Note: Descriptions are shown in the official language in which they were submitted.
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CELL ADHESION AND EXTRACELLULAR MATRIX PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of cell
adhesion and
extracellular matrix proteins and to the use of these sequences in the
diagnosis, treatment, and
prevention of immune system disorders, neurological disorders, developmental
disorders, connective
tissue disorders, and cell proliferative disorders, including cancer, and in
the assessment of the effects
of exogenous compounds on the expression of nucleic acid and amino acid
sequences of cell adhesion
and extracellular matrix proteins.
BACKGROUND OF THE INVENTION
Cell Adhesion Proteins
The surface of a cell is rich in transmembrane proteoglycans, glycoproteins,
glycolipids, and
receptors. These macromolecules mediate adhesion with other cells and with
components of the
ECM. The interaction of the cell with its surroundings profoundly influences
cell shape, stxength,
flexibility, motility, and adhesion. These dynamic properties are intimately
associated with signal
transduction pathways controlling cell proliferation and differentiation,
tissue construction, and .
embryonic development. Families of cell adhesion molecules include the
cadherins, integrins, lectins,
neural cell adhesion proteins, and some members of the proline-rich proteins.
Cadherins comprise a family of calcium-dependent glycoproteins that function
in mediating
cell-cell adhesion in virtually all solid tissues of multicellular organisms.
These proteins share multiple
repeats of a cadherin-specific motif, and the repeats form the folding units
of the cadherin
extracellular domain. Cadherin molecules cooperate to form focal contacts, or
adhesion plaques,
between adjacent epithelial cells. The cadherin family includes the classical
cadherins and
protocadherins. Classical cadherins include the E-cadherin, N-cadherin, and P-
cadherin subfamilies.
E-cadherin is present on many types of epithelial cells and is especially
important for embryonic
development. N-cadherin is present on nerve, muscle, and lens cells and is
also critical for embryonic
development. P-cadherin is present on cells of the placenta and epidermis.
Recent studies report that
protocadherins are involved in a variety of cell-cell interactions (Suzuki,
S.T. (1996) J. Cell Sci.
109:2609-2611). The intracellular anchorage of cadherins is regulated by their
dynamic association
with catenins, a family of cytoplasmic signal transduction proteins associated
with the actin
cytoskeleton. The anchorage of cadherins to the actin cytoskeleton appears to
be regulated by protein
tyrosine phosphorylation, and the cadherins are the target of phosphorylation-
induced functional
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disassembly (Aberle, H. et al. (1996) J. Cell. Biochem. 61:514-523).
Integrins are ubiquitous transmembrane adhesion molecules that link the ECM to
the internal
cytoskeleton. Integrins are composed of two noncovalently associated
transmembrane glycoprotein
subunits called a and (3. Integrins function as receptors that play a role in
signal transduction. For
example, binding of integrin to its extracellular ligand may stimulate changes
in intracellular calcium
levels or protein kinase activity (Sjaastad, M.D. and Nelson, W.J. (1997)
BioEssays 19:47-55). At
least ten cell surface receptors of the integrin family recognize the ECM
component fibronectin, which
is involved in many different biological processes including cell migration
and embryogenesis
(Johansson, S. et al. (1997) Front. Biosci. 2:D126-D146).
Lectins comprise a ubiquitous family of extracellular glycoproteins which bind
cell surface
carbohydrates specifically and reversibly, resulting in the agglutination of
cells (reviewed in
Drickamer, K. and Taylor, M. E. (1993) Annu. Rev. Cell Biol. 9:237-264). This
function is
particularly important for activation of the immune response. Lectins mediate
the agglutination and
mitogenic stimulation of lymphocytes at sites of inflammation (Lasky, L. A.
(1991) J. Cell. Biochem.
45:139-146; Paietta, E. et al. (1989) J. Tmmunol. 143:2850-2857).
Leetins are further classified into subfamilies based on carbohydrate-binding
specificity and
other criteria. The galectin subfamily, in particular, includes lectins that
bind (3-galactoside .
carbohydrate moieties in a thiol-dependent manner (reviewed in Hadari, Y. R.
et al. (1998) J. Biol.
Chem. 270:3447-3453). Galectins are widely expressed and developmentally
regulated. Galectins
contain a characteristic carbohydrate recognition domain (CRD). The CRD
comprises about 140
amino acids and contains several stretches of about 1 - 10 amino acids which
are highly conserved
among all galectins. A particular 6-amino acid motif within the CRD contains
conserved tryptophan
and arginine residues which are critical for carbohydrate binding. The CRD of
some galectins also
contains cysteine residues which may be important for disulfide bond
formation. Secondary structure
predictions indicate that the CRD forms several (3-sheets.
Galectins play a number of roles in diseases and conditions associated with
cell-cell and cell-
matrix interactions. For example, certain galectins associate with sites of
inflammation and bind to cell
surface immunoglobulin E molecules. In addition, galectins may play an
important role in cancer
metastasis. Galectin overexpression is correlated with the metastatic
potential of cancers in humans
and mice. Moreover, anti-galectin antibodies inhibit processes associated with
cell transformation,
such as cell aggregation and anchorage-independent growth (see, for example,
Su, Z.-Z. et al. (1996)
Proc. Natl. Acad. Sci. USA 93:7252-7257).
Selectins, or LEC-CAMS, comprise a specialized lectin subfamily involved
primarily in
2
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inflammation and leukocyte adhesion (Reviewed in Lasky, supra). Selectins
mediate the recruitment
of leukocytes from the circulation to sites of acute inflammation and are
expressed on the surface of
vascular endothelial cells in response to cytokine signaling. Selectins bind
to specific ligands on the
leukocyte cell membrane and enable the leukocyte to adhere to and migrate
along the endothelial
surface. Binding of selectin to its ligand leads to polarized rearrangement of
the actin cytoskeleton
and stimulates signal transduction within the leukocyte (Brenner, B. et al.
(1997) Biochem. Biophys.
Res. Commun. 231:802-807; Hidari, K. I. et al. (1997) J. Biol. Chem. 272:28750-
28756). Members of
the selectin family possess three characteristic motifs: a lectin or
carbohydrate recognition domain; an
epidermal growth factor-like domain; and a variable number of short consensus
repeats (scr or "sushi"
repeats) which are also present in complement regulatory proteins.
Neural cell adhesion proteins (NCAPs) play roles in the establishment of
neural networks
during development and regeneration of the nervous system (Uyemura, K. et al.
(1996) Essays
Biochem. 31:37-48; Brummendorf, T., and Rathjen, F.G. (1996) Curr. Opin.
Neurobiol. 6:584-S93).
NCAP participates in neuronal cell migration, cell adhesion, neurite
outgrowth, axonal fasciculation,
path~nding, synaptic target-recognition, synaptic formation, myelination and
regeneration. NCAPs are
expressed on the surfaces of neurons associated with learning and memory.
Mutations in genes
encoding NCAPS are linked With neurological diseases, including hereditary
neuropathy,
Charcot-Marie-Tooth disease, Dejerine-Sottas disease, X-linked hydrocephalus,
MASA syndrome
(mental retardation, aphasia, shuffling gait and adducted thumbs), and spastic
paraplegia type I. In
2o some cases, expression of NCAP is not restricted to the nervous system. L1,
for example, is
expressed in melanoma cells and hematopoietic tumor cells where it is
implicated in cell spreading and
migration, and may play a role in tumor progression (Montgomery, A.M. et al.
(1996) J. Cell Biol.
132:475-485).
NCAPs have at least one immunoglobulin constant or variable domain (Uyemura,
supra).
They are generally linked to the plasma membrane through a transmembrane
domain and/or a
glycosyl-phosphatidylinositol (GPI) anchor. The GPI linkage can be cleaved by
GPI phospholipase C.
Most NCAPs consist of an extracellular region made up of one or more
immunoglobulin domaius, a
membrane spanning domain, and an intracellular region. Many NCAPs contain post-
translational .
modifications including covalently attached oligosaccharide, glucuronic acid,
and sulfate. NCAPs fall
into three subgroups: simple-type, complex-type, and mixed-type. Simple-type
NCAPs contain one or
more variable or constant immunoglobulin domains, but lack other types of
domains. Members of the
simple-type subgroup include Schwann cell myelin protein (SMP), limbic system-
associated membrane
protein (LAMP), opiate binding cell-adhesion molecule (OBCAM), and myelin-
associated glycoproteiu
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(MAG). The complex-type NCAPs contain fibronectin type III domains in addition
to the
immunoglobulin domains. The complex-type subgroup includes neural cell-
adhesion molecule
(NCAM), axonin-1, F11, Bravo, and L1. Mixed-type NCAPs contain a combination
of
immunoglobulin domains and other motifs such as tyrosine kinase and epidermal
growth factor-like
domains. This subgroup includes Trk receptors of nerve growth factors such as
nerve growth factor
(NGF) and neurotropin 4 (NT4), Neu differentiation factors such as glial
growth factor II (GGFII) and
acetylcholine receptor-inducing factor (ARIA), and the semaphorin/collapsin
family such as
semaphorin B and collapsin.
Semaphorins are a large group of axonal guidance molecules consisting of at
least 30 different
members and are found in vertebrates, invertebrates, and even certain viruses.
All semaphorins
contain the sema domain which is approximately 500 amino acids in length.
Neuropilin, a semaphorin
receptor, has been shown to promote neurite outgrowth in vitro. The
extracellular region of
neuropilins consists of three different domains: CUB, discoidin, and MAM
domains. The CUB and
the MAM motifs of neuropilin have been proposed to have roles in protein-
protein interactions and are
suggested to be involved in the binding of semaphorins through the sema and
the C-terminal domains
(reviewed in Raper, J.A. (2000) C~rr. Opin. Neurobiol. 10:88-94).
An NCAP subfamily, the NCAP-LON subgroup, includes cell adhesion proteins
expressed on
distinct subpopulations of brain neurons. Members of the NCAP-LON subgroup
possess three
immunoglobulin domains and bind to cell membranes through GPI anchors. I~ilon
(a kindred of
NCAP-LON), for example, is expressed in the brain cerebral cortex and
hippocampus (Funatsu, N. et
al. (1999) J. Biol. Chem. 274:8224-8230). Trmmunostaining localizes Kilon to
the dendrites and soma of
pyramidal neuxons. Kilon has three C2 type immunoglobulin-like domains, six
pxedicted glycosylation
sites, and a GPI anchor. Expression of Kilon is developmentally regulated. It
is expressed at higher
levels in adult brain in comparison to embryonic and early postnatal brains.
Confocal microscopy
shows the presence of Kilon in dendrites of hypothalamic magnocellular neurons
secreting
neuropeptides, oxytocin or arginine vasopressin (Miyata, S. et al. (2000) J.
Comp. Neurol. 424:74-85).
Arginine vasopressin regulates body fluid homeostasis, extracellular
osmolarity and intravascular
volume. Oxytocin induces contractions of uterine smooth muscle during child
birth and of
myoepithelial cells in mammary glands during lactation. In magnocellular
neurons, Kilon is proposed to
play roles in the reorganization of dendritic connections during neuropeptide
secretion.
The neurexophilins are ligands for the neuron-specific cell surface proteins,
the a-neurexins.
Neurexophilins and neurexins may participate in a neuron signaling pathway
(Missler, M. and T.C.
Sudhof (1998) J. Neurosci. 18:3630-3638; Missler, M. et al. (1998) J. Biol.
Chem. 273:34716-34723).
4
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Ninjurin is a neuron cell surface protein which plays a role in cell adhesion
and in nerve regeneration
following injury. Ninjurin is up-regulated after nerve injury in dorsal root
ganglion neurons and in
Schwann cells (Araki, T. and Milbrandt, J. (1996) Neuron 17:353-361).
Ninjurin2 is expressed in
mature sensory and enteric neurons and promotes neurite outgrowth. Ninjurin2
is upregulated in
S Schwann. cells surrounding the distal segment of injured nerve with a time
course similar to that of
ninjurinl, neural CAM, and L1 (Araki, T. and Milbrandt, J. (2000) J. Neurosci.
20:187-19S).
Cell adhesion proteins also include some members of the proline-rich proteins
(PRPs). PRPs
are defined by a high frequency of proline, ranging from 20-SO% of the total
amino acid content.
Some PRPs have short domains which are rich in proline. These proline-rich
regions are associated
with protein-protein interactions. One family of PRPs are the proline-rich
synapse-associated proteins
(ProSAPs) which have been shown to bind to members of the postsynaptic density
(PSD) protein
family and subtypes of the somatostatin receptor (Yao, I. et al. (1999) J.
Biol. Chem. 274:
27463-27466; Zitzer, H. et al. (1999) J. Biol. Chem. 274:32997-33001). Members
of the ProSAP
family contain six to seven ankyrin repeats at the N-terminus, followed by an
SH3 domain, a PDZ
1S domain, and seven proline-rich regions and a SAM domain at the C terminus.
Several groups of
ProSAPs are important structural constituents. of synaptic structures in human
brain (Zitzer, supra).
Another member of the PRP family is the HLA-B-associated transcript 2 protein
(BAT2) which is
rich in proline and includes short tracts of polyproline, polyglycine, and
charged amino acids. BAT2
also contains four RGD (Arg-Gly-Asp) motifs typical of integrins (Banerji, J.
et al. (1990) Proc. Natl.
Acad. Sci. USA 87:2374-2378).
Toposome is a cell-adhesion glycoprotein isolated from mesenchyme-blastula
embryos.
Toposome precursors including vitellogenin promote cell adhesion of
dissociated blastula cells.
There are additional specific domains characteristic of cell adhesion
proteins. One such
domain is the MAM domain, a domain of about 170 amino acids found in the
extracellular region of
diverse proteins. These proteins all share a receptor-like architecture
comprising a signal peptide,
followed by a large N-terminal extracellular domain, a transmembrane region,
and an intracellular
domain (PROSITE document PDOC00604 MAM domain signature and profile). MAM
domain
proteins include zonadhesin, a sperm-specific membrane protein that binds to
the zona pellucida of the
egg; neuropilin, a cell adhesion molecule that functions during the formation
of certain neuronal
circuits, and Xenopus laevis thyroid hormone induced protein B, which contains
four MAM domains
and is involved in metamorphosis (Brown, D.D. et al. (1996) Proc. Natl. Acad.
Sci. USA 93:1924-
1929).
The WSC domain was originally found in the yeast WSC (cell-wall integrity and
stress
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response component) proteins which act as sensors of environmental stress. The
WSC domains are
extracellular and are thought to possess a carbohydrate binding role (Pouting,
C.P. et al. (1999) Curr.
Biol. 9:S 1-S2). A WSC domain. has recently been identified in polycystin-1, a
human plasma
membrane protein. Mutations in polycystin-1 are the cause of the commonest
form of autosomal
dominant polycystic kidney disease (Pouting, C.P. et al. (1999) C~rr. Biol.
9:8585-8588).
Leucine rich repeats (L88) are short motifs found in numerous proteins from a
wide range of
species. LRR motifs are of variable length, most commonly 20-29 amino acids,
and multiple xepeats
are typically present in tandem. LRR motifs are important for proteinlprotein
interactions and cell
adhesion, and LRR proteins are involved in cell/cell interactions,
moxphogenesis; and development
(Kobe, B. and Deisenhofer, J. (1995) C~rr. Opin. Struct. Biol. 5:409-416). The
human ISLR
(immunoglobulin superfamily containing leucine-rich repeat) protein contains a
C2-type
immunoglobulin domain as well as LRR motifs. The ISLR gene is linked to the
critical region for
Bardet-Biedl syndrome, a developmental disorder of which the most common
feature is retinal
dystrophy (Nagasawa, A. et al. (1999) Genomics 61:37-43).
The sterile alpha motif (SAM) domain is a conserved protein binding domain,
approximately
70 amino acids in length, and is involved in the regulation of many
developmental processes in
eukaryotes. The SAM domain can potentially function as a protein interaction
module through its
ability to form homo- or hetero-oligomers with other SAM domains (Schultz, J.
et al. (1997) Protein
Sci. 6:249-253).
Extracellular Matrix Proteins
The extracellular matrix (ECM) is a complex network of glycoproteins,
polysaccharides,
proteoglycans, and other macromolecules that are secreted from the cell into
the extracellular space.
The ECM remains in close association with the cell surface and provides a
supportive meshwork that
profoundly influences cell shape, motility, strength, flexibility, and
adhesion. In fact, adhesion of a cell
to its surrounding matrix is required for cell survival except in the case of
metastatic tumor cells, which
have overcome the need for cell-ECM anchorage. This phenomenon suggests that
the ECM plays a
critical role in the molecular mechanisms of growth control and metastasis.
(Reviewed in Ruoslahti,
E. (1996) Sci. Am. 275:72-77.) Furthermore, the ECM determines the structure
and physical
properties of connective tissue and is particularly important for
morphogenesis and other processes
associated with embryonic development and pattern formation.
The collagens comprise a family of ECM proteins that provide structure to
bone, teeth, skin,
ligaments, tendons, cartilage, blood vessels, and basement membranes. Multiple
collagen proteins
have been identified. Three collagen molecules fold together in a triple helix
stabilized by interchain
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disulfide bonds. Bundles of these triple helices then associate to form
fibrils.
Elastin and related proteins conifer elasticity to tissues such as skin, blood
vessels, and lungs.
Elastin is a highly hydrophobic protein of about 750 amino acids that is rich
in proline and glycine
residues. Elastin molecules are highly cross-linked, forming an extensive
extracellular network of
fibers and sheets. Elastin fibers are surrounded by a sheath of microfibrils
which are composed of a
number of glycoproteins, including fibrillin.
Fibronectin is a large ECM glycoprotein found in all vertebrates. Fibronectin
exists as a dimer
of two subunits, each containing about 2,500 amino acids. Each subunit folds
into a rod-like structure
containing multiple domains. The domains each contain multiple repeated
modules, the most common
of which is the type 111 fibronectin repeat. The type III fibxonectin repeat
is about 90 amino acids in
length and is also found in other ECM proteins and in some plasma membrane and
cytoplasrnic
proteins. Furthermore, some type DI fibronectin repeats contain a
characteristic tripeptide consisting
of Arginine-Glycine-Aspartic acid (RGD). The RGD sequence is recognized by the
integrity family of
cell surface receptors and is also found in other ECM proteins. (Reviewed in
Alberts, et al. (1994)
Molecular Biology of the Cell, Garland Publishing, New York, NY, pp. 986-987.)
Laminin is a major glycoprotein component of the basal lamina which underlies
and supports
epithelial cell sheets. Laminin is one of the first ECM proteins synthesized
in the developing embryo.
Laminin is an 850 kilodalton protein composed of three~polypeptide chains
joined in the shape of a
cross by disulfide bonds. La ini is especially important for angiogenesis and,
in particular, for guiding
the formation of capillaries. (Reviewed in Alberts, supra, pp. 990-991.)
Many proteinaceous ECM components are proteoglycans. Proteoglycans are
composed of
unbranched polysaccharide chains (glycosaminoglycans) attached to protein
cores. Common
proteoglycans include aggrecan, betaglycan, decorin, perlecan, serglycin, and
syndecan-1. Some of
these molecules not only provide mechanical support, but also bind to
extracellular signaling molecules,
such as fibroblast growth factor and transforming growth factor (3, suggesting
a role for proteoglycans
in cell-cell communication. (Reviewed in Alberts, supra, pp. 973-978.)
Dentin phosphoryn (DPP) is a major component of the dentin ECM. DPP is a
proteoglycan
that is synthesized and expressed by odontoblasts (Gu, K., et al. (1998) Eur.
J. Oral Sci. 106:1043-
1047). DPP is believed to nucleate or modulate the formation of hydroxyapatite
crystals.
Mucins are highly glycosylated glycoproteins that are the major structural
component of the
mucus gel. The physiological functions of mucins are cytoprotection,
mechanical protection,
maintenance of viscosity in secretions, and cellular recognition. MUC6 is a
human gastric mucin that
is also found in gall bladder, pancreas, seminal vesicles, and female
reproductive tract (Toribara,
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N.W., et al. (1997) J. Biol. Chem. 272:16398-16403). The MUC6 gene has been
mapped to human
chromosome 11 (Toribaxa, N.W., et al. (1993) J. Biol. Chem. 268:5879-5885).
Hemomucin is a novel
Dr~osopltila surface mucin that may be involved in the induction of
antibacterial effectox molecules
(Theopold, U., et al. (1996) J. Biol. Chem. 217:12708-12715).
Olfactomedin was originally identified as the major component of the mucus
layer surrounding
the chemosensory dendrites of olfactory neurons. Olfactomedin-related proteins
are secreted
glycoproteins with conserved C-terminal motifs. The TIGR/myocilin protein, an
olfactomedin-related
protein expressed in the eye, is associated with the pathogenesis of glaucoma
(Kulkarni, N.H. et al.
(2000) Genet. Res. 76:41-50).
Ankyrin (ANK) repeats mediate protein-protein interactions associated with
diverse
intracellular functions. ANK repeats are composed of about 33 amino acids that
form a helix-turn-
helix core preceded by a protruding "tip." These tips are of variable sequence
and may play a role in
protein-protein interactions. The helix-turn-helix region of the ANK repeats
stack on top of one
another and are stabilized by hydrophobic interactions (Yang, Y. et al. (1998)
Structure 6:619-626).
Sushi repeats, also called short consensus repeats (SCR), are found in a
number of proteins
that share the common feature of binding to other proteins. For example, in
the C-terminal domain of
versican, the sushi domain is important for heparin binding. Sushi domains
contain basic amino acid
residues, which may play a role in binding (Oleszewski, M. et al. (2000) J.
Biol. Chem. 275:34478-
34485).
2o Link, or X-link, modules are hyaluronan-binding domains found in proteins
involved in the
assembly of extracellular matrix, cell adhesion, and migration. The Link
module superfamily includes
CD44, cartilage link protein, and aggrecan. There is close similarity between
the Link module and the
C-type lectin domain, with the predicted hyaluronan-binding site at an
analogous position to the
carbohydrate binding pocket in E-selectin (Kohda, D. et al. (1996) Cell, Vol.
86, 767-775).
Multidomain or mosaic proteins play an. important role in the diverse
functions of the
extracellular matrix (Engel, J. et al. (1994) Development (Camb.) S35-42). ECM
proteins are
frequently characterized by the presence of one or more domains which may
contain a number of
potential intracellular disulfide bridge motifs. For example, domains which
match the epidermal growth
factor (EGF) tandem repeat consensus are present within several known
extracellular proteins that
promote cell growth, development, and cell signaling. This signature sequence
is about forty amino
acid residues in length and includes six conserved cysteine residues, and a
calcium binding site near
the N-terminus of the signature sequence. The main structure is a two-stranded
beta-sheet followed
by a loop to a C-terminal short two-stranded sheet. Subdomains between the
conserved cysteines
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vary in length (Davis, C.G. New Biol (1990) May;2(5):410-9). Post-
translationalhydroxylation of
aspartic acid or asparagine residues has been associated with EGF-like domains
in several proteins
(Prosite PDOC00010 Aspartic acid and asparagine hydroxylation site).
A number of proteins that contain calcium-binding EGF-like domain signature
sequences are
involved in growth and differentiation. Examples include bone morphogenic
protein 1, which induces
the formation of cartilage and bone; crumbs, which is a Dr-osophila epithelial
development protein;
Notch and a number of its homologs, which are involved in neural growth and
differentiation, and
transforming growth factor beta-1 binding protein (Expasy PROSITE document
PDOC00913; Soler,
C. and Carpenter, G., in Nicola, N.A. (1994) The Cytokine Facts Book, Oxford
University Press,
_ Oxford, UK, pp 193-197). EGF-like domains mediate protein-protein
interactions for a variety of
proteins. For example, EGF-like domains in. the ECM glycoprotein fibulin-1
have been shown to
mediate both self association and binding to fibronectin (Trap, H. et al.
(1997) J. Biol. Chem.
272:22600-22606). Point mutations in the EGF-like domains of ECM proteins have
been identified as
the cause of human disorders such as Marfan syndrome and pseudochondroplasia
(Maurer, P. et al.
(1996) Curr. Opin. Cell Biol. 8:609-617).
The CUB domain is an extracellular domain of approximately 110 amino acid
residues found
mostly in developmentally regulated proteins. The CUB domain contains four
conserved cysteine
residues and is predicted to have a structure similar to that of
immunoglobulins. Vertebrate bone
morphogenic protein 1, which induces cartilage and bone formation, and
fibropellins I and III from sea
urchin, which form the apical lamina component of the ECM, are examples of
proteins that contain
both CUB and EGF domains (PROSITE PDOC00908 CUB domain profile).
Other ECM proteins are members of the type A domain of von Willebrand factor
(vWFA)-
like module superfamily, a diverse group of proteins with a module sharing
high sequence similarity.
The vWFA-like module is found not only in plasma proteins but also in plasma
membrane and ECM
proteins (Colombatti, A. and Bonaldo, P. (1991) Blood 77:2305-2315). Crystal
structure analysis of an
integrin vWFA-like module shows a classic "Rossmann" fold and suggests a metal
ion-dependent
adhesion site for binding protein ligands (Lee, J.-O. et al. (1995) Cell
80:631-638). This family
includes the protein matrilin-2, an extracellular matrix protein that is
expressed in a broad range of
mammalian tissues and organs. Matrilin-2 is thought to play a role in ECM
assembly by bridging
3o collagen fibrils and the aggrecan network (Desk, F. et al. (1997) J. Biol.
Chem. 272:9268-9274).
The thrombospondins are multimeric, calcium-binding extracellular
glycoproteins found widely
in the embryonic extracellular matrix. These proteins are expressed in the
developing nervous system
or at specific sites in the adult nervous system after injury. Thrombospondins
contain multiple EGF-
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type repeats, as well as a motif known as the thrombospondiu type 1 repeat
(TSR). The TSR is
approximately 60 amino acids in length and contains six conserved cysteine
residues. Motifs within
TSR domains are involved in mediating cell adhesion through binding to
proteoglycaus and sulfated
glycolipids. Thrombospondin-1 inhibits angiogenesis and modulates endothelial
cell adhesion, motility,
and growth. TSR domains are found in a diverse group of other proteins, most
of which are
expressed in the developing nervous system and have potential roles in the
guidance of cell and growth
cone migration. Proteins that contain TSRs include the F-spondin gene family,
the semaphorin 5
family, UNC-5, and SCO-spondin. The TSR superfamily includes the ADAMTS
proteins which
contain an ADAM (A Disintegriu and Metalloproteinase) domain as well as one or
more TSRs. The
ADAMTS proteins have roles in regulating the turnover of cartilage matrix,
regulation of blood vessel
growth, and possibly development of the nervous system. (Reviewed in Adams,
J.C. and Tucker, R.
P. (2000) Dev. Dyn. 218:280-299.)
Fibrinogen, the principle protein of vertebrate blood clotting, is a hexamer
consisting of two
sets of three different chains (alpha, beta, and gamma). The C-terminal domain
of the beta and
gamma chains comprises about 270 amino acid residues and contains four
cysteines involved in two
disulfide bonds. This domain has also been found in mammalian tenascin-X, an
ECM protein that
appears to be involved in cell adhesion (Prosite PDOC00445 Fibrinogen beta and
gamma chains C-
terminal domain signature).
Expression~rofiliu~
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
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.
Colorectal cancer is the fourth most common cancer and the second most common
cause of
cancer death in the United States with approximately 130,000 new cases and
55,000 deaths per year.
Colon and rectal cancers share many environmental risk factors and both are
found in individuals with
specific genetic syndromes. (See Potter, J.D. {1999) J. Natl. Cancer Institute
91:916-932 for a review
of colorectal cancer.) Colon cancer is the only cancer that occurs with
approximately equal
frequency in men and women, and the five-year survival rate following
diagnosis of colon cancer is
around 55%o in the United States (Ries et al. (1990) National Institutes of
Health, DHHS Publ No.
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
(NIFI)90-2789).
Colon cancer is causally related to both genes and the environment. Several
molecular
pathways have been linked to the development of colon cancer, and the
expression of key genes in
any of these pathways may be lost by inherited or acquired mutation or by
hypermethylation. There
is a particular need to identify genes for which changes in expression may
provide an early indicator of
colon cancer or a predisposition for the development of colon cancer.
For example, it is well known that abnormal patterns of DNA methylation occur
consistently
in human tumors and include, simultaneously, widespread genomic
hypomethylation and localized areas
of increased methylation. In colon cancer in particular, it has been found
that these changes occur
early in tumor progression such as in premalignant polyps that precede colon
cancer. Indeed, DNA
methyltransferase, the enzyme that performs DNA methylation, is significantly
increased in
histologically normal mucosa from patients with colon cancer or the benign
polyps that precede
cancer, and this increase continues during the progression of colonic
neoplasms (Wafik, S. et al.
(1991) Proc. Natl. Acad. Sci. USA 88:3470-3474). Increased DNA methylation
occurs in G+C rich
areas of genomic DNA termed "CpG islands" that are important for maintenance
of an "open"
transcriptional conformation around genes, and that hypermethylation of these
regions results in a
"closed" conformation that silences gene transcription. It has been suggested
that the silencing or
downregulation of differentiation genes by such abnormal methylation of CpG
islands may prevent
differentiation in immortalized cells (Antequera, F. et al. (1990) Cell 62:503-
514).
Familial Adenomatous Polyposis (FAP) is a rare autosomal dominant syndrome
that precedes
colon cancer and is caused by an inherited mutation in the adenomatous
polyposis coli (APC) gene.
FAP is characterized by the early development of multiple colorectal adenomas
that progress to
cancer at a mean age of 44 years. The APC gene is a part of the APC-13-catenin-
Tcf (T-cell factor)
pathway. Impairment of this pathway results in the loss of orderly
replication, adhesion, and migration
of colonic epithelial cells that results in the growth of polyps. A series of
other genetic changes follow
activation of the APC-13-catenin-Tcf pathway and accompanies the transition
from normal colonic
mucosa to metastatic carcinoma. These changes include mutation of the K-Ras
proto-oncogene,
changes in methylation patterns, and mutation or loss of the tumor suppressor
genes p53 and Smad4/
DPC4. While the inheritance of a mutated APC gene is a rare event, the loss or
mutation of APC
and the consequent effects on the APC-l3-catenin-Tcf pathway is believed to be
central to the
majority of colon cancers in the general population.
Hereditary nonpolyposis Colorectal Cancer (HNPCC) is another inherited
autosomal dominant
syndrome with a less well defined phenotype than FAP. HNPCC, which accounts
for about 2% of
11
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colorectal cancer cases, is distinguished by the tendency to early onset of
cancer and the development
of other cancers, particularly those involving the endometrium, urinary tract,
stomach and biliary
system. HNPCC results from the mutation of one or more genes in the DNA mis-
match repair
(MMR) pathway. Mutations in two human MMR genes, MSH2 and MLH1, are found in a
large
majority of HNPCC families identified to date. The DNA MMR pathway identifies
and repairs errors
that result from the activity of DNA polymerase during replication.
Furthermore, loss of MMR
activity contributes to cancer progression through accumulation of other gene
mutations and deletions,
such as loss of the BAX gene which controls apoptosis, and the TGFIi receptor
1I gene which controls
cell growth. Because of the potential for irreparable damage to DNA in an
individual with a DNA
MMR defect, progression to carcinoma is more rapid than usual.
Although ulcerative colitis is a minor contributor to colon cancer, affected
individuals have
about a 20-fold increase in risk for developing cancer. Progression is
characterized by loss of the p53
gene which may occur early, appearing even in histologically normal tissue.
The progression of the
disease from ulcerative colitis to dysplasia/carcinoma without an intermediate
polyp state suggests a
high degree of mutagenic activity resulting from the exposure of proliferating
cells in the colonic
mucosa to the colonic contents.
Almost all colon cancers arise from cells in which the estrogen receptor (ER)
gene has been
silenced. The silencing of ER gene transcription is age related and linked to
hypermethylation of the
ER gene (Issa, J-P. J. et al. (1994) Nature Genetics 7:536-540). Introduction
of an exogenous ER
gene into cultured colon carcinoma cells results in marked growth suppression.
The connection
between loss of the ER protein in colonic epithelial cells and the consequent
development of cancer
has not been established.
Clearly there are a number of genetic alterations associated with colon cancer
and with the
development and progression of the disease, particularly the downregulation or
deletion of genes, that
potentially provide early indicators of cancer development, and which may also
be used to monitor
disease progression or provide possible therapeutic targets. The specific
genes affected in a given
case of colon cancer depend on the molecular progression of the disease.
Identification of additional
genes associated with colon cancer and the precancerous state would provide
more reliable diagnostic
patterns associated with the development and progression of the disease.
The discovery of new cell adhesion and extracellular matrix proteins, and the
polynucleotides
encoding them, satisfies a need in the art by providing new compositions which
are useful in the
diagnosis, prevention, and treatment of immune system disorders, neurological
disorders,
developmental disorders, connective tissue disorders, and cell proliferative
disorders, including cancer,
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WO 02/088322 PCT/US02/13874
and in the assessment of the effects of exogenous compounds on the expression
of nucleic acid and
amino acid sequences of cell adhesion and extracellular matrix proteins.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, cell adhesion and extracellular
matrix proteins,
referred to collectively as "CADECM" and individually as "CADECM-1," "CADECM-
2,"
"CADECM-3," "CADECM-4," "CADECM-5," "CADECM-6," "CADECM-7," "CADECM-8,"
"CADECM-9," "CADECM-10," and "CADECM-11." In one aspect, the invention
provides an
isolated polypeptide selected from the group consisting,of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ 1D N0:1-11, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ll~ N0:1-11, c) a biologically active
fragment of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ 1D
N0:1-11, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m N0:1-11. In one alternative, the invention provides an
isolated polypeptide
comprising the amino acid sequence of SEQ m NO:1-11.
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 N0:1-11, b) a polypeptide comprising a naturally
occurring amino acid sequence
at least 90% identical to an amino acid sequence selected from the group
consisting of SEQ ID N0:1-
11, c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-11, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ m N0:1-11. In
one alternative, the
polynucleotide encodes a polypeptide selected from the group consisting of SEQ
ID NO:1-11. In
another alternative, the polynucleotide is selected from the group consisting
of SEQ ID N0:12-22.
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 B7 N0:1-11, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ m N0:1-11, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m N0:1-11, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ 117 N0:1-11. In one
alternative, the
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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 ll~ N0:1-11, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ m N0:1-11, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-11, and d) an immunogenic fragment of a polypeptide
having au amino
acid sequence selected from the group consisting of SEQ m N0:1-11. 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 N0:1-11, 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:1-11, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ m N0:1-11, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
2o ID N0:1-11.
The invention further provides an isolated polynucleotide selected from the
group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
m N0:12-22, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consistixtg
of SEQ D? N0:12-22,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
the polynucleotide of b), and e) an RNA equivalent of a)-d). In one
alternative, the polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
m N0:12-22, b) a polynucleotide comprising a naturally occurriug
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:12-22,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
14
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WO 02/088322 PCT/US02/13874
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
N0:12-22, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ m NO:12-22, 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 m N0:1-11, 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:1-11, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ m NO:1-11, and d) an
irnmunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ m N0:1-11,
and a pharmaceutically acceptable excipient. In one embodiment, the
composition comprises an amino
acid sequence selected from the group consisting of SEQ 1D N0:1-11. The
invention additionally
provides a method of treating a disease or condition associated with decreased
expression of
functional CADECM, 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 7D N0:1-11, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-11, c) a biologically active fragment
of a polypeptide
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
having an amino acid sequence selected from the group consisting of SEQ m N0:1-
11, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID N0:1-11. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional CADECM,
comprising administering to a patient in need of such treatment the
composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1-11, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ m N0:1-11, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-11,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID N0:1-11. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
Tn 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
CADECM, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds to
a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID N0:1-11, b) a
polypeptide comprising a
naturally occurring amino acid sequence at Ieast 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-11, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
N0:1-11, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID N0:1-11. 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
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WO 02/088322 PCT/US02/13874
acid sequence selected from the group consisting of SEQ ID N0:1-11, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to au amino
acid sequence selected
from the group consisting of SEQ ID N0:1-11, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
N0:1-11, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID N0:1-11. 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 ll~ N0:12-
22, 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
117 N0:12-22, 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 N0:12-22,
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 1D N0:12-
22, u) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ 1D N0:12-22,
iii) a polynucleotide
complementary to the polynucleotide of i), iv) a polynucleotide complementary
to the polynucleotide of
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CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
ii), and v) an RNA equivalent of i)-iv). Alternatively, the target
polynucleotide comprises a fragment
of a polynucleotide sequence selected from the group consisting of i)-v)
above; c) quantifying the
amount of hybridization complex; and d) comparing the amount of hybridization
complex in the treated
biological sample with the amount of hybridization complex in an untreated
biological sample, wherein
a difference in the amount of hybridization complex in the treated biological
sample is indicative of
toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog, and the PROTEOME database identification numbers and annotations of
PROTEOME
database homologs, 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.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
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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
"CADECM" refers to the amino acid sequences of substantially purified CADECM
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
CADECM. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of CADECM either by
directly interacting with
CADECM or by acting on components of the biological pathway in which CA1?ECM
participates.
An "allelic variant" is an alternative form of the gene encoding CADECM.
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 CADECM include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as CADECM or
a polypeptide with at least one functional characteristic of CADECM. Included
within this definition
are polymorphisms which may or may not be readily detectable using a
particular oligonucleotide
probe of the polynucleotide encoding CADECM, and improper or unexpected
hybridization to allelic
variants, with a locus other than the normal chromosomal locus for the
polynucleotide sequence
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WO 02/088322 PCT/US02/13874
encoding CADECM. The encoded protein may also be "altered," and may contain
deletions,
insertions, or substitutions of amino acid residues which produce a silent
change and result in a
functionally equivalent CADECM. Deliberate amino acid substitutions may be
made on the basis of
similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity,
andlor the amphipathic nature of
the residues, as long as the biological or immunological activity of CADECM 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 CADECM. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates,
small molecules, or any other compound or composition which modulates the
activity of CADECM
either by directly interacting with CADECM or by acting on components of the
biological pathway in
which CADECM 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 CADECM 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 carnets that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLIT). 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
CA 02446023 2003-10-28
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makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen used
to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or ofigonucleotide 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 for 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), wluch 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-liuked 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 aptamer 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; nor
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
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WO 02/088322 PCT/US02/13874
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 CADECM, or
of any oligopeptide
thereof, to induce a specific immune response in appropriate animals or cells
and to bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising a
given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation ox an
aqueous solution.
Compositions comprising polynucleotide sequences encoding CADECM or fragments
of CADECM
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
xesequenced, 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 GELV>EW 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 anvno acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
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Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
lle Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide 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 ox 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 au
untreated sample, or a
diseased and a normal sample.
"Exon shuft7ing" 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.
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A "fragment" is a unique portion of CADECM or the polynucleotide encoding
CADECM
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
amiuo acid residues.
A fragment used as a probe, primer, autigen, therapeutic molecule, or for
other purposes, may be at
least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500
contiguous nucleotides or
amino acid residues in length. Fragments may be preferentially selected from
certain regions of a
molecule. For example, a polypeptide fragment may comprise a certain length of
contiguous amino
acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of
a polypeptide as shown
in a certain defined sequence. Clearly these lengths are exemplary, and any
length that is supported
by the specification, including the Sequence Listing, tables, and figures, may
be encompassed by the
present embodiments.
A fragment of SEQ m N0:12-22 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ 117 N0:12-22, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:12-22 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish SEQ
m NO:12-22 from related polynucleotide sequences. The precise length of a
fragment of SEQ m
N0:12-22 and the region of SEQ m N0:12-22 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 ll~ N0:1-11 is encoded by a fragment of SEQ m N0:12-22. A -
fragment of SEQ m NO:1-11 comprises a region of unique amino acid sequence
that specifically
identifies SEQ ID N0:1-11. For example, a fragment of SEQ ll~ N0:1-11 is
useful as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ m N0:1-11.
The precise length of a fragment of SEQ m N0:1-11 and the region of SEQ m N0:1-
11 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
trauslation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full
length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology' refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity". and "% identity," as applied to polynucleotide
sequences, refer to
the percentage of residue matches between at least two polynucleotide
sequences aligned using a
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WO 02/088322 PCT/US02/13874
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps in
the sequences being compared in order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity' between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol: 215:403-410),
which is available from
several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.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.nhn.nih.gov/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for matclz: 1
Penalty for mismatch: -2
0perz Gap: S arzd Extension Gap: 2 penalties
Gap x drop-off SO
Expect: 10
Word Size: 11
Filter-: orz
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity' and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally pxeserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=S. 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:
Matf~ix: BLOSUM62
3o Open Gap: 11 afad Extension Gap: 1 penalties
Gap x drop-off.' S0
Expect: 10
Word Size: 3
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WO 02/088322 PCT/US02/13874
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, de~.ned
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 rnicrochromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under deftned 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 (T"~ for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al. (1989)
Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor
Press, Plainview NY;
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WO 02/088322 PCT/US02/13874
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°l0. 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-SO% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
1o 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, filtexs, chips, pins or glass slides, or any
othex appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of CADECM
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 CADECM which is useful in any of the antibody production methods disclosed
herein or known in
the art.
The term "microarra~' 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.
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The term "modulate" refers to a change in the activity of CADECM. For example,
modulation may cause an increase or a decrease in protein activity, binding
characteristics, or any
other biological, functional, or immunological properties of CADECM.
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 CADECM may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in the
art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary
by cell type depending on the enzymatic milieu of CADECM.
"Probe" refers to nucleic acid sequences encoding CADECM, 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
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WO 02/088322 PCT/US02/13874
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo , Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Iuuis, 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
1o 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/MTT
Center for Genome.
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for example,
as PCR or sequencing primers, microarray elements, or specific probes to
identify fully or partially
complementary polynucleotides in a sample of nucleic acids. Methods of
oligonucleotide selection are
not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occuiTing or
has a sequence
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
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
uCADECMslated regions of a gene and includes enhancers, promoters, introns,
and 5' and 3'
uCADECMslated regions (UTRs). Regulatory elements interact with host or viral
proteins which
control transcription, translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of
the nitrogenous base thymine are replaced with uracil, and the sugar backbone
is composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing
CADECM, nucleic acids encoding CADECM, 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.
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WO 02/088322 PCT/US02/13874
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 kuown 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
au autonomously replicating plasmid or as part of the host chromosome, as well
as transiently
transformed cells which express the inserted DNA or RNA for limited periods of
time.
A "transgenic organism," as used herein, is any organism, including but not
limited to animals
and plants, in which one or more of the cells of the organism contains
heterologous nucleic acid
introduced by way of human intervention, such as by transgenic techniques well
known in the art. The
nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor of the cell,
by way of deliberate genetic manipulation, such as by microinjection or by
infection with a
recombinant virus. In one alternative, the nucleic acid can be introduced by
infection with a
recombinant viral vector, such as a lentiviral vector (Loin, C. et al. (2002)
Science 295:868-872). 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 trausconjugation.
Techniques for
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WO 02/088322 PCT/US02/13874
transferring the DNA of the present invention into such organisms are widely
known and provided in
references such as Sambrook et al. (1989), suura.
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.
THE INVENTION
The invention is based on the discovery of new human cell adhesion and
extracellular matrix
proteins (CADECM), the polynucleotides encoding CADECM, and the use of these
compositions for
the diagnosis, treatment, or prevention of immune system disorders,
neurological disorders,
developmental disorders, connective tissue disorders, and cell proliferative
disorders, including cancer.
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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 ll~). 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 117 NO:) and an
Incyte polynucleotide consensus sequence number (Incyte Polynucleotide m) 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 and the PROTEOME
database.
Columns 1 and 2 show the polypeptide sequence identification number
(Polypeptide SEQ )D NO:) and
the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID)
for polypeptides of the
invention. Column 3 shows the GenBank identification number (GenBank ID NO:)
of the nearest
GenBank homolog and the PROTEOME database identification numbers (PROTEOME )D
NO:) of
the nearest PROTEOME database homologs. Column 4 shows the probability scores
for the matches
between each polypeptide and its homolog(s). Column 5 shows the annotation of
the GenBank and
PROTEOME database 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 and
2 show the polypeptide sequence identification number (SEQ I7? 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 cell adhesion and
extracellular matrix proteins.
For example, SEQ ID N0:2 is 92% identical, from residue M1 to residue S828, to
murine PB-cadherin
(GenBank ID g4760578) 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 N0:2 also contains
cadherin and
cadherin cytoplasmic domains as determined by searching for statistically
significant matches in the
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hidden Markov model (I~VIM)-based PFAM database of conserved protein family
domains. (See
Table 3.) Data from BLllVll'S, MOTIFS, and PROFILESCAN analyses provide
further
corroborative evidence that SEQ LD N0:2 is a cadherin. In an alternative
example, SEQ JD N0:4 is
27% identical, from residue E2 to residue A1230, to chicken connectin/titin
(GenBank )D g1513030)
as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
The BLAST
probability score is 2.0e-177, which indicates the probability of obtaining
the observed polypeptide
sequence alignment by chance. SEQ ll~ NO:4 also contains 25 immunoglobulin
domains as
determined by searching for statistically significant matches in the hidden
Markov model (I~4M)-
based PFAM database of conserved protein family domains. (See Table 3.) Data
from BLAST
DOMO, BLAST PRODOM, and MOTIFS analysis and from blast analysis using the
PRODOM and
DOMO databases provide further corroborative evidence that SEQ LD N0:4 is a
thin, a muscle
protein containing of repetitive modules of immunoglobulin and fibronectin
motifs interspersed with
unique sequences. In an alternative example, SEQ ID NO:S is 42% identical,
from residue L4 to
residue 8705, to human protocadherin alpha C2 short form protein (GenBank ID
g545699.1) as
determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
The BLAST
probability scoxe is 1.3e-144, which indicates the probability of obtaining
the observed polypeptide
sequence alignment by chance. SEQ ID N0:5 also contains cadherin domains as
determined by
searching for statistically significant matches in the hidden Markov model
(I~VIM)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from BLM'S,
MOTIFS, and
PROFILESCAN analyses and BLAST analyses of the PRODOM and DOMO databases
provide
further corroborative evidence that SEQ ID N0:5 contains cadherin domains and
is a cell adhesion
protein. In addition, SPSCAN and HIVIIVIER analyses indicate that SEQ ID N0:5
contains a signal
peptide and TMAP analysis indicates that SEQ lD N0:5 contains three
transmembrane domains. In
an alternative example, SEQ LD NO:10 is 97% identical, from residue M1 to
residue V666, to neurexin
II beta-a (GenBank ~ g205719) 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 N0:10 also contains
a laminin G
domain as determined by searching for statistically significant matches in the
hidden Markov model
(I~VIM)-based PFAM database of conserved protein family domains. (See Table
3.) Data from
additional BLAST analysis provide further corroborative evidence that SEQ ll~
N0:3 is a neurexin.
SEQ lD N0:2-3, SEQ LD NO:6-9, and SEQ ID N0:11 were analyzed and annotated in
a similar
manner. The algorithms and parameters for the analysis of SEQ ID N0:1-11 are
described in Table
7.
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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
identification number (Polynucleotide SEQ ll~ NO:), the corresponding Incyte
polynucleotide
consensus sequence number (Incyte 117) 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 eDNA 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 117
N0:12-22 or that
distinguish between SEQ ID N0:12-22 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 XX~YYXXX NI NZ YYYI'Y 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 YY1'Y1' is the
number of the prediction generated by the algorithm, and N1,2~3..., 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
FT .XXXXXX_gAAAAA-gBBBBB_1 1V is a "stretched" sequence, with ' ' 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
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WO 02/088322 PCT/US02/13874
as a protein homolog for the "exon-stretching" algorithm, a RefSeq identifier
(denoted by "NM,"
"NP," or "NT") maybe used in place of the GenBank identifier (i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs
GNN, GFG, Exon prediction from genomic sequences using,
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 CADECM variants. A preferred CADECM 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 CADECM amino acid sequence, and which contains
at least one
functional or structural characteristic of CADECM.
The invention also encompasses polynucleotides which encode CADECM. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:12-22, which encodes CADECM. The
polynucleotide
sequences of SEQ ID N0:12-22, as presented in the Sequence Listing, embrace
the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced
with uracil, and the
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WO 02/088322 PCT/US02/13874
sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
CADECM.
Iu 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 CADECM. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ )D NO:12-
22 which has at least about 70%, or alternatively at least about 85%, or even
at least about 95%
polynucleotide sequence identity to a nucleic acid sequence selected from the
group consisting of SEQ
ID N0:12-22. Any one of the polynucleotide variants described above can encode
an amino acid
sequence which contains at least one functional or structural characteristic
of CADECM.
In addition, or in the alternative, a polynucleotide variant of the invention
is a splice variant of a
polynucleotide sequence encoding CADECM. A splice variant may have portions
which have
significant sequence identity to the polynucleotide sequence encoding CADECM,
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 60%, or alternatively less than
about 50% polynucleotide
sequence identity to the polynucleotide sequence encoding CADECM 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 CADECM. For example, a polynucleotide
comprising a
sequence of SEQ 1D N0:22 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 ox structural characteristic of CADECM.
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 CADECM, 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 CADECM, and all such variations
are to be considered
as being specifically disclosed.
Although nucleotide sequences which encode CADECM and its variants are
generally
capable of hybridizing to the nucleotide sequence of the naturally occurring
CADECM under
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appropriately selected conditions of stringency, it may be advantageous to
produce nucleotide
sequences encoding CADECM 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 CADECM and its derivatives without
altering the encoded
amino acid sequences include the production of RNA transcripts having more
desirable properties,
such as a greater half life, than transcripts produced from the naturally
occurring sequence.
The invention also encompasses production of DNA sequences which encode CADECM
and
CADECM 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 CADECM 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 1D
NO:12-22 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kirnlnel, 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 Biology and Biotechnolo$y, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding CADECM may be extended utilizing a partial
nucleotide
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WO 02/088322 PCT/US02/13874
sequence and employing various PCR based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent
to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et
al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme digestions and
legations may be used to insert an engineered double-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids
Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 primer analysis software
(National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in length,
to have a GC content of about 50% or more, and to anneal to the template at
temperatures of about
68°C to 72°C. ,
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. 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
CA 02446023 2003-10-28
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which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode CADECM may be cloned in recombinant DNA molecules that direct
expression of
CADECM, 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 CADECM.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter CADECM-encoding sequences for a variety of
purposes including,
but not limited to, modification of the cloning, processing, and/or expression
of the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuft7iug
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara 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.
Biotechuol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or improve
the biological properties of CADECM, 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 CADECM 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, CADECM 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
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WO 02/088322 PCT/US02/13874
techniques. (See, e.g., Creighton, T. (1984) Proteins, Structures and
Molecular Properties, WH
Freeman, New York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science
269:202-204.)
Automated synthesis may be achieved using the ABI 431A peptide synthesizer
(Applied Biosystems).
Additionally, the amino acid sequence of CADECM, or any part thereof, may be
altered during direct
synthesis andlor combined with sequences from other proteins, or any part
thereof, to produce a
variant polypeptide or a polypeptide having a sequence of a naturally
occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active CADECM, the nucleotide sequences
encoding
CADECM or derivatives thereof may be inserted into an appropriate expression
vector, i.e., a vector
which contains the necessary elements for transcriptional and translational
control of the inserted
coding sequence in a suitable host. These elements include regulatory
sequences, such as enhancers,
constitutive and inducible promoters, and 5' and 3' uCADECMslated regions in
the vector and in
polynucleotide sequences encoding CADECM. Such elements may vary in their
strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding CADECM. Such signals include the ATG initiation codon and
adjacent
sequences, e.g. the Kozak sequence. In cases where sequences encoding CADECM
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 CADECM 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.)
42
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WO 02/088322 PCT/US02/13874
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding CADECM. These include, but are not limited to, microorganisms such as
bacteria
transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast
transformed with yeast expression vectors; insect cell systems infected with
viral expression vectors
(e.g., baculovirus); plant cell systems transformed with viral expression
vectors (e.g., cauliflower
mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression
vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. (See, e.g., Sambrook, supra;
Ausubel, su ra; Van Heeke,
G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et
al. (1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-
1945; Takamatsu, N.
(1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y
(1992) McGraw
Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl.
Acad. Sci. USA
81:3655-3659; and Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.)
Expression vectors derived
from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from
various bacterial plasmids, may
be used for delivery of nucleotide sequences to the targeted organ, tissue, or
cell population. (See,
e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et
al. (1993) Proc. Natl.
Acad. Sci. USA 90(13):6340-6344; Bullet, R.M. et al. (1985) Nature
317(6040):813-815; McGregor,
D.P. et al. (1994) Mol. Tmmunol. 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 CADECM. For
example, routine
cloning, subcloning, and propagation of polynucleotide sequences encoding
CADECM can be achieved
using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La
Jolla CA) or
PSP~RT1 plasmid (Life Technologies). Ligation of sequences encoding CADECM
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 CADECM are
needed, e.g. for the
production of antibodies, vectors which direct high level expression of CADECM
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 CADECM. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
43
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
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 CADECM. Transcription of
sequences
encoding CADECM may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV
used alone or in combination with the omega leader sequence from TMV
(Takamatsu, N. (1987)
EMBO J. 6:307-311). Alternatively, plant promoters such as the small subunit
of RUBISCO or heat
shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J.
3:1671-1680; Brogue, R.
et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
Cell Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of
Science and Technoloay
(1992) McGraw Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding CADECM
may be ligated
into an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses CADECM 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
CADECM in cell lines is preferred. For example, sequences encoding CADECM 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
44.
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
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 apt 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; tieo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
l0 Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g.~ Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest
is also present, the presence and expression of the gene may need to be
confirmed. For example, if.
2o the sequence encoding CADECM is inserted within a marker gene sequence,
transformed cells
containing sequences encoding CADECM can be identified by the absence of
marker gene function.
Alternatively, a marker gene can be placed in tandem with a sequence encoding
CADECM 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 CADECM
and that
express CADECM 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.
T_mmunological methods for detecting and measuring the expression of CADECM
using either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioiznmunoassays
(RIAs), and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
monoclonal antibodies reactive to two non-interfering epitopes on CADECM 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 Tm_m__unology,
Greene Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Itmnunochemical
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
CADECM include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, the sequences encoding CADECM, 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 CADECM may be
cultured under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors containing
polynucleotides which encode CADECM may be designed to contain signal
sequences which direct
secretion of CADECM 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.
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CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding CADECM 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 CADECM protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of CADECM
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-trausferase (GST),
maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG, c-
myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification
of their cognate
fusion proteins on immobilized glutathione, maltose, phenylarsine oxide,
calmodulin, and metal-chelate
resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffinity purification of
fusion proteins using commercially available monoclonal and polyclonal
antibodies that specifically
recognize these epitope tags. A fusion protein may also be engineered to
contain a proteolytic
cleavage site located between the CADECM encoding sequence and the
heterologous protein
. sequence, so that CADECM may be cleaved away from the heterologous moiety
following
purification. Methods for fusion protein expression and purification are
discussed in Ausubel (1995,
su ra, 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 CADECM may
be
achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ
extract system (Promega).
These systems couple transcription and translation of protein-coding sequences
operably associated
with the T7, T3, or SP6 promoters. Translation takes place in the presence of
a radiolabeled amino
acid precursor, for example, 35S-methionine.
CADECM of the present invention or fragments thereof may be used to screen for
compounds that specifically bind to CADECM. At least one and up to a plurality
of test compounds
may be screened for specific binding to CADECM. 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
CADECM, 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) C~xrrent
Protocols in T_m_m__unolouy 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which
CADECM 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
47
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
these compounds involves producing appropriate cells which express CADECM,
either as a secreted
protein br on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing CADECM or cell membrane fractions which contain CADECM
are then
contacted with a test compound and binding, stimulation, or inhibition of
activity of either CADECM 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
CADECM, either in
solution or affixed to a solid support, and detecting the binding of CADECM 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.
CADECM of the present invention or fragments thereof may be used to screen for
compounds that modulate the activity of CADECM. Such compounds may include
agonists,
antagonists, or partial or inverse agonists. In one embodiment, an assay is
performed under conditions
permissive for CADECM activity, wherein CADECM is combined with at least one
test compound,
and the activity of CADECM in the presence of a test compound is compared with
the activity of
CADECM in the absence of the test compound. A change in the activity of CADECM
in the
presence of the test compound is indicative of a compound that modulates the
activity of CADECM.
Alternatively, a test compound is combined with an in vitro or cell-free
system comprising CADECM
under conditions suitable fox CADECM activity, and the assay is performed. In
either of these
assays, a test compound which modulates the activity of CADECM may do so
indirectly and need not
come in direct contact with the test compound. At least one and up to a
plurality of test compounds
may be screened.
In another embodiment, polynucleotides encoding CADECM 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
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CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
genome by homologous recombination. Alternatively, homologous recombination
takes place using the
Cre-loxP system to knockout a gene of interest in a tissue- or developmental
stage-specific manner
(March, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, I~.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 CADECM 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 CADECM 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 CADECM 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 CADECM, e.g., by secreting CADECM 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 CADECM and cell adhesion and extracellular matrix proteins. In
addition, examples of
tissues expressing CADECM can be found in Table 6 and can also be found in
Example XI.
Therefore, CADECM appears to play a role in immune system disorders,
neurological disorders,
developmental disorders, connective tissue disorders, and cell proliferative
disorders, including cancer.
Iu the treatment of disorders associated with increased CADECM expression or
activity, it is desirable
to decrease the expression or activity of CADECM. Iu the treatment of
disorders associated with
decreased CADECM expression or activity, it is desirable to increase the
expression or activity of
CADECM.
Therefore, in one embodiment, CADECM or a fragment or derivative thereof may
be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
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CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
activity of CADECM. Examples of such disorders include, but are not limited
to, au immune system
disorder, such as acquired immunodeficiency syndrome (AIDS), X-linked
agammaglobinemia of
Bruton, common variable immunodeficiency (CVI), DiGeorge's syndrome (thymic
hypoplasia), thymic
dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease
(SCID),
immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome),
Chediak-Higashi
syndrome, chronic granulomatous diseases, hereditary angioneurotic edema,
immunodeficiency
associated with Cushing's disease, Addison's disease, adult respiratory
distress syndrome, allergies,
ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,
autoimmune hemolytic anemia,
autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy
(APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease,
atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel syndrome,
multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis,
osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid artlwitis, 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 helininthic
infections, and trauma; a neurological disorder, such as epilepsy, ischemic
cerebrovascular disease,
stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's
disease, dementia,
Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral
sclerosis and other motor
neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa,
hereditary ataxias, multiple
sclerosis and other demyelinating diseases, bacterial and viral meningitis,
brain abscess, subdural
empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis
and radiculitis, viral
central nervous system disease, priors diseases including kuru, Creutzfeldt-
Jakob disease, and
Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional
and metabolic diseases
of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system including Down syndrome, cerebral palsy, neuroskeletal
disorders, autonomic nervous
system disorders, cranial nerve disorders, spinal cord diseases, muscular
dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD),
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,
dystonias, paranoid psychoses,
postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy,
corticobasal degeneration,
and familial frontotemporal dementia; a developmental disorder, such as renal
tubular acidosis, anemia,
C~shing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular
dystrophy, epilepsy,
gonadal dysgenesis, WAGR syndrome (Wilins' 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, spine bifida, anencephaly, craniorachischisis, congenital
glaucoma, cataract, and
sensorineural hearing loss; a connective tissue disorder, such as osteogenesis
imperfecta, Ehlers-
Danlos syndrome, chondrodysplasias, Marfan syndrome, Alport syndrome, familial
aortic aneurysm,
achondroplasia, mucopolysaccharidoses, osteoporosis, osteopetrosis, Paget's
disease, rickets,
osteomalacia, hyperparathyroidism, renal osteodystrophy, osteonecrosis,
osteomyelitis, osteoma,
osteoid osteoma, osteoblastoma, osteosarcoma, osteochondroma, chondroma,
chondroblastoma,
chondromyxoid fibroma, chondrosarcoma, fibrous cortical defect, nonossifying
fibroma, fibrous
dysplasia, hbrosarcoma, malignant fibrous histiocytoma, Ewing's sarcoma,
primitive neuroectodermal
tumor, giant cell tumor, osteoarrhritis, rheumatoid arthritis, ankylosiug
spondyloarthritis, Reiter's
syndrome, psoriatic arthritis, enteropathic arthritis, infectious artluitis,
gout, gouty arthritis, calcium
pyrophosphate crystal deposition disease, ganglion, synovial cyst,
villonodular synovitis, systemic
sclerosis, Dupuytren's contracture, hepatic fibrosis, lupus erythematosus,
mixed connective tissue
disease, epidermolysis bullosa simplex, bullous congenital ichthyosiform
erythroderma (epidermolytic
hyperkeratosis), non-epidermolytic and epidermolytic palmoplantar keratoderma,
ichthyosis bullosa of
Siemens, pachyonychia congenita, and white sponge nevus; and a cell
proliferative disorder, such as
actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue
disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera, psoriasis,
primary thrombocythemia, and cancers including adenocarcinoma, leukemia,
lymphoma, melanoma,
myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal
gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal
tract, heart, kidney, liver, lung,
muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus,
thyroid, and uterus.
In another embodiment, a vector capable of expressing CADECM 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 CADECM including, but not limited to, those
described above.
51
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WO 02/088322 PCT/US02/13874
In a further embodiment, a composition comprising a substantially purified
CADECM 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 CADECM
including, but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of CADECM
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of CADECM including, but not limited to, those listed above.
In a further embodiment, an antagonist of CADECM may be administered to a
subject to treat
or prevent a disorder associated with increased expression or activity of
CADECM. Examples of
such disorders include, but are not limited to, those inunune system
disorders, neurological disorders,
developmental disorders, connective tissue disorders, and cell proliferative
disorders, including cancer
described above. In one aspect, an antibody which specifically binds CADECM
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 CADECM.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding CADECM maybe administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of CADECM 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 CADECM may be produced using methods which are generally
known in
the art. In particular, purified CADECM may be used to produce antibodies or
to screen libraries of
pharmaceutical agents to identify those which specifically bind CADECM.
Antibodies to CADECM
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
(Muyldermans, S. (2001) J.
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WO 02/088322 PCT/US02/13874
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
CADECM or with any
fragment or oligopeptide thereof which has immunogenic properties. Depending
on the host species,
various adjuvants may be used to increase immunological response. Such
adjuvants include, but are
not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface
active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH,
and dinitrophenol. Among
adjuvants used in humans, BCG (bacilli Calinette-Guerin) and Corynebacterium
parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
CADECM 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 CADECM 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 CADECM may be prepared using any technique which
provides for
the production of antibody molecules by continuous cell lines in culture.
These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
T_mmunol. 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
CADECM-specific
single chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may
be generated by chain shuft7ing from random combinatorial immunoglobulin
libraries. (See, e.g.,
Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter,
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CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for CADECM may also be
generated. For example, such fragments include, but are not limited to, F(ab~2
fragments produced
by pepsin digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide
bridges of the F(ab~2 fragments. Alternatively, Fab expression libraries may
be constructed to allow
rapid and easy identification of monoclonal Fab fragments with the desired
specificity. (See, e.g.,
Huse, W.D. et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
CADECM and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering CADECM epitopes is generally used, but a competitive
binding assay may also
be employed (Pound, su ra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for CADECM. Affinity is
expressed as an
association constant, Ka, which is defined as the molar concentration of
CADECM-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 CADECM epitopes, represents the average affinity, or
avidity, of the antibodies
for CADECM. The K~ determined for a preparation of monoclonal antibodies,
which are
monospecific for a particular CADECM epitope, represents a true measure of
affinity. High-affinity
antibody preparations with K~ ranging from about 109 to 1012 L/mole are
preferred for use in
immunoassays in which the CADECM-antibody complex must withstand rigorous
manipulations.
Low-affinity antibody preparations with Ka ranging from about 106 to 10'
L/mole are preferred for use
in immunopurification and similar procedures which ultimately require
dissociation of CADECM,
preferably in active form, from the antibody (Catty, D. (1988) Antibodies,
Volume I: A Practical
Approach,1RL 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 CADECM-antibody
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CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
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, su ra, and
Coligan et al. su ra.)
In another embodiment of the invention, the polynucleotides encoding CADECM,
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 CADECM. 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 CADECM. (See, e.g., Agrawal, S., ed. (1996) Antisense
Therapeutics, Humana
Press Inc., Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Tmmunol. ~ 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 CADECM may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combitzed
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),
3o cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G, et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VICI or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
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 CADECM expression or regulation causes
disease, the expression
of CADECM 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
CADECM are treated by constructing mammalian expression vectors encoding
CADECM and
introducing these vectors by mechanical means into CADECM-deficient cells.
Mechanical transfer
technologies for use with cells in vivo or ex vitro include (i) direct DNA
microinjection into individual
cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated
transfection, (iv) receptor-mediated
gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F.
Anderson (1993) Annu.
Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and
H. Recipon (1998)
Curr. Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of CADECM 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).
CADECM 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) C~rr. Opin. Biotechnol. 9:451-456), commercially
available in the T-REX
plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the
plasmids PVGRXR and
PIND; Invitrogen); the FK506/rapamycin inducible promoter; or the
RU486/mifepristone inducible
promoter (Rossi, F.M.V. and H.M. Blau, supra)), or (iii) a tissue-specific
promoter or the native
promoter of the endogenous gene encoding CADECM from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KTT, 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
56
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
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.
(19$2) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of these
standardized mammalian trausfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to CADECM expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding CADECM under the control of an independent promoter or
the retrovirus
long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals,
and (iii) a Rev-
responsive element (RRE) along with additional retrovirus cis-acting RNA
sequences and coding
sequences required for efficient vector propagation. Retrovirus vectors (e.g.,
PFB and PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R. et
al. (1998) J. Virol. 72:9873-9880). U.S. Patent 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 retxovirus 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 CADECM to cells which have one or more genetic
abnormalities with
respect to the expression of CADECM. 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
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CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding CADECM to target cells which have one or more genetic
abnormalities with
respect to the expression of CADECM. The use of herpes simplex virus (HSV)-
based vectors may
be especially valuable for introducing CADECM 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 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 CADECM to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
CADECM into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
CADECM-coding RNAs and the synthesis of high levels of CADECM 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
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CA 02446023 2003-10-28
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therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide
host range of
alphaviruses will allow the introduction of CADECM 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 eDNA clones of alphaviruses, performing
alphavirus cDNA
and RNA transfections, and performing alphavirus infections, are well known to
those with ordinary
skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions -10
and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can
be achieved using triple helix base-pairing methodology. Triple helix pairing
is useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in
Huber, B.E. and B.I. Carr,
Molecular and Immunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-
177.) A
complementary sequence or antisense molecule may also be designed to block
trauslation of mRNA
by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding CADECM.
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 CADECM. 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
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CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
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 CADECM.
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 CADECM
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding CADECM may be therapeutically useful, and in the treatment of
disorders associated with
decreased CADECM expression or activity, a compound which specifically
promotes expression of
the polynucleotide encoding CADECM 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 polynueleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding CADECM 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
CADECM 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
CA 02446023 2003-10-28
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complementary to the sequence of the polynucleotide encoding CADECM. 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 are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g:, Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition which
generally comprises an active ingredient formulated with a pharmaceutically
acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and
proteins. Various
formulations are commonly known and are thoroughly discussed in the latest
edition of Remin~ton's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of
CADECM, antibodies to CADECM, and mimetics, agonists, antagonists, or
inhibitors of CADECM.
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,
iCADECMasal, enteral,
topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
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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). Pulinonary 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 CADECM or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, CADECM 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
CADECM or fragments thereof, antibodies of CADECM, and agonists, antagonists
or inhibitors of
CADECM, 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
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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 /,cg 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 tie 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 CADECM may be used
for the
diagnosis of disorders characterized by expression of CADECM, or in assays to
monitor patients being,
treated with CADECM or agonists, antagonists, or inhibitors of CADECM.
Antibodies useful for
diagnostic purposes may be prepared in the same manner as described above for
therapeutics.
Diagnostic assays for CADECM include methods which utilize the antibody and a
label to detect
CADECM in human body fluids or in extracts of cells or tissues. The antibodies
may be used with or
without modification, and may be labeled by covalent or non-covalent
attachment of a reporter
molecule. A wide variety of reporter molecules, several of which are described
above, are known in
the art and may be used.
A variety of protocols for measuring CADECM, including ELISAs, RIAs, and FACS,
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of CADECM
expression. Normal or standard values for CADECM expression are established by
combining body
fluids or cell extracts taken from normal mammalian subjects, for example,
human subjects, with
antibodies to CADECM under conditions suitable for complex formation. The
amount of standard
complex formation may be quantitated by various methods, such as photometric
means. Quantities of
CADECM 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.
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In another embodiment of the invention, the polynucleotides encoding CADECM
may be used
for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA rilolecules, and PNAs. The polynucleotides may be
used to detect
and quantify gene expression in biopsied tissues in which expression of CADECM
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of CADECM, and to monitor regulation of CADECM levels during
therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding CADECM or closely related
molecules may be
used to identify nucleic acid sequences which encode CADECM. 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
CADECM, allelic variants,
or related sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the CADECM encoding sequences. The hybridization
probes of the
subject invention may be DNA or RNA and may be derived from the sequence of
SEQ ID N0:12-22
or from genomic sequences including promoters, enhaucers, and introns of the
CADECM gene.
Means for producing specific hybridization probes for DNAs encoding CADECM
include the.
cloning of polynucleotide sequences encoding CADECM or CADECM 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 CADECM may be used for the diagnosis of
disorders
associated with expression of CADECM. Examples of such disorders include, but
are not limited to,
an immune system disorder, such as acquired immunodeficiency syndrome (AIDS),
X-linked
agammaglobinemia of Bruton, common variable immunode~.ciency (CVI), DiGeorge's
syndrome
(thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe
combined immunodeficiency
disease (SC7D), immunodeficiency with thrombocytopenia and eczema (Wiskott-
Aldrich syndrome),
Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary
angioneurotic edema,
r_mmunodeftciency associated with Cushing's disease, Addison's disease, adult
respiratory distress
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syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis, autoimmune
hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-
candidiasis-ectodermal
dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohu'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,
osteoat-thritis,
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 heltninthic
infections, and trauma; a neurological disorder, such as epilepsy, ischemic
cerebrovascular disease,
stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's
disease, dementia,
Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral
sclerosis and other motor
neuron disorders, progressive neural muscular atrophy, xetinitis 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 sclexosis, 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 nexvous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD),
akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,
dystonias, paranoid psychoses,
postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy,
corticobasal degeneration,
and familial frontotemporal dementia; a developmental disorder, such as renal
tubular acidosis, anemia,
Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular
dystrophy, epilepsy,
gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary
abnormalities, and mental
retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary
mucoepithelial dysplasia,
hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth
disease and
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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 connective tissue disorder, such as osteogenesis
imperfecta, Ehlers-
Danlos syndrome, chondrodysplasias, Marfan syndrome, Alport syndrome, familial
aortic aneurysm,
achondroplasia, mucopolysaccharidoses, osteoporosis, osteopetrosis, Paget's
disease, rickets,
osteomalacia, hyperparathyroidism, renal osteodystrophy, osteonecrosis,
osteomyelitis, osteoma,
osteoid osteoma, osteoblastoma, osteosarcoma, osteochondroma, chondroma,
chondroblastoma,
chondromyxoid fibroma, chondrosarcoma, fibrous cortical defect, nonossifying
fibroma, fibrous
dysplasia, hbrosarcoma, malignant fibrous histiocytoma, Ewing's sarcoma,
primitive neuroectodermal
tumor, giant cell tumor, osteoarthritis, rheumatoid arthritis, ankylosing
spondyloarthritis, Reiter's
syndrome, psoriatic arthritis, enteropathic arthritis, infectious arthritis,
gout, gouty arthritis, calcium
pyrophosphate crystal deposition disease, ganglion, synovial cyst,
villonodular synovitis, systemic
sclerosis, Dupuytren's contracture, hepatic fibrosis, lupus erythematosus,
mixed connective tissue
disease, epidermolysis bullosa simplex, bullous congenital ichthyosiform
erythroderma (epidermolytic
hyperkeratosis), non-epidermolytic and epidermolytic palmoplantar keratoderma,
ichthyosis bullosa of
Siemens, pachyonychia congenita, and white sponge nevus; and a cell
proliferative disorder, such as
actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue ,
disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera, psoriasis,
primary thrombocythemia, and cancers including adenocarcinoma, leukemia,
lymphoma, melanoma,
myeloma, sarcoma, teratocarciuoma, and, in particular, cancers of the adrenal
gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal
tract, heart, kidney, liver, lung,
muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus,
thyroid, and uterus. The polynucleotide sequences encoding CADECM 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 CADECM expression. Such qualitative or quantitative methods are
well known in the
art.
In a particular aspect, the nucleotide sequences encoding CADECM may be useful
in assays
that detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding CADECM 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
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control sample then the presence of altered levels of nucleotide sequences
encoding CADECM 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
CADECM, 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 CADECM, under conditions suitable
for hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from normal
subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding
CADECM 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 CADECM, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
CADECM, and will be employed under optimized conditions for identification of
a specific gene or
3o 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 CADECM may be used to detect single nucleotide polymorplusms (SNPs).
SNPs are
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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 CADECM are used to amplify
DNA using the
polymerase chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the
secondary and tertiary structures of PCR products in single-stranded form, and
these differences are
detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such as
DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of
individual
overlapping DNA fragments which assemble into a common consensus sequence.
These computer- .
based methods filter out sequence variations due to laboratory preparation of
DNA and sequencing
errors using statistical models and automated analyses of DNA sequence
chromatograms. In the
alternative, SNPs may be detected and characterized by mass spectrometry
using, for example, the
high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
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 ALOXS gene results in
diminished clinical response to treatment with au 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 CADECM 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.
T_m_m__unol. Methods 159:235-244;
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Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed of
quantitation of multiple samples
may be accelerated by running the assay in a high-throughput format where the
oligomer or
polynucleotide of interest is presented in various dilutions and a
spectrophotometric or colorimetric
response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic
profile.
In another embodiment, CADECM, fragments of CADECM, or antibodies specific for
CADECM maybe used as elements~on a microarray. The microarray maybe 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
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case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a
signature similar to that of a compound with known toxicity, it is likely to
share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain
expression information
from a large number of genes and gene families. Ideally, a genome-wide
measurement of expression
provides the highest quality signature. Even genes whose expression is not
altered by any tested
compounds are important as well, as the levels of expression of these genes
are used to normalize the
rest of the expression data. The normalization procedure is useful for
comparison of expression data
after treatment with different compounds. While the assignment of gene
function to elements of a
toxicant signature aids in interpretation of toxicity mechanisms, knowledge of
gene function is not
necessary for the statistical matching of signatures which leads to prediction
of toxicity. (See, for
example, Press Release 00-02 from the National Institute of Environmental
Health Sciences, released .
February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.)
Therefore, it is
important and desirable in toxicological screening using toxicant signatures
to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological sample
containing nucleic acids with the test compound. Nucleic acids that are
expressed in the treated
biological sample are hybridized with one or more probes specific to the
polynucleotides of the present
invention, so that transcript levels corresponding to the polynucleotides of
the present invention may be
quantified. The transcript levels in the treated biological sample are
compared with 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
CA 02446023 2003-10-28
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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 determined by
comparing its partial
sequence, preferably of at least 5 contiguous amino acid residues, to the
polypeptide sequences of the
present invention: In some cases, further sequence data may be obtained for
definitive protein
identification.
A proteomic profile may also be generated using antibodies specific for CADECM
to quantify
the levels of CADECM 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
maybe performed by
a variety of methods known in the art, for example, by reacting the proteins
in the sample with a thiol-
or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at each
array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. Iu 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
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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 comparittg these partial sequences to
the polypeptides of the
presentinvention.
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
txeated 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; Shalon, D, et
al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA
94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
Various types of
microarrays are well known and thoroughly described in DNA Microarrays: A
Practical Approach,
M. Schena, ed. (1999) Oxford University Press, London, hereby expressly
incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding CADECM
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences may
be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a multi-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial chromosomes
(HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
(See, for example, Larder, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.)
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Fluorescent in situ hybridization (FISIT) may be correlated with other
physical and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, su ra, 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 CADECM on
a physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to 11q22-23, any
sequences mapping to that area may represent associated or regulatory genes
for further investigation.
(See, e.g., Gatti, R.A. et 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, CADECM, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between CADECM and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with CADECM,
or fragments
thereof, and washed. Bound CADECM is then detected by methods well known in
the art. Purified
CADECM 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
3o immobilize it on a solid support.
Iu another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding CADECM specifically compete with a test compound
for binding
CADECM. In this manner, antibodies can be used to detect the presence of any
peptide which
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shares one or more antigenic determinants with CADECM.
In additional embodiments, the nucleotide sequences which encode CADECM may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are, therefore,
to be construed as merely illustrative, and not limitative of the remainder of
the disclosure in any way
whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below,
including U.S. Ser. No.60/288,290, U.S. Ser. No.60/292,468, U.S. Ser.
No.60/298,616, U.S. Ser.
No.60/301,672, and U.S. Ser. No.60/345,008 are expressly incorporated by
reference herein.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the L1FESEQ 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
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
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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 XL1-Blue, XL1-BIueMRF, or
SOLR from
Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were resuspended in 0.1-
ml of distilled water and stored, with or without lyoplulization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation such
as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200
thermal cycler
(MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
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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 progr~~rnming, and dinucleotide nearest
neighbor analysis. The
lncyte 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
Sapiens, Rattus norve -gtcus, Mus musculus, Caenorhabditis elegy,
Saccharomyces cerevisiae,
Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto
CA); hidden Markov
model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM
(Haft, D.H. et
al. (2001) Nucleic Acids Res. 29:41-43); and I~VIM-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, BLM'S, and
I~~VIER.
The Incyte cDNA sequences were assembled to produce full length polynucleotide
sequences.
Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched
sequences, or
Genscan-predicted coding sequences (see Examples IV and V) were used to extend
Incyte cDNA
assemblages to full length. Assembly was performed using programs based on
Phred, Phrap, and
Consed, and cDNA assemblages were screened for open reading frames using
programs based on
GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were
translated to derive
the corresponding full length polypeptide sequences. Alternatively, a
polypeptide of the invention may
begin at any of the methionine residues of the full length translated
polypeptide. ' Full length polypeptide
sequences were subsequently analyzed by querying against databases such as the
GenBank protein
databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO,
PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such
as PFAM,
INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART. Full
length
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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
N0:12-22. 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 cell adhesion and extracellular matrix proteins 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 cell adhesion and extracellular
matrix proteins, the
encoded polypeptides were analyzed by querying against PFAM models for cell
adhesion and
extracellular matrix proteins. Potential cell adhesion and extracellular
matrix proteins were also
identified by homology to Incyte cDNA sequences that had been annotated as
cell adhesion and
extracellular matrix proteins. 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
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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 lncyte cDNA coverage was available, this
information was used to
correct or confirm the Genscan predicted sequence. Full length polynucleotide
sequences were
obtained by assembling Genscan-predicted coding sequences with Incyte cDNA
sequences and/or
public cDNA sequences using the assembly process described in Example III.
Alternatively, full
length polynucleotide sequences were derived entirely from edited or unedited
Genscan-predicted
coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
~~Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example 1V. 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 au algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
~~Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, manunalian, vertebrate, and
eukaryote databases
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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 trauslated 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.
1o VI. Chromosomal Mapping of CADECM Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N012-22 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:12-22 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 Iustitute 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 centiMorgau (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances
are based on genetic markers mapped by Genethon which provide boundaries for
radiation hybrid
markers whose sequences were included in each of the clusters. Human genome
maps and other
resources available to the public, such as the NCBI "GeneMap'99" World Wide
Web site
(http://www.ncbi.nlm.nih.gov/genemap~, can be employed to determine if
previously identified disease
genes map within or in proximity to the intervals indicated above.
3o 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,
su ra, ch. 7; Ausubel
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(1995) su ra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or L1FESEQ (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 he 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 SO%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding CADECM 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 ITI). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following organ/tissue categories: cardiovascular
system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female; genitalia,
male; germ cells; hemic and immune system; liver; musculoskeletal system;
nervous system;
pancreas; respiratory system; sense organs; skin; stomatognathic system;
unclassifiedlmixed; 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
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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 CADECM. cDNA sequences and cDNA
library/tissue information are found in the LIFESEQ GOLD database (Incyte
Genomics, Palo Alto
CA).
VIII. Extension of CADECM 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 moxe, and to
anneal to the target
sequence at temperatures of about 68 °C to about 72 °C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR.using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)2504,
and 2-mercaptoethanol, Taq DNA polytizerase (Amersham Pharmacia Biotech),
ELONGASE
enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the
following parameters
for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 60°C, 1 min;
Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step
6: 68 °C, 5 min; Step 7: storage
at 4°C. In the alternative, the parameters for primer pair T7 and SK+
were as follows: Step 1: 94°C,
3 min; Step 2: 94 °C, 15 sec; Step 3: 57 °C, 1 min; Step 4: 68
°C, 2 min; Step 5: Steps 2, 3, and 4
repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~Cl
PICOGREEN
quantitation reagent (0.25°l0 (v/v) PICOGREEN; Molecular Probes, Eugene
OR) dissolved in 1X TE
and 0.5 ~.1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~1 to 10 ,u1 aliquot of the reaction mixture was
analyzed by
81
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WO 02/088322 PCT/US02/13874
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 polymerase (Stratagene) to
fill-in restriction
, site overhangs, and transfected into competent E. coli cells. Transformed
cells were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37 °C in 384-
well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60,,°C, 1 min; Step 4: 72°C, 2 min; Step
5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7:
storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with
20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5'regulatory sequences using the above procedure along with
oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Identification of Single Nucleotide Polymorphisms in CADECM Encoding
Polynucleotides
Common DNA sequence variants known as single nucleotide polymorphisms (SNPs)
were
identified in SEQ ll~ N0:12-22 using the LIFESEQ 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.
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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 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 or 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, S% 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 were not further tested in the other
three populations.
X. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:12-22 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled,by combining 50 pmol of each
oligomer, 250 /.cCi of
~y-32p] adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases: Ase
I, Bgl lI, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40 °C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
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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
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, W, chemical, or mechanical bonding procedures. A
typical array may
be produced using available methods and machines well known to those of
ordinary skill in the art and
may contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science
270:467-470; Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and
J. Hodgson (1998)
Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described
in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~.l oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/~,1 RNase inhibitor, 500 p.M dATP, 500 p.M dGTP, 500
p,M dTTP, 40 ACM
dCTP, 40 ACM 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
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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, Iuc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and resuspended
in 14 ~.15X 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 ~,l of the array
element DNA, at an average
concentration of 100 ng/~.1, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER W-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.
3o Hybridization
Hybridization reactions contain 9 ~1 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
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WO 02/088322 PCT/US02/13874
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
p1 of SX SSC in a corner of the chamber. The chamber containing the arrays is
incubated for about
6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in
a first wash buffer (1X SSC, 0.1%
SDS), three times for 10 minutes each at 45° C in a second wash buffer
(0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
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 1BM-
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
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WO 02/088322 PCT/US02/13874
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).
For example, SEQ ID NO:17 and SEQ ID N0:18 showed differential expression in
colon
tissues from patients with colon cancer compared to matched microscopically
normal tissues from the
same donors as determined by microarray analysis. Therefore, SEQ ID N0:17 and
SEQ ID N0:18
are useful in diagnostic assays for cell proliferative diseases, particularly
colon cancer.
In an alternative example, SEQ ID N0:19 showed differential expression in
mammary
epithelial cells versus various breast carcinoma lines as determined by
microarray analysis. The
expression of SEQ ll~ N0:19 was decreased by at least two fold in the breast
carcinoma lines relative
to normal mammary epithelial cells. Therefore, SEQ ID NO:19 is useful in
diagnostic assays for
detection of breast cancer.
In addition, SEQ ID N0:19 showed differential expression in inflammatory
responses as
determined by microarray analysis. The expression of SEQ ID N0:19 was
decreased by at least two -,
fold in an acute T cell leukemia cell line treated with PMA (a broad activator
of protein kinase C-
dependent pathways) and with ionomycin (a calcium ionophore that causes a
rapid rise in cytosolic
Ca2+ due to both a release of cytosolic Ca2+ stores and Ca2+ influx) compared
to untreated cells from
the same cell line. Therefore, SEQ ID N0:19 is useful in diagnostic assays for
inflammatory
responses.
In an alternative example, SEQ 1D N0:20 showed differential expression in
inflammatory
responses as determined by microarray analysis. The expression of SEQ ID N0:20
was increased by
at least two fold in human umbilical vein endothelial cells treated with tumor
necrosis factor-alpha
(TNF-a) relative to untreated umbilical vein endothelial cells. TNF-a is a
pleiotropic cytokine that
plays a central role in mediation of the inflammatory response through
activation of multiple signal
transduction pathways. TNF-a is produced by activated lymphocytes,
macrophages, and other white
blood cells, and is known to activate endothelial cells. Therefore, SEQ lD
N0:20 is useful in
diagnostic assays for inflammatory responses.
XII. Complementary Polynucleotides
Sequences complementary to the CADECM-encoding sequences, or any parts
thereof, are
used to detect, decrease, or inhibit expression of naturally occurring CADECM.
Although use of
~7
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WO 02/088322 PCT/US02/13874
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 CADECM.
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 CADECM-
encoding transcript.
XIII. Expression of CADECM
Expression and purification of CADECM is achieved using bacterial or virus-
based expression
systems. For expression of CADECM 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 CADECM upon induction with isopropyl
beta-D-
thiogalactopyranoside (IPTG). Expression of CADECM in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autogr~phica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding CADECM 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 Spodo~tera fru ip~ erda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K. et
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, CADECM is synthesized as a fusion protein with,
e.g.,
glutathione S-taransferase (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
CADECM at specifically engineered sites. FLAG, an 8-amino acid peptide,
enables immunoaf~nity
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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WO 02/088322 PCT/US02/13874
resins (QIAGEN). Methods for protein expression and purification are discussed
in Ausubel (1995,
supra, ch. 10 and 16). Purified CADECM obtained by these methods can be used
directly in the
assays shown in Examples XV1I and XVIII where applicable.
XIV. Functional Assays
CADECM function is assessed by expressing the sequences encoding CADECM 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 ~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 noCADECMsfected cells and is a
reliable predictor of
cDNA expression from the recombinant vector. Marker proteins of choice
include, e.g., Green
Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow
cytometry (FCM),
an automated, laser optics-based technique, is used to identify transfected
cells expressing GFP or
CD64-GFP and to evaluate the apoptotic state of the cells and other cellular
properties. FCM detects
and quantifies the uptake of fluorescent molecules that diagnose events
preceding or coincident with
cell death. These events include changes in nuclear DNA content as measured by
staining of DNA
with propidium iodide; changes in cell size and granularity as measured by
forward light scatter and 90
degree side light scatter; down-regulation of DNA synthesis as measured by
decrease in
bromodeoxyuridine uptake; alterations in expression of cell surface and
intracellular proteins as
measured by reactivity with specific antibodies; and alterations in plasma
membrane composition as
measured by the binding of fluorescein-conjugated Annexin V protein to the
cell surface. Methods in
flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford,
New York NY.
The influence of CADECM on gene expression can be assessed using highly
purified
populations of cells transfected with sequences encoding CADECM 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
noCADECMsfected 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 CADECM and
other genes of
interest can be analyzed by northern analysis or microarray techniques.
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XV. Production of CADECM Specific Antibodies
CADECM substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using
standard protocols.
Alternatively, the CADECM amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, su ra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, su ra.) Rabbits are
immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide and anti-CADECM activity by, for example, binding the peptide or
CADECM to a
substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and
reacting with radio-
iodinated goat anti-rabbit IgG.
XVI. Purification of Naturally Occurring CADECM Using Specific Antibodies
Naturally occurring or recombinant CADECM is substantially purified by
immunoaffinity
chromatography using antibodies specific for CADECM: An immunoafbnity column
is constructed by
covalently coupling anti-CADECM 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 CADECM are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of CADECM
(e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/CADECM 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 CADECM is collected.
XVII. Identification of Molecules Which Interact with CADECM
CADECM, or biologically active fragments thereof, are labeled with 1uI Bolton-
Hunter
reagent. (See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-
539.) Candidate
molecules previously arrayed in the wells of a multi-well plate are incubated
with the labeled
CADECM, washed, and any wells with labeled CADECM complex are assayed. Data
obtained using
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WO 02/088322 PCT/US02/13874
different concentrations of CADECM are used to calculate values for the
number, affinity, and
association of CADECM with the candidate molecules.
Alternatively, molecules interacting with CADECM are analyzed using the yeast
two hybrid
system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or
using commercially
available kits based on the two hybrid system, such as the MATCHMAKER system
(Clontech).
CADECM 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).
1o XVIII. Demonstration of CADECM Activity
An assay for CADECM activity measures the expression of CADECM on the cell
surface.
cDNA encoding CADECM is transfected into a non-leukocytic cell line. Cell
surface proteins are
labeled with biotin (de la Fuente, M.A. et al. (1997) Blood 90:2398-2405).
T_mmunoprecipitations are
performed using CADECM-specific antibodies, and immunoprecipitated samples are
analyzed using
SDS-PAGE and immunoblotting techniques. The ratio of labeled immunoprecipitant
to unlabeled
immunoprecipitant is proportional to the amount of CADECM expressed on the
cell surface.
Alternatively, an assay for CADECM activity measures the amount of cell
aggregation
induced by overexpression of CADECM. In this assay, cultured cells such as
NI133T3 are
transfected with cDNA encoding CADECM contained within a suitable mammalian
expression vector
under control of a strong promoter. Cotransfection with cDNA encoding a
fluorescent marker
protein, such as Green Fluorescent Protein (CLONTECH), is useful for
identifying stable
transfectants. The amount of cell agglutination, or clumping, associated with
trahsfected cells is
compared with that associated with uCADECMsfected cells. The amount of cell
agglutination is a
direct measure of CADECM activity.
Alternatively, an assay for CADECM activity measures the disruption of
cytoskeletal filament
networks upon overexpression of CADECM in cultured cell lines (Rezniczek, G.
A. et al. (1998) J.
Cell Biol. 141:209-225). cDNA encoding CADECM is subcloned into a mammalian
expression vector
that drives high levels of cDNA expression. This construct is transfected into
cultured cells, such as
rat kangaroo PtK2 or rat bladder carcinoma 8046 cells. Actin filaments and
intermediate filaments
such as keratin and vimentin are visualized by immunofluorescence microscopy
using antibodies and
techniques well known in the art. The configuration and abundance of
cyoskeletal filaments can be
assessed and quantified using confocal imaging techniques. In particular, the
bundling and collapse of
cytoskeletal filament networks is indicative of CADECM activity.
91
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
Alternatively, cell adhesion activity in CADECM is measured in a 96-well plate
in which wells
are first coated with CADECM by adding solutions of CADECM of varying
concentrations to the
wells. Excess CADECM is washed off with saline, and the wells incubated with a
solution of 1%
bovine serum albumin to block non-specific cell binding. Aliquots of a cell
suspension of a suitable cell
type are then added to the wells and incubated for a period of time at 37
°C. Non-adherent cells are
washed off with saline and the cells stained with a suitable cell stain such
as Coomassie blue. The
intensity of staining is measured using a variable wavelength multi-well plate
reader and compared to a
standard curve to determine the number of cells adhering to the CADECM coated
plates. The degree
of cell staining is proportional to the Bell adhesion activity of CADECM in
the sample.
Alternatively, measures of CADECM activity include tracer fluxes and
electrophysiological
approaches. Tracer fluxes are demonstrated by measuring uptake of labeled
substrates into Xenopus
Iaevis oocytes. Oocytes at stages V and VI are injected with CADECM mRNA (10
ng per oocyte)
and incubated for three days at 18 °C in OR2 medium (82.SmM NaCl, 2.5
mM KCl, 1mM CaCl2,
1mM MgCl2, 1mM NazHP04, 5 mM Hepes, 3.8 mM NaOH , SO~Cg/ml gentamycin, pH 7.8)
to allow
expression of CADECM protein. Oocytes are then transferred to standard uptake
medium (100mM
NaCl, 2 mM KCl, 1mM CaCh, 1mM MgClz, 10 mM HepeslTris pH 7.5). Uptake of
various
neurotransmitters is initiated by adding a 3H substrate to the oocytes. After
incubating for 30 minutes,
uptake is terminated by washing the oocytes three times in Na+-free medium,
measuring the
incorporated 3H, and comparing with controls. CADECM. activity is proportional
to the level of
internalized 3H substrate.
Alternatively, CADECM activity can be demonstrated using an
electrophysiological assay for
ion conductance. Capped CADECM mRNA transcribed with T7 polymerase is injected
into
defolliculated stage V Xenopus oocytes, similar to the previously described
method. Two to seven
days later, transport is measured by two-electrode voltage clamp recording.
Two-electrode voltage
clamp recordings are performed at a holding potential of 50 mV. The data are
filtered at 10 Hz and
recorded with the MacLab digital-to-analog converter and software for data
acquisition and analysis
(AD Instruments, Castle Hill, Australia). To study the dependence of CADECM on
external ions,
_,
sodium can be replaced by choline or N-methyl-D=glucamine and chloride by
gluconate, N03, or S04
(Kavanaugh, M.P. et al. (1992) J. Biol. Chem. 267:22007-22009).
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
92
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
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.
93
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
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WO 02/088322 PCT/US02/13874
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CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
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122
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
<110> INCYTE GENOMICS, INC.
YUE, Henry
LEE, Ernestine A.
DUGGAN, Brendan M.
THANGAVELU, Kavitha
HONCHELL, Cynthia D.
DING, Li
HILLMAN, Jennifer L.
BAUGHN, Mariah R.
KALLICK, Deborah A.
LEE, Sally
WARREN, Bridget A.
' XU, Yuming
TRAN, Uyen K.
LAL, Preeti G.
THORNTON, Michael
HAFALIA, April J.A.
YAO, Monique G.
NGUYEN, Danniel B.
GANDHI, Ameena R.
KHAN, Farrah A.
WALIA, Narinder K.
GRIFFIN, Jennifer A.
CHINN, Anna M.
ELLIOTT. Vicki S.
RAMKUMAR, Jayalaxmi
ARVIZU, Chandra S.
FORSYTHE, Ian J.
<120> CELL ADHESION AND EXTRACELLULAR MATRIX PROTEINS
<130> PF-0968 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/288,290; 60/292,468; 60/298,616; 60/301,672; 60/345,008
<151> 2001-05-02; 2001-05-21; 2001-06-15; 2001-06-28; 2002-01-04
<160> 22
<170> PERL Program
<210> 1
<211> 2053
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2707785CD1
<400> 1
Met Trp Leu Val Thr Phe Leu Leu Leu Leu Asp Ser Leu His Lys
1 5 10 15
Ala Arg Pro Glu Asp Val Gly Thr Ser Leu Tyr Phe Val Asn Asp
1/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
20 25 30
Ser Leu Gln Gln Val Thr Phe Ser Ser Ser Val Gly Val Val Val
35 40 45
Pro Cys Pro Ala Ala Gly Ser Pro Ser Ala Ala Leu Arg Trp Tyr
50 55 60
Leu Ala Thr Gly Asp Asp Ile Tyr Asp Val Pro His Ile Arg His
65 70 75
Val His Ala Asn Gly Thr Leu Gln Leu Tyr Pro Phe Ser Pro Ser
80 85 90
Ala Phe Asn Ser Phe Ile His Asp Asn Asp Tyr Phe Cys Thr Ala
95 100 105
Glu Asn Ala Ala Gly Lys Ile Arg Ser Pro Asn Ile Arg Val Lys
210 125 120
Ala Val Phe Arg Glu Pro Tyr Thr Val Arg Val Glu Asp Gln Arg
125 130 135
Ser Met Arg Gly Asn Val Ala Val Phe Lys Cys Leu Ile Pro Ser
140 145 150
Ser Val Gln Glu Tyr Val Ser Val Val Ser Trp Glu Lys Asp Thr
155 160 165
Val Ser Ile Ile Pro Glu His Arg Phe Phe Tle Thr Tyr His Gly
170 275 180
Gly Leu Tyr Ile Ser Asp Val Gln Lys Glu Asp Ala Leu Ser Thr
185 190 195
Tyr Arg Cys Ile Thr Lys His Lys Tyr Ser Gly Glu Thr Arg Gln
200 205 210
Ser Asn Gly Ala Arg Leu Ser Val Thr Asp Pro Ala Glu Ser Ile
215 220 225
Pro Thr Ile Leu Asp Gly Phe His Ser Gln Glu Val Trp Ala Gly
230 235 240
His Thr Val Glu Leu Pro Cys Thr Ala Ser Gly Tyr Pro Ile Pro
245 250 255
Ala Ile Arg Trp Leu Lys Asp Gly Arg Pro Leu Pro Ala Asp Ser
260 265 270
Arg Trp Thr Lys Arg Ile Thr Gly Leu Thr Ile Ser Asp Leu Arg
275 280 285
Thr Glu Asp Ser Gly Thr Tyr Ile Cys Glu Val Thr Asn Thr Phe
290 295 300
Gly Ser Ala Glu Ala Thr Gly Ile Leu Met Val Ile Asp Pro Leu
305 310 315
His Val Thr Leu Thr Pro Lys Lys Leu Lys Thr Gly Ile Gly Ser
320 325 330
Thr Val Ile Leu Ser Cys Ala Leu Thr Gly Ser Pro Glu Phe Thr
335 340 345
Ile Arg Trp Tyr Arg Asn Thr Glu Leu Val Leu Pro Asp Glu Ala
350 355 360
Ile Ser Ile Arg GIy Leu Ser Asn Glu Thr Leu Leu Ile Thr Ser
365 370 375
Ala Gln Lys Ser His Ser Gly Ala Tyr Gln Cys Phe Ala Thr Arg
380 385 390
Lys Ala Gln Thr Ala Gln Asp Phe Ala Ile Ile Ala Leu Glu Asp
395 400 405
Gly Thr Pro Arg Ile Val Ser Ser Phe Ser Glu Lys Val Val Asn
410 415 420
Pro Gly Glu Gln Phe Ser Leu Met Cys Ala Ala Lys Gly Ala Pro
425 430 435
Pro Pro Thr Val Thr Trp Ala Leu Asp Asp Glu Pro Ile Val Arg
2/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
440 445 450
Asp Gly Ser His Arg Thr Asn Gln Tyr Thr Met Ser Asp Gly Thr
455 460 465
Thr Ile Ser His Met Asn Val Thr Gly Pro Gln Ile Arg Asp Gly
470 475 480
Gly Val Tyr Arg Cys Thr Ala Arg Asn Leu Val Gly Ser Ala Glu
485 490 495
Tyr Gln Ala Arg Ile Asn Val Arg Gly Pro Pro Ser Ile Arg Ala
500 505 510
Met Arg Asn Ile Thr Ala Val Ala Gly Arg Asp Thr Leu Ile Asn
515 520 525
Cys Arg Val Ile Gly Tyr Pro Tyr Tyr Ser Ile Lys Trp Tyr Lys
530 535 540
Asp Ala Leu Leu Leu Pro Asp Asn His Arg Gln Val Val Phe Glu
545 550 555
Asn Gly Thr Leu Lys Leu Thr Asp Val Gln Lys Gly Met Asp Glu
560 565 570
Gly Glu Tyr Leu Cys Ser Val Leu Ile Gln Pro Gln Leu Ser Ile
575 580 585
Ser Gln Ser Val His Val Ala Val Lys Val Pro Pro Leu Ile Gln
590 595 , 600
Pro Phe Glu Phe Pro Pro Ala Ser Ile Gly Gln Leu Leu Tyr Ile
605 610 615
Pro Cys Val Val Ser Ser Gly Asp Met Pro Ile Arg Ile Thr Trp
620 625 630
Arg Lys Asp Gly Gln Val Ile Ile Ser Gly Ser Gly Val Thr Ile
635 640 645
Glu Ser Lys Glu Phe Met Ser Ser Leu Gln Ile Ser Ser Val Ser
650 655 660
Leu Lys His Asn Gly Asn Tyr Thr Cys Ile Ala Ser Asn'Ala Ala
665 670 675
Ala Thr Val Ser Arg Glu Arg Gln Leu Ile Val Arg Val Pro pro
680 685 690
Arg Phe Val Val Gln Pro Asn Asn Gln Asp Gly Ile Tyr Gly Lys
695 700 705
Ala Gly Val Leu Asn Cys Ser Val Asp Gly Tyr Pro Pro Pro Lys
710 715 720
Val Met Trp Lys His Ala Lys Gly Ser Gly Asn Pro Gln Gln Tyr
725 730 735
His Pro Val Pro Leu Thr Gly Arg Ile Gln Ile Leu Pro Asn Ser
740 745 750
Ser Leu Leu Ile Arg His Val Leu Glu Glu Asp Ile Gly Tyr Tyr
755 760 765
Leu Cys Gln Ala Ser Asn Gly Val Gly Thr Asp Ile Ser Lys Ser
770 775 780
Met Phe Leu Thr Val Lys Ile Pro Ala Met Ile Thr Ser His Pro
785 790 795
Asn Thr Thr Ile Ala Ile Lys Gly His Ala Lys Glu Leu Asn Cys
800 805 810
Thr Ala Arg Gly Glu Arg Pro Ile Ile Ile Arg Trp Glu Lys Gly
815 820 825
Asp Thr Val Ile Asp Pro Asp Arg Val Met Arg Tyr Ala Ile Ala
830 835 840
Thr Lys Asp Asn Gly Asp Glu Val Val Ser Thr Leu Lys Leu Lys
845 850 855
Pro Ala Asp Arg Gly Asp Ser Val Phe Phe Ser Cys His Ala Ile
3/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
860 865 870
Asn Ser Tyr Gly Glu Asp Arg Gly Leu Ile Gln Leu Thr Val Gln
875 880 885
Glu Pro Pro Asp Pro Pro Glu Leu Glu Ile Arg Glu Val Lys Ala
890 895 900
Arg Ser Met Asn Leu Arg Trp Thr Gln Arg Phe Asp Gly Asn Ser
905 910 915
Ile Ile Thr Gly Phe Asp Ile Glu Tyr Lys Asn Lys Ser Asp Ser
920 925 930
Trp Asp Phe Lys Gln Ser Thr Arg Asn Ile Ser Pro Thr Ile Asn
935 940 945
Gln Ala Asn Ile Val Asp Leu His Pro Ala Ser Val Tyr Ser Tle
950 955 960
Arg Met Tyr Ser Phe Asn Lys Ile Gly Arg Ser Glu Pro Ser Lys
965 970 975
Glu Leu Thr Ile Ser Thr Glu Glu Ala Ala Pro Asp Gly Pro Pro
980 985 990
Met Asp Val Thr Leu Gln Pro Val Thr Ser Gln Ser Ile Gln Val
995 1000 1005
Thr Trp Lys Ala Pro Lys Lys Glu Leu Gln Asn Gly Val Ile Arg
1010 1015 1020
Gly Tyr Gln Ile Gly Tyr Arg Glu Asn Ser Pro Gly Ser Asn Gly
1025 1030 1035
Gln Tyr Ser Ile Val Glu Met Lys Ala Thr Gly Asp Ser Glu Val
1040 1045 1050
Tyr Thr Leu Asp Asn Leu Lys Lys Phe Ala Gln Tyr Gly Val Val
1055 1060 1065
Val Gln Ala Phe Asn Arg Ala Gly Thr Gly Pro Ser Ser Ser Glu
1070 1075 1080
Ile Asn Ala Thr Thr Leu Glu Asp Val Pro Ser Gln Pro Pro Glu
1085 1090 1095
Asn Val Arg Ala Leu Ser Ile Thr Ser Asp Val Ala Val Ile Ser
1100 1105 1110
Trp Ser Glu Pro Pro Arg Ser Thr Leu Asn Gly Val Leu Lys Gly
1115 1120 1125
Tyr Arg Val Ile Phe Trp Ser Leu Tyr Val Asp Gly Glu Trp Gly
1130 ' 1135 1140
Glu Met Gln Asn Ile Thr Thr Thr Arg Glu Arg Val Glu Leu Arg
1145 1150 1155
Gly Met Glu Lys Phe Thr Asn Tyr Ser Val Gln Val Leu Ala Tyr
1160 1165 1170
Thr Gln Ala Gly Asp Gly Val Arg Ser Ser Val Leu Tyr Ile Gln
1175 1180 1185
Thr Lys Glu Asp Val Pro Gly Pro Pro Ala Gly Ile Lys Ala Val
1190 1195 1200
Pro Ser Ser Ala Ser Ser Val Val Val Ser Trp Leu Pro Pro Thr
1205 1210 1215
Lys Pro Asn Gly Val Ile Arg Lys Tyr Thr Ile Phe Cys Ser Ser
1220 1225 1230
Pro Gly Ser Gly Gln Pro Ala Pro Ser Glu Tyr Glu Thr Ser Pro
1235 1240 1245
Glu Gln Leu Phe Tyr Arg Ile Ala His Leu Asn Arg Gly Gln Gln
1250 1255 1260
Tyr Leu Leu Trp Val Ala Ala Val Thr Ser Ala Gly Arg Gly Asn
1265 1270 1275
Ser Ser Glu Lys Val Thr Ile Glu Pro Ala Gly Lys Ala Pro Ala
4/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
1280 1285 1290
Lys Ile Ile Ser Phe Gly Gly Thr Val Thr Thr Pro Trp Met Lys
1295 ~ 1300 1305
Asp Val Arg Leu Pro Cys Asn Ser Val Gly Asp Pro Ala Pro Ala
1310 1325 1320
Val Lys Trp Thr Lys Asp Ser Glu Asp Ser Ala Ile Pro Val Ser
1325 1330 1335
Met Asp Gly His Arg Leu Ile His Thx Asn Gly Thr Leu Leu Leu
1340 1345 1350
Arg Ala Val Lys Ala Glu Asp Ser Gly Tyr Tyr Thr Cys Thr Ala
1355 1360 1365
Thr Asn Thr Gly Gly Phe Asp Thr Ile Ile Val Asn Leu Leu Val
1370 1375 1380
Gln Val Pro Pro Asp Gln Pro Arg Leu Thr Val Ser Lys Thr Ser
1385 1390 1395
Ala Ser Ser Ile Thr Leu Thr Trp Ile Pro Gly Asp Asn Gly Gly
1400 ~ 1405 1410
Ser Ser Ile Arg Gly Phe Val Leu Gln Tyr Ser Val Asp Asn Ser
1415 1420 1425
Glu Glu Trp Lys Asp Val Phe Ile Ser Ser Ser Glu Arg Ser Phe
1430 1435 1440
Lys Leu Asp Ser Leu Lys Cys Gly Thr Trp Tyr Lys Val Lys Leu
1445 1450 1455
Ala Ala Lys Asn Ser Val Gly Ser Gly Arg Ile Ser Glu Ile Ile
1460 1465 1470
Glu AIa Lys Thr His Gly Arg Glu Pro Ser Phe Ser Lys Asp Gln
1475 1480 1485
His Leu Phe Thr His Ile Asn Ser Thr His Ala Arg Leu Asn Leu
1490 1495 1500
Gln Gly Trp Asn Asn Gly Gly Cys Pro Ile Thr Ala Ile Val Leu
1505 1510 1515
Glu Tyr Arg Pro Lys Gly Thr Trp Ala Trp Gln Gly Leu Arg Ala
1520 1525 1530
Asn Ser Ser Gly Glu Val Phe Leu Thr Glu Leu Arg Glu Ala Thr
1535 1540 1545
Trp Tyr Glu Leu Arg Met Arg Ala Cys Asn Ser Ala Gly Cys Gly
1550 1555 ~ 1560
Asn Glu Thr Ala Gln Phe Ala Thr Leu Asp Tyr Asp Gly Ser Thr
1565 1570 1575
Ile Pro Pro Ile Lys Ser Ala Gln Gly Glu Gly Asp Asp Val Lys
1580 1585 1590
Lys Leu Phe Thr Ile Gly Cys Pro Val Ile Leu Ala Thr Leu Gly
1595 1600 1605
Val Ala Leu Leu Phe Ile Val Arg Lys Lys Arg Lys Glu Lys Arg
1610 1615 1620
Leu Lys Arg Leu Arg Asp Ala Lys Ser Leu Ala Glu Met Leu Ile
1625 1630 1635
Ser Lys Asn Asn Arg Ser Phe Asp Thr Pro Val Lys Gly Pro Pro
1640 1645 1650
Gln Gly Pro Arg Leu His Ile Asp Ile Pro Arg Val Gln Leu Leu
1655 1660 1665
Ile Glu Asp Lys Glu Gly Ile Lys Gln Leu Gly Asp Asp Lys Ala
1670 1675 1680
Thr Ile Pro Val Thr Asp Ala Glu Phe Ser Gln Ala Val Asn Pro
1685 1690 1695
Gln Ser Phe Cys Thr Gly Val Ser Leu His His Pro Thr Leu Ile
5/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
1700 1705 1710
Gln Ser Thr Gly Pro Leu Ile Asp Met Ser Asp Ile Arg Pro Gly
1715 1720 1725
Thr Asn Pro Val Ser Arg Lys Asn Val Lys Ser Ala His Ser Thr
2730 1735 1740
Arg Asn Arg Tyr Ser Ser Gln Trp Thr Leu Thr Lys Cys Gln Ala
1745 1750 1755
Ser Thr Pro Ala Arg Thr Leu Thr Ser Asp Trp Arg Thr Val Gly
1760 1765 1770
Ser Gln His Gly Val Thr Val Thr Glu Ser Asp Ser Tyr Ser Ala
1775 1780 1785
Ser Leu Ser Gln Asp Thr Asp Lys Gly Arg Asn Ser Met Val Ser
1790 1795 1800
Thr Glu Ser Ala Ser Ser Thr Tyr Glu Glu Leu Ala Arg Ala Tyr
1805 1810 1815
Glu His Ala Lys Leu Glu Glu Gln Leu Gln His Ala Lys Phe Glu
1820 1825 1830
Ile Thr Glu Cys Phe Ile Ser Asp Ser Ser Ser Asp Gln Met Thr
1835 1840 1845
Thr Gly Thr Asn Glu Asn Ala Asp Ser Met Thr Ser Met Ser Thr
1850 1855 1860
Pro Ser Glu Pro Gly Ile Cys Arg Phe Thr Ala Ser Pro Pro Lys
1865 1870 1875
Pro Gln Asp Ala Asp Arg Gly Lys Asn Val AIa'Val Pro Ile Pro
1880 1885 1890
His Arg Ala Asn Lys Ser Asp Tyr Cys Asn Leu Pro Leu Tyr Ala
1895 1900 1905
Lys Ser Glu Ala Phe Phe Arg Lys Ala Asp Gly Arg Glu Pro Cys
1910 1915 1920
Pro Val Val Pro Pro Arg Glu Ala Ser Ile Arg Asn Leu Ala Arg
1925 1930 1935
Thr Tyr His Thr Gln Ala Arg His Leu Thr Leu Asp Pro Ala Ser
1940 1945 1950
Lys Ser Leu Gly Leu Pro His Pro Gly Ala Pro Ala Ala Ala Ser
1955 1960 1965
Thr Ala Thr Leu Pro Gln Arg Thr Leu Ala Met Pro Ala Pro Pro
1970 1975 1980
Ala Gly Thr Ala Pro Pro Ala Pro Gly Pro Thr Pro Ala Glu Pro
1985 1990 1995
Pro Thr Ala Pro Ser Ala Ala Pro Pro Ala Pro Ser Thr Glu Pro
2000 2005 2010
Pro Arg Ala Gly Gly Pro His Thr Lys Met Gly Gly Ser Arg Asp
2015 2020 2025
Ser Leu Leu Glu Met Ser Thr Ser Gly Val Gly Arg Ser Gln Lys'
2030 2035 2040
Gln Gly Ala Gly Ala Tyr Ser Lys Ser Tyr Thr Leu Val
2045 2050
<210> 2
<211> 828
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1414780CD1
6/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
<400> 2
Met Arg Pro Arg Pro Glu Gly Arg Gly Leu Arg Ala Gly Val Ala
1 5 10 15
Leu Ser Pro Ala Leu Leu Leu Leu Leu Leu Leu Pro Pro Pro Pro
20 25 30
Thr Leu Leu Gly Arg Leu Trp Ala Ala Gly Thr Pro Ser Pro Ser
35 40 45
Ala Pro Gly Ala Arg Gln Asp Gly Ala Leu Gly Ala Gly Arg Val
50 55 60
Lys Arg Gly Trp Val Trp Asn Gln Phe Phe Val Val Glu Glu Tyr
65 70 75
Thr Gly Thr Glu Pro Leu Tyr Val Gly Lys Ile His Ser Asp Ser
80 85 90
Asp Glu Gly Asp Gly Ala Ile Lys Tyr Thr Ile Ser Gly Glu Gly
95 100 105
Ala Gly Thr Ile Phe Leu Ile Asp Glu Leu Thr Gly Asp Ile His
110 115 120
Ala Met Glu Arg Leu Asp Arg Glu Gln Lys Thr Phe Tyr Thr Leu
125 130 135
Arg Ala Gln Ala Arg Asp Arg Ala Thr Asn Arg Leu Leu Glu Pro
140 145 150
Glu Ser Glu Phe Ile Ile Lys Val Gln Asp Ile Asn Asp Ser Glu
155 160 165
Pro Arg Phe Leu His-Gly Pro Tyr Ile Gly Ser Val Ala Glu Leu
170 175 180
Ser Pro Thr Gly Thr Ser Val Met Gln Val Met Ala Ser Asp Ala
185 190 195
Asp Asp Pro Thr Tyr Gly Ser Ser Ala Arg Leu Val Tyr Ser Val
200 205 210
Leu Asp Gly Glu His His Phe Thr Val Asp Pro Lys Thr Gly Val
215 220 225
Ile Arg Thr Ala Val Pro Asp Leu Asp Arg Glu Ser Gln Glu Arg
230 235 240
Tyr Glu Val Val Ile Gln Ala Thr Asp Met Ala Gly Gln Leu Gly
245 ' 250 255
Gly Leu Ser Gly Ser Thr Thr Val Thr Ile Val Val Thr Asp Val
260 265 270
Asn Asp Asn Pro Pro Arg Phe Pro Gln Lys Met Tyr Gln Phe Ser
275 280 285
Ile Gln Glu Ser Ala Pro Ile Gly Thr Ala Val Gly Arg Val Lys
290 295 300
Ala Glu Asp Ser Asp Val Gly Glu Asn Thr Asp Met Thr Tyr His
305 310 315
Leu Lys Asp Glu Ser Ser Ser Gly Gly Asp Val Phe Lys Val Thr
320 325 330
Thr Asp Ser Asp Thr Gln Glu Ala Ile Ile Val Val Gln Lys Arg
335 340 345
Leu Asp Phe Glu Ser Gln Pro Val His Thr Val Ile Leu Glu Ala
350 355 360
Leu Asn Lys Phe Val Asp Pro Arg Phe Ala Asp Leu Gly Thr Phe
365 370 375
Arg Asp Gln Ala Ile Val Arg Val Ala Val Thr Asp Val Asp Glu
380 385 390
Pro Pro Glu Phe Arg Pro Pro Ser Gly Leu Leu Glu Val Gln Glu
395 400 405
Asp Ala Gln Val Gly Ser Leu Val Gly Val Val Thr Ala Arg Asp
7/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
410 415 420
Pro Asp Ala Ala Asn Arg Pro Val Arg Tyr Ala Ile Asp Arg Glu
425 430 435
Ser Asp Leu Asp Gln Ile Phe Asp Ile Asp Ala Asp Thr Gly Ala
440 445 450
Ile VaI Thr Gly Lys Gly Leu Asp Arg Glu Thr Ala Gly Trp His
455 460 465
Asn Ile Thr Val Leu Ala Met Glu Ala Asp Asn His Ala Gln Leu
470 475 480
Ser Arg Ala Ser Leu Arg Ile Arg Ile Leu Asp Val Asn Asp Asn
485 490 495
Pro Pro Glu Leu Ala Thr Pro Tyr Glu Ala Ala Val Cys Glu Asp
500 505 510
Ala Lys Pro Gly Gln Leu Ile Gln Thr Ile Ser Val Val Asp Arg
515 520 525
Asp Glu Pro Gln Gly Gly His Arg Phe Tyr Phe Arg Leu Val Pro
530 535 540
Glu Ala Pro Ser Asn Pro His Phe Ser Leu Leu Asp Ile Gln Asp
545 550 555
Asn Thr Ala Ala Val His Thr GIn His Val Gly Phe Asn Arg Gln
560 565 570
Glu Gln Asp Val Phe Phe Leu Pro Ile Leu Val Val Asp Ser Gly
575 580 585
Pro Pro Thr Leu Ser Ser Thr Gly Thr Leu Thr Ile Arg Ile Cys
590 595 600
Gly Cys Asp Ser Ser Gly Thr Ile Gln Ser Cys Asn Thr Thr Ala
605 610 615
Phe Val Met Ala Ala Ser Leu Ser Pro Gly Ala Leu Ile AIa Leu
620 625 630
Leu Val Cys Val Leu Ile Leu Val Val Leu Va1 Leu Leu Ile Leu
635 640 645
Thr Leu Arg Arg His His Lys Ser His Leu Ser Sex Asp Glu Asp
650 655 660
Glu Asp Met Arg Asp Asn Val Ile Lys Tyr Asn Asp Glu Gly Gly
665 670 675
Gly Glu Gln Asp Thr Glu Ala Tyr Asp Met Ser Ala Leu Arg Ser
680 685 690
Leu Tyr Asp Phe Gly Glu Leu Lys Gly Gly Asp Gly Gly Gly Ser
695 700 705
Ala Gly Gly Gly Ala Gly Gly Gly Ser Gly Gly Gly Ala Gly Ser
710 715 720
Pro Pro Gln Ala His Leu Pro Ser Glu Arg His Ser Leu Pro Gln
725 730 735
Gly Pro Pro Ser Pro Glu Pro Asp Phe Ser Val Phe Arg Asp Phe
740 745 750
Ile Ser Arg Lys Val Ala Leu Ala Asp Gly Asp Leu Ser Val Pro
755 760 765
Pro Tyr Asp Ala Phe Gln Thr Tyr AIa Phe Glu Gly AIa Asp Ser
770 775 780
Pro Ala Ala Ser Leu Ser Ser Leu His Ser Gly Ser Ser Gly Ser
785 790 795
Glu Gln Asp Phe Ala Tyr Leu Ser Ser Trp Gly Pro Arg Phe Arg
800 805 810
Pro Leu Ala Ala Leu Tyr Ala Gly His Arg Gly Asp Asp Glu Ala
815 820 825
Gln Ala Ser
8/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
<210> 3
<211> 1003
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3109513CD1
<400> 3
Met Asn Gly Gly Asn Glu Sex Ser Gly Ala Asp Arg Ala Gly Gly
1 5 10 15
Pro Val Ala Thr Ser Val Pro Ile Gly Trp Gln Arg Cys Val Arg
20 25 30
Glu Gly Ala Val Leu Tyr Ile Ser Pro Ser Gly Thr Glu Leu Ser
35 40 45
Ser Leu Glu Gln Thr Arg Ser Tyr Leu Leu Ser Asp Gly Thr Cys
50 55 60
Lys Cys Gly Leu Glu Cys Pro Leu Asn Val Pro Lys Val Phe Asn
65 70 75
Phe Asp Pro Leu Ala Pro Val Thr Pro Gly Gly Ala Gly Val Gly
80 85 90
Pro Ala Ser Glu Glu Asp Met Thr Lys Leu Cys Asn His Arg Arg
95 100 105
Lys Ala Val Ala Met Ala Thr Leu Tyr Arg Ser Met Glu Thr Thr
110 115 120
Cys Ser His Ser Ser Pro Gly Glu Gly Ala Ser Pro Gln Met Phe
125 130 135
His Thr Val Ser Pro Gly Pro Pro Ser Ala Arg Pro Pro Cys Arg
140 145 150
Val Pro Pro Thr Thr Pro Leu Asn Gly Gly Pro Gly Ser Leu Pro
155 160 165
Pro Glu Pro Pro Ser Val Ser Gln Ala Phe Pro Thr Leu Ala Gly
170 175 180
Pro Gly Gly Leu Phe Pro Pro Arg Leu Ala Asp Pro Val Pro Ser
185 190 195
Gly Gly Ser Ser Ser Pro Arg Phe Leu Pro Arg Gly Asn Ala Pro
200 205 210
Ser Pro Ala Pro Pro Pro Pro Pro Ala Ile Ser Leu Asn Ala Pro
215 220 225
Ser Tyr Asn Trp Gly Ala Ala Leu Arg Ser Ser Leu Val Pro Ser
230 235 240
Asp Leu Gly Ser Pro Pro Ala Pro His Ala Ser Ser Ser Pro Pro
245 250 255
Ser Asp Pro Pro Leu Phe His Cys Ser Asp Ala Leu Thr Pro Pro
260 265 270
Pro Leu Pro Pro Ser Asn Asn Leu Pro Ala His Pro Gly Pro Ala
275 280 285
Ser Gln Pro Pro Val Ser Ser Ala Thr Met His Leu Pro Leu Val
290 295 300
Leu Gly Pro Leu Gly Gly Ala Pro Thr Val Glu Gly Pro Gly Ala
305 310 315
Pro Pro Phe Leu Ala Ser Ser Leu Leu Ser Ala Ala Ala Lys Ala
320 325 330
9/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
Gln His Pro Pro Leu Pro Pro Pro Ser Thr Leu Gln Gly Arg Arg
335 340 345
Pro Arg Ala Gln Ala Pro Ser Ala Ser His Ser Ser Ser Leu Arg
350 355 360
Pro Ser Gln Arg Arg Pro Arg Arg Pro Pro Thr Val Phe Arg Leu
365 370 375
Leu Glu Gly Arg Gly Pro Gln Thr Pro Arg Arg Ser Arg Pro Arg
380 385 390
Ala Pro Ala Pro Val Pro Gln Pro Phe Ser Leu Pro Glu Pro Ser
395 400 405
Gln Pro Ile Leu Pro Ser Val Leu Ser Leu Leu~Gly Leu Pro Thr
410 415 420
Pro Gly Pro Ser His Ser Asp Gly Ser Phe Asn Leu Leu Gly Ser
425 430 435
Asp Ala His Leu Pro Pro Pro Pro Thr Leu Ser Ser Gly Ser Pro
440 445 450
Pro Gln Pro Arg His Pro Ile Gln Pro Ser Leu Pro Gly Thr Thr
455 460 465
Ser Gly Ser Leu Ser Ser Val Pro Gly Ala Pro Ala Pro Pro Ala
470 475 480
Ala Ser Lys Ala Pro Val Val Pro 5er Pro Val Leu Gln Ser Pro
485 490 495
Ser Glu Gly Leu Gly Met Gly Ala Gly Pro Ala Cys Pro Leu Pro
500 505 510
Pro Leu Ala Gly Gly Glu Ala Phe Pro Phe Pro Ser Pro Glu Gln
515 520 525
Gly Leu Ala Leu Ser Gly AIa Gly Phe Pro Gly Met Leu Gly AIa
530 535 540
Leu Pro Leu Pro Leu Ser Leu Gly Gln Pro Pro Pro Ser Pro Leu
545 550 555
Leu Asn His Ser Leu Phe Gly Val Leu Thr Gly Gly Gly Gly Gln
560 565 570
Pro Pro Pro Glu Pro Leu Leu Pro Pro Pro Gly Gly Pro Gly Pro
575 580 585
Pro Leu Ala Pro Gly Glu Pro Glu Gly Pro Ser Leu Leu Val Ala
590 595 600
Ser Leu Leu Pro Pro Pro Pro Ser Asp Leu Leu Pro Pro Pro Ser
605 610 615
Ala Pro Pro Ser Asn Leu Leu Ala Ser Phe Leu Pro Leu Leu Ala
620 625 630
Leu Gly Pro Thr Ala Gly Asp Gly Glu Gly Ser Ala Glu Gly Ala
635 640 645
Gly Gly Pro Ser Gly Glu Pro Phe Ser Gly Leu Gly Asp Leu Ser
650 655 660
Pro Leu Leu Phe Pro Pro Leu Ser Ala Pro Pro Thr Leu Ile Ala
665 670 675
Leu Asn Ser AIa Leu Leu Ala AIa Thr Leu Asp Pro Pro Ser Gly
680 685 690
Thr Pro Pro Gln Pro Cys Val Leu Ser Ala Pro Gln Pro Gly Pro
695 700 705
Pro Thr Ser Ser Val Thr Thr Ala Thr Thr Asp Pro Gly Ala Ser
710 715 720
Ser Leu Gly Lys Ala Pro Ser Asn Ser Gly Arg Pro Pro Gln Leu
725 730 735
Leu Ser Pro Leu Leu Gly Ala Ser Leu Leu Gly Asp Leu Ser Ser
740 745 750
7.0/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
Leu Thr Ser Ser Pro Gly Ala Leu Pro Ser Leu Leu Gln Pro Pro
755 760 765
Gly Pro Leu Leu Ser Gly Gln Leu Gly Leu Gln Leu Leu Pro Gly
770 775 780
Gly Gly Ala Pro Pro Pro Leu Ser Glu Ala Ser Ser Pro Leu Ala
785 790 795
Cys Leu Leu Gln Ser Leu Gln Ile Pro Pro Glu Gln Pro Glu Ala
800 805 810
Pro Cys Leu Pro Pro Glu Ser Pro Ala Ser Ala Leu Glu Pro Glu
815 820 825
Pro Ala Arg Pro Pro Leu Ser Ala Leu Ala Pro Pro His Gly Ser
830 835 840
Pro Asp Pro Pro Val Pro Glu Leu Leu Thr Gly Arg Gly Ser Gly
845 850 855
Lys Arg Gly Arg Arg Gly Gly Gly Gly Leu Arg Gly Ile Asn Gly
860 865 870
Glu Ala Arg Pro Ala Arg Gly Arg Lys Pro Gly Ser Arg Arg Glu
875 880 885
Pro Gly Arg Leu Ala Leu Lys Trp Gly Thr Arg Gly Gly Phe Asn
890 895 900
Gly Gln Met Glu Arg Ser Pro Arg Arg Thr His His Trp Gln His
905 910 915
Asn Gly Glu Leu Ala Glu Gly Gly Ala Glu Pro Lys Asp Fro Pro
920 925 930
Pro Pro Gly Pro His Ser Glu Asp Leu Lys Val Pro Pro Gly Val
935 940 945
Val Arg Lys Ser Arg Arg Gly Arg Arg Arg Lys Tyr Asn Pro Thr
950 955 960
Arg Asn Ser Asn Ser Ser Arg Gln Asp Ile Thr Leu Glu Pro Ser
965 970 975
Pro Thr Ala Arg Ala Ala Val Pro Leu Pro Pro Arg Ala Arg Pro
980 985 990
Gly Arg Pro Ala Lys Asn Lys Arg Arg Lys Leu Ala Pro
995 1000
<210> 4
<211> 2328
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7326129CD1
<400> 4
Met Glu Val Gln Leu Ser His Ala Asp Val Glu Gly Ser Trp Thr
1 5 10 15
Arg Asp Gly Leu Arg Leu Gln Gln Gly Pro Thr Cys His Leu Ala
20 25 30
Val Arg Gly Pro Met His Thr Leu Thr Leu Ser Gly Leu Arg Pro
35 40 45
Glu Asp Ser Gly Leu Met Val Phe Lys Ala Glu Gly Val His Thr
50 55 60
Ser Ala Arg Leu Val Val Thr Glu Leu Pro Val Ser Phe Ser Arg
65 70 75
Pro Leu Gln Asp Val Val Thr Thr Glu Lys Glu Lys Val Thr Leu
11/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
80 85 90
Glu Cys Glu Leu Ser Arg Pro Asn Val Asp Val Arg Trp Leu Lys
95 100 105
Asp Gly Val Glu Leu Arg Ala Gly Lys Thr Met Ala Ile Ala Ala
110 115 120
Gln Gly Ala Cys Arg Ser Leu Thr Ile Tyr Arg Cys Glu Phe Ala
125 130 135
Asp Gln Gly Val Tyr Val Cys Asp Ala His Asp Ala Gln Ser Ser
140 145 150
Ala Ser Val Lys Val Gln Gly Arg Asn Ile Gln Ile Val Arg Pro
155 160 165
Leu Glu Asp Val Glu Val Met Glu Lys Asp Gly Ala Thr Phe Ser
170 175 180
Cys Glu Val Ser His Asp Glu Val Pro Gly Gln Trp Phe Trp Glu
185 190 . 195
Gly Ser Lys Leu Arg Pro Thr Asp Asn Val Arg Ile Arg Gln Glu
200 205 210
Gly Arg Thr Tyr Thr Leu Ile Tyr Arg Arg Val Leu Ala Glu Asp
215 220 225
Ala Gly Glu Ile Gln Phe Val Ala Glu Asn Ala Glu Ser Arg Ala
230 235 240
Gln Leu Arg Val Lys Glu Leu Pro Val Thr Leu Val Arg Pro Leu
245 250 255
Arg Asp Lys Ile Ala Met Glu Lys His Arg Gly Val Leu Glu Cys
260 265 270
Gln Val Ser Arg Ala Ser Ala Gln Val Arg Trp Phe Lys Gly Ser
275 280 285
Gln Glu Leu Gln Pro Gly Pro Lys Tyr Glu Leu Val Ser Asp Gly
290 295 300
Leu Tyr Arg Lys Leu Ile Ile Ser Asp Val His Ala Glu Asp Glu
305 310 315
Asp Thr Tyr Thr Cys Asp Ala Gly Asp Val Lys Thr Ser Ala Gln
320 325 330
Phe Phe Val Glu Glu Gln Ser Ile Thr Ile Val Arg Gly Leu Gln
335 340 345
Asp Val Thr Val Met Glu Pro Ala Pro Ala Trp Phe Glu Cys Glu
350 355 360
Thr Ser Ile Pro Ser Val Arg Pro Pro Lys Trp Leu Leu Gly Lys
365 370 375
Thr Val Leu Gln Ala Gly Gly Asn Val Gly Leu Glu Gln Glu Gly
380 385 390
Thr Val His Arg Leu Met Leu Arg Arg Thr Cys Ser Thr Met Thr
395 400 405
Gly Pro Val His Phe Thr Val Gly Lys Ser Arg Ser Ser Ala Arg
410 415 420
Leu Val Val Ser Asp Ile Pro Val Val Leu Thr Arg Pro Leu Glu
425 430 435
Pro Lys Thr Gly Arg Glu Leu Gln Ser Val Val Leu Ser Cys Asp
440 445 450
Phe Arg Pro Ala Pro Lys Ala Val Gln Trp Tyr Lys Asp Asp Thr
455 460 4&5
Pro Leu Ser Pro Ser Glu Lys Phe Lys Met Ser Leu Glu Gly Gln
470 475 480
Met Ala Glu Leu Arg Ile Leu Arg Leu Met Pro Ala Asp Ala Gly
485 490 495
Val Tyr Arg Cys Gln Ala Gly Ser Ala His Ser Ser Thr Glu Val
12/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
500 505 510
Thr Val Glu Ala Arg Glu Val Thr Val Thr Gly Pro Leu Gln Asp
515 520 525
Ala Glu Ala Thr Glu Glu Gly Trp Ala Ser Phe Ser Cys Glu Leu
530 535 540
Ser His Glu Asp Glu Glu Val Glu Trp Ser Leu Asn Gly Met Pro
545 550 555
Leu Tyr Asn Asp Ser Phe His Glu Ile Ser His Lys Gly Arg Arg
560 565 570
His Thr Leu Val Leu Lys Ser Ile Gln Arg Ala Asp Ala Gly Ile
575 580 585
Val Arg Ala Ser Ser Leu Lys Val Ser Thr Ser Ala Arg Leu Glu
590 595 600
Val Arg Val Lys Pro Val Val Phe Leu Lys Ala Leu Asp Asp Leu
605 610 615
Ser Ala Glu Glu Arg Gly Thr Leu Ala Leu Gln Cys Glu Val Ser
620 625 630
Asp Pro Glu Ala His Val Val Trp Arg Lys Asp Gly Val Gln Leu
635 640 645
Gly Pro Ser Asp Lys Tyr Asp Phe Leu His Thr Ala Gly Thr Arg
650 655 660
Gly Leu Val Val His Asp Val Ser Pro Glu Asp Ala Gly Leu Tyr
665 670 675
Thr Cys His Met Gly Ser Glu Glu Thr Arg Ala Arg Val Arg Val
680 685 690
His Asp Leu His Val Gly IIe Thr Lys Arg Leu Lys Thr Met Glu
695 700 705
Val Leu Glu Gly Glu Ser Cys Ser Phe Glu Cys Val Leu Ser His
710 715 720
Glu Ser Ala Ser Asp Pro Ala Met Trp Thr Val Gly Gly Lys Thr
725 730 735
Val Gly Ser Ser Ser Arg Phe Gln Ala Thr Arg Gln Gly Arg Lys
740 745 750
Tyr Ile Leu Val Val Arg Glu Ala Ala Pro Ser Asp Ala Gly Glu
755 760 765
Val Val Phe Ser Val Arg Gly Leu Thr Ser Lys Ala Ser Leu Ile
T70 775 '780
Val Arg Glu Arg Pro Ala Ala Ile Ile Lys Pro Leu Glu Asp GIn
785 790 795
Trp Val Ala Pro Gly Glu Asp Val Glu Leu Arg Cys Glu Leu Ser
800 805 810
Arg Ala Gly Thr Pro Val His Trp Leu Lys Asp Arg Lys Ala Ile
815 820 825
Arg Lys Ser Gln Lys Tyr Asp Val Val Cys Glu Gly Thr Met Ala
830 835 840
Met Leu Val Ile Arg Gly Ala Ser Leu Lys Asp Ala Gly Glu Tyr
845 850 855
Thr Cys Glu Val Glu Ala Ser Lys Ser Thr AIa Ser Leu His Val
860 865 870
Glu GIu Lys Ala Asn Cys Phe Thr Glu GIu Leu Thr Asn Leu Gln
875 880 885
Val Glu Glu Lys Gly Thr Ala Val Phe Thr Cys Lys Thr Glu His
890 895 900
Pro Ala Ala Thr Val Thr Trp Arg Lys Gly Leu Leu Glu Leu Arg
905 910 915
Ala Ser Gly Lys His Gln Pro Ser Gln Glu Gly Leu Thr Leu Arg
13/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
920 925 930
Leu Thr Ile Ser Ala Leu Glu Lys Ala Asp Ser Asp Thr Tyr Thr
935 940 ' 945
Cys Asp Ile Gly Gln Ala Gln Ser Arg Ala Gln Leu Leu Val Gln
950 955 960
Gly Arg Arg Val His Ile Ile Glu Asp Leu Glu Asp Val Asp Val
965 970 975
Gln Glu Gly Ser Ser Ala Thr Phe Arg Cys Arg Ile Ser Pro Ala
980 985 990
Asn Tyr Glu Pro Val His Trp Phe Leu Asp Lys Thr Pro Leu His
995 1000 1005
Ala Asn Glu Leu Asn Glu Ile Asp Ala Gln Pro Gly Gly Tyr His
1010 1015 1020
Val Leu Thr Leu Arg Gln Leu Ala Leu Lys Asp Ser Gly Thr Ile
1025 1030 1035
Tyr Phe Glu Ala Gly Asp Gln Arg Ala Ser Ala Ala Leu Arg Val
1040 1045 1050
Thr Glu Lys Pro Ser Val Phe Ser Arg Glu Leu Thr Asp Ala Thr
1055 1060 1065
Ile Thr Glu Gly Glu Asp Leu Thr Leu Val Cys Glu Thr Ser Thr
1070 1075 1080
Cys Asp Ile Pro Val Cys Trp Thr Lys Asp Gly Lys Thr Leu Arg
1085 1090 1095
Gly Ser Ala Arg Cys Gln Leu Ser His Glu Gly His Arg Ala Gln
1100 1105 1110
Leu Leu Ile Thr Gly Ala Thr Leu Gln Asp Ser Gly Arg Tyr Lys
1115 1120 1125
Cys Glu Ala Gly Gly Ala Cys Ser Ser Ser Ile Val Arg Val His
1130 1135 1140
Ala Arg Pro Val Arg Phe Gln Glu Ala Leu Lys Asp Leu Glu Val
1145 1150 1155
Leu Glu Gly Gly Ala Ala Thr Leu Arg Cys Val Leu Ser Ser Val
1160 1165 1170
Ala Ala Pro Val Lys Trp Cys Tyr Gly Asn Asn Val Leu Arg Pro
1175 1180 1185
Gly Asp Lys Tyr Ser Leu Arg Gln Glu Gly Ala Met Leu Glu Leu
1190 1195 1200
Val Val Arg Asn Leu Arg Pro Gln Asp Ser Gly Arg Tyr Ser Cys
1205 1210 1215
Ser Phe Gly Asp Gln Thr Thr Ser Ala Thr Leu Thr Val Thr Ala
1220 1225 1230
Leu Pro Ala Gln Phe Ile Gly Lys Leu Arg Asn Lys Glu Ala Thr
1235 1240 1245
Glu Gly Ala Thr Ala Thr Leu Arg Cys Glu Leu Ser Lys Ala Ala
1250 1255 1260
Pro Val Glu Trp Arg Lys Gly Ser Glu Thr Leu Arg Asp Gly Asp
1265 1270 1275
Arg Tyr Cys Leu Arg Gln Asp Gly Ala Met Cys Glu Leu Gln Ile
1280 1285 1290
Arg Gly Leu Ala Met Val Asp Ala Ala Glu Tyr Ser Cys Val Cys
'1295 1300 1305
Gly Glu Glu Arg Thr Ser Ala Ser Leu Thr Ile Arg Pro Met Pro
1310 1315 1320
Ala His Phe Ile Gly Arg Leu Arg His Gln Glu Ser Ile Glu Gly
1325 1330 1335
Ala Thr Ala Thr Leu Arg Cys Glu Leu Ser Lys Ala Ala Pro Val
14/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
1340 1345 1350
Glu Trp Arg Lys Gly Arg Glu Ser Leu Arg Asp Gly Asp Arg His
1355 1360 1365
Ser Leu Arg Gln Asp Gly Ala Val Cys Glu Leu Gln Ile Cys Gly
1370 1375 1380
Leu Ala Val Ala Asp Ala Gly Glu Tyr Ser Cys Val Cys Gly Glu
1385 1390 1395
Glu Arg Thr Ser Ala Thr Leu Thr Val Lys Ala Leu Pro Ala Lys
1400 1405 1420
Phe Thr Glu Gly Leu Arg Asn Glu Glu Ala Val Glu Gly Ala Thr
1415 1420 1425
Ala Met Leu Trp Cys Glu Leu Ser Lys Val Ala Pro Val Glu Trp
1430 1435 1440
Arg Lys Gly Pro Glu Asn Leu Arg Asp Gly Asp Arg Tyr Ile Leu
1445 1450 1455
Arg Gln Glu Gly Thr Arg Cys Glu Leu Gln Ile Cys Gly Leu Ala
1460 1465 1470
Met Ala Asp Ala Gly Glu Tyr Leu Cys Val Cys Gly Gln Glu Arg
1475 1480 1485
Thr Ser Ala Thr Leu Thr Ile Arg Ala Leu Pro Ala Arg Phe Ile
1490 1495 1500
Glu Asp Val Lys Asn Gln Glu Ala Arg Glu Gly Ala Thr Ala Val
1505 1510 1515
Leu Gln Cys Glu Leu Asn Ser Ala Ala Pro Val Glu Trp Arg Lys
1520 1525 1530
GIy Ser Glu Thr Leu Arg Asp Gly Asp Arg Tyr Ser Leu Arg Gln
1535 1540 1545
Asp Gly Thr Lys Cys Glu Leu Gln Ile Arg Gly Leu Ala Met Ala
1550 1555 1560
Asp Thr Gly Glu Tyr Ser Cys Val Cys Gly Gln Glu Arg Thr Ser
1565 1570 1575
Ala Met Leu Thr Val Arg Ala Leu Pro Ile Lys Phe Thr Glu Gly
1580 1585 2590
Leu Arg Asn Glu Glu Ala Thr Glu Gly Ala Thr Ala Val Leu Arg
1595 1600 1605
Cys Glu Leu Ser Lys Met Ala Pro Val Glu Trp Trp Lys Gly His
1610 1615 1620
Glu Thr Leu Arg Asp Gly Asp Arg His Ser Leu Arg Gln Asp Gly
1625 1630 1635
Ala Arg Cys Glu Leu Gln Ile Arg Gly Leu Val Ala Glu Asp Ala
1640 1645 1650
Gly Glu Tyr Leu Cys Met Cys Gly Lys Glu Arg Thr Ser Ala Met
1655 1660 1665
Leu Thr Val Arg Ala Met Pro Ser Lys Phe Ile Glu Gly Leu Arg
1670 1675 1680
Asn Glu Glu Ala Thr Glu Gly Asp Thr Ala Thr Leu Trp Cys Glu
1685 1690 1695
Leu Ser Lys Ala Ala Pro Val Glu Trp Arg Lys Gly His Glu Thr
1700 1705 1710
Leu Arg Asp Gly Asp Arg His Ser Leu Arg Gln Asp Gly Ser Arg
1715 1720 1725
Cys Glu Leu Gln Ile Arg Gly Leu Ala Val Val Asp Ala Gly Glu
1730 1735 1740
Tyr Ser Cys Va1 Cys Gly Gln Glu Arg Thr Ser Ala Thr Leu Thr
1745 1750 1755
Val Arg Ala Leu Pro Ala Arg Phe Ile Glu Asp Val Lys Asn Gln
15/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
1760 1765 1770
Glu Ala Arg Glu Gly Ala Thr Ala Val Leu Gln Cys Glu Leu Ser
1775 1780 1785
Lys Ala Ala Pro Val Glu Trp Arg Lys Gly Ser Glu Thr Leu Arg
1790 1795 1800
Gly Gly Asp Arg Tyr Ser Leu Arg Gln Asp Gly Thr Arg Cys Glu
1805 1810 1815
Leu Gln Ile His Gly Leu Ser Val Ala Asp Thr Gly Glu Tyr Ser
1820 1825 1830
Cys Val Cys Gly Gln Glu Arg Thr Ser Ala Thr Leu Thr Val Arg
1835 1840 1845
Ala Leu Pro Ala Arg Phe Thr Gln Asp Leu Lys Thr Lys Glu Ala
1850 1855 1860
Ser Glu GIy Ala Thr Ala Thr Leu GIn Cys Glu Leu Ser Lys VaI
1865 1870 1875
Ala Pro Val Glu Trp Lys Lys Gly Pro Glu Thr Leu Arg Asp Gly
1880 1885 1890
Gly Arg Tyr Ser Leu Lys Gln Asp Gly Thr Arg Cys Glu Leu Gln
1895 1900 1905
Ile His Asp Leu Ser Val Ala Asp Ala Gly Glu Tyr Ser Cys Met
1910 1915 1920
Cys Gly Gln Glu Arg Thr Ser Ala Met Leu Thr Val Arg Ala Leu
1925 1930 1935
Pro Ala Arg Phe Thr Glu Gly Leu Arg Asn Glu Glu Ala Met Glu
1940 1945 1950
Gly Ala Thr Ala Thr Leu Gln Cys Glu Leu Ser Lys Ala Ala Pro
1955 1960 1965
Val Glu Trp Arg Lys Gly Leu Glu Ala Leu Arg Asp Gly Asp Lys
1970 1975 1980
Tyr Ser Leu Arg Gln Asp Gly Ala Val Cys Glu Leu Gln Ile His
1985 1990 1995
Gly Leu Ala Met Ala Asp Asn Gly Val Tyr Ser Cys Val Cys Gly
2000 2005 2010
Gln Glu Arg Thr Ser Ala Thr Leu Thr Val Arg Ala Leu Pro Ala
2015 2020 2025
Arg Phe Ile Glu Asp Met Arg Asn Gln Lys Ala Thr Glu Gly Ala
2030 2035 2040
Thr Val Thr Leu Gln Cys Lys Leu Arg Lys Ala Ala Pro Val Glu
2045 2050 2055
Trp Arg Lys Gly Pro Asn Thr Leu Lys Asp Gly Asp Arg Tyr Ser
2060 2065 2070
Leu Lys Gln Asp Gly Thr Ser Cys Glu Leu Gln Ile Arg Gly Leu
2075 2080 2085
Val Ile Ala Asp Ala Gly Glu Tyr Ser Cys Ile Cys Glu Gln Glu
2090 2095 2100
Arg Thr Ser Ala Thr Leu Thr Val Arg Ala Leu Pro Ala Arg Phe
2105 2110 2115
Ile Glu Asp Val Arg Asn His Glu Ala Thr Glu Gly Ala Thr Ala
2120 2125 2130
Val Leu Gln Cys Glu Leu Ser Lys Ala Ala Pro Val Glu Trp Arg
2135 2140 2145
Lys Gly Ser Glu Thr Leu Arg Asp Gly Asp Arg Tyr Ser Leu Arg
2150 2155 2160
Gln Asp Gly Thr Arg Cys Glu Leu Gln Ile Arg Gly Leu Ala Val
2165 2170 2175
Glu Asp Thr Gly Glu Tyr Leu Cys Val Cys Gly Gln Glu Arg Thr
16/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
2180 2185 2190
Ser Ala Thr Leu Thr Val Arg Ala Leu Pro Ala Arg Phe Ile Asp
2295 2200 2205
Asn Met Thr Asn Gln Glu Ala Arg Glu Gly Ala Thr Ala Thr Leu
2210 2215 2220
His Cys Glu Leu Ser Lys Ala Ala Pro Val Glu Trp Arg Lys Gly
2225 2230 2235
Arg Glu Ser Leu Arg Asp Gly Asp Arg His Ser Leu Arg Gln Asp
2240 2245 2250
Gly Ala Val Cys Glu Leu Gln Ile Cys Gly Leu Ala Val Ala Asp
2255 2260 2265
Ala Gly Glu Tyr Ser Cys Val Cys Gly Glu Glu Arg Thr Ser Ala
2270 2275 2280
Thr Leu Thr Val Lys Gly Asn Asp Cys Ser Trp Pro Arg Ala Trp
2285 2290 2295
Val Ala Met Ser Glu Arg Val Cys Thr Phe Leu Leu Cys Ala His
2300 2305 2310
Val Cys Ala Val Ala Phe Pro Val Phe Leu Arg Val Val Pro Ser
2315 2320 2325
Phe Leu Gln
<210> 5
<211> 1148
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 8065556CD1
<400> 5
Met Glu Ser Leu Leu Leu Pro Val Leu Leu Leu Leu Ala Ile Leu
1 5 10 15
Trp Thr Gln Ala Ala Ala Leu Ile Asn Leu Lys Tyr Ser Val Glu
20 25 30
Glu Glu Gln Arg Ala Gly Thr Val Ile Ala Asn Val Ala Lys Asp
35 40 45
Ala Arg Glu Ala Gly Phe Ala Leu Asp Pro Arg Gln Ala Ser Ala
50 55 60
Phe Arg Val Val Ser Asn Ser Ala Pro His Leu Val Asp Ile Asn
65 70 75
Pro Ser Ser Gly Leu Leu Val Thr Lys Gln Lys Ile Asp Arg Asp
80 85 90
Leu Leu Cys Arg Gln Ser Pro Lys Cys Ile Ile Ser Leu Glu Val
95 100 105
Met Ser Ser Ser Met Glu Ile Cys Val Ile Lys Val Glu Ile Lys
110 115 120
Asp Leu Asn Asp Asn Ala Pro Ser Phe Pro Ala Ala Gln Ile Glu
125 130 135
Leu Glu Ile Ser Glu Ala Ala Ser Pro Gly Thr Arg Ile Pro Leu
140 145 150
Asp Ser Ala Tyr Asp Pro Asp Ser Gly Ser Phe Gly Val Gln Thr
155 160 165
Tyr Glu Leu Thr Pro Asn Glu Leu Phe Gly Leu Glu Ile Lys Thr
170 175 180
17/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
Arg Gly Asp Gly Ser Arg Phe Ala Glu Leu Val Val Glu Lys Ser
185 190 195
Leu Asp Arg Glu Thr Gln Ser His Tyr Ser Phe Arg Ile Thr Ala
200 205 210
Leu Asp Gly Gly Asp Pro Pro Arg Leu Gly Thr Val Gly Leu Ser
215 220 225
Ile Lys Val Thr Asp Ser Asn Asp Asn Asn Pro Val Phe Ser Glu
230 235 240
Ser Thr Tyr Ala Val Ser Val Pro Glu Ile Ser Pro Pro Asn Thr
245 250 255
Pro Val Ile Arg Leu Asn Ala Ser Asp Pro Asp Glu Gly Thr Asn
260 265 270
Gly Gln Val Val Tyr Ser Phe Tyr Gly Tyr Val Asn Asp Arg Thr
275 280 285
Arg Glu Leu Phe Gln Ile Asp Pro His Ser Gly Leu Val Thr Val
290 295 300
Thr Gly Ala Leu Asp Tyr Glu Glu GIy His VaI Tyr Glu Leu Asp
305 310 315
Val Gln Ala Lys Asp Leu Gly Pro Asn Ser Ile Pro Ala His Cys
320 325 330
Lys Val Thr Val Ser Val Leu Asp Thr Asn Asp Asn Pro Pro Val
335 340 345
Ile Asn Leu Leu Ser Val Asn Ser Glu Leu Val Glu Val Ser Glu
350 355 360
Ser Ala Pro Pro Gly Tyr Val Ile Ala Leu Val Arg Val Ser Asp
365 370 375
Arg Asp Ser Gly Leu Asn Gly Arg Val Gln Cys Arg Leu Leu Gly
380 385 390
Asn Val Pro Phe Arg Leu Gln Glu Tyr Glu Ser Phe Ser Thr Ile
395 400 405
Leu Val Asp Gly Arg Leu Asp Arg Glu Gln His Asp Gln Tyr Asn
410 415 420
Leu Thr Ile Gln Ala Arg Asp Gly Gly Val Pro Met Leu Gln Ser
425 430 435
Ala Lys Ser Phe Thr Val Leu Ile Thr Asp Glu Asn Asp Asn His
440 445 450
Pro His Phe Ser Lys Pro Tyr Tyr Gln Val Ile Val Gln Glu Asn
455 460 465
Asn Thr Pro Gly Ala Tyr Leu Leu Ser Val Ser Ala Arg Asp Pro
470 475 480
Asp Leu Gly Leu Asn Gly Ser Val Ser Tyr Gln Ile Val Pro Ser
485 490 495
Gln Val Arg Asp Met Pro Val Phe Thr Tyr Val Ser Ile Asn Pro
500 505 510
Asn Ser Gly Asp Ile Tyr Ala Leu Arg Ser Phe Asn His Glu Gln
515 520 525
Thr Lys Ala Phe Glu Phe Lys Val Leu Ala Lys Asp Gly Gly Leu
530 535 540
Pro Ser Leu Gln Ser Asn Ala Thr Val Arg Val Ile Ile Leu Asp
545 550 555
Val Asn Asp Asn Thr Pro Val Ile Thr Ala Pro Pro Leu Ile Asn
560 565 570
Gly Thr Ala Glu Val Tyr Ile Pro Arg Asn Ser Gly Ile Gly Tyr
575 580 585
Leu Val Thr Val Val Lys Ala Glu Asp Tyr Asp Glu Gly Glu Asn
590 595 600
18/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
Gly Arg Val Thr Tyr Asp Met Thr Glu Gly Asp Arg Gly Phe Phe
605 610 615
Glu Ile Asp Gln Val Asn Gly Glu Val Arg Thr Thr Arg Thr Phe
620 625 630
Gly Glu Ser Ser Lys Ser Ser Tyr Glu Leu Tle Val Val Ala His
635 640 645
Asp His Gly Lys Thr Ser Leu Ser Ala Ser Ala Leu Val Leu Tle
650 655 660
Tyr Leu Ser Pro Ala Leu Asp Ala Gln Glu Ser Met Gly Ser Val
665 - 670 675
Asn Leu Ser Leu Ile Phe Ile Ile Ala Leu Gly Ser Ile Ala Gly
680 685 690
Ile Leu Phe Val Thr Met Ile Phe Val Ala Ile Lys Cys Lys Arg
695 700 705
Asp Asn Lys Glu Ile Arg Thr Tyr Asn Cys Ser Asn Cys Leu Thr
710 715 720
Ile Thr Cys Leu Leu Gly Cys Phe Ile Lys Gly Gln Asn Ser Lys
725 730 735
Cys Leu His Cys Ile Ser Val Ser Pro Ile Ser Glu Glu Gln Asp
740 745 750
Lys Lys Thr Glu Glu Lys Val Ser Leu Arg Gly Lys Arg Ile Ala
755 760 765
Glu Tyr Ser Tyr Gly His Gln Lys Lys Ser Ser Lys Lys Lys Lys
770 775 780
Ile Ser Lys Asn Asp Ile Arg Leu Val Pro Arg Asp Val Glu Glu
785 790 795
Thr Asp Lys Met Asn Val Val Ser Cys Ser Ser Leu Thr Ser Ser
800 805 810
Leu Asn Tyr Phe Asp Tyr His Gln Gln Thr Leu Pro Leu Gly Cys
815 820 825
Arg Arg Ser Glu Ser Thr Phe Leu Asn Val Glu Asn Gln Asn Thr
830 835 840
Arg Asn Thr Ser Ala Asn His Ile Tyr His His Ser Phe Asn Ser
845 850 855
Gln Gly Pro Gln Gln Pro Asp Leu Ile Ile Asn Gly Val Pro Leu
860 865 870
Pro Glu Thr Glu Asn Tyr Ser Phe Asp Ser Asn Tyr Val Asn Ser
875 880 885
Arg Ala His Leu Ile Lys Ser Ser Ser Thr Phe Lys Asp Leu Glu
890 895 900
Gly Asn Ser Leu Lys Asp Ser Gly His Glu Glu Ser Asp Gln Thr
905 910 915
Asp Ser Glu His Asp Val Gln Arg Ser Leu Tyr Cys Asp Thr Ala
920 925 930
Val Asn Asp Val Leu Asn Thr Ser Val Thr Ser Met Gly Ser Gln
935 940 945
Met Pro Asp His Asp Gln Asn Glu Gly Phe His Cys Arg Glu Glu
950 955 960
Cys Arg Ile Leu Gly His Ser Asp Arg Cys Trp Met Pro Arg Asn
965 970 975
Pro Met Pro Ile Arg Ser Lys Ser Pro Glu His Val Arg Asn Ile
980 985 990
Ile Ala Leu Ser Ile Glu Ala Thr Ala Ala Asp Val Glu Ala Tyr
995 1000 1005
Asp Asp Cys Gly Pro Thr Lys Arg Thr Phe Ala Thr Phe Gly Lys
1010 1015 1020
19/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
Asp Val Ser Asp His Pro Ala Glu Glu Arg Pro Thr Leu Lys Gly
1025 1030 1035
Lys Arg Thr Val Asp Val Thr Ile Cys Ser Pro Lys Val Asn Ser
1040 1045 1050
Val Ile Arg Glu Ala Gly Asn Gly Cys Glu Ala Ile Ser Pro Val
1055 1060 1065
Thr Ser Pro Leu His Leu Lys Ser Ser Leu Pro Thr Lys Pro Ser
1070 1075 1080
Val Ser Tyr Thr Ile Ala Leu Ala Pro Pro Ala Arg Asp Leu Glu
1085 1090 1095
Gln Tyr Val Asn Asn Val Asn Asn Gly Pro Thr Arg Pro Ser Glu
1100 1105 1110
Ala Glu Pro Arg Gly Ala Asp Ser Glu Lys Val Met His Glu Val
1115 1120 1125
Ser Pro Ile Leu Lys Glu Gly Arg Asn Lys Glu Ser Pro Gly Val
1130 1135 1140
Lys Arg Leu Lys Asp Ile Val Leu
1145
<210> 6
<211> 1026
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7037678CD1
<400> 6
Met Arg Leu Pro Trp Glu Leu Leu Val Leu Gln Ser Phe Ile Trp
1 5 10 15
Cys Leu Ala Asp Asp Ser Thr Leu His Gly Pro Ile Phe Ile Gln
20 25 30
Glu Pro Ser Pro Val Met Phe Pro Leu Asp Ser Glu Glu Lys Lys
35 40 45
Val Lys Leu Asn Cys Glu Val Lys Gly Asn Pro Lys Pro His Ile
~50 55 60
Arg Trp Lys Leu Asn Gly Thr Asp Val Asp Thr Gly Met Asp Phe
65 70 75
Arg Tyr Ser Val Val Glu Gly Ser Leu Leu Ile Asn Asn Pro Asn
80 85 90
Lys Thr Gln Asp Ala Gly Thr Tyr Gln Cys Thr Ala Thr Asn Ser
95 100 105
Phe Gly Thr Ile Val Ser Arg Glu Ala Lys Leu Gln Phe Ala Tyr
110 115 120
Leu Asp Asn Phe Lys Thr Arg Thr Arg Ser Thr Val Ser Val Arg
125 130 135
Arg Gly Gln Gly Met Val Leu Leu Cys Gly Pro Pro Pro His Ser
140 145 150
Gly Glu Leu Ser Tyr Ala Trp Ile Phe Asn Glu Tyr Pro Ser Tyr
155 160 165
Gln Asp Asn Arg Arg Phe Val Ser Gln Glu Thr Gly Asn Leu Tyr
170 175 180
Ile Ala Lys Val Glu Lys Ser Asp Val Gly Asn Tyr Thr Cys Val
185 190 195
Val Thr Asn Thr Val Thr Asn His Lys Val Leu Gly Pro Pro Thr
20/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
200 205 210
Pro Leu Ile Leu Arg Asn Asp Gly Val Met Gly Glu Tyr Glu Pro
215 220 225
Lys Ile Glu Val Gln Phe Pro Glu Thr Val Pro Thr Ala Lys Gly
230 235 240
Ala Thr Val Lys Leu Glu Cys Phe Ala Leu Gly Asn Pro Val Pro
245 250 255
Thr Ile Ile Trp Arg Arg Ala Asp Gly Lys Pro Ile Ala Arg Lys
260 265 270
Ala Arg Arg His Lys Ser Asn Gly Ile Leu Glu Ile Pro Asn Phe
275 280 285
Gln Gln Glu Asp Ala Gly Leu Tyr Glu Cys Val Ala Glu Asn Ser
290 295 300
Arg Gly Lys Asn Val Ala Arg Gly Gln Leu Thr Phe Tyr Ala Gln
305 310 315
Pro Asn Trp Ile Gln Lys Ile Asn Asp Ile His Val Ala Met Glu
320 325 330
Glu Asn Val Phe Trp Glu Cys Lys Ala Asn Gly Arg Pro Lys Pro
335 340 345
Thr Tyr Lys Trp Leu Lys Asn Gly Glu Pro Leu Leu Thr Arg Asp
350 355 360
Arg Ile Gln Ile Glu Gln Gly Thr Leu Asn Ile Thr Ile Val Asn
365 370 375
Leu Ser Asp Ala Gly Met Tyr Gln Cys Leu Ala Glu Asn Lys His
380 385 390
Gly Val Ile Phe Ser Asn Ala Glu Leu Ser Val Ile Ala Val Gly
395 400 405
Pro Asp Phe Ser Arg Thr Leu Leu Lys Arg Val Thr Leu Val Lys
410 415 420
Val Gly Gly Glu Val Val Ile Glu Cys Lys Pro Lys Ala Ser Pro
425 430 435
Lys Pro Val Tyr Thr Trp Lys Lys Gly Arg Asp Ile Leu Lys Glu
440 445 450
Asn Glu Arg Ile Thr Ile Ser Glu Asp Gly Asn Leu Arg Ile Ile
455 460 465
Asn Val Thr Lys Ser Asp Ala Gly Ser Tyr Thr Cys Ile Ala Thr
470 475 480
Asn His Phe Gly Thr Ala Ser Ser Thr Gly Asn Leu Val Val Lys
485 490 495
Asp Pro Thr Arg Val Met Val Pro Pro Ser Ser Met Asp Val Thr
500 505 510
Val Gly Glu Ser Ile Val Leu Pro Cys Gln Val Thr His Asp His
515 520 525
Ser Leu Asp I1e Val Phe Thr Trp Ser Phe Asn Gly His Leu Ile
530 535 540
Asp Phe Asp Arg Asp Gly Asp His Phe Glu Arg Val Gly Gly Gln
545 550 555
Asp Ser Ala Gly Asp Leu Met Ile Arg Asn Ile Gln Leu Lys His
560 565 570
Ala Gly Lys Tyr Val Cys Met Val Gln Thr Ser Val Asp Arg Leu
575 580 585
Ser Ala Ala Ala Asp Leu Ile Val Arg Gly Pro Pro Gly Pro Pro
590 595 600
Glu Ala Val Thr Ile Asp Glu Ile Thr Asp Thr Thr Ala Gln Leu
605 610 615
Ser Trp Arg Pro Gly Pro Asp Asn His Ser Pro Ile Thr Met Tyr
21/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
620 625 630
Val Ile Gln Ala Arg Thr Pro Phe Ser Val Gly Trp Gln Ala Val
635 640 645
Ser Thr Val Pro Glu Leu Ile Asp Gly Lys Thr Phe Thr Ala Thr
650 655 660
Val Val Gly Leu Asn Pro Trp Val Glu Tyr Glu Phe Arg Thr Val
665 670 675
Ala Ala Asn Val Ile Gly Ile Gly Glu Pro Ser Arg Pro Ser Glu
680 685 690
Lys Arg Arg Thr Glu Glu Ala Leu Pro Glu Val Thr Pro Ala Asn
695 700 705
Val Ser Gly Gly Gly Gly Ser Lys Ser Glu Leu Val Ile Thr Trp
710 715 720
Glu Thr Val Pro Glu Glu Leu Gln Asn Gly Arg Gly Phe Gly Tyr
725 730 735
Val Val Ala Phe Arg Pro Tyr Gly Lys Met Ile Trp Met Leu Thr
740 745 750
Val Leu Ala Ser Ala Asp Ala Ser Arg Tyr Val Phe Arg Asn Glu
755 760 765
Ser Val His Pro Phe Ser Pro Phe Glu Vah Lys Val Gly Val Phe
770 775 780
Asn Asn Lys Gly Glu Gly Pro Phe Ser Pro Thr Thr Val Val Tyr
785 790 795
Ser Ala Glu Glu Glu Pro Thr Lys Pro Pro Ala Ser Ile Phe Ala
800 805 810
Arg Ser Leu Ser Ala Thr Asp Ile Glu Val Phe Trp Ala Ser Pro
815 820 825
Leu Glu Lys Asn Arg Gly Arg Ile Gln Gly Tyr Glu Val Lys Tyr
830 835 840
Trp Arg His Glu Asp Lys Glu Glu Asn Ala Arg Lys Ile Arg Thr
845 850 855
Val Gly Asn Gln Thr Ser Thr Lys Ile Thr Asn Leu Lys Gly Ser
860 865 870
Val Leu Tyr His Leu Ala Val Lys Ala Tyr Asn Ser Ala Gly Thr
875 880 885
Gly Pro Ser Ser Ala Thr Val Asn Val Thr Thr Arg Lys Pro Pro
890 895 900
Pro Ser Gln Pro Pro Gly Asn Ile Ile Trp Asn Ser Ser Asp Ser
905 910 915
Lys Ile Ile Leu Asn Trp Asp Gln Val Lys Ala Leu Asp Asn Glu
920 925 930
Ser Glu Val Lys Gly Tyr Lys Val Leu Tyr Arg Trp Asn Arg Gln
935 940 945
Ser Ser Thr Ser Val Ile Glu Thr Asn Lys Thr Ser Val Glu Leu
950 955 960
Ser Leu Pro Phe Asp Glu Asp Tyr Ile Ile Glu Ile Lys Pro Phe
965 970 975
Ser Asp Gly Gly Asp Gly Ser Ser Ser Glu Gln Ile Arg Ile Pro
980 985 990
Lys Ile Ser Asn Ala Tyr Ala Arg Gly Ser Gly Ala Ser Thr Ser
995 1000 1005
Asn Ala Cys Thr Leu Ser Ala Ile Ser Thr Ile Met Ile Ser Leu
1010 1015 1020
Thr Ala Arg Ser Ser Leu
1025
22/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
<210> 7
<211> 607
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1428867CD1
<400> 7
Met Ala Gln Leu Trp Leu Ser Cys Phe Leu Leu Pro Ala Leu Val
1 5 10 15
Val Ser Val Ala Ala Asn Val Ala Pro Lys Phe Leu Ala Asn Met
20 25 30
Thr Ser Val Ile Leu Pro Glu Asp Leu Pro Val Gly Ala Gln Ala
35 40 45
Phe Trp Leu Val Ala Glu Asp Gln Asp Asn Asp Pro Leu Thr Tyr
50 55 60
Gly Met Ser Gly Pro Asn Ala Tyr Phe Phe Ala Val Thr Pro Lys
~65 70 75
Thr Gly Glu Val Lys Leu Ala Ser Ala Leu Asp Tyr Glu Thr Leu
80 85 90
Tyr Thr Phe Lys Val Thr Ile Ser Val Ser Asp Pro Tyr Ile Gln
95 100 105
Val Gln Arg Glu Met Leu Val Ile Val Glu Asp Arg Asn Asp Asn
110 115 120
Ala Pro Val Phe Gln Asn Thr Ala Phe Ser Thr Ser Ile Asn Glu
125 130 135
Thr Leu Pro Val Gly Ser Val Val Phe Ser Val Leu Ala Val Asp
140 145 150
Lys Asp Met Gly Ser Ala Gly Met VaI Val Tyr Ser Ile Glu Lys
155 160 165
Val Ile Pro Ser Thr Gly Asp Ser Glu His Leu Phe Arg Ile Leu
170 175 180
Ala Asn Gly Ser Ile Val Leu Asn Gly Ser Leu Ser Tyr Asn Asn
185 190 195
Lys Ser Ala Phe Tyr Gln Leu Glu Leu Lys Ala Cys Asp Leu Gly
200 205 210
Gly Met Tyr His Asn Thr Phe Thr Ile Gln Cys Ser Leu Pro Val
225 220 225
Phe Leu Ser Ile Ser Val Val Asp Gln Pro Asp Leu Asp Pro Gln
230 235 240
Phe Val Arg Glu Phe Tyr Ser Ala Ser Val Ala Glu Asp Ala Ala
245 250 255
Lys Gly Thr Ser Val Leu Thr Val GIu Ala VaI Asp GIy Asp Lys
260 265 270
Gly Ile Asn Asp Pro Val Tle Tyr Ser Ile Ser Tyr Ser Thr Arg
275 280 285
Pro Gly Trp Phe Asp Ile Gly Ala Asp Gly Val Ile Arg Val Asn
290 295 300
Gly Ser Leu Asp Arg Glu Gln Leu Leu Glu Ala Asp Glu Glu Val
305 310 315
Gln Leu Gln Val Thr Ala Thr Glu Thr His Leu Asn Ile Tyr Gly
320 325 330
Gln Glu Ala Lys Val Ser Ile Trp Val Thr Val Arg Val Met Asp
335 340 345
23/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
Val Asn Asp His Lys Pro Glu Phe Tyr Asn Cys Ser Leu Pro Ala
350 355 360
Cys Thr Phe Thr Pro Glu Glu Ala Gln Val Asn Phe Thr Gly Tyr
365 370 375
Val Asp Glu His Ala Ser Pro Arg Ile Pro Tle Asp Asp Leu Thr
380 385 390
Met Val Val Tyr Asp Pro Asp Lys Gly Ser Asn Gly Thr Phe Leu
395 400 405
Leu Ser Leu Gly Gly Pro Asp Ala Glu Ala Phe Ser Val Ser Pro
410 415 420
Glu Arg Ala Ala Gly Ser Ala Ser Val Gln Val Leu Val Arg Val
425 430 435
Ser Ala Leu Val Asp Tyr Glu Arg Gln Thr Ala Met Ala Val Gln
440 445 450
Val Val Ala Thr Asp Ser Val Ser Gln Asn Phe Ser Val Ala Met
455 460 465
Val Thr Ile His Leu Arg Asp Ile Asn Asp His Arg Pro Thr Phe
470 475 480
Pro Gln Ser Leu Tyr Val Leu Thr Val Pro Glu His Ser Ala Thr
485 490 495
Gly Ser Val Val Thr Asp Ser Ile His Ala Thr Asp Pro Asp Thr
500 505 510
Gly Ala Trp Gly Gln Ile Thr Tyr Ser Leu Leu Pro Gly Asn Gly
515 520 525
Ala Asp Leu Phe Gln Val Asp Pro Val Ser Gly Thr Val Thr Val
530 535 540
Arg Asn Gly Glu Leu Leu Asp Arg Glu Ser Gln Ala Val Tyr Tyr
545 550 555
Leu Thr Leu Gln Ala Thr Asp Gly Gly Asn Leu Ser Ser Ser Thr
560 ' 565 570
Thr Leu Gln Ile His Leu Leu Asp Ile Asn Asp Asn Ala Pro Val
575 580 585
Val Ser Gly Ser Tyr Asn Ile Phe Val Gln Glu Glu Glu Gly Asn
590 595 600
Val Ser Val Thr Ile Gln Val
605
<210> 8
<211> 671
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2736276CD1
<400> 8
Met Ser Arg Leu Phe Asp Met Pro Cys Asp Glu Thr Leu Cys Ser
1 5 10 15
Ala Asp Ser Phe Cys Val Asn Asp Tyr Thr Trp Gly Gly Ser Axg
20 25 30
Cys Gln Cys Thr Leu Gly Lys Gly Gly Glu Ser Cys Ser Glu Asp
35 40 45
Ile Val Ile Gln Tyr Pro Gln Phe Phe Gly His Ser Tyr Val Thr
50 55 60
Phe Glu Pro Leu Lys Asn Ser Tyr Gln Ala Phe Gln Ile Thr Leu
24/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
65 70 75
Glu Phe Arg Ala Glu Ala Glu Asp Gly Leu Leu Leu Tyr Cys Gly
80 85 90
Glu Asn Glu His Gly Arg Gly Asp Phe Met Ser Leu Ala Ile Ile
95 100 105
Arg Arg Ser Leu Gln Phe Arg Phe Asn Cys Gly Thr Gly Val Ala
110 115 120
Ile Ile Val Ser Glu Thr Lys Ile Lys Leu Gly Gly Trp His Thr
125 130 135
Val Met Leu Tyr Arg Asp Gly Leu Asn Gly Leu Leu Gln Leu Asn
140 145 150
Asn Gly Thr Pro Val Thr Gly Gln Ser Gln Gly Gln Tyr Ser Lys
155 160 165
Ile Thr Phe Arg Thr Pro Leu Tyr Leu Gly Gly Ala Pro Ser Ala
170 175 180
Tyr Trp Leu Val Arg Ala Thr Gly Thr Asn Arg Gly Phe Gln Gly
185 190 195
Cys Val Gln Sex Leu Ala Val Asn Gly Arg Arg Ile Asp Met Arg
200 205 210
Pro Trp Pro Leu Gly Lys Ala Leu Ser Gly Ala Asp Val Gly Glu
215 220 225
Cys Ser Ser Gly Ile Cys Asp Glu Ala Ser Cys Ile His Gly Gly
230 235 240
Thr Cys Thr Ala Ile Lys Ala Asp Ser Tyr Ile Cys Leu Cys Pro
245 250 255
Leu Gly Phe Lys Gly Arg His Cys Glu Asp Ala Phe Thr Leu Thr
260 265 270
Ile Pro Gln Phe Arg Glu Ser Leu Arg Ser Tyr Ala Ala Thr Pro
275 280 285
Trp Pro Leu Glu Pro Gln His Tyr Leu Ser Phe Met Glu Phe Glu
290 295 300
Ile Thr Phe Arg Pro Asp Ser Gly Asp Gly Val Leu Leu Tyr Ser
305 310 315
Tyr Asp Thr Gly Ser Lys Asp Phe Leu Ser Ile Asn Leu Ala Gly
320 325 330
Gly His Val Glu Phe Arg Phe Asp Cys Gly Ser Gly Thr Gly Val
335 340 345
Leu Arg Ser Glu Asp Pro Leu Thr Leu Gly Asn Trp His Glu Leu
350 355 360
Arg Val Ser Arg Thr Ala Lys Asn Gly Ile Leu Gln Val Asp Lys
365 370 375
Gln Lys Ile Val Glu Gly Met Ala Glu Gly Gly Phe Thr Gln Ile
380 385 390
Lys Cys Asn Thr Asp Ile Phe Ile Gly Gly Val Pro Asn Tyr Asp
395 400 405
Asp Val Lys Lys Asn Ser Gly Val Leu Lys Pro Phe Ser Gly Ser
410 415 420
Ile Gln Lys Ile Ile Leu Asn Asp Arg Thr Ile His Val Lys His
425 430 435
Asp Phe Thr Ser Gly Val Asn Val Glu Asn Ala Ala His Pro Cys
440 445 450
Val Arg Ala Pro Cys Ala His Gly Gly Ser Cys Arg Pro Arg Lys
455 460 465
Glu Gly Tyr Asp Cys Asp Cys Pro Leu Gly Phe Glu Gly Leu His
470 475 480
Cys Gln Lys Ala Ile Ile Glu Ala Ile Glu Ile Pro Gln Phe Ile
25/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
485 490 495
Gly Arg Ser Tyr Leu Thr Tyr Asp Asn Pro Asp Ile Leu Lys Arg
500 505 510
Val Ser Gly Ser Arg Ser Asn Val Phe Met Arg Phe Lys Thr Thr
515 520 525
Ala Lys Asp Gly Leu Leu Leu Trp Arg Gly Asp Ser Pro Met Arg
530 535 540
Pro Asn Ser Asp Phe Ile Ser Leu Gly Leu Arg Asp Gly Ala Leu
545 550 555
Val Phe Ser Tyr Asn Leu Gly Ser Gly Val Ala Ser Ile Met Val
560 565 570
Asn Gly Ser Phe Asn Asp Gly Arg Trp His Arg Val Lys Ala Val
575 580 585
Arg Asp Gly Gln Ser Gly Lys Ile Thr Val Asp Asp Tyr Gly Ala
590 595 600
Arg Thr Gly Lys Ser Pro Gly Met Met Arg Gln Leu Asn Ile Asn
605 610 615
Gly Ala Leu Tyr Val Gly Gly Met Lys Glu Ile Ala Leu His Thr
620 625 630
Asn Arg Gln Tyr Met Arg Gly Leu Val Gly Cys Ile Ser His Phe
635 640 645
Thr Leu Ser Thr Asp Tyr His Ile Ser Leu Val Glu Asp Ala Val
650 655 660
Asp Gly Lys Asn Ile Asn Thr Cys Gly Ala Lys
665 670
<210> 9
<211> 247
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 3683719CD1
<400> 9
Met Arg Gly Asn Leu Ala Leu Val Gly Val Leu Ile Ser Leu Ala
1 5 10 15
Phe Leu Ser Leu Leu Pro Ser Gly His Pro Gln Pro Ala Gly Asp
20 25 30
Asp Ala Cys Ser Val Gln Ile Leu Val Pro Gly Leu Lys Gly Asp
35 40 45
Met Gly Asp Lys Gly Gln Lys Gly Ser Val Gly Arg His Gly Lys
50 55 60
Ile Gly Pro Ile Gly Ser Lys Gly Glu Lys Gly Asp Ser Gly Asp
65 70 75
Ile Gly Pro Pro Gly Pro Asn Gly Glu Pro Gly Leu Pro Cys Glu
80 85 90
Cys Ser Gln Leu Arg Lys Ala Ile Gly Glu Met Asp Asn Gln Val
95 100 105
Ser Gln Leu Thr Ser Glu Leu Lys Phe Ile Lys Asn Ala Val Ala
110 115 120
Gly Val Arg Glu Thr Glu Ser Lys Ile Tyr Leu Leu Val Lys Glu
125 130 135
Glu Lys Arg Tyr Ala Asp Ala Gln Leu Ser Cys Gln Gly Arg Gly
140 145 150
26/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
Gly Thr Leu Ser Met Pro Lys Asp Glu Ala Ala Asn Gly Leu Met
155 160 165
Ala Ala Tyr Leu Ala Gln Ala Gly Leu Ala Arg Val Phe Ile Gly
170 175 180
Ile Asn Asp Leu Glu Lys Glu Gly Ala Phe Val Tyr Ser Asp His
185 190 195
Ser Pro Met Arg Thr Phe Asn Lys Trp Arg Ser Gly Glu Pro Asn
200 205 210
Asn Ala Tyr Asp Glu Glu Asp Cys Val Glu Met Val Ala Ser Gly
215 220 225
Gly Trp Asn Asp Val Ala Cys His Thr Thr Met Tyr Phe Met Cys
230 235 240
Glu Phe Asp Lys Glu Asn Met
245
<210> 10
<211> 666
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6988448CD1
<400> 10
Met Pro Pro Gly Gly Ser Gly Pro Gly Gly Cys Pro Arg Arg Pro
1 5 10 15
Pro Ala Leu Ala Gly Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro
20 25 30
Pro Pro Leu Leu Pro Leu Leu Pro Leu Leu Leu Leu Leu Leu Leu
35 40 45
Gly Ala Ala Glu Gly Ala Arg Val Ser Ser Ser Leu Ser Thr Thr
50 55 60
His His Val His His Phe His Ser Lys His Gly Thr Val Pro Ile
65 70 75
Ala Ile Asn Arg Met Pro Phe Leu Thr Arg Gly Gly His Ala Gly
80 85 90
Thr Thr Tyr Ile Phe Gly Lys Gly Gly Ala Leu Ile Thr Tyr Thr
95 100 105
Trp Pro Pro Asn Asp Arg Pro Ser Thr Arg Met Asp Arg Leu Ala
110 115 120
Val Gly Phe Ser Thr His Gln Arg Ser Ala Val Leu Val Arg Val
125 130 135
Asp Ser Ala Ser Gly Leu Gly Asp Tyr Leu Gln Leu His Ile Asp
140 145 150
Gln Gly Thr Val Gly Val Ile Phe Asn Val Gly Thr Asp Asp Ile
155 160 165
Thr Ile Asp Glu Pro Asn Ala Ile Val Ser Asp Gly Lys Tyr His
170 175 180
Val Val Arg Phe Thr Arg Ser Gly Gly Asn Ala Thr Leu Gln Val
185 190 195
Asp Ser Trp Pro Val Asn Glu Arg Tyr Pro Ala Gly Asn Phe Asp
200 205 210
Asn Glu Arg Leu Ala Ile Ala Arg Gln Arg Ile Pro Tyr Arg Leu
215 220 225
Gly Arg Val Val Asp Glu Trp Leu Leu Asp Lys Gly Arg Gln Leu
27/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
230 235 240
Thr Ile Phe Asn Ser Gln Ala Ala Ile Lys Ile Gly Gly Arg Asp
245 250 255
Gln Gly Arg Pro Phe Gln Gly Gln Val Ser Gly Leu Tyr Tyr Asn
260 265 270
Gly Leu Lys Val Leu Ala Leu Ala Ala Glu Ser Asp Pro Asn Val
275 280 285
Arg Thr Glu Gly His Leu Arg Leu Val Gly Glu Gly Pro Ser Val
290 295 300
Leu Leu Ser Ala Glu Thr Thr Ala Thr Thr Leu Leu Ala Asp Met
305 310 315
Ala Thr Thr Ile Met Glu Thr Thr Thr Thr Met Ala Thr Thr Thr
320 325 330
Thr Arg Arg Gly Arg Ser Pro Thr Leu Arg Asp Ser Thr Thr Gln
335 340 345
Asn Thr Asp Asp Leu Leu Val Ala Ser Ala Glu Cys Pro Ser Asp
350 355 360
Asp Glu Asp Leu Glu Glu Cys Glu Pro Ser Thr Gly Gly Glu Leu
365 370 375
Ile Leu Pro Ile Ile Thr Glu Asp Ser Leu Asp Pro Pro Pro Val
380 385 390
Ala Thr Arg Ser Pro Phe Val Pro Pro Pro Pro Thr Phe Tyr Pro
395 400 405
Phe Leu Thr Gly Val Gly Ala Thr Gln Asp Thr Leu Pro Pro Pro
410 415 420
Ala Ala Arg Arg Pro Pro Ser Gly Gly Pro Cys Gln Ala Glu Arg
425 430 435
Asp Asp Ser Asp Cys Glu Glu Pro Ile Glu Ala Ser Gly Phe Ala
440 445 450
Ser Gly Glu Val Phe Asp Ser Ser Leu Pro Pro Thr Asp Asp Glu
455 460 465
Asp Phe Tyr Thr Thr Phe Pro Leu Val Thr Asp Arg Thr Thr Leu
470 475 480
Leu Ser Pro Arg Lys Pro Ala Pro Arg Pro Asn Leu Arg Thr Asp
485 490 495
Gly Ala Thr Gly Ala Pro Gly Val Leu Phe Ala Pro Ser Ala Pro
500 505 510
Ala Pro Asn Leu Pro Ala Gly Lys Met Asn His Arg Asp Pro Leu
515 520 525
Gln Pro Leu Leu Glu Asn Pro Pro Leu Gly Pro Gly Ala Pro Thr
530 535 540
Ser Phe Glu Pro Arg Arg Pro Pro Pro Leu Arg Pro Gly Val Thr
545 550 555
Sex Ala Pro Gly Phe Pro His Leu Pro Thr Ala Asn Pro Thr Gly
560 565 570
Pro Gly Glu Arg Gly Pro Pro Gly Ala Val Glu Val Ile Arg Glu
575 580 585
Ser Ser Ser Thr Thr Gly Met Val Val Gly Ile Val Ala Ala Ala
590 595 600
Ala Leu Cys Ile Leu Ile Leu Leu Tyr Ala Met Tyr Lys Tyr Arg
605 6l0 615
Asn Arg Asp Glu Gly Ser Tyr Gln Val Asp Gln Ser Arg Asn Tyr
620 625 630
Ile Ser Asn Ser Ala Gln Ser Asn Gly Ala Val Val Lys Glu Lys
635 640 645
Ala Pro Ala Ala Pro Lys Thr Pro Ser Lys Ala Lys Lys Asn Lys
28/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
650 655 660
Asp Lys Glu Tyr Tyr Val
665
<210> 11
<211> 472
<212> PRT
<213> Homo Sapiens
<220>
<222> misc_feature
<223> Incyte ID No: 7500307CD1
<400> 11
Met Pro Pro Gly Gly Ser Gly Pro Gly Gly Cys Pro Arg Arg Pro
1 5 10 15
Pro Ala Leu Ala Gly Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro
20 25 30
Pro Pro Leu Leu Pro Leu Leu Pro Leu Leu Leu Leu Leu Leu Leu
35 40 45
Gly Ala Ala Glu Gly Ala Arg Val Ser Ser Ser Leu Ser Thr Thr
50 55 60
His His Val His His Phe His Ser Lys His Gly Thr Val Pro Ile
65 70 75
Ala Ile Asn Arg Met Pro Phe Leu Thr Arg Gly Gly His Ala Gly
80 85 90
Thr Thr Tyr Ile Phe Gly Lys Gly Gly Ala Leu Ile Thr Tyr Thr
95 100 105
Trp Pro Pro Asn Asp Arg Pro Ser Thr Arg Met Asp Arg Leu Ala
110 115 120
Val Gly Phe Ser Thr His Gln Arg Ser Ala Val Leu Val Arg Val
125 130 ~ 135
Asp Ser Ala Ser Gly Leu Gly Asp Tyr Leu Gln Leu His Ile Asp
140 145 150
Gln Gly Thr Val Gly Val Ile Phe Asn Val Gly Thr Asp Asp Ile
155 160 165
Thr Ile Asp Glu Pro Asn Ala Ile Val Ser Asp Gly Lys Tyr His
170 ~ 175 180
Val Val Arg Phe Thr Arg Ser Gly Gly Asn Ala Thr Leu Gln Val
185 190 195
Asp Ser Trp Pro Val Asn Glu Arg Tyr Pro Ala Gly Asn Phe Asp
200 205 210
Asn Glu Arg Leu Ala Ile Ala Arg Gln Arg Ile Pro Tyr Arg Leu
215 220 225
Gly Arg Val Val Asp Glu Trp Leu Leu Asp Lys Gly Arg Gln Leu
230 235 240
Thr Tle Phe Asn Ser Gln Ala Ala Ile Lys Tle Gly Gly Arg Asp
245 250 255
Gln Gly Arg Pro Phe Gln Gly Gln Val Ser Gly Leu Tyr Tyr Asn
260 265 270
Gly Leu Lys Val Leu Ala Leu Ala Ala Glu Ser Asp Pro Asn Val
275 280 285
Arg Thr GIu Gly His Leu Arg Leu Val GIy GIu Gly Pro Ser Val
290 295 300
Leu Leu Ser AIa Glu Thr Thr Ala Thr Thr Leu Leu AIa Asp Met
305 310 315
29/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
Ala Thr Thr Ile Met Glu Thr Thr Thr Thr Met Ala Thr Thr Thr
320 325 330
Thr Arg Arg Gly Arg Ser Pro Thr Leu Arg Asp Ser Thr Thr Gln
335 340 345
Asn Thr Asp Asp Leu Leu Val Ala Ser Ala Glu Cys Pro Ser Asp
350 355 360
Asp Glu Asp Leu Glu Glu Cys Glu Pro Ser Thr Ala Asn Pro Thr
365 370 375
Gly Pro Gly Glu Arg Gly Pro Pro Gly Ala Val Glu Val Ile Arg
380 385 390
Glu Ser Ser Ser Thr Thr Gly Met Val Val Gly Ile Val Ala Ala
395 400 405
Ala Ala Leu Cys Ile Leu Ile Leu Leu Tyr Ala Met Tyr Lys Tyr
410 415 420
Arg Asn Arg Asp Glu Gly Ser Tyr Gln Val Asp Gln Ser Arg Asn
425 430 435
Tyr Ile Ser Asn Ser Ala Gln Ser Asn Gly Ala Val Val Lys Glu
440 445 450
Lys Ala Pro Ala Ala Pro Lys Thr Pro Ser Lys Ala Lys Lys Asn
455 460 465
Lys Asp Lys Glu Tyr Tyr Val
470
<210> 12
<212> 6849
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No; 2707785CB1
<400> 12
gactgaggag cagagagagc ggggcgccga gtgcgggcgg ctgggagcgc gctgagcggg 6o-
ggagaggcgc tgccgcacgg ccggccacag gaccacctcc ccggagaata gggcctcttt 120
atggcatgtg gctggtaact ttcctcctgc tcctggactc tttacacaaa gcccgccctg 180
aagatgttgg caccagcctc tactttgtaa atgactcctt gcagcaggtg accttttcca 240
gctccgtggg ggtggtggtg ccctgcccgg ccgcgggctc ccccagcgcg gcccttcgat 300
ggtacctggc cacaggggac gacatctacg acgtgccgca catccggcac gtccacgcca 360
acgggacgct gcagctctac cccttctccc cctccgcctt caatagcttt atccacgaca 420
atgactactt ctgcaccgcg gagaacgctg ccggcaagat ccggagcccc aacatccgcg 480
tcaaagcagt tttcagggaa ccctacaccg tccgggtgga .ggatcaaagg tcaatgcgtg 540
gcaacgtggc cgtcttcaag tgcctcatcc cctcttcagt gcaggaatat gttagcgttg 600
tatcttggga gaaagacaca gtctccatca tcccagaaca caggtttttt attacctacc 660
acggcgggct gtacatctct gacgtacaga aggaggacgc cctctccacc tatcgctgca 720
tcaccaagca caagtatagc ggggagaccc ggcagagcaa tggggcacgc ctctctgtga 780
cagaccctgc tgagtcgatc cccaccatcc tggatggctt ccactcccag gaagtgtggg 840
ccggccacac cgtggagctg ccctgcaccg cctcgggcta ccctatcccc gccatccgct 900
ggctcaagga tggccggccc ctcccggctg acagccgctg gaccaagcgc atcacagggc 960
tgaccatcag cgacttgcgg accgaggaca gcggcaccta catttgtgag gtcaccaaca 1020
ccttcggttc ggcagaggcc acaggcatcc tcatggtcat tgatcccctt catgtgaccc 1080
tgacaccaaa gaagctgaag accggcattg gcagcacggt catcctctcc tgtgccctga 1140
cgggctcccc agagttcacc atccgctggt atcgcaacac ggagctggtg ctgcctgacg 1200
aggccatctc catccgcggg ctcagcaacg agacgctgct catcacctcg gcccagaaga 1260
gccattccgg ggcctaccag tgcttcgcta cccgcaaggc ccagaccgcc caggactttg 1320
ccatcattgc acttgaggat ggcacgcccc gcatcgtctc gtccttcagc gagaaggtgg 1380
30/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
tcaaccccgg ggagcagttc tcactgatgt gtgcggccaa gggcgccccg ccccccacgg 1440
tcacctgggc cctcgacgat gagcccatcg tgcgggatgg cagccaccgc accaaccagt 1500
acaccatgtc ggacggcacc accatcagcc acatgaacgt cacaggcccc cagatccgcg 1560
acgggggcgt gtaccggtgc acagcgcgga acttggtggg cagtgctgaa tatcaggcgc 1620
gaataaacgt aagaggccca cccagcatcc gggctatgcg gaacatcaca gcagtcgccg 1680
ggcgggacac ccttatcaac tgcagggtca tcggctatcc ctactactcc atcaagtggt 1740
acaaggatgc cctgctgctg ccagacaacc accgccaggt ggtgtttgag aatgggaccc 1800
tcaagctgac tgacgtgcag aagggcatgg atgaggggga gtacctgtgc agtgtcctca 1860
tccagcccca gctctccatc agccagagcg ttcacgtagc cgtcaaagtg ccccctctga 1920
tccagccctt cgaattccca cccgcctcca tcggccagct gctctacatt ccctgtgtgg 1980
tgtcctcggg ggacatgccc atccgtatca cctggaggaa ggacggacag gtgatcatct 2040
caggctcggg cgtgaccatc gagagcaagg aattcatgag ctccctgcag atctctagcg 2100
tctccctcaa gcacaacggc aactatacat gcatcgccag caacgcagcc gccaccgtga 2160
gccgggagcg ccagctcatc gtgcgtgtgc cccctcgatt tgtggtgcaa cccaacaacc 2220
aggatggcat ctacggcaaa gctggtgtgc tcaactgctc ggtggacggc taccccccac 2280
ccaaggtcat gtggaagcat gccaagggga gcgggaaccc ccagcagtac caccctgtgc 2340
ccctcactgg ccgcatccag atcctgccca acagctcgct gctgatccgc cacgtcctag 2400
aagaggacat cggctactac ctctgccagg ccagcaacgg cgtaggcacc gacatcagca 2460
agtccatgtt cctcacagtc aagatcccgg ccatgatcac ttcccacccc aacaccacca 2520
tcgccatcaa gggccatgcg aaggagctaa actgcacggc acggggtgag cggcccatca 2580
tcatccgctg ggagaagggg gacacagtca tcgaccctga ccgcgtcatg cggtatgcca 2640
tcgccaccaa ggacaacggc gacgaggtcg tctccacact gaagctcaag cccgctgacc 2700
gtggggactc tgtgttcttc agctgccatg ccatcaactc gtatggggag gaccggggct 2760
tgatccaact cactgtgcaa gagccccccg accccccaga gctggagatc cgggaggtga 2820
aggcccggag catgaacctg cgctggaccc agcgattcga cgggaacagc atcatcacgg 2880
gcttcgacat tgaatacaag aacaaatcag attcctggga cttcaagcag tccacacgca 2940
acatctcccc caccatcaac caggccaaca ttgtggactt gcacccggca tctgtgtaca 3000
gcatccgcat gtactctttc aacaagattg gccgcagtga accaagcaag gagctcacca 3060
tcagcactga ggaggccgct cccgatgggc cccccatgga tgttaccttg cagccagtga 3120
cctcacagag catccaggtg acctggaagg cacccaagaa ggagctgcag aacggtgtca 3180
tccggggcta ccagattggc tacagagaga acagccccgg cagcaacggg cagtacagca 3240
tcgtggagat gaaggccacg ggggacagcg aggtctacac cctggacaac ctcaagaagt 3300
tcgcccagta tggggtggtg gtccaagcct tcaatcgggc tggcacgggg ccctcttcca 3360
gcgagatcaa tgccaccact ctggaggatg tgcccagcca gccccctgag aacgtccggg 3420
ccctgtccat cacttctgac gtggccgtca tctcctggtc agagcccccg cgcagcaccc 3480
tcaatggcgt cctcaaaggc tatcgggtca tcttctggtc cctctatgtt gatggggagt 3540
~ggggcgagat gcagaacatc accaccacgc gggagcgggt ggagctgcgg ggcatggaga 3600
agttcaccaa ctacagcgtc caggtgctgg cctacaccca ggctggggac ggcgtacgca 3660
gcagtgtgct ctacatccag accaaggagg acgttccagg tccccctgct ggcatcaaag 3720
ctgtcccttc atcagctagc agtgtggttg tgtcttggct cccccctacc aagcccaacg 3780
gggtgatccg caagtacacc atcttctgtt ccagccccgg gtctggccag ccggctccca 3840
gcgagtacga gacgagtcca gagcagctct tctaccggat cgcccaccta aaccgcggtc 3900
agcagtatct gctgtgggtg gccgccgtca cetctgccgg ccggggcaac agcagcgaga 3960
aggtgaccat cgagcetgct ggcaaggccc cagcaaagat catctccttt gggggcaccg 4020
tgacaacacc ttggatgaaa gatgttcggc tgccttgcaa ttcagtggga gatccagccc 4080
ctgctgtgaa gtggaccaag gacagtgaag actcggccat tccagtgtcc atggatgggc 4140
accggctcat ccacaccaat ggcacactgc tgctgcgtgc agtgaaggct gaggactctg 4200
gctactacac gtgcacggcc accaacactg gtggctttga caccatcatc gtcaaccttc 4260
tggtgcaagt tcccccggac cagccccgcc tcactgtctc caaaacctca gcttcgtcca 4320
tcaccctgac ctggattcca ggtgacaatg ggggcagctc catccgaggc ttcgtgctac 4380
agtactcggt ggacaacagc gaggagtgga aggatgtgtt catcagctcc agcgagcgct 4440
ccttcaagct ggacagcctc aagtgtggca cgtggtacaa ggtgaagctg gcagccaaga 4500
acagcgtggg ctctgggcgc atcagcgaga tcatcgaggc caagacccac gggcgggagc 4560
cctccttcag caaagaccaa cacctcttca cccacatcaa ctccacgcat gctcggctta 4620
acctgcaggg ctggaacaat gggggctgcc ctatcacagc catcgttctg gagtaccggc 4680
ccaaggggac ctgggcctgg cagggcctcc gggccaacag ctccggggag gtgtttctga 4740
31/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
cggaactgcg agaggccacg tggtacgagc tgcgcatgag ggcttgcaac agtgcgggct 4800
gcggcaatga aacagcccag ttcgccaccc tggactacga tggcagcacc attccaccca 4860
tcaagtctgc tcaaggtgaa ggggatgatg tgaagaagct gttcaccatc ggctgccctg 4920
tcatcctggc cacactgggg gtggcactgc tcttcatcgt acgcaagaag aggaaggaga 4980
aacggctgaa gcgactccga gatgcaaaga gtttggcaga aatgttgata agcaagaaca 5040
atagaagctt tgacacccct gtgaaagggc caccccaggg cccacggcta cacattgaca 5100
tccccagggt ccagctgctc atcgaggaca aagaaggcat caagcaactg ggagatgaca 5160
aggccaccat ccctgtgaca gatgctgagt tcagccaagc tgtcaaccca cagagcttct 5220
gtactggcgt ctccttgcac cacccaaccc tcatccagag cacaggaccc ctcatcgaca 5280
tgtctgacat ccggccagga accaatccag tgtccaggaa gaatgtgaag tcagcccaca 5340
gcacccggaa ccggtactca agccagtgga ccctgaccaa gtgccaggcc tccacacctg 5400
cccgcaccct cacctccgac tggcgcaccg tgggctccca gcatggtgtc acggtcactg 5460
agagtgacag ctacagtgcc agcctgtccc aggacacaga caaaggaagg aacagcatgg 5520
tgtccactga gagtgcctct tccacctacg aggagctggc ccgggcctat gagcatgcca 5580
agctggagga gcagctgcag cacgccaagt ttgagatcac cgagtgcttc atctctgaca 5640
gttcctctga ccagatgacc acaggcacca acgagaacgc cgacagcatg acatccatga 5700
gcacaccctc agagcctggc atctgccgct ttaccgcctc accacccaag ccccaggatg 5760
cggaccgggg caaaaacgtg gctgtgccca tccctcaccg ggccaacaag agtgactact 5820
gcaacctgcc cctgtatgcc aagtcagagg ccttctttcg aaaggcagat ggacgtgagc 5880
cctgccccgt ggtcccaccc cgtgaggcct ccatccggaa cctggctcga acctaccaca 5940
cccaggctcg ccacctgacc ctggaccctg ccagcaagtc cttgggcctt ccccacccag 6000
gggcccccgc tgccgcctcc acagccacct tacctcagag gactctggcc atgccagccc 6060
ccccagccgg cacagccccc ccagcccccg gccccacccc tgctgagcca cccaccgccc 6120
ccagcgctgc ccctccggcc cccagcaccg agcctccacg agccgggggc ccacacacca 6180
aaatgggggg ctccagggac tcgcttctcg agatgagcac atcgggggta gggaggtctc 6240
agaagcaggg ggccggggcc tactccaaat cctacaccct ggtgtagggc ccgcaggaag 6300
agcagccacg cctgggccgc gccgcgccgc agccccacac gccagctcgg ctgtttttct 6360
gcattattta tattcaactg acagacaaaa accaaccaac gacaaaacaa aaacccccaa 6420
tcatgaacgc ctgtacatag aactcttttg tacaaatgaa actattttct tcttctccat 6480
gaagccaggg cacaaagaat ttgacagtac aagtcaaatc ccccacccca caaaatatgt 6540
gtggagatat atatacatat atagacagac aggaacgcgt ccacgagcta tatatctata 6600
tatttctctc accctatttt gagacagagg cacaaagact cagcaatttt tttccctcct 6660
cctcaccttc cccccagtct aggtggtttt gacaaagacc aaaatcccaa ctcagagaca 6720
ctgcatgcga ttttactgtt ccaagaaaac caggagttgc ttcaatttgc agatgcttat 6780
gtgttaatac ctttttctat gaaaaaagac ccagcgccgt gtgcaataaa ggttatgttt 6840
ctaaaaaaa 6849
<210> 13
<211> 3267
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1414780CB1
<220>
<221> unsure
<222> 3210
<223> a, t, c, g, or other
<400> 13
atgaggccga ggcccgaagg tagggggctc cgggcgggag tcgcgctgtc ccccgcgcta 60
ctgctgctgc tgctgctgcc gccgccgccg acgctgctgg ggcgcctgtg ggcagcgggc 120
acaccctcgc cgtcggcgcc cggagctcgg caggacggcg cgctgggagc cggccgcgtc 180
aaacgcggct gggtgtggaa ccagttcttc gtggtagagg agtacacggg cacggagccc 240
32/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
ctgtatgtgg gcaagatcca ctccgactca gacgagggtg acggggccat caagtacacc 300
atctcaggcg agggtgctgg gaccatcttc ctgatcgacg agctgacagg cgacattcat 360
gccatggagc gcctggaccg cgagcagaaa accttctaca cgctgcgggc ccaggctcgg 420
gatcgcgcca ccaaccgcct actggagccc gagtcggagt tcatcatcaa ggtgcaggac 480
atcaatgaca gtgagccccg cttcctgcac ggcccctata ttggcagcgt ggccgagctc 540
tcacctacag gcacgtcggt gatgcaggtg atggcctcgg atgcggatga ccccacgtac 600
ggcagcagcg ctcggctggt gtacagcgtg ctggacggcg agcaccactt caccgtggac 660
cccaagaccg gcgtaatccg gacggctgtg cctgaccttg accgcgagag ccaggagcgc 720
tacgaggtgg tgatccaggc cacagacatg gcgggtcagc tgggtggcct ctcgggctcc 780
actaccgtca ccatcgtagt caccgacgtc aatgacaacc cgccccgttt cccgcagaag 840
atgtaccagt tcagcatcca ggagtcagcc cccattggaa cggctgtggg acgtgtgaag 900
gctgaggact cagacgtggg agagaacaca gacatgactt accaccttaa ggacgagagc 960
agcagcggcg gcgatgtgtt caaggtcacc acagacagcg acactcagga ggccatcatc 1020
gtagtgcaga agcgcctgga cttcgaatcc cagcccgtgc acaccgtgat cctggaggcc 1080
ctcaacaagt tcgtggaccc ccgcttcgcc gacctgggca cgttccgcga ccaggcgatc 1140
gtgcgcgtgg ccgtgaccga cgtggacgag ccccccgagt tccggccgcc ctccggcctc 1200
ctggaggtgc aggaggacgc gcaggtgggc tccctggtcg gcgtggtgac ggcgcgggac 1260
cccgacgccg ccaaccggcc cgtccggtac gccattgacc gcgaatcaga tttggaccag 1320
atcttcgata tcgatgcgga cacaggcgcc atcgtgactg gcaaggggct ggaccgcgag 1380
acggccggct ggcacaacat cacagtgctg gccatggagg cggacaatca tgcacagcta 1440
tcccgggcat ccctaaggat ccgaatcctg gatgtgaacg acaatccccc agaactggcc 1500
acaccctacg aggcagctgt atgcgaggat gccaagccag gccagctcat ccagaccatc 1560
agcgtggtgg acagagacga gccccaaggc gggcaccgct tctatttccg cctggtgcct 1620
gaagctccca gcaaccctca tttctctctg cttgacatcc aagacaacac cgctgcagtg 1680
cacacgcagc acgtgggctt caaccggcag gagcaggacg tgttcttcct gcccatcctg 1740
gtggtagaca gtgggccgcc cacactgagc agcacaggca cgctcaccat ccgcatctgt 1800
ggctgcgaca gctccggcac catccagtcc tgcaacacca cggcctttgt catggccgcc 1860
tccctcagcc ccggcgccct catcgccctc ttggtctgcg ttctcatcct ggttgtgctg 1920
gtgctgctga tcctcaccct caggcgccac cacaagagcc acctgagctc ggacgaggat 1980
gaagacatgc gggacaacgt catcaaatac aacgacgaag gcggcggcga gcaggacacc 2040
gaagcctacg acatgtcggc gctgcggagc ctctacgact tcggcgagct caagggcggc 2100
gacgggggcg gcagcgcggg cgggggagcg ggcgggggct cgggcggggg cgcgggcagc 2160
cccccgcagg cccacctgcc ctccgagcgc cactcgctgc cgcaggggcc gccgagcccc 2220
gagccagact tctcagtgtt cagggacttc atcagccgca aggtggcact ggcggacggg 2280
gacctgtcgg tgccgcccta cgacgccttc cagacctacg ccttcgaggg cgcggactcg 2340
ccggccgcct cgctcagctc cctgcacagc ggctcgtcgg gctccgagca ggacttcgcc 2400
tatctcagca gc,tggggtcc gcgcttccgg cccctggccg cgctctacgc cggccaccgc 2460
ggggacgacg aggcccaggc ctcctagccc ctcgccctgc cgtcggggcg cggctgctca 252'0
cccgcccagc acacgccggg gccccaggac aacgcgtttc ccccgcggac cccctttcct 2580
gccctccccc aaccctccct tggcggctgg acggaggggg gacttgacta ggagcggact 2640
cttccattcc tcctcctcta ggggtgcagc tttggagccc agaggtgcgg gattctgacc 2700
aacggcatta aaactgaggc gagaccgggc acggtgtggc tctggggtta gaatgggaga 2760
tgggggtggc gttgcagagt cgggaagggg cgggtcactc aatcctggcc tgggggagaa 2820
tgctggaggg agcacgccgc tgagatgccc ccaccccagg tttcccccat cagagttaag 2880
aggaaagaag gctgttcact tactaagcac ctactgtgtg ctgggacgcc tgtacagaga 2940
ccagctcagt cgtcagagaa accatgaggt ggtgtcctgc acggaataga aggggaaagg 3000
acccccagag aagtggctgt gacccgtcca agggcacctg gccagtgaat ggcagagcct 3060
ctggcactga ccagcctgcc tggcctcggg caagtcactt cacttctctg gtcctcagtt 3120
tcctcatctg tcaaatgggg ttaataacag aacctacctc gaagagttgt gaggctaaaa 3180
agggttcata tgtgtcaagt ggttaggacn agtcctggca catagtaggt gttcaataaa 3240
tgctagcctt tgttactatt aaaaaaa 3267
<210> 14
<211> 3713
<212> DNA
<213> Homo sapiens
33/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
<220>
<221> misc_feature
<223> Incyte ID No: 3109513CB1
<400> 14
ccggccgggg cccctggccc ccatggcgca tgcgcgggag gccgctcggt gatccgcggc 60
ggcggcagcg gcgcttcctg ctaggaccgg ccggggccgt accggaggct cgggctccac 120
cgaccctcct cccaccccct cccactcacc ctctgggccg cgactgcgca gggcggggcc 180
ggccgaacca tgggccgcgg tgtgggctaa gctggtggcc ccggctttag actggacccc 240
acaatgtttg cagagatgtt caggcacgcg ggagctgatt acacacaatg aatgggggca 300
atgagagcag tggagcagac agagctgggg gccctgtggc cacatctgtc cccatcggct 360
ggcagcgctg tgtgcgagag ggtgctgtgc tctacatcag tccaagtggc acagagctgt 420
cttccttgga gcaaacccgg agctacctcc tcagcgatgg gacctgcaag tgcggtctgg 480
agtgtccact taatgtcccc aaggttttca actttgaccc tttggccccg gtgaccccgg 540
gtggggctgg ggtggggcca gcatcagagg aggacatgac caagctgtgc aaccaccggc 600
ggaaagctgt tgctatggca actctgtacc gcagcatgga gaccacctgc tcacactctt 660
ctcctggaga gggagcgagc ccccaaatgt tccacactgt gtccccaggg cceccctctg 720
cccgccctcc ctgtcgagtt cctcctacaa ctccacttaa tgggggtcct ggctcccttc 780
ccccagaacc accctcagtt tcccaggcct ttcccactct agcaggccct ggggggcttt 840
tccccccaag gcttgctgac ccagtccctt ctgggggcag tagcagcccc cgtttcctcc 900
caaggggcaa tgccccctct ccagccccac ctcctccacc tgctatcagc ctcaatgctc 960
cctcatacaa ctggggagct gccctcagat ccagcctggt gccctctgac ctgggctctc 1020
ctccggcccc tcatgcctcc tcctcaccac cttcagaccc tcctctcttc cactgtagtg 1080
atgccttaac accccctccc ctgcccccga gcaataatct ccccgcccac cctggtcctg 1140
cctctcagcc accagtgtct tcagccacta tgcacctgcc cctggtcctg gggcccctgg 1200
gaggggcccc cacggtggag gggcctgggg cacccccctt ccttgctagc agcctactct 1260
ctgcagcggc caaggcacag catcccccac taccccctcc cagcacttta cagggccgaa 1320
ggccccgtgc ccaggcaccc tcagcttccc actcetcatc acttcgtccc tctcagcgtc 1380
gtccccgcag accccctact gtatttcgat tgctagaagg gagaggccct caaaccccta 1440
gacggagccg tcctcgggcc cctgctcctg tcccccaacc cttttctctc ccggagccat 1500
cccaaccaat tctcccttct gtgctgtccc tgctgggact ccccacccct ggcccttccc 1560
actctgatgg aagctttaac cttttggggt cagatgcaca ccttcctcct cccccaaccc 1620
tctcctcagg gagccctccc cagcccaggc accccatcca gccctccctg cctgggacca 1680
ccagtggcag cctcagcagt gtgccaggtg cccctgcccc accagctgcc tccaaagccc 1740
cagtagtccc cagccctgtg cttcaaagcc catccgaagg actggggatg ggggcaggcc 1800
cggcctgccc tctgcctccc ctggctggtg gagaggcttt ccctttcccc agccctgagc 1860
agggcctggc actgagtgga gctggcttcc ctgggatgct tggggccttg cctctccctc 1920
tgagtctggg gcagcctcca ccttctccat tgctcaacca cagtttattt ggtgtgctga 1980
ctgggggagg aggacaacct ccccctgagc ccctgctacc cccaccagga ggacctggtc 2040
ctcccctagc cccaggagag cctgaagggc cttcgctttt ggtggcttcc ttgcttcctc 2100
caccaccctc agaccttctt ccacctcctt cagcacctcc cagcaacctc cttgcctctt 2160
tcctgcccct gttggctctg ggccccacag ctggggatgg ggagggatct gcagagggag 2220
ccgggggtcc aagtggggag ccattttcag gcttgggaga cctgtccccc ctacttttcc 2280
ccccactttc agccccccct accctcatag ctttaaattc tgcgctgctg gctgccaccc 2340
tggatccccc ctcggggaca cccccccagc cctgtgtcct gagtgccccc caacctggac 2400
cacctacctc cagtgtcacc acggcaacta ctgacccggg ggcctcctct ctgggcaagg 2460
ccccctccaa ctcagggaga cccccccaac tccttagccc tctgctgggt gccagcctgc 2520
tgggtgacct gtcttcactg accagcagcc ctggagccct ccccagcctg ttgcagcctc 2580
ctggccctct tctctctggc cagttggggc tgcagctcct ccctgggggg ggagctcctc 2640
cacccctctc agaggcttct agtcccctag cctgcctgct acagagtctc cagatccctc 2700
cagagcagcc agaagccccc tgtctacccc ccgagagccc tgcctcagcc ctcgaaccag 2760
agcctgccag gcctcccctc agtgccttag ccccacccca tggttctccc gaccccccag 2820
tccctgagct gctcactggg agggggtcag ggaaacgggg ccggagggga ggagggggac 2880
ttaggggcat taatggtgag gccaggccag cccggggccg aaagcctggc agccggcggg 2940
agcctggccg actggccctc aaatggggga cacgtggtgg cttcaatgga caaatggaaa 3000
ggtccccaag aagaacccac cattggcagc ataatgggga gctggctgaa gggggtgctg 3060
34/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
agcccaagga tccaccccct cccgggcccc attctgagga ccttaaggtg cccccgggag 3120
tagtcagaaa gtctcgtcgt ggccgtagga gaaaatacaa ccctacccgg aacagcaata 3180
gctcccgcca ggacattacc ttggaaccca gccctacagc ccgagcagct gtccctctgc 3240
ctccccgggc ccgccctggc cgtcctgcca aaaacaagag gaggaaactg gccccatagc 3300
agccatacct ggagctggat ctgaccctga ttggggagag ctgagtgctg agccttggga 3360
gcccctgcca gccacctgca cctgtggaca gtggccaaca ggctcagttt acaaacctgt 3420
gagctactgt tggctgctgc cctccttccc agtgaaagag acgttgtgat gatgcgactg 3480
aggattatgc aacgtggtcc aaccggagcg gccagcatga ccagctgtcc aggggctgcc 3540
tcctgccttt tcttttgtaa agacaagacc cttgggagtt ttaattctgt tttgtacttg 3600
ccctgtgggg cctccactgc ttttctatgg gagacactct taatttaaca gatgagaata 3660
ttttgaaact ctgaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 3713
<210> 15
<211> 7564
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7326129CB1
<400> 15
ccgtagggtt gggagagggg caggtctccc gcccgagggg cgagcggtgg gggtaggcgt 60
ggggcggctt cccggccctc cccgagctcc tggggaagcc ggctgtgccc caggggttct 120
aaaggcggtg gtctcagagc agcggctgac ccgtgacacc gcgtgtgcac cgcagtgcgc 180
caggtacggc tcgtacgggg cctgcaggca gtggaggcac gggagcaggg cacggctacc 240
atggaggtgc agctgtcgca tgcggacgtg gagggcagct ggactcgtga'cggtctgcgg 300
ctccagcagg ggcccacgtg ccacctggct gtgcggggcc ccatgcacac cctcacactc 360
tcggggctgc ggccagagga tagtggcctt atggtcttca aggccgaagg agtgcacacg 420
tcggcgcggc tcgtggtcac cgagcttccc gtgagcttca gccgcccgct gcaggacgtg 480
gtgaccactg agaaggagaa ggttaccctg gagtgcgagc tgtcgcgtcc taatgtggat 540
gtgcgctggc tgaaggacgg tgtggagctg cgggcaggca agacgatggc catcgcagcc 600
cagggcgcct gcaggagcct caccatttac cggtgcgagt tcgcggatca gggagtgtat 660
gtgtgtgatg cccatgatgc ccagagctct gcctccgtga aggtacaagg ccgcaacatc 720
cagatcgtga ggcccctgga ggatgtggaa gtgatggaga aggacggtgc caccttctcc 780
tgtgaggtct cccacgacga agtgcctggc cagtggttct gggagggcag taaactgcgg 840
cccactgaca acgtgcgcat ccgccaggaa ggaaggacat acactctcat etaccggaga 900
gtcctggcgg aagatgcagg agagatccaa tttgtagccg aaaatgcaga atcgcgagcc 960
cagctccgag tgaaggagct gccagtgacc ctcgtgcgcc cgctgcggga caagattgcc 1020
atggagaagc accgcggtgt gctggagtgt caggtgtccc gggccagcgc ccaggtgcgg 1080
tggttcaagg gcagtcagga gctgcagccc gggcccaagt acgagctggt cagtgatggc 1140
ctctaccgca agctgatcat cagtgatgtc cacgcagagg acgaggacac ctacacctgt 1200
gacgccggtg atgtcaagac cagtgcacag ttcttcgtgg aagagcaatc catcaccatt 1260
gtgcggggtc tgcaggacgt gacagtgatg gagcccgctc ctgcctggtt tgagtgtgag 1320
acctccatcc cctcagtgcg gccacctaag tggctcctgg ggaagacggt gttgcaggct 1380
ggggggaacg tgggcctgga gcaggagggc acggtgcacc ggctgatgct gcggcggacc 1440
tgctccacca tgaccgggcc cgtgcacttc accgttggca agtcgcgctc ctctgcccgc 1500
ctggtggtct cagacatccc cgtagtcctc acacggccgt tggagcccaa gacagggcgt 1560
gagctgcagt cagtggtcct gtcctgcgac ttccggccag cccccaaggc tgtgcagtgg 1620
tacaaggatg acacgcccct gtctccctct gagaagttta agatgagcct ggagggtcag 1680
atggctgagc tgcgcatcct ccggctcatg cctgctgatg ctggtgtcta ccggtgccag 1740
gcgggcagtg cccacagcag cactgaggtc actgtggaag cgcgggaggt gacagtgaca 1800
gggccgctac aggatgcaga ggccacggag gagggctggg ccagcttctc ctgtgagctg 1860
tcccacgagg atgaggaggt cgagtggtcg ctcaacggga tgcccctgta caacgacagc 1920
ttccatgaga tctcacacaa gggccggcgc cacacgctgg tactgaagag catccagcgg 1980
gctgatgcgg gcatagtacg cgcctcctcc ctgaaggtgt cgacctctgc ccgcctggag 2040
35/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
gtccgagtga agccggtggt gttcctgaag gcgctggatg acctgtccgc agaggagcgc 2100
ggcaccctgg ccctgcagtg tgaagtctct gaccccgagg cccatgtggt gtggcgcaaa 2160
gatggcgtgc agctgggccc cagtgacaag tatgacttcc tgcacacggc gggcacgcgg 2220
gggctcgtgg tgcatgacgt gagccctgaa gacgccggcc tgtacacctg ccacatgggc 2280
tccgaggaga cccgggcccg ggtccgcgtg cacgatctgc acgtgggcat caccaagagg 2340
ctgaagacaa tggaggtgct ggaaggggaa agctgcagct ttgagtgcgt cctgtcccac 2400
gagagtgcca gcgacccggc catgtggaca gtcggtggga agacagtggg cagctccagc 2460
cgcttccagg ccacacgtca gggccgaaaa tacatcctgg tggtccggga ggctgcacca 2520
agtgatgccg gggaggtggt cttctctgtg cggggcctca cctccaaggc ctcactcatt 2580
gtcagagaga ggccggccgc catcatcaag cccctggaag accagtgggt ggcgccaggg 2640
gaggacgtgg agctgcgctg tgagctgtca cgggcgggaa cgcccgtgca ctggctgaag 2700
gacaggaagg ccatccgcaa gagccagaag tatgatgtgg tctgcgaggg cacgatggcc 2760
atgctggtca tccgcggggc ctcgctcaag gacgcgggcg agtacacgtg tgaggtggag 2820
gcttccaaga gcacagccag cctccatgtg gaagaaaaag caaactgctt cacagaggag 28$0
ctgaccaatc tgcaggtgga ggagaaaggc acagctgtgt tcacgtgcaa gacggagcac 2940
cccgcggcca cagtgacctg gcgcaagggc ctcttggagc tacgggcctc agggaagcac 3000
cagcccagcc aggagggcct gaccctgcgg ctcaccatca gtgccctgga gaaggcagac 3060
agcgacacct atacctgcga cattggccag gcccagtccc gggcccagct cctagtgcaa 3120
ggccggagag tgcacatcat cgaggacctg gaggatgtgg atgtgcagga gggctcctcg 3180
gccaccttcc gttgccggat ctccccggcc aactacgagc ctgtgcactg gttcctggac 3240
aagacacccc tgcatgccaa cgagctcaat gagatcgatg cccagcccgg gggctaccac 3300
gtgctgaccc tgcggcagct ggcgctcaag gactcgggca ccatctactt tgaggcgggt 3360
gaccagcggg cctcggccgc cctgcgggtc actgagaagc caagcgtctt ctcccgggag 3420
ctcacagatg ccaccatcac agagggtgag gacttgaccc tggtgtgcga gaccagcacc 3480
tgcgacattc ctgtgtgctg gaccaaggat gggaagaccc tgcgggggtc tgcccggtgc 3540
cagctgagcc atgagggcca ccgggcccag ctgctcatca ctggggccac cctgcaggac 3600
agtggacgct acaagtgtga ggctgggggc gcctgcagca gctccattgt cagggtgcat 3660
gcgcggccag tgcggttcca ggaggccctg aaggacctgg aggtgctgga gggtggtgct 3720
gccacactgc gctgtgtgct gtcatctgtg gctgcgcccg tgaagtggtg ctatggaaac 3780
aacgtcctga ggccaggtga caaatacagc ctacgccagg agggtgccat gctggagctg 3840
gtggtccgga acctccggcc gcaggacagc gggcggtact catgctcctt cggggaccag 3900
actacttctg ccaccctcac agtgactgcc ctgcctgccc agttcatcgg gaaactgaga 3960
aacaaggagg ccacagaagg ggccacggcc acgctgcggt gtgagctgag caaggcagcc 4020
cctgtggagt ggagaaaggg gtccgagacc ctcagagatg gggacagata ctgtctgagg 4080
caggacgggg ccatgtgtga gctgcagatc cgtggcctgg ccatggtgga tgccgcggag 4140
tactcgtgtg tgtgtggaga ggagaggacc tcagcctcac tcaccatcag gcccatgcct 4200
gcccacttca taggaagact gagacaccaa gagagcatag aaggggccac agccacgctg 4260
cggtgtgagc tgagcaaggc ggcccccgtg gagtggagga aggggcgtga gagcctcaga 4320
gatggggaca gacatagcct gaggcaggac ggggctgtgt gcgagctgca gatctgtggc 4380
ctggctgtgg cagatgctgg ggagtactcc tgtgtgtgtg gggaggagag gacctctgcc 4440
actctcaccg tgaaggccct gccagccaag ttcacagagg gtctgaggaa tgaagaggcc 4500
gtggaagggg ccacagccat gttgtggtgt gaactgagca aggtggcccc tgtggagtgg 4560
aggaaggggc ccgagaacct cagagatggg gacagataca tcctgaggca ggaggggacc 4620
aggtgtgagc tgcagatctg tggcctggcc atggcggacg ccggggagta cttgtgtgtg 4680
tgcgggcagg agaggacctc agccacgctc accatcaggg ctctgcctgc caggttcata 4740
gaagatgtga aaaaccagga ggccagagaa ggggccacgg ctgtgctgca gtgtgagctg 4800
aacagtgcag cccctgtgga gtggagaaag gggtctgaga cccttagaga tggggacaga 4860
tacagcctga ggcaggacgg gactaaatgt gagctgcaga ttcgtggcct ggccatggca 4920
gacactgggg agtactcgtg cgtgtgcggg caggagagga cctcggctat gctcaccgtc 4980
agggctctac ccatcaagtt cacagagggt ctgaggaacg aagaggccac agaaggggca 5040
acagccgtgc tgcggtgtga gctgagcaag atggcccccg tggagtggtg gaaggggcat 5100
gagaccctca gagatggaga cagacacagc ctgaggcagg acggggccag gtgtgagctg 5160
cagatccgcg gcctcgtggc agaggacgct ggggagtacc tgtgcatgtg cgggaaggag 5220
aggacctcag ccatgctcac cgtcagggcc atgccttcca agttcataga gggtctgagg 5280
aatgaagagg ccacagaagg ggacacggcc acgctgtggt gtgagctgag caaggcggca 5340
ccggtggagt ggaggaaggg gcatgagacc ctcagagatg gggacagaca cagcctgagg 5400
36/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
caggatgggt ccaggtgtga gctgcagatc cgtggcctgg ctgtggtgga tgccggggag 5460
tactcgtgtg tgtgcgggca ggagaggacc tcagccacac tcactgtcag ggccctgcct 5520
gccagattca tagaagatgt gaaaaaccag gaggccagag aaggggccac ggccgtgctg 5580
caatgtgagc tgagcaaggc ggcccccgtg gagtggagga aggggtctga gaccctcaga 5640
ggtggggaca gatacagcct gaggcaggat gggaccagat gtgagctgca gattcatggc 5700
ctgtctgtgg cagacactgg ggagtactcg tgtgtgtgcg ggcaggagag gacctcggcc 5760
acactcaccg tcagggccct gcctgcacga ttcactcaag atctgaagac caaggaggcc 5820
tcagaagggg ccacagctac actgcagtgt gagctgagca aggtggcccc tgtggaatgg 5880
aagaagggtc ctgagaccct cagagatggg ggcagataca gcctgaagca ggatgggacg 5940
aggtgtgagc tgcagatcca tgacctgtct gtggcggatg ctggggaata ctcatgcatg 6000
tgtggacaag agaggacctc ggccatgctc actgtcaggg ccctgcctgc caggttcaca 6060
gagggtctga ggaatgaaga ggccatggaa ggggccacag ccacactgca atgtgagctg 6120
agcaaggcag cccctgtgga gtggaggaaa ggccttgagg ctctcagaga tggggacaaa 6180
tacagcctga gacaagacgg ggctgtgtgt gagctgcaga ttcatggcct ggctatggca 6240
gataacgggg tgtactcatg tgtgtgtggg caggagagga cctcagctac actcactgtc 6300
agggccctgc ctgccagatt catagaggat atgagaaacc agaaggccac agaaggggct 6360
acagtcacat tgcaatgtaa gctgagaaag gcggcccccg tggagtggag aaaggggccc 6420
aacaccctca aagatgggga caggtacagc ctgaagcagg atgggaccag ttgtgagctg 6480
cagattcgtg gcctggtcat agcagatgct ggagaatact cgtgcatatg tgagcaggag 6540
aggacctcgg ccacgctcac tgtcagggcc ctgccggcca gattcataga agatgtgaga 6600
aatcacgagg ccacagaagg ggccacagct gtgctgcagt gtgagctgag caaggcggcc 6660
cccgtggagt ggcggaaggg gtctgagacc ctcagagatg gggacagata tagcctgagg 6720
caggacggga cgaggtgtga gctgcagatt cgtggcctgg ctgtggagga cactggagag 6780
tatttgtgtg tgtgcgggca ggagagaacc tcagctacac tcactgtcag ggccctgcca 6840
gccagattca tagacaacat gacaaaccag gaagccagag aaggggccac ggccacactg 6900
cactgtgaac tgagcaaggc ggcccccgtg gagtggagga aggggcgtga gagcctcaga 6960
gatggggaca gacatagcct gaggcaggac ggggctgtgt gcgagctgca gatctgtggc 7020
ctggctgtgg cagatgctgg ggagtactcc tgtgtgtgtg gggaggagag gacctctgcc 7080
actctcaccg tgaagggtaa tgactgctcc tggccacgtg catgggtggc tatgtctgag 7140
cgggtgtgca cattcctgct ttgtgctcac gtctgcgctg tggccttccc tgtctttctg 7200
cgtgtggttc cttcattcct tcagtaggga ttccccacac ctgctgcgtg ttacccgtct 7260
caggagcagg aagacagcag agaagagagg gctgtttgag ccctacagag ttgtaggcag 7320
agacagagga tgtggggaga accaaatcat tacaaaagtg agatcacaga tcttctccag 7380
ggtaacacta ggaaggcagg aaggcaatca ggaggagccc cagaagcgga acacagacgg 7440
gagtgggggg atggcaggtg ggctggagtg cacatcctgg aaggggaggg tccattgtga 7500
ggaggagaca ggcagggtca ctgtggcgga ggatggtgat gggggtcgac tgtggagagg 7560
aggg 7564
<210> 16
<211> 5998
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 8065556CB1
<400> 16
atggagtcgc tcctgctgcc ggtgctgctg ctgctggcca tactgtggac gcaggctgcc 60
gccctcatta atctcaagta ctcggtagaa gaggagcagc gcgccgggac ggtgattgcc 120
aacgtggcca aagacgcgcg agaggcgggc ttcgcgctgg acccccggca ggcttcagcc 180
tttcgcgtgg tgtccaactc ggctccacac ctagtggaca tcaatcccag ctctggcctg 240
ctggtcacca agcagaagat tgaccgtgat ctgctgtgcc gccagagccc caagtgcatc 300
atctcgctcg aggtcatgtc cagctcaatg gaaatctgcg tgataaaggt ggagatcaag 360
gacctgaacg acaatgcgcc cagtttcccg gcagcacaga tcgagctgga gatctcggag 420
gcagccagcc ctggcacgcg catcccgctg gacagcgctt acgatccaga ctcaggaagc 480
37/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
tttggcgtgc agacttacga gctcacgccc aacgagctgt tcggcctgga gatcaagacg 540
cgcggcgacg gctcccgctt tgccgaactc gtggtggaaa agagcctgga ccgcgagacg 600
cagtcgcact acagcttccg aatcactgcg ctagacggtg gcgacccgcc gcgcctgggc 660
accgttggcc ttagtatcaa ggtgaccgac tccaatgaca acaacccggt gtttagcgag 720
tccacctacg cggtgagcgt gccagaaatc tcgcctccca acacacccgt catccgcctc 780
aacgccagcg atccagacga gggcaccaac ggccaggtgg tctactcctt ctatggctac 840
gtcaacgacc gcacgcgcga gctctttcag atcgacccgc acagtggcct ggtcactgtc 900
actggcgctt tagactacga agaggggcac gtgtacgaac tggacgtgca ggctaaggac 960
ttggggccca attccatccc ggcacactgc aaggtcaccg tcagcgtgct ggacaccaat 1020
gacaatccgc cggtcatcaa cctgctgtca gtcaacagtg agcttgtgga ggtcagcgag 1080
agcgcccccc cgggctacgt gatcgccttg gtgcgggtgt ctgatcgcga ctcaggcctc 1140
aatggacgtg tgcagtgccg tttgctgggc aatgtgccct ttcgactgca ggaatatgag 1200
agcttctcca ctattctggt ggacggacgg ctggaccgcg agcagcacga ccaatacaac 1260
ctcacaattc aggcacgcga cggcggcgtg cccatgctgc agagtgccaa gtcctttacc 1320
gtgctcatca ctgacgaaaa tgacaaccac ccgcactttt ccaagcccta ctaccaggtc 1380
attgtgcagg agaacaacac gcctggcgcc tatctgctct ctgtgtctgc tcgcgacccc 2440
gacctgggtc tcaacggcag tgtctcctac cagatcgtgc cgtcgcaggt gcgggacatg 1500
cctgtcttca cctatgtctc catcaatccc aactcaggcg acatctacgc gctgcgatcc 1560
tttaaccacg agcagaccaa ggcgttcgaa ttcaaggtgc tggccaagga cggcggcctt 1620
ccctcactgc aaagcaacgc tacggtgcgg gtcatcatcc tcgacgtcaa cgacaacacc 1680
ccggtcatca cagccccacc tctgattaac ggcactgccg aggtctacat accccgcaac 1740
tctggcatag gctacctggt gactgttgtc aaggcagaag actacgatga gggcgaaaat 1800
ggccgagtca cctacgacat gaccgagggc gaccgcggct tctttgaaat agaccaggtc 1860
aatggcgaag tcagaaccac ccgcaccttc ggggagagct ccaagtcctc ctatgagctt 1920
atcgtggtgg ctcacgacca cggcaagaca tctctctctg cctctgctct cgtcctaatc 1980
tacttgtccc ctgctctcga tgcccaagag tcaatgggct ctgtgaactt gtccttgatt 2040
ttcattattg ccctgggctc cattgcgggc atcctctttg taactatgat cttcgtggca 2100
atcaagtgca agcgagacaa caaagagatc cggacctaca actgcagtaa ttgtttaacc 2160
atcacttgtc tectcggctg ttttataaaa ggacaaaaca gcaagtgtct gcattgcatc 2220
tcggtttctc ccattagcga,ggagcaagac aaaaagacag aggagaaagt gagcctaagg 2280
ggaaagagaa ttgctgagta ctcctatggg catcaaaaga aatcaagcaa aaagaaaaaa 2340
atcagtaaga atgacatccg cctggtaccc cgggatgtgg aggagacaga caagatgaac 2400
gttgtcagtt gctcttccct gacctcctcc ctcaactatt ttgactacca ccagcagacg 2460
ctgcccctgg gctgccgccg ctctgagagc actttcctga atgtggagaa ccagaatacc 2520
cgcaacacca gtgctaacca catctaccat cactctttca acagccaggg gccccagcag 2580
cctgacctga ttatcaacgg tgtgcctctg cctgagactg aaaactattc ttttgactcc 2640
aactacgtga atagccgagc ccatttaatc aagagcagct ccaccttcaa ggacttagag 2700
ggcaacagcc tgaaggatag tggacatgag gagagtgacc aaactgacag tgagcatgat 2760
gtccagcgga gcctgtattg tgatactgct gtcaacgatg tgctgaacac cagtgtgacc 2820
tccatgggat ctcagatgcc tgatcatgat cagaatgaag gatttcattg ccgggaagaa 2880
tgccggattc ttggecactc tgacaggtgc tggatgcccc ggaaccccat gcccatccgt 2940
tccaagtccc ctgagcatgt gaggaacatc atcgcgctgt ctattgaagc tactgctgct 3000
gatgtcgagg cttatgacga ctgcggcccc accaaacgga ctttcgcaac ctttgggaaa 3060
gatgtcagcg accacccggc tgaggagagg cctaccctga aaggcaagag gactgtcgat 3120
gtgaccatct gcagccccaa ggtcaacagc gttatccggg aggcaggcaa tggctgtgag 3180
gcgattagcc ctgtcacctc ccccctccac ctcaagagct ctctgcccac caagccttcc 3240
gtgtcttaca ccattgccct ggctccccca gcccgtgatc tggagcagta tgtcaacaat 3300
gtcaacaatg gccctactcg tccctctgaa gctgagcccc gtggagctga tagcgagaaa 3360
gtcatgcatg aggtcagccc cattctgaag gaaggtcgca acaaagagtc ccctggtgtg 3420
aagcgtctga aggatatcgt tctctaaacc agtctccagg aagaagagaa agaaaccaca 3480
ctggctagtg aagaagcagg agcttcttgt tttaattgct caccaatggt tggttcttga 3540
gtggctatat ttcagagctt ttcctaaatg tattgtttat aggtgattat cattctgtga 3600
cagtcccttg tttcaacagg cagcaggggt gttcagttgg agcaaattag ctttggcttg 3660
agttgttcat ggggccttga tgttggggaa acagagacaa attcagttgt gaaaagtatt 3720
atgtattaag tgtttgaatt tatatatttt tctatgtcaa aattataata taaattacca 3780
ttgtttgtgg aggattacat ttaaaaaagc aaaaagtgaa aaaaaaaagc tctggacatc 3840
38/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
tttaataagc tcgccactgt tttttgtagt gtagacaagt taatggtcat ttattgtgta 3900
ctattcattg attcagtgat gtgaaattga gcccccaaaa ggttgtttct gaagctcgag 3960
tacttcccaa tgccgctact ttgctgtgga cacccctgct ttaaaaaccc ttttgctagc 4020
tgtgctattg ttgtatttga tattgcaaat tgctatgtgt gtggtgatgg caaaaggctt 4080
ttaaaagttg gtgttttctt tttcttaaaa aaataataaa actacagaca aaaacaaagt 4140
gcaaacaact gagacagcaa atgaatgagc aacaccatgc atatagattg agtacttgga 4200
gaatactcac gttttttaac tcatgtagtg attgctcacc actttgcaaa gtatttttcc 4260
tgcctttcag tggtctgccc aggtccatga tagatcattg cagaaagtca tttttggata 4320
ggctgctatc taatttgcag ttgtcagaaa tactgtgctg aaagttttat gacatccatc 4380
ttttaaattt tcaggccctg aacaggaaag ttgctcctga agttatgttt ccaggcttag 4440
aaattccctc tctccaatca tctaaaattg aagatgactg aatctatttt aagatctaaa 4500
ttagcatctc ttcagacaca cacttcctga ttcagtgttt cccaatcatg attagaatta 4560
gactgcaaag cacgtcatgg gctgggattg agaactccat gttgctctgt atcttaggct 4620
tatatacgta gggatagagt aagcagtaca aagtgtatat tttggaaaag acaggtaaca 4680
ggtaacagca tcgctaatcc aattactgtg ccttctaaac ggagacactc agacacttga 4740
actcatcttt ttatgactaa tagttttttg attaaaaatg tgaacaagaa aatactaaaa 4800
taaaaatcct ttcgatttag ccaactcttt ttgctgtagg ctaggaatac acacttttta 4860
aattaacaca ctgtagatcc tttttttctg ttttaacatt cctctaactc ccccatttat 4920
cttttgtcta ttaatattca ctagccaaca tagtattttt gcagcctaag agttcattat 4980
ttaaaaataa actaaagaaa tatgtcacct ttgttctgcc ttggtcttaa gagagttgtt 5040
tctagagaaa caagttttat ggttctgttt tcatttgttt cattttttga aattcaggaa 5100
tacacagaga gaaggcctag aggttagagc actagaatgc aaaagaggat ttattttctt 5160
aagtttaggt agataagtca gctcttttga atgttttact tatttttgcc tgagttccta 5220
gctttacgca ttactaggaa tattttcttt tacaggaggg agaagggttt tgagggaggg 5280
ggtgggtaag gcagtaggga tggggttagg taggggagaa catttctgaa aaagaactta 5340
taatgaagct acaaatccat ccttatttct tattcaggct gaatactacc tcatgcaaaa 5400
tatggtgtgg ctgcaaattt ccctctgaag ctgacacaat taaggatgaa ttaccataac 5460
atcttcattt ttcctcattt catgtgcctc tcatacagtt ttctccctct gatttatttc 5520
atttggtagt ggatttgaat taagtattta tttctctttg caagtgacta tttgattaac 5580
acattaaaaa ttttttaaaa atttcccctt taagttatga tggtgctata gaatttagac 5640
tgtttctcac ctgatccatc ctgatattat gttattagct accgatttca aggtcacttt 5700
gaagtcagac ttcacagttt ctacaggtgt atttctgcta tgtccagggg accacggcgc 5760
gaacaatttg gggcgtggac ctcattgccg ttggtgttgg cccaagagcc tggtggacag 5820
cctacagcca gttaatgtag gggcgggtga atggcgccgg aggccacctg aatacttagg 5880
atagaccccc tcccacaaca tggccggggg caattggccc cccgggggga gaactagaat 5940
tcccgcgtgt gcccacctaa taattgcggc ctcacataat cccctaatac aaaatctc 5998
<210> 17
<211> 3593
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7037678CB1
<400> 17
gccgcgccag acggagcccg gggttcgacg ccaggattgg ctagcaagta gggagctttc 60
gccgccgccc cgggcccctc ggactgtgcc ggcgccgcac ccgaggctct cgccagcccg 120
gcgccccggt gctgaggaat cattgacata gagtaactcc acagcatgtg tcttcaagag 180
cttccctaaa agattaaagg ttatacaaaa cttaaaagaa gcagcaattc tattcgcttg 240
ttattggact tgaaactccc tttgacctcg gaaactgaag atgaggttgc catgggaact 300
gctggtactg caatcattca tttggtgcct tgcagatgat tccacactgc atggcccgat 360
ttttattcaa gaaccaagtc ctgtaatgtt ccctttggat tctgaggaga aaaaagtgaa 420
gctcaattgt gaagttaaag gaaatccaaa acctcatatc aggtggaagt taaatggaac 480
agatgttgac actggtatgg atttccgcta cagtgttgtt gaagggagct tgttgatcaa 540
39/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
taaccccaat aaaacccaag atgctggaac gtaccagtgc acagcgacaa actcgtttgg 600
aacaattgtt agcagagaag caaagcttca gtttgcttat cttgacaact ttaaaacaag 660
aacaagaagc actgtgtctg tccgtcgagg tcaaggaatg gtgctactgt gtggcccgcc 720
accccattct ggagagctga gttatgcctg gatcttcaat gaataccctt cctatcagga 780
taatcgccgc tttgtttctc aagagactgg gaatctgtat attgccaaag tagaaaaatc 840
agatgttggg aattatacct gtgtggttac caataccgtg acaaaccaca aggtcctggg 900
gccacctaca ccactaatat tgagaaatga tggagtgatg ggtgaatatg agcccaaaat 960
agaagtgcag ttcccagaaa cagttccgac tgcaaaagga gcaacggtga agctggaatg 1020
ctttgcttta ggaaatccag taccaactat tatctggcga agagctgatg gaaagccaat 1080
agcaaggaaa gccagaagac acaagtcaaa tggaattctt gagatcccta attttcagca 1140
ggaggatgct ggtttatatg aatgtgtagc tgaaaattcc agagggaaaa atgtagcaag 1200
gggacagcta actttctatg ctcaacctaa ttggattcaa aaaataaatg atattcacgt 1260
ggccatggaa gaaaatgtct tttgggaatg taaagcaaat ggaaggccta agcctacata 1320
caagtggcta aaaaatggcg aacctctgct aactcgggat agaattcaaa ttgagcaagg 1380
aacactcaac ataacaatag tgaacctctc agatgctggc atgtatcagt gtttggcaga 1440
gaataaacat ggagttatct tttccaacgc agagcttagt gttatagctg taggtccaga 1500
tttttcaaga acactcttga aaagagtaac tcttgtcaaa gtgggaggtg aagttgtcat 1560
tgagtgtaag ccaaaagcgt ctccaaaacc tgtttacacc tggaagaaag gaagggatat 1620
attaaaagaa aatgaaagaa ttaccatttc tgaagatgga aacctcagaa tcatcaacgt 1680
tactaaatca gacgctggga gttatacctg tatagccact aaccattttg gaactgctag 1740
cagtactgga aacttggtag tgaaagatcc aacaagggta atggtacccc cttccagtat 1800
ggatgtcact gttggagaga gtattgtttt accgtgccag gtaacgcatg atcactcgct 1860
agacatcgtg tttacttggt catttaatgg acacctgata gactttgaca gagatgggga 1920
ccactttgaa agagttggag ggcaggattc agctggtgat ttgatgatcc gaaacatcca 1980
actgaagcat gctgggaaat atgtctgcat ggtccaaaca agtgtggaca ggctatctgc 2040
tgctgcagac ctgattgtaa gaggtcctcc aggtccccca gaggctgtga caatagacga 2100
aatcacagat accactgctc agctctcctg gagacccggg cctgacaatc acagccccat 2160
caccatgtat gtcattcaag ccaggactcc attctccgtg ggctggcaag cagtcagtac 2220
agtcccagaa ctcattgatg ggaagacatt cacagcgacc gtggtgggtt tgaacccttg 2280
ggttgaatat gaattccgca cagttgcagc caacgtgatt gggattgggg agcccagccg 2340
cccctcagag aaacggagaa cagaagaagc tctccccgaa gtcacaccag cgaatgtcag 2400
tggtggcgga ggcagcaaat ctgaactggt tataacctgg gagacggtcc ctgaggaatt 2460
acagaatggt cgaggctttg gttatgtggt ggccttccgg ccctacggta aaatgatctg 2520
gatgctgaca gtgctggcct cagctgatgc ctctagatac gtgttcagga atgagagcgt 2580
gcaccccttc tctccctttg aggttaaagt aggtgtcttc aacaacaaag gagaaggccc 2640
tttcagtccc accacggtgg tgtattctgc agaagaagaa cccaccaaac caccagccag 2700
tatctttgcc agaagtcttt ctgccacaga tattgaagtt ttctgggcct ccccactgga 2760
gaagaataga ggacgaatac aaggttatga ggttaaatat tggagacatg aagacaaaga 2820
agaaaatgct agaaaaatac gaacagttgg aaatcagaca tcaacaaaaa tcacgaactt 2880
aaaaggcagt gtgctgtatc acttagctgt caaggcatat aattctgctg ggacaggccc 2940
ctctagtgca acagtcaatg tgacaacccg aaagccacca ccaagtcaac cccccggaaa 3000
catcatatgg aattcatcag actccaaaat tatcctgaat tgggatcaag tgaaggccct 3060
ggataatgag tcggaagtaa aaggatacaa agtcttgtac agatggaaca gacaaagcag 3120
cacatctgtc attgaaacaa ataaaacatc ggtggagctt tctttgcctt tcgatgaaga 3180
ttatataata gaaattaagc cattcagcga cggaggagat ggcagcagca gtgaacaaat 3240
tcgaattcca aagatatcaa atgcctacgc gagaggatct ggggcttcca cttcgaatgc 3300
atgtacgctg tcagccatca gtacaataat gatttccctc acagctaggt ccagtttatg 3360
acaaaagtta tctgaaggac ttgctgttta taatataagc aacatttagc tagttgtttg 3420
gaagacaccc agtactaagt aatattgttg ttcaagtaca tcttattact ggaataaaaa 3480
tgttttttgc ttctttagga atggcattat acagtacttc ctcaaagcaa atctagcttg 3540
gtctgaagtt tcttgggaaa ctctgcaatg cactgaagac atctgtaata tga 3593
<210> 18
<211> 4565
<212> DNA
<213> Homo Sapiens
40/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
<220>
<221> misc_feature
<223> Incyte ID No: 1428867CB1
<400> 18
cggctcgagc ctgtgtgcca gcgcctgtcc ggcgcctgcc tgccgcctcc gtggcgaagg 60
ggacacaggt ccctgcggat gtgatggccc agctatggct gtcctgcttc ctccttcctg 120
ccctcgtggt gtctgtggca gccaacgtgg ccccgaagtt cctagccaac atgacgtcag 180
tgatcctgcc tgaggacctg cctgtgggtg cccaggcctt ctggttggta gcggaagacc 240
aggacaatga ccctctgacc tatgggatga gcggccccaa tgcctacttc ttcgctg°~ca 300
ctccgaaaac tggggaagtg aagctggcca gcgctctgga ctacgagaca ctctacacat 360
tcaaagtcac catctccgtg agcgacccct acatccaggt gcagagggag atgctggtga 420
ttgtggaaga tagaaacgac aacgcacccg ttttccagaa caccgctttc tccaccagca 480
tcaacgagac cctgcccgtg ggcagtgtgg tgttctccgt gctggccgtg gataaagaca 540
tggggtctgc aggcatggtc gtgtactcca tagagaaggt catccctagc actggggaca 600
gcgagcatct cttccggatc ctggccaatg gctccatcgt cctcaatggc agcctcagct 660
acaacaacaa gagcgctttc taccagctgg agctgaaggc ctgtgacttg ggcggcatgt 720
accacaacac cttcaccatc cagtgctccc tgcctgtctt cctgtccatc tccgtggtgg 780
accagcctga ccttgacccc cagtttgtca gggagtttta ctcggcctct gtggctgagg 840
atgcagccaa gggaacctcg gtgctgacgg tggaggctgt ggatggcgac aaaggcatca 900
atgaccctgt gatctacagc.atctcctact ccacgcggcc cggctggttt gacatcgggg 960
cagatggggt gatcagggtc aacggctccc tggaccgtga gcagctgctg gaggcggatg 1020
aggaggtgca gctgcaggtc acggccaccg agacacacct caacatctac gggcaggagg 1080
ccaaggtgag catctgggtg acagtgagag tgatggacgt caatgaccac aaacctgagt 1140
tttacaactg cagcctccca gcctgcacct tcacccccga agaggcccaa gtgaacttca 1200
ctggctacgt ggacgagcat gcctcccccc gcatccccat cgatgacctc accatggtgg 1260
tctacgaccc ggacaagggc agcaatggca ccttcctgtt gtcgctgggg ggccccgatg 1320
cagaagcctt cagcgtctcc ccggagcggg cagcgggctc agcctccgtt caggtgctgg 1380
tgagagtatc cgcgctggtg gactacgaga ggcagacggc gatggcggtg caggttgtgg 1440
ccacagactc cgtcagccag aacttctccg tcgccatggt gaccatccac cttagagaca 1500
ttaatgacca caggcccacg tttccccaga gcttgtacgt cctcacggtg ccagagcaca 1560
gcgccaccgg ctctgtggtc accgacagca tccacgccac ggacccagac acgggcgcgt 1620
ggggccaaat tacctacagc ctgctcccag gaaatggggc agacctcttc caagtggatc 1680
ccgtctcagg gacggtgacg gtgaggaacg gtgagctgct ggaccgggag agccaggccg 1740
tgtactacct gacgctgcag gccacagatg gcgggaacct gtcctcctcc accacactgc 1800
agatccacct gctggacatc aacgacaatg cacccgtggt tagcggctcc tacaacatct 1860
tcgtccagga ggaggagggc aatgtctccg tgaccatcca ggtgtgagcc tgctggacct 1920
ggtgggccca cgacaatgat gagccgggca ccaacaacag ccgtctgctc ttcaacctgc 1980
tgcctggccc ctacagccac aacttctcct tggaccctga cacagggctc ctcagaaacc 2040
tggggcccct ggacagagag gccatcgacc ccgccctgga gggccgcatt gtgctgacag 2100
tgcttgtgtc tgactgcggc gagcctgtcc tcggcaccaa agtcaatgtc accatcactg 2160
tggaggacat caatgataac ctgcccatct tcaatcagtc cagctacaac tttacggtga 2220
aggaggagga tccaggagtg ctagtgggcg tggtgaaggc ctgggacgcg gaccagacgg 2280
aagccaacaa ccgcatcagc ttcagcctgt cggggagtgg tgccaactac ttcatgatcc 2340
gaggcttggt gctgggggct gggtgggctg agggctacct ccggctgccc ccggacgtga 2400
gcctggatta cgagacacag cccgtcttca acttgacagt gagtgctgag aacccagacc 2460
cccagggggg tgagaccata gtagacgtct gcgtgaatgt gaaagacgtg aacgacaatc 2520
cccccaccct ggatgtagcc tcactccggg gcatccgtgt ggctgagaat ggctcacagc 2580
acggccaggt ggctgtggtg gttgcctcgg atgtggacac cagtgcccag ctggagatac 2640
agcttgtgaa cattctctgc accaaggccg gggtcgatgt gggcagccta tgctggggct 2700
ggttctcagt ggcagccaac ggctctgtgt acatcaacca gagcaaagcc atcgactacg 2760
aggcctgtga cctggtcacg ctggttgtgc gggcctgtga cctagccacg gaccccggct 2820
tccaggccta cagcaacaat ggaagcctcc tcattaccat tgaggacgtg aatgacaatg 2880
caccctattt tctgcctgag aataagactt ttgtgatcat ccctgaactc gtgctgccca 2940
accgggaggt ggcttctgtc cgggccagag acgatgattc agggaacaat ggcgtcatcc 3000
tgttctccat cctccgagta gacttcatct ctaaggacgg ggccaccatc cctttccagg 3060
41/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
gtgtcttctc gatcttcacc tcctccgagg ccgacgtgtt cgctgggagc attcagccgg 3120
tgaccagcct cgactccact ctccaaggca cctaccaagt gacagtccag gccagggaca 3180
gaccttcctt gggtcctttc ctggaagcca ccaccaccct gaatctcttc accgtggacc 3240
agagttaccg ctcgcggctg cagttctcca caccgaagga ggaggtgggc gccaacagac 3300
aggcgattaa tgcggctctt acccaggcaa ccaggactac agtatacatt gtggacattc 3360
aggacataga ttctgcagct cgggcccgac ctcactccta cctcgatgcc tactttgtct 3420
tccccaatgg gtcagccctg acccttgatg agctgagtgt gatgatccgg aatgatcagg 3480
actcgctgac gcagctgctg cagctggggc tggtggtgct gggctcccag gagagccagg 3540
agtcagacct gtcgaaacag ctcatcagtg tcatcatagg attgggagtg gctttgctgc 3600
tggtccttgt gatcatgacc atggccttcg tgtgtgtgcg gaagagctac aaccggaagc 3660
ttcaagctat gaaggctgcc aaggaggcca ggaagacagc agcaggggtg atgccctcag 3720
cccctgccat cccagggact aacatgtaca acactgagcg agccaacccc atgctgaacc 3780
tccccaacaa agacctgggc ttggagtacc tctctccctc caatgacctg gactctgtca 3840
gcgtcaactc cctggacgac aactctgtgg atgtggacaa gaacagtcag gaaatcaagg 3900
agcacaggcc accacacaca ccaccagagc cagatccaga gcccctgagc gtggtcctgt 3960
taggacggca ggcaggcgca agtggacagc tggaggggcc atcctacacc aacgctggcc 4020
tggacaccac ggacctgtga caggggcccc cactcttctg gaccccttga agaggcccta 4080
ccacacccta actgcacctg tctccctgga gatgaaaata tatgacgctg ccctgcctcc 4140
tgcttttggc caatcacggc agacaggggt tggggaaata ttttattacc aatgtatact 4200
gtgacagttt gtagccaaaa actgcggctg gaggggtggg gacgggacac tgagtggtca 4260
caagggactt gggctcacag cacagggggg acaaggggct ggagagggtg gcctttaaaa 4320
gacaactgtg gttatagaat gagcccagct gtgacctcca gaccttcctg agaccctctg 4380
gcctttctgt gactctctct cagctgagcc cccagggtac ttcctgtagc tgtctttggc 4440
ctctctggga atctcaaacc tgtgactcag tgggagaggg gatggggctg gaaccaggcg 4500
ggtgggagat aggaactggg gaaggaccac caacagcatg caagagacgc cccggccacg 4560
gggcc 4565
<220> 19
<211> 2847
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2736276CB1
<400> 19
gggagcaaaa aagggggtga atccgggggg gtgcgcgccg cggttgttat aataacggtc 60
taggtacgag aagatgttat atctaaccca aagacatttc taggctcatc ccccctacct 120
cagcatctct ccctgtgacc acggtggctc cccagcccat tcccatacag agaaagggga 180
agaatggtgt ggccataatg tcaaggctct ttgacatgcc ttgtgatgaa actctctgct 240
ctgctgacag cttctgtgtc aatgactaca cctggggggg ctcgcgatgc cagtgcaccc 300
tgggcaaagg tggtgagagc tgctcagaag atattgttat ccagtatcct cagttctttg 360
gccactccta tgtaacgttt gaacctctga agaattctta tcaggcattt caaattactc 420
ttgaatttag ggcggaggca gaggatggct tactgctcta ctgtggggag aacgaacacg 480
ggagggggga tttcatgtcc ctggctatca tccgacgctc cctgcagttc aggtttaatt 540
gtggaactgg ggttgccatc atcgtaagtg agaccaaaat caaactaggg ggttggcaca 600
cggttatgct ctacagagat gggctgaacg ggctgctgca gctgaacaat ggcaccccag 660
tgacaggcca gtctcagggc caatacagta aaattacttt ccggacacct ctctatcttg 720
gtggcgctcc cagcgcttac tggttggtta gagcaacagg gacaaaccga ggctttcaag 780
gctgtgtgca gtcgctcgct gtgaatggga ggagaattga catgaggccc tggcccctgg 840
gaaaagcact cagtggggct gatgtggggg aatgcagcag tggaatctgt gatgaggcct 900
cgtgcatcca tggtggcacc tgcacagcaa tcaaagccga ctcctacatt tgcctctgtc 960
cccttgggtt taaaggtcga cactgtgaag atgctttcac cttgaccatt cctcagttca 1020
gagagtctct gagatcttac gctgcaactc cctggccact ggagccccag cattaccttt 1080
ccttcatgga atttgagatc acatttcggc cagactcagg agatggtgtc ctcctgtaca 1140
42/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
gctatgacac aggcagcaaa gacttcctgt ccatcaactt ggcagggggc cacgtggagt 1200
tccgctttga ctgtggctct gggaccggtg tcctcaggag tgaagatccc ctcaccctgg 1260
gcaactggca cgagcttcgt gtatctcgca cagcaaagaa tggaatctta caggtggata 1320
agcagaagat agtggaggga atggcagagg gaggcttcac acagattaag tgcaacacag 1380
acattttcat tggcggagtc cccaattatg atgatgtgaa gaagaactcg ggtgtcctga 1440
agcctttcag cgggagcatc cagaagatca tcctgaatga ccgaaccatc catgtgaagc 1500
atgacttcac ctccggagtg aatgtggaga atgcggccca cccctgtgtg agagcccctt 1560
gtgcccatgg gggcagctgc cggcccagga aggagggcta tgactgtgac tgccccttgg 1620
gctttgaggg gcttcactgc cagaaagcga tcatagaagc cattgagatc ccgcagttta 1680
tcggccgcag ttacctgacg tatgacaacc cagatatctt gaagagggtg tcaggatcaa 1740
gatcaaatgt gttcatgagg tttaaaacaa ctgccaagga tggccttttg ctgtggaggg 1800
gagacagccc catgagaccc aacagcgact tcatttcctt gggccttcgg gatggagccc 1860
tcgtgttcag ctataacctg ggcagtggtg tggcatccat catggtgaat ggctccttca 1920
acgatggtcg gtggcaccga gttaaggccg ttagggatgg ccagtcagga aagataaccg 1980
tggatgacta tggagccaga acaggcaaat ccccaggcat gatgcggcag cttaacatca 2040
atggagctct gtatgtgggt ggaatgaagg aaattgctct gcacactaac aggcaatata 2100
tgagagggct cgtgggctgt atctctcact tcaccctgtc caccgattac cacatttccc 2160
tcgtggaaga tgccgtggat gggaaaaaca tcaacacttg tggagccaag taacaccagc 2220
tggccttgtc caagggacag agccttctat tctgagaatc ccaggggccc tcagaccctg 2280
cctgatgcta tatgcagagg cccagggacc aggtgtgttt cctctcacca agaagaaagt 2340
acacactgat gagaaactga gaaccaagac aggcatccct gggtggcctt tcctgctgac 2400
actccacgag ctgacccagc agaattctct gtgtaggaag catcggactt tgtccattga 2460
atatgtagcg gctgccagag atcacacatc aatgcaaatt ccagagcctg tctgctatag 2520
ctcagtgact gtgttgtgat tcatagtaca ttaaaaagag agagagagag aaagaatccc 2580
acagggcact attaaaatac ttctctcctt ccctgactca tgacactctt cctgacagca 2640
gaatgactgt gtgaccttga acttcacatt tcccacattg gcccttggat tgttcggatt 2700
aaccccttcc actcctcact ggctggttca ctgtgttctg actagtccat aaaaataaag 2760
atggaaggag atcaaaccaa aaaaaaaaaa aagggggggc cccgaatagt ggagccggaa 2820
acccggggaa taatccggac cggactg 2847
<210> 20
<211> 1147
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3683719CB1
<400> 20
ggcgacgggc aggacgcccc gttcgcctta gcgcgtgctc aggagttggt gtcctgcctg 60
cgctcaggat gagggggaat ctggccctgg tgggcgttct aatcagcctg gccttcctgt 120
cactgctgcc atctggacat cctcagccgg ctggcgatga cgcctgctct gtgcagatcc 180
tcgtccctgg cctcaaagga gacatggggg acaaaggaca gaaaggcagt gtgggtcgtc 240
atggaaaaat tggtcccatt ggctctaaag gtgagaaagg agattccggt gacataggac 300
cccctggtcc taatggagaa ccaggcctcc catgtgagtg cagccagctg cgcaaggcca 360
tcggggagat ggacaaccag gtctctcagc tgaccagcga gctcaagttc atcaagaatg 420
ctgtcgccgg tgtgcgcgag acggagagca agatctacct gctggtgaag gaggagaagc 480
gctacgcgga cgcccagctg tcctgccagg gccgcggggg cacgctgagc atgcccaagg 540
acgaggctgc caatggcctg atggccgcat acctggcgca agccggcctg gcccgtgtct 600
tcatcggcat caacgacctg gagaaggagg gcgccttcgt gtactctgac cactccccca 660
tgcggacctt caacaagtgg cgcagcggtg agcccaacaa tgcctacgac gaggaggact 720
gcgtggagat ggtggcctcg ggcggctgga acgacgtggc ctgccacacc accatgtact 780
tcatgtgtga gtttgacaag gagaacatgt gagcctcagg ctggggctgc ccattggggg 840
ccccacatgt ccctgcaggg ttggcaggga cagagcccag accatggtgc cagccaggga 900
gctgtccctc tgtgaagggt ggaggctcac tgagtagagg gctgttgtct aaactgagaa 960
43/45
aggcctgtga cctggtcacg
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
aatggcctat gcttaagagg aaaatgaaag tgttcctggg gtgctgtctc tgaagaagca 1020
gagtttcatt acctgtattg tagccccaat gtcattatgt aattattacc cagaattgct 1080
cttccataaa gcttgtgcct ttgtccaagc tatacaataa aatctttaag tagtgcagta 1140
aaaaaaa 1147
<210> 21
<211> 2001
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6988448CB1
<400> 21
atgccccccg gggggagcgg gccggggggg tgcccgcgcc gccccccggc cctggctggg 60
cccctgccgc cgcctccacc gccgccgccg ccacctctgc tgccgctgtt gccgctgttg 120
ctgctgttgc tgctgggggc ggccgagggg gcccgggtct cctccagcct cagcaccacc 180
caccacgtcc accacttcca cagcaagcac ggcaccgtgc ccatcgccat caaccgcatg 240
cccttcctca cccgcggcgg ccacgccggg accacataca tctttgggaa ggggggagcg 300
ctcatcacct acacgtggcc ccccaatgac aggcccagca cgaggatgga tcgcctggcc 360
gtgggcttca gcacccacca gcggagcgct gtgctggtgc gggtggacag cgcctccggc 420
cttggagact acctgcagct gcacatcgac cagggcaccg tgggggtgat ctttaacgtg 480
ggcacggacg acattaccat cgacgagccc aacgccatag taagcgacgg caaataccac 540
gtggtgcgct tcactcgaag cggcggcaac gccaccctgc aggtggacag ctggccggtc 600
aacgagcggt acccggcagg aaactttgat aacgagcgcc tggcgattgc tagacagaga 660
atcccctacc ggcttggtcg agtagtagat gagtggctgc tcgacaaagg ccgccagctg 720
accatcttca acagccaggc tgccatcaag atcgggggcc gggatcaggg ccgccccttc '780
cagggccagg tgtccggcct ctactacaat gggctcaagg tgctggcgct ggccgccgag 840
agcgacccca atgtgcggac tgagggtcac ctgcgcctgg tgggggaggg gccgtccgtg 900
ctgctcagtg cggagaccac ggccaccacc ctgctggctg acatggccac caccatcatg 960
gagactacca ccaccatggc cactaccacc acgcgccggg gccgctcccc cacactgagg 1020
gacagcacca cccagaacac agatgacctg ctggtggcct ctgctgagtg tccaagcgat 1080
gatgaggacc tggaggagtg tgagcccagt actggaggag agttaatatt gcccattatc 1140
acggaggact ccttagaccc ccctcccgtg gccacccgat cccccttcgt gcccccgccc 1200
cctaccttct accccttcct cacgggagtg ggcgccaccc aagacacgct gcccccgccc 1260
gccgcgcgcc gcccgccctc tgggggcccg tgccaggccg agcgggacga cagcgactgc 1320
gaggagccca tcgaggcctc gggcttcgcc tccggggagg tctttgactc cagcctcccc 1380
cccacggacg acgaggactt ttacaccacc tttcccctgg tcacggaccg caccaccctc 1440
ctgtcacccc gcaaacccgc tccccggccc~aacctcagga cagatggggc cacgggcgcc 1500
cctggggtgc tgtttgcccc ctccgccccg gcccccaacc tgccggcggg caaaatgaac 1560
caccgagacc cgcttcagcc cttgctggag aacccgccct tggggcccgg ggcccccacg 1620
tcctttgagc cgcggaggcc ccctcccctg cgccccggcg tgacctcagc ccccggcttc 1680
ccccatctgc ccacagccaa ccccacaggg cctggggagc ggggcccgcc gggcgcagtg 1740
gaggtgatcc gggagtccag cagcaccacg ggcatggtgg tgggcattgt ggcggcggcg 1800
gcgctctgca tcctcatcct cctctacgcc atgtataagt accgcaatcg tgatgagggc 1860
tcctaccagg tggaccagag ccgaaactac atcagtaact cggcccagag caatggggcg 1920
gtggtgaaag agaaggcccc ggctgccccc aagacgccca gcaaggccaa gaagaacaaa 1980
gacaaggagt attatgtctg a 2001
<210> 22
<211> 1419
<212> DNA
<213> Homo Sapiens
<220>
44/45
CA 02446023 2003-10-28
WO 02/088322 PCT/US02/13874
<221> misc feature
<223> Incyte ID No: 7500307CB1
<400> 22
atgccccccg gggggagcgg gccggggggg tgcccgcgcc gccccccggc cctggctggg 60
cccctgccgc cgcctccacc gccgccgccg ccacctctgc tgccgctgtt gccgctgttg 120
ctgctgttgc tgctgggggc ggccgagggg gcccgggtct cctccagcct cagcaccacc 180
caccacgtcc accacttcca cagcaagcac ggcaccgtgc ccatcgccat caaccgcatg 240
cccttcctca cccgcggcgg ccacgccggg accacataca tctttgggaa ggggggagcg 300
ctcatcacct acacgtggcc ccccaatgac aggcccagca cgaggatgga tcgcctggcc 360
gtgggcttca gcacccacca gcggagcgct gtgctggtgc gggtggacag cgcctccggc 420
cttggagact acctgcagct gcacatcgac cagggcaccg tgggggtgat ctttaacgtg 480
ggcacggacg acattaccat cgacgagccc aacgccatag taagcgacgg caaataccac 540
gtggtgcgct tcactcgaag cggcggcaac gccaccctgc aggtggacag ctggccggtc 600
aacgagcggt acccggcagg aaactttgat aacgagcgcc tggcgattgc tagacagaga 660
atcccctacc ggcttggtcg agtagtagat gagtggctgc tcgacaaagg ccgccagctg 720
accatcttca acagccaggc tgccatcaag atcgggggcc gggatcaggg ccgccccttc 780
cagggccagg tgtccggcct ctactacaat gggctcaagg tgctggcgct ggccgccgag 840
agcgacccca atgtgcggac tgagggtcac ctgcgcctgg tgggggaggg gccgtccgtg 900
ctgctcagtg cggagaccac ggccaccacc ctgctggctg acatggccac caccatcatg 960
gagactacca ccaccatggc cactaccacc acgcgccggg gccgctcccc cacactgagg 1020
gacagcacca cccagaacac agatgacctg ctggtggcct ctgctgagtg tccaagcgat 1080
gatgaggacc tggaggagtg tgagcccagt actgccaacc ccacagggcc tggggagcgg 1140
ggcccgccgg gcgcagtgga ggtgatccgg gagtccagca gcaccacggg catggtggtg 1200
ggcattgtgg cggcggcggc gctctgcatc ctcatcctcc tctacgccat gtataagtac 1260
cgcaatcgtg atgagggctc ctaccaggtg gaccagagcc gaaactacat cagtaactcg 1320
gcccagagca atggggcggt ggtgaaagag aaggccccgg ctgcccccaa gacgcccagc 1380
aaggccaaga agaacaaaga caaggagtat tatgtctga 1419
45/45
