgcaga taacccggac cctggtggct 840
gcccgagcct ttgtgcaggg cctggagact ggaagaaatg tggtcagcga agcgcttaag 900
gtgccggtgt ctgaaggctg cagccaggct ctgatgcgtc tcatcggctg tcccctgtgc 960
cggggggtcc cctcacttat gccctgccag ggcttctgcc tcaacgtggt tcgtggctgt 1020
ctcagcagca ggggactgga gcctgactgg ggcaactatc tggatggtct cctgatcctg 1080
gctgataagc tccagggccc cttttccttt gagctgacgg ccgagtccat tggggtgaag 1140
atctcggagg gtttgatgta cctgcaggaa aacagtgcga aggtgtccgc ccaggtgttt 1200
caggagtgcg gcccccccga cccggtgcct gcccgcaacc gtcgagcccc gccgccccgg 1260
gaagaggcgg gccggctgtg gtcgatggtg accgaggagg agcggcccac gacggccgca 1320
ggcaccaacc tgcaccggct ggtgtgggag ctccgcgagc gtctggcccg gatgcggggc 1380
ttctgggccc ggctgtccct gacggtgtgc ggagactctc gcatggcagc ggacgcctcg 1440
ctggaggcgg cgccctgctg gaccggagcc gggcggggcc ggtacttgcc gccagtggtc 1500
gggggctccc cggccgagca ggtcaacaac cccgagctca aggtggacgc ctcgggcccc 1560
gatgtcccga cacggcggcg tcgactacag ctccgggcgg ccacggccag aatgaaaacg 1620
gccgcactgg gacacgacct ggacgggcag gacgcggatg aggatgccag cggctctgga 1680
gggggacagc agtatgcaga tgactggatg gctggggctg tggctccccc agcccggcct 1740
32!41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
cctcggcctc cataccctcc tagaagggat ggttctgggg gcaaaggagg aggtggcagt 1800
gcccgctaca accagggccg gagcaggagt gggggggcat ctattggttt tcacacccaa 1860
accatcctca ttctctccct ctcagccctg gccctgcttg gacctcgata acgggggact 1920
gagggtgctt gagtaggatg tgagacttca tgggcctggg tcctgttgag ttttttcagt 1980
atcaatttct taaaccaaat tttaaaaaaa acaaggtggg ggggtgctca tctcgtgacc 2040
tctgccaccc acatccttca caaactccat gtttcagtgt ttgagtccat gtttattctg 2100
caaataaatg gtaatgtatt ggacccctaa aaa 2133
<210> 20
<211> 5162
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7488313CB1
<400> 20
tgctcgtctg aggctgctga ggcgacggcc ggtgtcgtgg tcgcggtacc tgttccaaca 60
cggctcgcgg gcccgtgccg gctccggtcc ccggcgcggc tgtccgagcc cctgcggcgg 120
gcggacgatg gtgtggcgga gcacgcggac gcgggcggcg cggcggcggg catgaaggag 180
gatggaaggg caggacgagg tgtcggcgcg ggagcagcac ttccacagcc aagtgcggga 240
gtccacgata tgtttccttc tttttgccat tctctacgtt gtttcctact tcatcatcac 300
aagatacaag agaaaatcag atgaacaaga agatgaagat gccatcgtca acaggatttc 360
gttgtttttg agcacgttca ctctcgcagt gtcagctggg gctgttttgc ttttaccctt 420
ctcaatcatc agcaatgaaa tcctgctttc ttttcctcag aactactata ttcagtggct 480
aaatggctcc ctgattcatg gtttgtggaa tcttgcttcc cttttttcca acctttgttt 540
atttgtattg atgccctttg cctttttctt tctggaatca gaaggctttg ctggcctgaa 600
aaagggaatc cgagcccgca ttttagagac tttggtcatg cttcttcttc ttgcgttact 660
cattcttggg atagtgtggg tagcttcagc actcattgac aacgatgccg caagcatgga 720
atctttatat gatctctggg agttctatct accctattta tattcctgta tatcattgat 780
gggatgtttg ttacttctct tgtgtacacc agttggcctt tctcgtatgt tcacagtgat 840
gggtcagttg ctagtgaagc caacaattct tgaagacctg gatgaacaaa tttatatcat 900
taccttagag gaagaagcac tccagagacg actaaatggg ctgtcttcat cggtggaata 960
caacataatg gagttggaac aagaacttga aaatgtaaag actcttaaga caaaattaga 1020
gaggcgaaaa aaggcttcag catgggaaag aaatttggtg tatcccgctg ttatggttct 1080
ccttcttatt gagacatcca tctcggtcct cttggtggct tgtaatattc tttgcctatt 1140
ggttgatgaa acagcaatgc caaaaggaac aagggggcct ggaataggaa atgcctctct 1200
ttctacgttt ggttttgtgg gagctgcgct tgaaatcatt ttgattttct atcttatggt 1260
gtcctctgtt gtcggcttct atagccttcg attttttgga aactttactc ccaagaaaga 1320
tgacacaact atgacaaaga tcattggaaa ttgtgtgtcc atcttggttt tgagctctgc 1380
tctgcctgtg atgtcgagaa cactgggaat cactagattt gatctacttg gcgactttgg 1440
aaggtttaat tggctgggaa atttctatat tgtattatcc tacaatttgc tttttgctat 1500
tgtgacaaca ttgtgtctgg tccgaaaatt cacctctgca gttcgagaag aacttttcaa 1560
ggccctaggg cttcataaac ttcacttacc aaatacttca agggattcag aaacagccaa 1620
gccttctgta aatgggcatc agaaagcact gtgagacgca cagacggcgt cttctgccac 1680
caagagaccc gagaactcca gattcacgac attcctgtcc catgtagaag catttccatt 1740
caaccgtggc ccctcttcag aacctagacc tatcagtgcc attttttttt cataatctac 1800
gaagaacttg gctatggctg atctttttta aatttaactt tctgatggac cctgtagttt 1860
ccagttaagt gcagattcct tacagacata tagaacagcg cattcttctg tagacatttg 1920
ctcatgttgg taaatacaat cacccatatg aaaaaattgt tttcacctga tatgaaaatg 1980
ttagaaaagg caaactccgg gacttctaaa gatttactta aatcccatta tgtactttat 2040
tcagaatgta gaagctgact tgaaaggcat ccttggtact aagtgaagct tattcagaaa 2100
atgcattttt caaatgcaat ggcaactgct tgtagatatc atttttgcag tgtatgttgg 2160
agctgtaatg gttgcaatta tgtttcttat ttccttaaaa gcaaaaagcg tagtttctga 2220
tttatgttat agaatgatac tgattagact ttgagccaag gggaaaatac taaattcttt 2280
33/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
taaacctgga gccttagaga gccacaggaa tatcttctgt tgtacagtct aataagctgt 2340
ggtaggaagt atcatgtaat cacagtttaa tgacagttta tgtatatata taattcagta 2400
ttccctctga taacatagtt gccagtgttt aatacacttg taacttggat ttttacctta 2460
taggctatat gtatactcag ttttttaaag catttttttc agagatcact taattcccca 2520
tgcttctgca atgcatataa aaactataaa tgccgagtgg tagaaactcc tctttcttca 2580
tagtcctcag gctttggtta catttgcata tgccatttga agcctccagc ttttaccagt 2640
ttaacatcca aagttcacag catcagcatt catggtgtaa gaacagtttt gcagtataac 2700
acgatctgat aatcattcag ttattaaatt gtaaataatt attgggatgg tttcttggct 2760
ttaagtccac tgaataaaaa ctatgaaatt gcactctgtg tcaaccatcc actaggatag 2820
aataccgaaa tctgtgcatg caaaaatagg agatgggccc atttgcacac aattcgtagt 2880
tatgcagtct gctatataaa tatgttcaca tgcactgtgt gtatgaaaat agatggtctg 2940
tgttcagaca aaagtaaaac atttttttca aattgttaca tttaaaggtt ttctgggaga 3000
aatttatgaa acgcaggctg tgtctatttg acatcagaaa tttccacttt aaaccaaaat 3060
aataagaaac tttaatctgt atatttacaa cctttgttga gtacacttcc cccttattta 3120
tacgtctgca tttccttccg agcttcacat ctttctaaaa tgcagcttgg ttttaaaata 3180
aaagaacatt cattttgtga ttctaaacaa gcttcagtaa ataccaccag tatagtactg 3240
gtgaatttct cagcataaaa tcgacatacc taaaaagtta ataaaattca gctcttttcc 3300
aatttcattg ttatgcctat tgaagtatta attgccaggt ttgattttta gtgaagcttg 3360
gagtccatac tttgagcaga ccaagtgaag ggaagaacag aaagaaactc aggagtagag 3420
taatatcact tctcacttac accactttca ggcacatcca aagagttcct agatacttgg 3480
aaaatgtctg aaaattttta agtaaaatac taaacttttc agtgtttagc tcaacttttt 3540
gttcatttgg aagtttctct ccatccgagg acttaagcca gttttggatt tgtaagccct 3600
gagtacaata cacttcctgg aggcatcctc actgctgttg aagcaaagga tatgcatggg 3660
gtggaaggac ggcttcgaac ctgggactca tatgccttga gaacaaatag attgttacag 3720
ccttgggctg ctgcgtaatc acggttcctc gaggctcttc ctgagcacat gcccaagcat 3780
ctgcctctgg agagactgac tccaaatgca ggtgcttcca ttggagctag gtcggaggct 3840
gctttatatg acgaactcca gaaatggatg ccagaatacg gaggccaaac gttctgagtc 3900
ctggtaagga cagtcgctct gggggtcctc attttactgc agttcctgca cgccagtgaa 3960
agagaggaga tagaccctgg aaggcagagc tgcagatgct catcatcagg tcaattctgg 4020
agctacagtt ttgtttctga ctggataggg atgcaccagt gactgtcaca tcaagcagtc 4080
cttttattct ctctccttta gtatcgattt taaagggcat taggcactat ggttccagag 4140
tttcttgggg aaaacttgca gattcttatt aattggttct gcaatactta aataaattat 4200
tttacaatta taagttttca gattataaca tttgtattaa tttttactga ttttccaaga 4260
tacttcttag atttactatt tacgtagctt tatgtacatt ctctgtaaaa atagacctct 4320
aaatatgagg ctttacatga aatttgtaca cacatacaca ctaatgttag ctccttaaat 4380
tgctgcacta aggtgctggt tagtagagat ggacggagcc tctcgcgttt tgctctcaga 4440
tgtgttaaag gcgcacgtgt acctgctctc agcggcagtg cggcctcccc atctgctggg 4500
tgCCCatggC CCtCCCtgCa gcctcagtga tgacctcgtc tgccagggac acaggttttc 4560
atcatttaca ggctcttatg tgctagtttt gttggtagca cgttatttaa tgcataaagg 4620
cagaattctt acaagttttt tttttttaat gtgaacatag atgcagcacc gactttttaa 4680
acttgaaaaa actggtataa tgttaacttt taaaaataac atttggacac actagtaatt 4740
gatttttgtt tacagattgt tttgtttaca aattgttagt ctttgtttct atgagatact 4800
tttagtgtga ctttttaaat gtcttagaaa ttaaaagttg tacaaaaagt gatttcatat 4860
ttggtttata agcatttata tgtggggttt atttgttctt ttgttttttc catcttaaat 4920
atcatcatgg ctaaaactta agggtattta tagtttaatt ccatttcagt tttatagagg 4980
gcagtaatta ttctgatgaa tgttgaatta agaaatggat attttctttc tctgttgtgc 5040
agttattggt agatcaattt cttataaccc acaatgtagc atcaataatt gatagcatgt 5100
attttattta attacttgaa ttatttagac ttgatttctc taattttttc cataaaagga 5160
ct 5162
<210> 21
<211> 1712
<212> DNA
<213> Homo sapiens
<220>
34/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
<221> misc_feature
<223> Incyte ID No: 6013113CB1
<400> 21
tcccggcttc cagaaagctc cccttgcttt ccgcggcatt ctttgggcag gcgtgcaaag 60
actccagaat tggaggcatg atgaagactc tgctgctgtt tgtggggctg ctgctgacct 120
gggagagtgg gcaggtcctg ggggaccaga cggtctcaga caatgagctc caggaaatgt 180
ccaatcaggg aagtaagtac gtcaataagg aaattcaaaa tgctgtcaac ggggtgaaac 240
agataaagac tctcatagaa aaaacaaacg aagagcgcaa gacactgctc agcaacctag 300
aagaagccaa gaagaagaaa gaggatgccc taaatgagac cagggaatca gagacaaagc 360
tgaaggagct cccaggagtg tgcaatgaga ccatgatggc cctctgggaa gagtgtaagc 420
cctgcctgaa acagacctgc atgaagttct acgcacgcgt ctgcagaagt ggctcaggcc 480
tggttggccg ccagcttgag gagttcctga accagagctc gcccttctac ttctggatga 540
atggtgaccg catcgactcc ctgctggaga acgaccggca gcagacgcac atgctggatg 600
tcatgcagga ccacttcagc cgcgcgtcca gcatcataga cgagctcttc caggacaggt 660
tcttcacccg ggagccccag gatacctacc actacctgcc cttcagcctg ccccaccgga 720
ggcctcactt cttctttccc aagtcccgca tcgtccgcag cttgatgccc ttctctccgt 780
acgagcccct gaacttccac gccatgttcc agcccttcct tgagatgata cacgaggctc 840
agcaggccat ggacatccac ttccacagcc cggccttcca gcacccgcca acagaattca 900
tacgagaagg cgacgatgac cggactgtgt gccgggagat ccgccacaac tccacgggct 960
gcctgcggat gaaggaccag tgtgacaagt gccgggagat cttgtctgtg gactgttcca 1020
ccaacaaccc ctcccaggct aagctgcggc gggagctcga cgaatccctc caggtcgctg 1080
agaggttgac caggaaatac aacgagctgc taaagtccta ccagtggaag atgctcaaca 1140
cctcctcctt gctggagcag ctgaacgagc agtttaactg ggtgtcccgg ctggcaaacc 1200
tcacgcaagg cgaagaccag tactatctgc gggtcaccac ggtggcttcc cacacttctg 1260
actcggacgt tccttccggt gtcactgagg tggtcgtgaa gctctttgac tctgatccca 1320
tcactgtgac ggtccctgta gaagtctcca ggaagaaccc taaatttatg gagaccgtgg 1380
cggagaaagc gctgcaggaa taccgcaaaa agcaccggga cagtttgctg aagctgctaa 1440
gccggagagc cacgtgggct gagctcagag gccctggagc tctcttggag cttctggctg 1500
ttcgccggaa ggtggcagga ttttgtgatg aaaagaggga ggaggagaag ggcaaggagc 1560
aacgagggtg tgtatgtgat gcccaagaga aagcagaggt ggcagtgaag ctcctaagag 1620
acgaaggtgg gagggcactg tgcaactgtc agagcaccga catgcagcag ggtcccttcc 1680
tcatcgtgac tgtcagccag agaaggcagt ga 1712
<210> 22
<211> 8645
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7488573CB1
<220>
<221> unsure
<222> 93
<223> a, t, c, g, or other
<400> 22
ggattatttg aaggactatt cttagaccct tttaagaaga tttaaaggaa aaccactcgg 60
ccctgagttc ggcgaggacc ctgtttgtgg atntggagga gcgcgggccg gaggccatgg 120
acgtgaagga gaggaagcct taccgctcgc tgacccggcg ccgcgacgcc gagcgccgct 180
acaccagctc gtccgcggac agcgaggagg gcaaagcccc gcagaaatcg tacagctcca 240
gcgagaccct gaaggcctac gaccaggacg cccgcctagc ctatggcagc cgcgtcaagg 300
acattgtgcc gcaggaggcc gaggaattct gccgcacagg tgccaacttc accctgcggg 360
agctggggct ggaagaagta acgccccctc acgggaccct gtaccggaca gacattggcc 420
35/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
tcccccactg cggctactcc atgggggctg gctctgatgc cgacatggag gctgacacgg 480
tgctgtcccc tgagcacccc gtgcgtctgt ggggccggag cacacggtca gggcgcagct 540
cctgcctgtc cagccgggcc aattccaatc tcacactcac cgacaccgag catgaaaaca 600
ctgagactcc gggcggcctg cagaaccacg cgcggctccg gacgccgccg ccgccgctct 660
cgcacgccca cacccccaac cagcaccacg cggcctccat taactccctg aaccggggca 720
acttcacgcc gaggagcaac cccagcccgg cccccacgga ccactcgctc tccggagagc 780
cccctgccgg cggcgcccag gagcctgccc acgcccagga gaactggctg ctcaacagca 840
acatccccct ggagaccaga aacctaggca agcagccatt cctagggaca ttgcaggaca 900
acctcattga gatggacatt ctcggcgcct cccgccatga tggggcttac agtgacgggc 960
acttcctctt caagcctgga ggcacctccc cgctcttctg caccacatca ccagggtacc 1020
cactgacgtc cagcacagtg tactctcctc cgccccgacc cctgccccgc agcaccttcg 1080
cccggccggc ctttaacctc aagaagccct ccaagtactg taactggaag tgcgcagccc 1140
tgagcgccat cgtcatctca gccactctgg tcatcctgct ggcatacttt gtgggtaagc 1200
acctcttcaa ctggcacctg cagccgatgg aggggcagat gtatgagatc acggaggaca 1260
cagccagcag ttggcctgtg ccaaccgacg tctccctata cccctcaggg ggcactggct 1320
tagagacccc tgacaggaaa ggcaaaggaa ccacagaagg aaagcccagt agtttctttc 1380
cagaggacag tttcatagat tctggagaaa ttgatgtggg aaggcgagct tcccagaaga 1440
ttcctcctgg cactttctgg agatctcaag tgttcataga ccatcctgtg catctgaaat 1500
tcaatgtgtc tctgggaaag gcagccctgg ttggcattta tggcagaaaa ggcctccctc 1560
cttcacatac acagtttgac tttgtggagc tgctggatgg caggaggctc ctaacccagg 1620
aggcgcggag cctagagggg accccgcgcc agtctcgggg aactgtgccc ccctccagcc 1680
atgagacagg cttcatccag tatttggatt caggaatctg gcacttggct ttttacaatg 1740
acggaaagga gtcagaagtg gtttcctttc tcaccactgc cattgagtcg gtggataact 1800
gccccagcaa ctgctatggc aatggtgact gcatctctgg gacctgccac tgcttcctgg 1860
gtttcctggg ccccgactgt ggcagagcct cctgccccgt gctctgtagc ggaaatggcc 1920
aatacatgaa aggcagatgc ttgtgccaca gtggctggaa aggcgctgag tgcgatgtgc 1980
ccaccaacca gtgtatcgat gtggcctgca gcaaccatgg cacctgcatc atgggcacct 2040
gcatctgcaa ccctggctac aagggcgaga gctgtgagga agtggactgc atggacccca 2100
catgttcagg ccggggtgtc tgcgtgagag gcgaatgcca ctgctctgtg ggatggggag 2160
gcaccaactg cgagaccccc agggccacat gcttagacca gtgttcaggc cacggaacct 2220
tcctcccgga caccgggctt tgcagctgtg acccaagctg gactggacac gactgttcta 2280
tcgagatctg tgctgccgac tgtggtggcc atggcgtgtg cgtagggggc acctgccgct 2340
gcgaggatgg ctggatgggg gcagcctgcg accagcgggc ctgccacccg cgctgtgccg 2400
agcatgggac ctgccgcgac ggcaagtgcg agtgcagccc tggctggaat ggcgaacact 2460
gcaccatcgc tcactatctg gatagggtag ttaaagaggg ttgccctggg ttgtgcaatg 2520
gcaacggcag atgtacctta gacctgaatg gttggcactg cgtctgccag ctgggctgga 2580
gaggagctgg ctgtgacact tccatggaga ctgcctgcgg tgacagcaaa gacaatgatg 2640
gagatggcct ggtggactgc atggaccctg actgctgcct ccagcccctg tgccatatca 2700
acccgctgtg CCttggCtCC CCtaaCCCtC tggacatcat ccaggagaca caggtccctg 2760
tgtcacagca gaacctacac tccttctatg accgcatcaa gttcctcgtg ggcagggaca 2820
gcacgcacat aatccccggg gagaacccct ttgatggagg gcatgcttgt gttattcgtg 2880
gccaagtgat gacatcagat ggaacccccc tggttggtgt gaacatcagt tttgtcaata 2940
accctctctt tggatataca atcagcaggc aagatggcag ctttgacttg gtgacaaatg 3000
gcggcatctc catcatcctg cggttcgagc gggcaccttt catcacacag gagcacaccc 3060
tgtggctgcc atgggatcgc ttctttgtca tggaaaccat catcatgaga catgaggaga 3120
atgagattcc cagctgtgac ctgagcaatt ttgcccgccc caacccagtc gtctctccat 3180
ccccactgac gtccttcgcc agctcctgtg cagagaaagg ccccattgtg ccggaaattc 3240
aggctttgca ggaggaaatc tctatctctg gctgcaagat gaggctgagc tacctgagca 3300
gccggacccc tggctacaaa tctgtcctga ggatcagcct cacccacccg accatcccct 3360
tcaacctcat gaaggtgcac ctcatggtag cggtggaggg ccgcctcttc aggaagtggt 3420
tcgctgcagc cccagacctg tcctattatt tcatttggga caagacagac gtctacaacc 3480
agaaggtgtt tgggctttca gaagcctttg tttccgtggg ttatgaatat gaatcctgcc 3540
cagatctaat cctgtgggaa aaaagaacaa cagtgctgca gggctatgaa attgatgcgt 3600
ccaagcttgg aggatggagc ctagacaaac atcatgccct caacattcaa agtggcatcc 3660
tgcacaaagg gaatggggag aaccagtttg tgtctcagca gcctcctgtc attgggagca 3720
tcatgggcaa tgggcgccgg agaagcatct cctgccccag ctgcaacggc cttgctgacg 3780
36/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
gcaacaagct cctggcccca gtggccctca cctgtggctc tgacgggagc ctctatgtgg 3840
gtgatttcaa ctacattaga aggatcttcc cctctggaaa tgtcaccaac atcctagagc 3900
tgagtcacag tccagcacac aaatactacc tggccacaga ccccatgagt ggggccgtct 3960
tcctttctga cagcaacagc cggcgggtct ttaaaatcaa gtccactgtg gtggtgaagg 4020
accttgtcaa gaactctgag gtggttgcgg ggacaggtga ccagtgcctc ccctttgatg 4080
acactcgctg cggggatggt gggaaggcca cagaagccac actcaccaat cccaggggca 4140
ttacagtgga caagtttggg ctgatctact tcgtggatgg caccatgatc agacgcatcg 4200
atcagaatgg gatcatctcc accctgctcg gctctaatga tctcacatca gcccggccac 4260
tcagctgtga ttctgtcatg gatatttccc aggttcacct ggagtggccc acagacttag 4320
ccatcaaccc aatggacaac tcactttatg tcctcgacaa caatgtggtc ctgcaaatct 4380
ctgaaaacca ccaggtgcgc attgtcgccg ggaggcccat gcactgccag gtccctggca 4440
ttgaccactt cctgctaagc aaggtggcca tccacgcaac cctggagtca gccaccgctt 4500
tggctgtttc acacaatggg gtcctgtata ttgctgagac tgatgagaaa aagatcaacc 4560
gcatcaggca ggtcaccact agtggagaga tctcactcgt tgctggggcc cccagtggct 4620
gtgactgtaa aaatgatgcc aactgtgatt gtttttctgg agacgatggt tatgccaagg 4680
atgcaaagtt aaatacccca tcttccttgg ctgtgtgtgt tgatggggag ctctacgtgg 4740
ccgaccttgg gaacatccga attcggttta tccggaagaa caagcctttc ctcaacaccc 4800
agaacatgta tgagctgtct tcaccaattg accaggagct ctatctgttt gataccaccg 4860
gcaagcacct gtacacccaa agcctgccca caggagacta cctgtacaac ttcacctaca 4920
ctggggacgg cgacatcaca ctcatcacag acaacaatgg caacatggta aatgtccgcc 4980
gagactctac tgggatgccc ctctggctgg tggtcccaga tggccaggtg tactgggtga 5040
ccatgggcac caacagtgca ctcaagagtg tgaccacaca aggacacgag ttggccatga-5100
tgacatacca tggcaattcc ggccttctgg caaccaaaag caatgaaaac ggatggacaa 5160
cattttatga gtacgacagc tttggccgcc tgacaaatgt gaccttccct actggccagg 5220
tgagcagttt ccgaagtgat acagacagtt cagtgcatgt ccaggtagag acctccagca 5280
aggatgatgt caccataacc accaacctgt ctgcctcagg cgccttctac acactgctgc 5340
aagaccaagt ccggaacagc tactacatcg gggccgatgg ctccttgcgg ctgctgctgg 5400
ccaacggcat ggaggtggcg ctgcagactg agccccactt gctggctggc accgtcaacc 5460
ccaccgtggg caagaggaat gtcacgctgc ccatcgacaa cggcctcaac ctggtggagt 5520
ggcgccagcg caaagagcag gctcggggcc aggtcactgt ctttgggcgc cggctgcggg 5580
tgcacaaccg aaatctccta tctctggact ttgatcgcgt aacacgcaca gagaagatct 5640
atgatgacca ccgcaagttc acccttcgga ttctgtacga ccaggcgggg cggcccagcc 5700
tctggtcacc cagcagcagg ctgaatggtg tcaacgtgac atactcccct gggggttaca 5760
ttgctggcat ccagaggggc atcatgtctg aaagaatgga atacgaccag gcgggccgca 5820
tcacatccag gatcttcgct gatgggaaga catggagcta cacatactta gagaagtcca 5880
tggtgctgct actacacagc cagaggcagt atatctttga gttcgacaag aatgaccgcc 5940
tctcttctgt gacgatgccc aacgtggcgc ggcagacact agagaccatc cgctcagtgg 6000
gctactacag aaacatctat cagccccctg agggcaatgc ctcagtcata caggacttca 6060
ctgaggatgg gcacctcctt cacaccttct acctgggcac tggccgcagg gtgatataca 6120
agtatggcaa actgtcaaag ctggcagaga cgctctatga caccaccaag gtcagtttca 6180
cctatgacga gacggcaggc atgctgaaga ccatcaacct acagaatgag ggcttcacct 6240
gcaccatccg ctaccgtcag attgggcccc tgattgaccg acagatcttc cgcttcactg 6300
aggaaggcat ggtcaacgcc,cgttttgact acaactatga caacagcttc cgggtgacca 6360
gcatgcaggc tgtgatcaac gagaccccac tgcccattga tctctatcgc tatgatgatg 6420
tgtcaggcaa gacagagcag tttgggaagt ttggtgtcat ttactatgac attaaccaga 6480
tcatcaccac agctgtcatg acccacacca agcattttga tgcatatggc aggatgaagg 6540
aagtgcagta tgagatcttc cgctcgctca tgtactggat gaccgtccag tatgataaca 6600
tggggcgagt agtgaagaag gagctgaagg taggacccta cgccaatacc actcgctact 6660
cctatgagta tgatgctgac ggccagctgc agacagtctc catcaatgac aagccactct 6720
ggcgctacag ctacgacctc aatgggaacc tgcacttact gagccctggg aacagtgcac 6780
ggctcacacc actacggtat gacatccgcg accgcatcac tcggctgggt gacgtgcaat 6840
acaagatgga tgaggatggc ttcctgaggc agcggggcgg tgatatcttt gagtacaact 6900
cagctggcct gctcatcaag gcctacaacc gggctggcag ctggagtgtc aggtaccgct 6960
acgatggcct ggggcggcgc gtgtccagca agagcagcca cagccaccac ctgcagttct 7020
tctatgcaga cctgaccaac cccaccaagg tcacccacct gtacaaccac tccagctctg 7080
agatcacctc cctctactac gacttgcaag gacacctctt tgccatggag ctgagcagtg 7140
37!41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
gtgatgagtt ttacatagct tgtgacaaca tcgggacccc tcttgctgtc tttagtggaa 7200
caggtttgat gatcaagcaa atcctgtaca cagcctatgg ggagatctac atggatacca 7260
accccaactt tcagatcatc ataggctacc atggtggcct ctatgatcca ctcaccaagc 7320
ttgtccacat gggccggcga gattatgatg tgctggccgg acgctggact agcccagacc 7380
acgagctgtg gaagcacctt agtagcagca acgtcatgcc ttttaatctc tatatgttca 7440
aaaacaacaa ccccatcagc aactcccagg acatcaagtg cttcatgaca gatgttaaca 7500
gctggctgct cacctttgga ttccagctac acaacgtgat ccctggttat cccaaaccag 7560
acatggatgc catggaaccc tcctacgagc tcatccacac acagatgaaa acgcaggagt 7620
gggacaacag caagtctatc ctcggggtac agtgtgaagt acagaagcag ctcaaggcct 7680
ttgtcacctt agaacggttt gaccagctct atggctccac aatcaccagc tgccagcagg 7740
ctccaaagac caagaagttt gcatccagcg gctcagtctt tggcaagggg gtcaagtttg 7800
ccttgaagga tggccgagtg accacagaca tcatcagtgt ggccaatgag gatgggcgaa 7860
gggttgctgc catcttgaac catgcccact acctagagaa cctgcacttc accattgatg 7920
gggtggatac ccattacttt gtgaaaccag gaccttcaga aggtgacctg gccatcctgg 7980
gcctcagtgg ggggcggcga accctggaga atggggtcaa cgtcactgtg tcccagatca 8040
acacagtact taatggcagg actagacgct acacagacat ccagctccag tacggggcac 8100
tgtgcttgaa cacacgctac gggacaacgt tggatgagga gaaggcacgg gtcctggagc 8160
tggcccggca gagagccgtg cgccaagcgt gggcccgcga gcagcagaga ctgcgggaag 8220
gggaggaagg cctgcgggcc tggacagagg gggagaagca gcaggtgctg agcacagggc 8280
gggtgcaagg ctacgacggc tttttcgtga tctctgtcga gcagtaccca gaactgtcag 8340
acagcgccaa caacatccac ttcatgagac agagcgagat gggccggagg tgacagagag 8400
gaccaaggac ttcttgccaa agacagctac tcttttgtgg ccgcatacct gactgtgttg 8460
tacttttaaa aaaatgattt tttaacaagt gcagaaacaa aaagatactg gttgcattgt 8520
aactcatgca acatcctttt ttttagaaaa gaaaaacaca gatttggcct tcgcacattt 8580
tttgcaaaga acagaaggta tttttttctg tagtgtgatc acaatgaaaa ctttattgtc 8640
aaaaa 8645
<210> 23
<211> 6812
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7506027CB1
<400> 23
gtctcagcct cacctcttag cttttccatc tgcacagccg ggccagatcc ccgcagccag 60
catcacgggc agccaggcca accgtcccgg cgtcttccta ttttagacat ctcgctgcct 120
cagtcccttc taatgtttcc agccaggctg cggggggagg aaaaagaggt tactgctact 180
ttaaatgtac tgtatgaagg cgagggctgg aaaggggcct gcttgcagga atacccagtc 240
atctagttgg aaaagccgcc agatggaata caaaaggagg aacccagacg ctcatggaga 300
cagcctcggt tcataaatca ggtggggcca ggggctgggg gcccacacgc catggagccc 360
gactcccttc tggaccaaga cgactcctac gagtcgcctc aagaaaggcc gggctctcgg 420
cgcagcctgc ctggcagcct ttccgagaag agccccagca tggagccctc ggccgccacg 480
ccgttccggg tcacgggctt cctcagccgc cgcctcaagg gctccatcaa gcgcaccaag 540
agccagccca agctggaccg caaccacagc ttccgccaca tcctgccggg gttccggagc 600
gccgccgccg ccgccgcgga caatgagagg tcccatctga tgccgaggct gaaggagtct 660
cgctcccacg agtccctgct cagccccagc agtgcggtgg aggcgctgga cctcagcatg 720
gaggaagagg tggtcatcaa gcccgtgcac agcagcatcc ttggccagga ctactgcttc 780
gaggtgacga cgtcatcagg aagcaagtgc ttttcctgcc ggtctgcagc tgagcgggat 840
aagtggatgg agaacctccg gcgagcggtg catcccaaca aggacaacag ccggcgtgtg 900
gagcacatcc tgaagctgtg ggtgatcgag gccaaggacc tgccagccaa gaagaagtac 960
ctgtgcgagc tgtgcctgga cgatgtgctc tatgcccgca ccacgggcaa gctcaagacg 1020
gacaatgttt tctggggcga gcacttcgag ttccacaact tgccgcctct gcgcacggtc 1080
actgtccacc tgtaccggga gaccgacaag aagaagaaga aggagcgcaa cagttacctg 1140
38/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
ggcctggtga gcctacctgc tgcctCggtg gccgggcggc agttcgtgga gaagtggtac 1200
ccggtggtga cgcccaaccc caagggcggc aagggccctg gacccatgat ccgcatcaag 1260
gcgcgctacc aaaccatcac catcctgccc atggagatgt acaaagagtt cgctgagcac 1320
atcaccaacc actacctggg gctgtgtgca gccctcgagc ccatcctcag tgccaagacc 1380
aaggaggaga tggcatctgc cctggtgcac atcctgcaga gcacgggcaa ggtgaaggac 1440
ttcctgacag acctgatgat gtcagaggtg gaccgctgcg gggacaacga gcacctcatc 1500
ttccgggaga acacactggc caccaaggcc attgaggagt acctcaagct agtgggccag 1560
aagtacctgc aggacgccct aggtgagttc atcaaagcgc tgtatgagtc agatgagaac 1620
tgcgaagtgg atcccagcaa gtgctcggcc gctgacctcc cagagcacca gggcaacctc 1680
aagatgtgct gcgagctggc cttctgcaag atcatcaact cctactgtgt cttcccacgg 1740
gagttgaaag aggtgtttgc ctcgtggagg caggagtgca gcagtcgcgg ccgcccggac 1800
atcagtgagc ggctcatcag cgcctccctc ttcctgcgct tcctctgccc agccatcatg 1860
tcgccctcac tcttcaacct gctgcaggag taccctgatg accgcactgc ccgcaccctc 1920
accctcatcg ccaaggtcac ccagaacctg gccaactttg ccaaatttgg cagcaaggag 1980
gaatacatgt ccttcatgaa ccagttccta gagcatgagt ggaccaacat gcagcgcttc 2040
ctgctggaga tctccaaccc cgagaccctc tccaatacag ccggcttcga gggctacatc 2100
gacctgggcc gcgagctctc cagcctgcac tcactgctct gggaggccgt cagccagctg 2160
gagcagagca tagtatccaa actgggaccc ctgcctcgga tcctgaggga cgtccacaca 2220
gcactgagca ccccaggtag cgggcagctc ccagggacca atgacctggc ctccacaccg 2280
ggctctggca gcagcagcat ctcagctggg ctgcagaaga tggtgattga gaacgatctt 2340
tccgggtcct ccggggtcca gccctcacct gcccgcagct cgagttactc ggaagccaac 2400
gagcctgatc ttcagatggc caacggtggc aagagcctct ccatggtgga cctccaggac 2460
gcccgcacgc tggatgggga ggcaggctcc ccggcgggcc ccgacgtcct ccccacagat 2520
gggcaggccg ctgcagctca gctggtggcc gggtggccgg cccgggcaac cccagtgaac 2580
ctggcagggc tggccacggt gcggcgggca ggccagacac caaccacacc aggcacctcc 2640
gagggcgcgc caggccggcc ccagctgttg gcaccgctct ccttccagaa ccctgtgtac 2700
cagatggcgg ctggcctgcc gctgtcaccc cgtggccttg gcgactcagg ctctgagggc 2760
cacagctccc tgagctcaca cagcaacagc gaggagttgg cggctgctgc caagctggga 2820
agtttcagca ctgccgcgga ggagctggct cggcggcccg gtgagctggc acggcgacag 2880
atgtcactga ctgaaaaagg cgggcagccc acggtgccac ggcagaacag tgctggcccc 2940
cagaggagga tcgaccagcc tccgccccca cccccgccgc cacctcctgc cccccgcggc 3000
cggacgcccc ccaacctgct gagcaccctg cagtacccaa gaccctcaag cggaaccctg 3060
gcgtcggcct cacctgattg ggtgggcccc agtacccgcc tgaggcagca gtcctcttcc 3120
tccaaggggg acagcccaga actgaagcca cgggcagtgc acaagcaggg cccttcacct 3180
gtgagcccca atgccctgga ccgcacagcc gcttggctct tgaccatgaa cgcgcagttg 3240
ttagaagacg agggcctggg cccagacccc ccccacaggg ataggctaag gagtaaggac 3300
gagctcagcc aagcagaaaa ggacctggcg gtgctgcagg acaagctgcg aatctccacc 3360
aagaagctgg aggagtatga gaccctgttc aagtgccagg aggagacgac gcagaagctg 3420
gtgctggagt accaggcacg gctggaggag ggcgaggagc ggctgcggcg gcagcaggag 3480
gacaaggaca tccagatgaa gggcatcatc agcaggttga tgtccgtgga ggaagaactg 3540
aagaaggacc acgcagagat gcaagcggct gtggactcca aacagaagat cattgatgcc 3600
caggagaagc gcattgcctc gttggatgcc gccaatgccc gcctcatgag tgccctgacc 3660
cagctgaaag agaggtacag catgcaagcC cgtaacggca tctcccccac caaccccacc 3720
aaattgcaga ttactgagaa cggcgagttc agaaacagca gcaattgtta acctgcctga 3780
ggagggagga agctacccaa ggagaggggg actatggtgg ccaagggcag ggtctcggcc 3840
tggggaggca cccacggttg cagccccagc gcgggtgtca ggaggccgag cctcccctcc 3900
ctgccgctgt ccaggaggcg gccgcagagg gagccaccag agactgaagc agcgtgaggc 3960
gaggtcacca gccgctccct gtggggtgcg ggcagaagag actgcacgct ggggagtggg 4020
gacagcctga tggggcaggg ggcctgccaa aaatatgtct gttggttcct gaatgtggtg 4080
tgtccttgtc ctcctggatc tggccgagtg catgtgtccc cccacacctg tgccagggag 4140
ggggcttcct ggagggggga ttcaagggct aggggcctac acctgtggct tcccctcgcc 4200
tccttggggg gcccgggact ccctggcagc caggccctgt catgtgggac ctggcacttg 4260
gcagatcagt tggcaggcag gaagatagga ggacacagag caggaggtca gtgtcccctg 4320
cctgtctcca tccgaagcac ctgccactgc atgcagcctg ttgggacctt cctggctgtg 4380
aggaactgag gattcctacc cacccacccc ctctgaacct gtccccagag caccacctgc 4440
taccttcttc cctgccttag ttgtattgcc agatagaccc agtgagggcc atggcttttt 4500
39!41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
cttgtgagct cttgtccctg tggggaggac ccacagcttc ccacacctcc cacacaggcc 4560
caggctgatg ctctagggct cccagaagcc agagatctgg gcggatctgg ccagatggct 4620
ctgagcactg tatctgcctt ctcctggggc ccagcacacc cagggcacag tggtcctgta 4680
gggagtgcca cctggtgctc accctgaaag aaaaggtgat ccttcctctg agtgatggtt 4740
taaaaaaaag attctaacgc ctgcaggccc tgagaaggtg gataactgtg attttttttc 4800
ctttcacagt atgcattaga aacaaaagcc cgcttgctcg cttgctggaa cacaggggcc 4860
ttttaagttg agcgtgcgca ctgcatggga aatagcggcc ctggaggatg ttagacttgc 4920
tccctctcca agacagcagc agcctgcacc tgccccgtgt gtgtggccgg cctcctcctc 4980
acccttcccg gcccccggcc aaggacccag gcgctgcata caggggaggg gcgcacccca 5040
cagctggggc cggttttcct cagctctagg ctgttctgta gcttatctgc ccctccccca 5100
ctttcaagac agatgagcag gagcttgggt ctctctcggc ccctgtctgt tcccagcccc 5160
tgcagattct gagcaaaggc cctgggtaag aagggtggga gtggggcctt tgccagcaga 5220
gccagggcag ggcgagctgc aggaatcacc cctctgcccc tgcagctgga atgtgccaca 5280
gaggccccac ctgaagggtg gatgtgctgg aggggtggcc cagagccata ctgcgtccac 5340
cctgagctcg gggacaggtg acagtggctg ctctgggaag gggcttttag atgtaaccta 5400
caattcagtt aggctagaga cagatgctgg tggaggaagg gctgggccac cagggatcac 5460
agaccacagg aagatgggag gtggaagcag aggccctgcc cccacccctt cctgtctcac 5520
tcttctgtct tgtccccacc catgcgcctt cgtgcctgag accagggtgg ccacacaggc 5580
agggcctggc tccagtctca tcctcccatt gcccagtgag ccctgctctt ctctccccag 5640
ccccctccca ccgctgcctc gtagagtgac ctcggacaga gcccccctag caatacaggg 5700
aggctcccgg ggcctggaca ggcgggctcg gaggctaccc gctgtggccg gtgccagctg 5760
cccttgcagg gtgggtgagc tctcaggccg agagccttat ttacctagtg caaaaactgt 5820
aaaagtgtac agactcttca cagattttta tcttaattgc aagtctgccg attttgtaaa 5880
tgttcttggt gtttgactgt aatgtaacta tctcacctaa tggttgtaca tatcctttgg 5940
tcctggtgct gccgagggct ggccgggact gctgctctcc caagggtttt attttatttc 6000
tgaatctaga gaacagtatt gggcaggagg aaaaggcttg gtgtctgcgg ggggtgtctt 6060
ccctgcctgt ggcatttgtg tgttggcttt gcagctgctg tctgagtagt ggccactggg 6120
gtgccttcac tgggccagtc aacggggggc tcctgcccag gccacagaga acctgagttc 6180
ccgggagctg ggccctgcct gcagccaggg ctggggttgc cagaggccct ggagggaagg 6240
acagtccctg ctggggaaga acagccccgg ggccccctgg tcaccgagac tcagcctctg 6300
ctggagaaag ccacgccctc cctgctagca cagaggcctg actgactttt ttgcttaact 6360
tccatgttct gggtgatgga aactgccaaa cctcctgtca gtgaggactc tttccgactg 6420.
cccagaaagt gggggtggag gaccgaggct acagctccac acgccccggt cccccagagc 6480
atctgcccca ggtacacctc cccctgcgcc ccgcacgact gcgggagcca gactgtccag 6540
ggaaacagcc tctctctttt ctacacactc agccacaaag ccccccagct cccacaccgc 6600
gtcccagctc ccctcttttg taagtatgtg aaaaggaaaa aatgcaaacg ttggagtttg 6660
ggctggagct cctccctcca gctgcgactt ttaactatgt aataatgtac agaggaagct 6720
gttggtgttc taagactctg tgtggctgtg caatttctgt acatttgcaa ttagaaatat 6780
taaagattta tttagctatt ttaaaaaaaa as 6812
<210> 24
<211> 1589
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503618CB1
<400> 24
tcccggcttc cagaaagctc cccttgcttt ccgcggcatt ctttgggcag gcgtgcaaag 60
actccagaat tggaggcatg atgaagactc tgctgctgtt tgtggggctg ctgctgacct 120
gggagagtgg gcaggtcctg ggggaccaga cggtctcaga caatgagctc caggaaatgt 180
ccaatcaggg aagtaagtac gtcaataagg aaattcaaaa tgctgtcaac ggggtgaaac 240
agataaagac tctcatagaa aaaacaaacg aagagcgcaa gacactgctc agcaacctag 300
aagaagccaa gaagaagaaa gaggatgccc taaatgagac cagggaatca gagacaaagc 360
40/41
CA 02441495 2003-08-06
WO 02/072830 PCT/US02/03715
tgaaggagct cccaggagtg tgc~atgaga ccatgatggc cctctgggaa gagtgtaagc 420
cctgcctgaa acagacctgc atgaagttct acgcacgcgt ctgcagaagt ggctcaggcc 480
tggttggccg ccagcttgag gagttcctga accagagctc gcccttctac ttctggatga 540
atggtgaccg catcgactcc ctgctggaga acgaccggca gcagacgcac atgctggatg 600
tcatgcagga ccacttcagc cgcgcgtcca gcatcataga cgagctcttc tctccgtacg 660
agcccctgaa cttccacgcc atgttccagc ccttccttga gatgatacac gaggctcagc 720
aggccatgga catccacttc cacagcccgg ccttccagca cccgccaaca gaattcatac 780
gagaaggcga cgatgaccgg actgtgtgcc gggagatccg ccacaactcc acgggctgcc 840
tgcggatgaa ggaccagtgt gacaagtgcc gggagatctt gtctgtggac tgttccacca 900
acaacccctc ccaggctaag ctgcggcggg agctcgacga atccctccag gtcgctgaga 960
ggttgaccag gaaatacaac gagctgctaa agtcctacca gtggaagatg ctcaacacct 1020
cctccttgct ggagcagctg aacgagcagt ttaactgggt gtcccggctg gcaaacctca 2080
cgcaaggcga agaccagtac tatctgcggg tcaccacggt ggcttcccac acttctgact 1140
cggacgttcc ttccggtgtc actgaggtgg tcgtgaagct ctttgactct gatcccatca 1200
ctgtgacggt ccctgtagaa gtctccagga agaaccctaa atttatggag accgtggcgg 1260
agaaagcgct gcaggaatac cgcaaaaagc accgggacag tttgctgaag ctgctaagcc 1320
ggagagccac gtgggctgag ctcagaggcc ctggagctct cttggagctt ctggctgttc 1380
gccggaaggt ggcaggattt tgtgatgaaa agagggagga ggagaagggc aaggagcaac 1440
gagggtgtgt atgtgatgcc caagagaaag cagaggtggc agtgaagctc ctaagagacg 1500
aaggtgggag ggcactgtgc aactgtcaga gcaccgacat gcagcagggt CCCttCCtCa 1560
tcgtgactgt cagccagaga aggcagtga 1589
41/41
