Note: Descriptions are shown in the official language in which they were submitted.
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IMMUNOGLOBULIN SUPERFAMILY PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of
immunoglobulin superfamily
proteins and to the use of these sequences in the diagnosis, treatment, and
prevention of immune
system, neurological, developmental, muscle, and cell proliferative disorders,
and in the assessment of
the effects of exogenous compounds on the expression of nucleic acid and amino
acid sequences of
immunoglobulin superfamily proteins.
1o BACKGROUND OF THE INVENTION
Most cell surface and soluble molecules that mediate functions such as
recognition, adhesion or
binding have evolved from a common evolutionary precursor (i.e., these
proteins have structural
homology). A number of molecules outside the immune system that have similar
functions are also
derived from this same evolutionary precursor. These molecules are classified
as members of the
immunoglobulin (Ig) superfamily. The criteria for a protein to be a member of
the Ig superfamily is to
have one or more Ig domains, which are regions of 70-110 amino acid residues
in length homologous
to either Ig variable-like (V) or Ig constant-like (C) domains. Members of the
Ig superfamily include
antibodies (Ab), T cell receptors (TCRs), class I and II major
histocompatibility (MHC) proteins, CD2,
CD3, CD4, CDB, poly-Ig receptors, Fc receptors, neural cell-adhesion molecule
(NCAM) and platelet-
derived growth factor receptor (PDGFR).
Ig domains (V and C) are regions of conserved amino acid residues that give a
polypeptide a
globular tertiary structure called an immunoglobulin (or antibody) fold, which
consists of two
approximately parallel layers of (3-sheets. Conserved cysteine residues form
an intrachain disulfide-
bonded loop, SS-75 amino acid residues in length, which connects the two
layers of the (3-sheets.
Each (3-sheet has three or four anti-parallel ~i-strands of 5-10 amino acid
residues. Hydrophobic and
hydrophilic interactions of amino acid residues within the (3-strands
stabilize the Ig fold (hydrophobic
on inward facing amino acid residues and hydrophilic on the amino acid
residues in the outward facing
portion of the strands). A V domain consists of a longer polypeptide than a C
domain, with an
additional pair of (3-strands in the Ig fold.
3o A consistent feature of Ig superfamily genes is that each sequence of an Ig
domain is
encoded by a single exon. It is possible that the superfamily evolved from a
gene coding for a single
Ig domain involved in mediating cell-cell interactions. New members of the
superfamily then arose by
exon and gene duplications. Modem Ig superfamily proteins contain different
numbers of V and/or C
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domains. Another evolutionary feature of this superfamily is the ability to
undergo DNA
rearrangements, a unique feature retained by the antigen receptor members of
the family.
Many members of the Ig superfamily are integral plasma membrane proteins with
extracellular Ig domains. The hydrophobic amino acid residues of their
transmembrane domains and
their cytoplasmic tails are very diverse, with little or no homology among Ig
family members or to
known signal-transducing structures. There are exceptions to this general
superfamily description.
For example, the cytoplasmic tail of PDGFR has tyrosine kinase activity. In
addition Thy-1 is a
glycoprotein found on thymocytes and T cells. This protein has no cytoplasmic
tail, but is instead
attached to the plasma membrane by a covalent glycophosphatidylinositol
linkage.
Another common feature of many Ig superfamily proteins is the interactions
between Ig
domains which are essential for the function of these molecules. Interactions
between Ig domains of
a multimeric protein can be either homophilic or heterophilic (i.e., between
the same or different Ig
domains). Antibodies are multimeric proteins which have both homophilic and
heterophilic interactions
between Ig domains. Pairing of constant regions of heavy chains forms the Fc
region of an antibody
and pairing of variable regions of light and heavy chains form the antigen
binding site of an antibody.
Heterophilic interactions also occur between Ig domains of different
molecules. These interactions
provide adhesion between cells for significant cell-cell interactions in the
immune system and in the
developing and mature nervous system. (Reviewed in Abbas, A.K. et al. (1991)
Cellular and
Molecular Immunolo~y, W.B. Saunders Company, Philadelphia, PA, pp.142-145.)
Antibodies
Antibodies are multimeric members of the Ig superfamily which are either
expressed on the
surface of B-cells or secreted by B-cells into the circulation. Antibodies
bind and neutralize foreign
antigens in the blood and other extracellular fluids. The prototypical
antibody is a tetramer consisting
of two identical heavy polypeptide chains (H-chains) and two identical light
polypeptide chains (L-
chains) interlinked by disulfide bonds. This arrangement confers the
characteristic Y-shape to
antibody molecules. Antibodies are classified based on their H-chain
composition. The five antibody
classes, IgA, IgD, IgE, IgG and IgM, are defined by the a, 8, s, 'y, and p. H-
chain types. There are
two types of L-chains, x and ~,, either of which may associate as a pair with
any H-chain pair. IgG,
the most common class of antibody found in the circulation, is tetrameric,
while the other classes of
antibodies are generally variants or multimers of this basic structure.
H-chains and L-chains each contain an N-terminal variable region and a C-
terminal constant
region. The constant region consists of about 110 amino acids in L-chains and
about 330 or 440 amino
acids in H-chains. The amino acid sequence of the constant region is nearly
identical among H- or L-
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chains of a particular class. The variable region consists of about 110 amino
acids in both H- and L-
chains. However, the amino acid sequence of the variable region differs among
H- or L-chains of a
particular class. Within each H- or L-chain variable region are three
hypervariable regions of
extensive sequence diversity, each consisting of about 5 to 10 amino acids. In
the antibody molecule,
the H- and L-chain hypervariable regions come together to form the antigen
recognition site.
(Reviewed in Alberts, B. et al. (1994) Molecular Biology of the Cell, Garland
Publishing, New York,
NY, pp. 1206-1213 and 1216-1217.)
Both H-chains and L-chains contain the repeated Ig domains of members of the
Ig
superfamily. For example, a typical H-chain contains four Ig domains, three of
which occur within the
constant region and one of which occurs within the variable region and
contributes to the formation of
the antigen recognition site. Likewise, a typical L-chain contains two Ig
domains, one of which occurs
within the constant region and one of which occurs within the variable region.
The immune system is capable of recognizing and responding to any foreign
molecule that
enters the body. Therefore, the immune system must be armed with a full
repertoire of antibodies
against all potential antigens. Such antibody diversity is generated by
somatic rearrangement of gene
segments encoding variable and constant regions. These gene segments are
joined together by site-
specific recombination which occurs between highly conserved DNA sequences
that flank each gene
segment. Because there are hundreds of different gene segments, millions of
unique genes can be
generated combinatorially. In addition, imprecise joining of these segments
and an unusually high rate
of somatic mutation within these segments further contribute to the generation
of a diverse antibody
population.
Neural Cell Adhesion Proteins
Neural cell adhesion proteins (NCAPs) play roles in the establishment of
neural networks
during development and regeneration of the nervous system (Uyemura et al.
(1996) Essays Biochem.
31:37-48; Brummendorf and Rathjen (1996) C~rr. Opin. Neurobiol. 6:584-593).
NCAP participates in
neuronal cell migration, cell adhesion, neurite outgrowth, axonal
fasciculation, pathfmding, 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 Charcot-Marie-Tooth disease (a
hereditary neuropathy),
Dejerine-Sottas disease, X-linked hydrocephalus, MASA syndrome (mental
retardation, aphasia,
shuffling gait and adducted thumbs), and spastic paraplegia type I. In 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
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tumor progression (Montgomery et al. (1996) J. Cell Biol. 132:475-485).
NCAPs have at least one immunoglobulin constant or variable domain (Uyemura et
al.,
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 domains, 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) and opiate-binding cell-adhesion molecule (OBCAM). 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 Ll. Mixed-type
NCAPs contain a combination of immunoglobulin domains and other motifs such as
tyrosine kinase,
epidermal growth factor-like, sema, and PSI (plexins, semaphorins, and
integrins) 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), the semaphorin/collapsin family
such as semaphorin B
and collapsin, and receptors for members of the semaphorin/collapsin family
such as plexin (for plexin,
see below).
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. Kilon
(a kindred of
NCAP-LON), for example, is expressed in the brain cerebral cortex and
hippocampus (Funatsu et al.
(1999) J. Biol. Chem. 274:8224-8230). Immunostaining localizes Kilon to the
dendrites and soma of
pyramidal neurons. Kilon has three C2 type immunoglobulin-like domains, six
predicted 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 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
4
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play roles in the reorganization of dendritic connections during neuropeptide
secretion.
Sidekick (SDK) is a member of the NCAP family. The extracellular region of SDK
contains
six immunoglobulin domains and thirteen fibronectin type ffI domains. SDK is
involved in cell-cell
interaction during eye development in Drosophila (Nguyen, D. N. T. et al.
(1997) Development 124:
3303).
Synaptic Membrane Glycoproteins
Specialized cell junctions can occur at points of cell-cell contact. Among
these cell junctions
are communicating junctions which mediate the passage of chemical and
electrical signals between
cells. In the central nervous system, communicating junctions between neurons
are known as synaptic
junctions. They are composed of the membranes and cytoskeletons of the pre-
and post-synaptic
neurons. Some glycoproteins, found in biochemically isolated synaptic
subfractions such as the
synaptic membrane (5M) and postsynaptic density (PSD) fractions, have been
identified and their
functions established. An example is the SM glycoprotein, gp50, identified as
the (32 subunit of the
Na+/K*-ATPase.
Two glycoproteins, gp65 and gp55, are major components of synaptic membranes
prepared
from rat forebrain. They are members of the Ig superfamily containing three
and two Ig domains,
respectively. As members of the Ig superfamily, it is proposed that a possible
function of these
proteins is to mediate adhesive interactions at the synaptic junction.
(Langnaese, K. et al. (1997) J.
Biol. Chem.272:821-827.)
Lectins
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. Immunol. 143:2850-2857).
Sialic acid binding Ig-like lectins (SIGLECs) are members of the Ig
superfamily that bind to
sialic acids in glycoproteins and glycolipids. SIGLECs include sialoadhesin,
CD22, CD33, myelin-
associated glycoprotein (MAG), SIGLEC-5, SIGLEC-6, SIGLEC-7, and SIGLEC-8. The
extracellular
3o region of SIGLEC has a membrane distal V-set domain followed by varying
numbers of C2-set
domains. The sialic acid binding domain is mapped to the V-set domain. Except
for MAG which is
expressed exclusively in the nervous system, most SIGLECs are expressed on
distinct subsets of
hemopoietic cells. For example, SIGLEC-8 is expressed exclusively in
eosinophils, one form of
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polymorphonuclear leucocyte (granulocyte) (Floyd, H. et al. (2000) J. Biol.
Chem. 275: 861-866).
Leucine-Rich Repeat Proteins
Leucine-rich repeat proteins (LRRPs) are involved in protein-protein
interactions. LRRPs
such as mammalian neuronal leucine-rich repeat proteins (NLLR-1 and NLLR-2),
Drosophila
connectin, slit, chaopin, and toll all play roles in neuronal development. The
extracellular region of
LRRPs contains varying numbers of leucine-rich repeats, immunoglobulin-like
domains, and fibronectin
type DI domains (Taguchi, A. et al. (1996) Brain Res. Mol. Brain Res. 35:31-
40).
In addition to the V and C2 sets of immunoglobulin-like domains, there is a D
set
immunoglobulin-like domain, named IPTlTIG (for immunoglobulin-like fold shared
by plexins and
transcription factors). IPT/TIG containing proteins include plexins, MET/ RON/
SEA (hepatocyte
growth factor receptor family), and the transcription factor XCoe2, a
transcription factor of the
Col/Olf 1/BBF family involved in the specification of primary neurons in
Xenopus (Bork, P. et al.
(1999) Trends in Biochem. 24:261-263; Santoro, N. M. et al. (1996) Mol. Cell
Biol. 16:7072-7083;
Dubois L. et al. (1998) Curr. Bio1.8:199-209). Plexins such as plexin A and
VESPR have been shown
to be neuronal semaphorin receptors that control axon guidance (Winberg M. L.
et al. (1998) Cell
95:903-916).
Expression~ro
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.
The discovery of new immunoglobulin superfamily 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, neurological, developmental,
muscle, and cell
proliferative disorders, and in the assessment of the effects of exogenous
compounds on the
expression of nucleic acid and amino acid sequences of immunoglobulin
superfamily proteins.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, immunoglobulin superfamily
proteins, referred to
collectively as "IGSFP" and individually as "IGSFP-l," "IGSFP-2," "IGSFP-3,"
"IGSFP-4," "IGSFP-
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5," "IGSFP-6," "IGSFP-7," "IGSFP-8," "IGSFP-9," "IGSFP-10," "IGSFP-11," and
"IGSFP-12." In
one aspect, the invention provides an isolated polypeptide selected from the
group consisting of a) a
polypeptide comprising an amino acid sequence selected from the group
consisting of SEQ 117 NO:1-
12, b) a polypeptide comprising a naturally occurring amino acid sequence at
least 90% identical to an
amino acid sequence selected from the group consisting of SEQ ID NO:l-12, c) a
biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ll~ NO:1-12, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ B7 NO:1-12. In one alternative, the
invention provides an
isolated polypeptide comprising the amino acid sequence of SEQ ID N0:1-12.
to 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 N0:1-12, b) a polypeptide comprising a naturally
occurring amino acid sequence
at least 90% identical to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
12, c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ ID NO:l-12, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID N0:1-12. In
one alternative, the
polynucleotide encodes a polypeptide selected from the group consisting of SEQ
ID NO:1-12. In
another alternative, the polynucleotide is selected from the group consisting
of SEQ ll~ N0:13-24.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID N0:1-12, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ D7 NO:1-12, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-12, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID N0:1-12. In one
alternative, the
invention provides a cell transformed with the recombinant polynucleotide. In
another alternative, the
invention provides a transgenic organism comprising the recombinant
polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:l-12, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO:1-12, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
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consisting of SEQ ID N0:1-12, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ >I7 NO:1-12. 'The
method comprises a)
culturing a cell under conditions suitable for expression of the polypeptide,
wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid sequence
selected from the group consisting of SEQ )D N0:1-12, b) a polypeptide
comprising a naturally
occurring amino acid sequence at least 90% identical to an amino acid sequence
selected from the
group consisting of SEQ B7 NO:l-12, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ m NO:l-12, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
)D N0:1-12.
The invention further provides an isolated polynucleotide selected from the
group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
>D N0:13-24, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:13-24,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
the polynucleotide of b), and e) an RNA equivalent of a)-d). 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
>D N0:13-24, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ m N0:13-24,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
the polynucleotide of b), and e) an RNA equivalent of a)-d). The method
comprises a) hybridizing the
sample with a probe comprising at least 20 contiguous nucleotides comprising a
sequence
complementary to said target polynucleotide in the sample, and which probe
specifically hybridizes to
said target polynucleotide, under conditions whereby a hybridization complex
is formed between said
probe and said target polynucleotide or fragments thereof, and b) detecting
the presence or absence of
said hybridization complex, and optionally, if present, the amount thereof. In
one alternative, the probe
comprises at least 60 contiguous nucleotides.
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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 )T7
N0:13-24, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ >D N0:13-24, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) amplifying said
target polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b)
detecting the presence or absence of said amplified target polynucleotide or
fragment thereof, and,
optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of
a polypeptide
selected from the group consisting of a) a polypeptide comprising an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-12, b) a polypeptide comprising a
naturally occurring
amino acid sequence at least 90% identical to an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-12, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-12, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-12,
and a pharmaceutically acceptable excipient. In one embodiment, the
composition comprises an amino
acid sequence selected from the group consisting of SEQ )D NO:1-12. The
invention additionally
provides a method of treating a disease or condition associated with decreased
expression of
functional IGSFP, comprising administering to a patient in need of such
treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-12, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:l-12, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:I-12, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ >D NO:1-12. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional IGSFP, comprising
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administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID N0:1-12, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ ID N0:1-12, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-12,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-12. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a composition comprising an antagonist compound identified
by the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
IGSFP, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds to
a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-12, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-12, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-12, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-12. The method comprises a) combining the
polypeptide with at least one
test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ll~ N0:1-12, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-12, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
N0:1-12, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-12. The method comprises a) combining the
polypeptide with at least one
test compound under conditions permissive for the activity of the polypeptide,
b) assessing the activity
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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 )D N0:13-24,
the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, b) detecting
altered expression of the target polynucleotide, and c) comparing the
expression of the target
polynucleotide in the presence of varying amounts of the compound and in the
absence of the
compound.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound; b)
hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
)D N0:13-24, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ )17 N0:13-
24, iii) a polynucleotide
having a sequence complementary to i), iv) a polynucleotide complementary to
the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions
whereby a specific
hybridization complex is formed between said probe and a target polynucleotide
in the biological
sample, said target polynucleotide selected from the group consisting of i) a
polynucleotide comprising
a polynucleotide sequence selected from the group consisting of SEQ )D N0:13-
24, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ )D N0:13-24,
iii) a polynucleotide
complementary to the polynucleotide of i), iv) a polynucleotide complementary
to the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Alternatively, the target
polynucleotide comprises a fragment
of a polynucleotide sequence selected from the group consisting of i)-v)
above; c) quantifying the
amount of hybridization complex; and d) comparing the amount of hybridization
complex in the treated
biological sample with the amount of hybridization complex in an untreated
biological sample, wherein
a difference in the amount of hybridization complex in the treated biological
sample is indicative of
toxicity of the test compound.
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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.
2o DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
3o 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
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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
"IGSFP" refers to the amino acid sequences of substantially purified IGSFP
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
IGSFP. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of IGSFP either by
directly interacting with
IGSFP or by acting on components of the biological pathway in which IGSFP
participates.
An "allelic variant" is an alternative form of the gene encoding IGSFP.
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 IGSFP include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as IGSFP or a
polypeptide with at least one functional characteristic of IGSFP. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding IGSFP, and improper or unexpected hybridization to
allelic variants, with a
locus other than the normal chromosomal locus for the polynucleotide sequence
encoding IGSFP. 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 IGSFP. Deliberate
amino acid substitutions may be made on the basis of similarity in polarity,
charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues,
as long as the biological
or immunological activity of IGSFP 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
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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 IGSFP. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of IGSFP either by
directly interacting with IGSFP or by acting on components of the biological
pathway in which IGSFP
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind IGSFP 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
Garners that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and
keyhole limpet hemocyanin (KLI~. The coupled peptide is then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen used
to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an in vitro evolutionary
process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
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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), which may improve a desired
property, e.g.,
resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
e.g., a high molecular weight carrier to slow clearance of the aptamer from
the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by
photo-activation of a
cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-
13.)
The term "intramer" refers to an 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; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic IGSFP, 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
CA 02440618 2003-09-11
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sequences that anneal by base-pairing. For example, 5 =AGT-3' pairs with its
complement,
3 =TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising a
given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding IGSFP or fragments
of IGSFP may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salinon sperm
DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to
repeated DNA sequence analysis to resolve uncalled bases, extended using the
XL-PCR kit (Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI) or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended
and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
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Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of IGSFP or the polynucleotide encoding IGSFP
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 S to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10, 15,
16, 20, 25, 30, 40, 50, 60, 75> 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
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from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:13-24 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:13-24, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:13-24 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish SEQ
ID N0:13-24 from related polynucleotide sequences. The precise length of a
fragment of SEQ >D
N0:13-24 and the region of SEQ ID N0:13-24 to which the fragment corresponds
are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ >D NO:1-12 is encoded by a fragment of SEQ ID N0:13-24. A
fragment of SEQ ID NO:1-12 comprises a region of unique amino acid sequence
that specifically
identifies SEQ ID NO:1-12. For example, a fragment of SEQ ID NO:1-12 is useful
as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ ID NO:1-12.
The precise length of a fragment of SEQ ID NO:1-12 and the region of SEQ B7
NO:1-12 to which
the fragment corresponds are routinely determinable by one of ordinary skill
in the art based on the
intended purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full
length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer to
the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps in
the sequences being compared in order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
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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.nlm.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 match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off.' S0
Expect: 10
Word Size: Il
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
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similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off. SO
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire.defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length,
for example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, 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.
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"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
to 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 p.g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carned 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"'~ ed., vol. 1-3, Cold Spring Harbor
Press, Plainview NY;
specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1% SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, SS°C, or
42°C may be used. SSC concentration may
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
sheared and denatured salmon sperm DNA at about 100-200 ~g/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
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such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
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 IGSFP
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 IGSFP which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of IGSFP. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
functional, or immunological properties of IGSFP.
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
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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.
to "Post-translational modification" of an IGSFP 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 IGSFP.
"Probe" refers to nucleic acid sequences encoding IGSFP, their complements, or
fragments
15 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
20 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,
25 or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2"d
ed., vol. 1-3, Cold
30 Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Bio- logy, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M.
et al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
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WO 02/072794 PCT/US02/09052
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas
South West Medical Center, Dallas TX) is capable of choosing specific primers
from megabase
l0 sequences and is thus useful for designing primers on a genome-wide scope.
The Primer3 primer
selection program (available to the public from the Whitehead Institute/MIT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for example,
as PCR or sequencing primers, microarray elements, or specific probes to
identify fully or partially
complementary polynucleotides in a sample of nucleic acids. Methods of
oligonucleotide selection are
not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, su ra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for
example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
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vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
l0 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 IGSFP,
nucleic acids encoding IGSFP, or fragments thereof may comprise a bodily
fluid; an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or cDNA,
in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recognized by the
binding molecule. For
example, if an antibody is specific for epitope "A," the presence of a
polypeptide comprising the
epitope A, or the presence of free unlabeled A, in a reaction containing free
labeled A and the
antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
CA 02440618 2003-09-11
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microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" or "expression profile" refers to the collective pattern
of gene expression
by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or viral
infection, electroporation, heat shock, lipofection, and particle bombardment.
The term "transformed
cells" includes stably transformed cells in which the inserted DNA is capable
of replication either as
an autonomously replicating plasmid or as part of the host chromosome, as well
as transiently
transformed cells which express the inserted DNA or RNA for limited periods of
time.
A "transgenic organism," as used herein, is any organism, including but not
limited to animals
and plants, in which one or more of the cells of the organism contains
heterologous nucleic acid
introduced by way of human intervention, such as by transgenic techniques well
known in the 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 (Lois, 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 transconjugation.
Techniques for
transfernng the DNA of the present invention into such organisms are widely
known and provided in
references such as Sambrook et al. (1989), supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
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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. Polyrnorphic 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 immunoglobulin
superfamily proteins
(IGSFP), the polynucleotides encoding IGSFP, and the use of these compositions
for the diagnosis,
treatment, or prevention of immune system, neurological, developmental,
muscle, and cell proliferative
disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project ll~). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ 177 NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide 1D) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ D7 NO:) and an
Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as
shown. Column 6
shows the Incyte ID numbers of physical, full length clones corresponding to
the polypeptide and
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polynucleotide sequences of the invention. The full length clones encode
polypeptides which have at
least 95% sequence identity to the polypeptide sequences shown in column 3.
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 l and 2 show the polypeptide sequence identification number
(Polypeptide SEQ D7 NO:) and
the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID)
for polypeptides of the
invention. Column 3 shows the GenBank identification number (GenBank 117 NO:)
of the nearest
GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID
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 ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide 117) 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 immunoglobulin
superfamily proteins. For
example, SEQ ID N0:2 is 50% identical, from residue Q34 to residue P563, to
Mus musculus Fca/m
receptor (GenBank ID g11071950) as determined by the Basic Local Alignment
Search Tool
(BLAST). (See Table 2.) The BLAST probability score is 9.6e-121, which
indicates the probability
of obtaining the observed polypeptide sequence alignment by chance. SEQ ID
N0:2 also contains an
immunoglobulin domain as determined by searching for statistically significant
matches in the hidden
Markov model (HIVIM)-based PFAM database of conserved protein family domains.
(See Table 3.)
Data from additional BLAST analyses provide further corroborative evidence
that SEQ D7 N0:2 is an
immunoglobulin. In an alternative example, SEQ D7 N0:3 is 40% identical, from
residue L30 to
residue V176, to surface protein MCA-32 (GenBank LD g1136501) as determined by
the Basic Local
Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is
6.9e-35, which
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indicates the probability of obtaining the observed polypeptide sequence
alignment by chance. SEQ
ID N0:3 also contains an immunoglobulin domain as determined by searching for
statistically
significant matches in the hidden Markov model (HMM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and additional BLAST
analyses
provide further corroborative evidence that SEQ ll~ N0:3 is a surface protein.
In an alternative
example, SEQ )D N0:8 is 86% identical, from residue Ml to residue 5433, to
cell-surface molecule
Ly-9 (GenBank ID g10197717) as determined by the Basic Local Alignment Search
Tool (BLAST).
(See Table 2.) The BLAST probability score is 7.4e-191, which indicates the
probability of obtaining
the observed polypeptide sequence alignment by chance. SEQ ID N0:8 also
contains
immunoglobulin domains as determined by searching for statistically
significant matches in the hidden
Markov model (HMM)-based PFAM database of conserved protein family domains.
(See Table 3.)
Data from additional BLAST analysis provide further corroborative evidence
that SEQ )D N0:8 is a
cell surface molecule which is a member of the immunoglobulin superfamily. In
an alternative
example, SEQ ID NO:11 is 52% identical, from residue N43 to residue Q604, to
human NEPHl
(GenBank ll~ 814572521) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 5.4e-158, which indicates the
probability of obtaining the
observed polypeptide sequence alignment by chance. As determined by BLAST
analysis using the
PROTEOME database, SEQ ID NO:11 is localized to the plasma membrane, is
homologous to a
human protein which contains an immunoglobulin domain and has a region of low
similarity to a region
of an opioid-binding cell adhesion molecule, which is a
glycosylphosphatidylinositol (GPI)-anchored
neural cell adhesion molecule (PROTEOME >D 598720~FLJ10845); SEQ >D N0:11 is
also
homologous to human Nephrin which is a member of the immunoglobulin
superfamily expressed in
renal glomeruli which may have a role in the development or function of the
kidney filtration barrier.
Mutation of the Nephrin gene causes congenital nephrotic syndrome (PROTEOME ID
340970~NPHS1). SEQ >D N0:11 also contains an immunoglobulin domain as
determined by searching
for statistically significant matches in the hidden Markov model (HMM)-based
PFAM database of
conserved protein family domains. (See Table 3.) Data from BLllVIPS, MOTIFS,
and additional
BLAST analyses provide further corroborative evidence that SEQ ID N0:11 is a
member of the
immunoglobulin superfamily. SEQ D7 NO:1, SEQ )D N0:4-7, SEQ >D N0:9-10 and SEQ
ID N0:12
were analyzed and annotated in a similar manner. The algorithms and parameters
for the analysis of
SEQ 117 NO:1-12 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
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combination of these two types of sequences. Column 1 lists the polynucleotide
sequence
identification number (Polynucleotide SEQ ID 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 cDNA and/or genomic sequences used to assemble the full
length polynucleotide
sequences of the invention, and of fragments of the polynucleotide sequences
which are useful, for
example, in hybridization or amplification technologies that identify SEQ )D
N0:13-24 or that
distinguish between SEQ )D N0:13-24 and related polynucleotide sequences.
The polynucleotide fragments described in Column 2 of Table 4 may refer
specifically, for
example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from
pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2
may refer to GenBank
cDNAs or ESTs which contributed to the assembly of the full length
polynucleotide sequences. In
addition, the polynucleotide fragments described in column 2 may identify
sequences derived from the
ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences
including the
designation "ENST"). Alternatively, the polynucleotide fragments described in
column 2 may be
derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those
sequences
including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence
Records (i.e., those
sequences including the designation "NP"). Alternatively, the polynucleotide
fragments described in
column 2 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together by
an "exon stitching" algorithm. For example, a polynucleotide sequence
identified as
FL XXXXXX NI NZ_YYYYY N3 NQ represents a "stitched" sequence in which XXxXXX
is the
identification number of the cluster of sequences to which the algorithm was
applied, and YYYYY is the
number of the prediction generated by the algorithm, and NI,Z,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
~~1AAAA~BBBBB_1 N is a "stretched" sequence, with ~'~~;~1XXX being the Incyte
project identification number, gAAAAA being the GenBank identification number
of the human
genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB
being the GenBank
identification number or NCBI RefSeq identification number of the nearest
GenBank protein homolog,
and N referring to specific exons (See Example V). In instances where a RefSeq
sequence was used
as a protein homolog for the "exon-stretching" algorithm, a RefSeq identifier
(denoted by "NM,"
"NP," or "NT") may be used in place of the GenBank identifier (i.e., gBBBBB).
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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).
to 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 IGSFP variants. A preferred IGSFP 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 IGSFP amino acid sequence, and which contains at
least one functional or
structural characteristic of IGSFP.
The invention also encompasses polynucleotides which encode IGSFP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:13-24, which encodes IGSFP. The
polynucleotide
sequences of SEQ ID N0:13-24, as presented in the Sequence Listing, embrace
the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced
with uracil, and the
sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
IGSFP. In
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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 IGSFP. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:13-
24 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 N0:13-24. Any one of the polynucleotide variants described above can encode
an amino acid
sequence which contains at least one functional or structural characteristic
of IGSFP.
In addition, or in the alternative, a polynucleotide variant of the invention
is a splice variant of a
polynucleotide sequence encoding IGSFP. A splice variant may have portions
which have significant
sequence identity to the polynucleotide sequence encoding IGSFP, 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 IGSFP 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 IGSFP. For example, a polynucleotide comprising a sequence
of SEQ ID N0:14
is a splice variant of a polynucleotide comprising a sequence of SEQ ID N0:24
and a polynucleotide
comprising a sequence of SEQ ID N0:16 is a splice variant of a polynucleotide
comprising a sequence
of SEQ ID N0:17. Any one of the splice variants described above can encode an
amino acid
sequence which contains at least one functional or structural characteristic
of IGSFP.
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 IGSFP, 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 IGSFP, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode IGSFP and its variants are
generally capable of
hybridizing to the nucleotide sequence of the naturally occurring IGSFP under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding IGSFP or
32
CA 02440618 2003-09-11
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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 IGSFP 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 IGSFP
and
IGSFP derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the
l0 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 IGSFP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:13-24 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol. 152:507-
511.) Hybridization conditions, including annealing and wash conditions, are
described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding IGSFP may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
33
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WO 02/072794 PCT/US02/09052
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
l0 ligations may be used to insert an engineered double-stranded sequence into
a region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids
Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 primer analysis software
(National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in length,
to have a GC content of about 50% or more, and to anneal to the template at
temperatures of about
68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the S' 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
3o software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and
the entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
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In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode IGSFP may be cloned in recombinant DNA molecules that direct expression
of IGSFP, 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 IGSFP.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter IGSFP-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or improve
the biological properties of IGSFP, 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 IGSFP 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,
IGSFP itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide
synthesis can be performed using various solution-phase or solid-phase
techniques. (See, e.g.,
Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp.
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
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 IGSFP, or any part thereof, may be altered during direct synthesis and/or
combined with sequences
from other proteins, or any part thereof, to produce a variant polypeptide or
a polypeptide having a
sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active IGSFP, the nucleotide sequences
encoding IGSFP or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in
a suitable host. These elements include regulatory sequences, such as
enhancers, constitutive and
inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotide sequences
encoding IGSFP. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
IGSFP. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding IGSFP 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 IGSFP and appropriate transcriptional
and translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. (1995)
Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, ch. 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding IGSFP. These include, but are not limited to, microorganisms such as
bacteria transformed
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WO 02/072794 PCT/US02/09052
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, su ra; Ausubel, su ra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw
Hill, New
York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and
l0 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; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al. (1994)
Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-
242.) The
invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding IGSFP. For
example, routine cloning,
subcloning, and propagation of polynucleotide sequences encoding IGSFP can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORTl
plasmid (Life Technologies). Ligation of sequences encoding IGSFP 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 IGSFP are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of IGSFP 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 IGSFP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, su ra;
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CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
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 IGSFP. Transcription of
sequences
encoding IGSFP 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 TechnoloQV
(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 IGSFP
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential El or E3 region of the viral genome may
be used to obtain
infective virus which expresses IGSFP 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
IGSFP in cell lines is preferred. For example, sequences encoding IGSFP can be
transformed into cell
lines using expression vectors which may contain viral origins of replication
and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
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Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic, or
herbicide resistance can be used as the basis for selection. For example, dhfr
confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech), B glucuronidase and its substrate B-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest
is also present, the presence and expression of the gene may need to be
confirmed. For example, if
the sequence encoding IGSFP is inserted within a marker gene sequence,
transformed cells containing
sequences encoding IGSFP can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding IGSFP 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 IGSFP
and that express
IGSFP 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.
Itnmunological methods for detecting and measuring the expression of IGSFP
using either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on IGSFP is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
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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 ItnmunoloQV, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Itnmunochemical
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
IGSFP include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, the sequences encoding IGSFP, or any fragments thereof, may be
cloned into a vector
to 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 polymerise
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 IGSFP 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 IGSFP may be designed to contain signal sequences
which direct
secretion of IGSFP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which cleaves a
"prepro" or "pro" form of the
protein may also be used to specify protein targeting, folding, and/or
activity. Different host cells
which have specific cellular machinery and characteristic mechanisms for post-
translational activities
(e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type
Culture
Collection (ATCC, Manassas VA) and may be chosen to ensure the correct
modification and
processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding IGSFP may be ligated to a heterologous sequence resulting
in translation of a
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fusion protein in any of the aforementioned host systems. For example, a
chimeric IGSFP protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of IGSFP
activity. Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available affinity
matrices. Such moieties include, but are not limited to, glutathione S-
transferase (GST), maltose
binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-
His, FLAG, c-myc, and
hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their
cognate fusion
proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
and metal-chelate resins,
respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
proteins using commercially available monoclonal and polyclonal antibodies
that specifically recognize
these epitope tags. A fusion protein may also be engineered to contain a
proteolytic cleavage site
located between the IGSFP encoding sequence and the heterologous protein
sequence, so that IGSFP
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 IGSFP 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.
IGSFP of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to IGSFP. At least one and up to a plurality of test
compounds may be screened
for specific binding to IGSFP. 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
IGSFP, e.g., a ligand or fragment thereof, a natural substrate, a structural
or functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunology 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which IGSFP
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express IGSFP,
either as a secreted
protein or on the cell-membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing IGSFP or cell membrane fractions which contain IGSFP
are then contacted
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with a test compound and binding, stimulation, or inhibition of activity of
either IGSFP 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
IGSFP, either in solution
or affixed to a solid support, and detecting the binding of IGSFP 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 axed to a
solid support.
IGSFP of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of IGSFP. Such compounds may inchzde agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for IGSFP
activity, wherein IGSFP is combined with at least one test compound, and the
activity of IGSFP in the
presence of a test compound is compared with the activity of IGSFP in the
absence of the test
compound. A change in the activity of IGSFP in the presence of the test
compound is indicative of a
compound that modulates the activity of IGSFP. Alternatively, a test compound
is combined with an
in vitro or cell-free system comprising IGSFP under conditions suitable for
IGSFP activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of IGSFP
may do so indirectly and need not come in direct contact with the test
compound. At least one and up
2o to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding IGSFP or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem (ES)
cells. Such techniques are well known in the art and are useful for the
generation of animal models of
human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No.
5,767,337.) For example,
mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the
early mouse embryo and
grown in culture. The ES cells are transformed with a vector containing the
gene of interest disrupted
by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi,
M.R. (1989) Science
244:1288-1292). The vector integrates into the corresponding region of the
host genome by
homologous recombination. Alternatively, homologous recombination takes place
using the Cre-loxP
system to knockout a gene of interest in a tissue- or developmental stage-
specific manner (Marth, J.D.
(1996) Clip. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids
Res. 25:4323-4330).
Transformed ES cells are identified and microinjected into mouse cell
blastocysts such as those from
the C57BL/6 mouse strain. The blastocysts are surgically transferred to
pseudopregnant dams, and
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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 IGSFP 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 IGSFP 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
l0 of a polynucleotide encoding IGSFP 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 IGSFP, e.g., by secreting IGSFP 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 IGSFP and immunoglobulin superfamily proteins. In addition, the
expression of IGSFP is
closely associated with brain, colon, diseased skin, diseased lung,
hippocampus, spleen, and diseased
2o vermis tissues, as well as, CD4+ T and peripheral blood cells. Therefore,
IGSFP appears to play a
role in immune system, neurological, developmental, muscle, and cell
proliferative disorders. In the
treatment of disorders associated with increased IGSFP expression or activity,
it is desirable to
decrease the expression or activity of IGSFP. In the treatment of disorders
associated with decreased
IGSFP expression or activity, it is desirable to increase the expression or
activity of IGSFP.
Therefore, in one embodiment, IGSFP 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 IGSFP. Examples of such disorders include, but are not limited to,
an immune system
disorder such as acquired immunodeficiency syndrome (AIDS), X-linked
agammaglobinemia of
Bruton, common variable immunodeficiency (CVI), DiGeorge's syndrome (thymic
hypoplasia), thymic
dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease
(SCID),
immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome),
Chediak-Higashi
syndrome, chronic granulomatous diseases, hereditary angioneurotic edema,
immunodeficiency
associated with C~shing's disease, Addison's disease, adult respiratory
distress syndrome, allergies,
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ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,
autoimmune hemolytic anemia,
autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy
(APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease,
atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel syndrome,
multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis,
osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and 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 demyefinating diseases, bacterial and viral meningitis,
brain abscess, subdural
empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis
and radiculitis, viral
central nervous system disease, prion diseases including kuru, Creutzfeldt-
Jakob disease, and
Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional
and metabolic diseases
of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system including Down syndrome, cerebral palsy, neuroskeletal
disorders, autonomic nervous
system disorders, cranial nerve disorders, spinal cord diseases, muscular
dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD),
akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,
dystonias, paranoid psychoses,
postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy,
corticobasal degeneration,
and familial frontotemporal dementia; a developmental disorder such as renal
tubular acidosis, anemia,
C~shing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular
dystrophy, epilepsy,
gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary
abnormalities, and mental
retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary
mucoepithelial dysplasia,
hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth
disease and
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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 muscle disorder such as cardiomyopathy,
myocarditis, Duchenne's
muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central
core disease, nemaline
myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy,
infectious myositis,
polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy,
and ethanol myopathy;
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 IGSFP 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 IGSFP including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
IGSFP 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 IGSFP
including, but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of IGSFP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of IGSFP including, but not limited to, those listed above.
In a further embodiment, an antagonist of IGSFP may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of IGSFP.
Examples of such
disorders include, but are not limited to, those immune system, neurological,
developmental, muscle,
and cell proliferative disorders described above. In one aspect, an antibody
which specifically binds
IGSFP 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 IGSFP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding IGSFP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of IGSFP including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
CA 02440618 2003-09-11
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sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of IGSFP may be produced using methods which are generally known
in the
art. In particular, purified IGSFP may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind IGSFP.
Antibodies to IGSFP may also
l0 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. 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
IGSFP or with any
fragment or oligopeptide thereof which has immunogenic properties. Depending
on the host species,
various adjuvants may be used to increase immunological response. Such
adjuvants include, but are
not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface
active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH,
and dinitrophenol. Among
adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium
parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
IGSFP 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 IGSFP 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 IGSFP 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
46
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technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
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
IGSFP-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g., Burton,
D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter,
G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for IGSFP may also be
generated.
For example, such fragments include, but are not limited to, F(ab')Z fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
IGSFP and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering IGSFP 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 IGSFP. Affinity is
expressed as an association
constant, Ke, which is defined as the molar concentration of IGSFP-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The Ka
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CA 02440618 2003-09-11
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determined for a preparation of polyclonal antibodies, which are heterogeneous
in their affinities for
multiple IGSFP epitopes, represents the average affinity, or avidity, of the
antibodies for IGSFP. The
Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a particular
IGSFP epitope, represents a true measure of affinity. High-affinity antibody
preparations with Ka
ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in
which the IGSFP-
antibody complex must withstand rigorous manipulations. Low-affinity antibody
preparations with Ke
ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of IGSFP, preferably in
active form, from the
antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRh
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 IGSFP-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and
Coligan et al. su ra.)
In another embodiment of the invention, the polynucleotides encoding IGSFP, 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
IGSFP. 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
IGSFP. (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.,
3o Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, su ra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
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CA 02440618 2003-09-11
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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 IGSFP 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 (SCTD)-Xl disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
l0 (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al.
(1995) Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
15 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
20 brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trypanosoma cruzi). In the
case where a genetic deficiency in IGSFP expression or regulation causes
disease, the expression of
IGSFP 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
25 IGSFP are treated by constructing mammalian expression vectors encoding
IGSFP and introducing
these vectors by mechanical means into IGSFP-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.
30 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon
(1998) C~rr. Opin.
Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of IGSFP include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
49
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(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).
IGSFP
may be expressed using (i) a constitutively active promoter, (e.g., from
cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes),
(ii) an inducible promoter
(e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992)
Proc. Natl. Acad. Sci.
USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi,
F.M.V. and H.M. Blau
(1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and H.M. Blau, su ra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding IGSFP from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of these
standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to IGSFP expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding IGSFP 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.
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Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ T-cells), and the
return of transduced cells to a patient are procedures well known to persons
skilled in the art of gene
therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et
al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al. (1998)
Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding IGSFP to cells which have one or more genetic
abnormalities with respect to
the expression of IGSFP. The construction and packaging of adenovirus-based
vectors are well
known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven to
be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for
gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999)
Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature
18:389:239-242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding IGSFP to target cells which have one or more genetic
abnormalities with
respect to the expression of IGSFP. The use of herpes simplex virus (HSV)-
based vectors may be
especially valuable for introducing IGSFP to cells of the central nervous
system, for which HSV has a
2o 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
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ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding IGSFP 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) C~rr. 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
IGSFP into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
IGSFP-coding RNAs and the synthesis of high levels of IGSFP in vector
transduced cells. While
alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
persistent infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN)
indicates that the lytic replication of alphaviruses can be altered to suit
the needs of the gene therapy
application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
allow the introduction of IGSFP into a variety of cell types. The specific
transduction of a subset of
cells in a population may require the sorting of cells prior to transduction.
The methods of
manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA
transfections, and performing alphavirus infections, are well known to those
with ordinary skill in the
art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions -10
and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can
be achieved using triple helix base-pairing methodology. Triple helix pairing
is useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in
Huber, B.E. and B.I. Carr,
Molecular and Itnmunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp.
163-177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA
by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
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endonucleolytic cleavage of sequences encoding IGSFP.
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
l0 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 IGSFP. Such DNA sequences may be incorporated into a wide
variety of
vectors with suitable RNA polymerase promoters such as T7 or SP6.
Alternatively, these cDNA
constructs that synthesize complementary RNA, constitutively or inducibly, can
be introduced into cell
lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified
forms of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding IGSFP. 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
3o compounds may alter polynucleotide expression by acting as either
inhibitors or promoters of
polynucleotide expression. Thus, in the treatment of disorders associated with
increased IGSFP
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding IGSFP may be therapeutically useful, and in the treatment of
disorders associated with
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decreased IGSFP expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding IGSFP may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding IGSFP 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
IGSFP are assayed by
any method commonly known in the art. Typically, the expression of a specific
nucleotide is detected
by hybridization with a probe having a nucleotide sequence complementary to
the sequence of the
polynucleotide encoding IGSFP. 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
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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 on's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of IGSFP,
antibodies to IGSFP, and mimetics, agonists, antagonists, or inhibitors of
IGSFP.
The compositions utilized in this invention may be administered by any number
of routes
l0 including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of. small molecules (e.g. traditional low molecular weight organic
drugs), aerosol delivery of fast-
acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and '
proteins), recent developments in the field of pulinonary 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 IGSFP or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, IGSFP 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
CA 02440618 2003-09-11
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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 IGSFP
or fragments thereof, antibodies of IGSFP, and agonists, antagonists or
inhibitors of IGSFP, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDso (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDso/EDso ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDso
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and response
to therapy. Long-acting compositions may be administered every 3 to 4 days,
every week, or
biweekly depending on the half life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 ~cg, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind IGSFP may be used
for the
diagnosis of disorders characterized by expression of IGSFP, or in assays to
monitor patients being
treated with IGSFP or agonists, antagonists, or inhibitors of IGSFP.
Antibodies useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic
assays for IGSFP include methods which utilize the antibody and a label to
detect IGSFP in human
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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 IGSFP, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
IGSFP expression. Normal
or standard values for IGSFP expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibodies to IGSFP under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of IGSFP
expressed in
l0 subject, control, and disease samples from biopsied tissues are compared
with the standard values.
Deviation between standard and subject values establishes the parameters for
diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding IGSFP may
be used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of IGSFP
may be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
IGSFP, and to monitor regulation of IGSFP levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding IGSFP or closely related
molecules may be used to
identify nucleic acid sequences which encode IGSFP. 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 IGSFP, 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 IGSFP encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ )D
N0:13-24 or from
genomic sequences including promoters, enhancers, and introns of the IGSFP
gene.
Means for producing specific hybridization probes for DNAs encoding IGSFP
include the
cloning of polynucleotide sequences encoding IGSFP or IGSFP 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
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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 IGSFP may be used for the diagnosis of
disorders
associated with expression of IGSFP. Examples of such disorders include, but
are not limited to, an
S 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 (SC>D), immunodeficiency with thrombocytopenia and eczema (Wiskott-
Aldrich syndrome),
. Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary
angioneurotic edema,
immunodeficiency associated with C~shing's disease, Addison's disease, adult
respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis, autoimmune
hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-
candidiasis-ectodermal
dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's
disease, atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel syndrome,
multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis,
osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and helminthic
infections, and trauma; a neurological disorder such as epilepsy, ischemic
cerebrovascular disease,
stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's
disease, dementia,
Parkinson's disease and other 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
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neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis,
periodic paralysis, mental
disorders including mood; anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD),
akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,
dystonias, paranoid psychoses,
postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy,
corticobasal degeneration,
and familial frontotemporal dementia; a developmental disorder such as renal
tubular acidosis, anemia,
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, spina bifida, anencephaly, craniorachischisis, congenital
glaucoma, cataract, and
sensorineural hearing loss; a muscle disorder such as cardiomyopathy,
myocarditis, Duchenne's
muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central
core disease, nemaline
myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy,
infectious myositis,
polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy,
and ethanol myopathy;
and a cell proliferative disorder such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis,
paroxysmal nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall bladder,
ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis,
prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
The polynucleotide
sequences encoding IGSFP 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 IGSFP expression.
Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding IGSFP may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding IGSFP may be labeled by standard methods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a suitable
incubation period, the sample is washed and the signal is quantified and
compared with a standard
value. If the amount of signal in the patient sample is significantly altered
in comparison to a control
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sample then the presence of altered levels of nucleotide sequences encoding
IGSFP 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
IGSFP, 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 IGSFP, 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 IGSFP
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 IGSFP, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
IGSFP, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding IGSFP may be used to detect single nucleotide polymorphisms (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 IGSFP 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 an anti-asthma drug that
targets the 5-lipoxygenase
pathway. Analysis of the distribution of SNPs in different populations is
useful for investigating
genetic drift, mutation, recombination, and selection, as well as for tracing
the origins of populations
and their migrations. (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y. and Z. Gu
(1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) C~rr. Opin.
Neurobiol. 11:637-641.)
Methods which may also be used to quantify the expression of IGSFP include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
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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
l0 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, IGSFP, fragments of IGSFP, or antibodies specific for
IGSFP may be
used as elements on a microarray. The microarray may be used to monitor or
measure protein-protein
interactions, drug-target interactions, and gene expression profiles, as
described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
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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
l0 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
conditions and at a given time. A profile of a cell's proteome may thus be
generated by separating
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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, su ra). 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 IGSFP
to quantify the
levels of IGSFP expression. In one embodiment, the antibodies are used as
elements on a microarray,
and protein expression levels are quantified by exposing the microarray to the
sample and detecting
the levels of protein bound to each array element (Lueking, A. et al. (1999)
Anal. Biochem. 270:103-
111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a
variety of methods known in the art, for example, by reacting the proteins in
the sample with a thiol- or
amino-reactive fluorescent compound and detecting the amount of fluorescence
bound at each array
element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
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each protein is compared to the amount of the corresponding protein in an
untreated biological sample.
A difference in the amount of protein between the two samples is indicative of
a toxic response to the
test compound in the treated sample. Individual proteins are identified by
sequencing the amino acid
residues of the individual proteins and comparing these partial sequences to
the polypeptides of the
present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared
l0 with the amount in an untreated biological sample. A difference in the
amount of protein between the
two samples is indicative of a toxic response to the test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad.
Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalom D. et
al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA
94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
Various types of
microarrays are well known and thoroughly described in DNA Microarrays: A
Practical Approach,
M. Schena, ed. (1999) Oxford University Press, London, hereby expressly
incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding IGSFP
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a mufti-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (PACs), 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
3o 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.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
CA 02440618 2003-09-11
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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 IGSFP 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
l0 valuable to investigators searching for disease genes using positional
cloning or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to l 1q22-23, any
sequences mapping to that area may represent associated or regulatory genes
for further investigation.
(See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide
sequence of the instant
invention may also be used to detect differences in the chromosomal location
due to translocation,
inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, IGSFP, 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 IGSFP 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.,
Geysers, 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 IGSFP,
or fragments thereof,
and washed. Bound IGSFP is then detected by methods well known in the art.
Purified IGSFP can
also be coated directly onto plates for use in the aforementioned drug
screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the peptide
and immobilize it on a
solid support.
3o In another embodiment, one may use competitive drug screening assays in
which neutralizing
antibodies capable of binding IGSFP specifically compete with a test compound
for binding IGSFP. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with IGSFP.
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In additional embodiments, the nucleotide sequences which encode IGSFP 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,
1o including U.S. Ser. No.60/275,249, U.S. Ser. No.60/316,810, U.S. Ser.
No.60/323,977, U.S. Ser.
No.60/348,447, and U.S. Ser. No.60/343,880, are expressly incorporated by
reference herein.
EXAMPLES
I. Construction of cDNA Libraries
15 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 CsCI cushions or
extracted with
2o 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
25 (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
30 UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life
Technologies), using
the recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, su ra,
units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
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appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), 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, XLl-BlueMRF, or
SOLR from
Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at 4
°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycfing steps were carned 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 Biosysterns) thermal cycler or the PTC-200
thermal cycler
(MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
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were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the
techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo
Sapiens, Rattus norveQicus, Mus musculus, Caenorhabditis ele~ans,
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 HMM-based protein domain
databases such as SMART
(Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic, I. et
al. (2002) Nucleic
Acids Res. 30:242-244). (HMM is a probabilistic approach which analyzes
consensus primary
structures of gene families. See, for example, Eddy, S.R. (1996) C~rr. Opin.
Struct. Biol. 6:361-365.)
The queries were performed using programs based on BLAST, FASTA, BL>IVVIPS,
and HIVIMER.
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 (HIVI1VI)-based protein family databases
such as PFAM,
INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART. Full
length
polynucleotide sequences are also analyzed using MACDNASIS PRO software
(Hitachi Software
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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 1D
N0:13-24. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative immunoglobulin superfamily 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 immunoglobulin superfamily proteins,
the encoded
polypeptides were analyzed by querying against PFAM models for immunoglobulin
superfamily
proteins. Potential immunoglobulin superfamily proteins were also identified
by homology to Incyte
cDNA sequences that had been annotated as immunoglobulin superfamily 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 sequence
predicted by
Genscan, such as extra or omitted exons. BLAST analysis was also used to find
any Incyte cDNA or
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public cDNA coverage of the Genscan-predicted sequences, thus providing
evidence for transcription.
When Incyte cDNA coverage was available, this information was used to correct
or confirm the
Genscan predicted sequence. Full length polynucleotide sequences were obtained
by assembling
Genscan-predicted coding sequences with Incyte cDNA sequences and/or public
cDNA sequences
using the assembly process described in Example III. Alternatively, full
length polynucleotide
sequences were derived entirely from edited or unedited Genscan-predicted
coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
DI were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" 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, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
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Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for homologous
genomic sequences from the public human genome databases. Partial DNA
sequences were
therefore "stretched" or extended by the addition of homologous genomic
sequences. The resultant
stretched sequences were examined to determine whether it contained a complete
gene.
VI. Chromosomal Mapping of IGSFP Encoding Polynucleotides
The sequences which were used to assemble SEQ >D N0:13-24 were compared with
l0 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 N0:13-24 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ 1D NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances
are based on genetic markers mapped by Genethon which provide boundaries for
radiation hybrid
markers whose sequences were included in each of the clusters. Human genome
maps and other
resources available to the public, such as the NCBI "GeneMap'99" World Wide
Web site
(http://www.ncbi.nlin.nih.gov/genemap~, can be employed to determine if
previously identified disease
genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel
(1995) supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
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molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
search can be modified to determine whether any particular match is
categorized as exact or similar.
The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the
l0 length of the sequence match. The product score is a normalized value
between 0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and SO%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding IGSFP are analyzed with
respect to the
tissue sources from which they were derived. For example, some full length
sequences are
assembled, at least in part, with overlapping Incyte cDNA sequences (see
Example III). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following organ/tissue categories: cardiovascular
system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female; genitalia,
male; germ cells; heroic and immune system; liver; musculoskeletal system;
nervous system;
pancreas; respiratory system; sense organs; skin; stomatognathic system;
unclassified/mixed; or
3o urinary tract. The number of libraries in each category is counted and
divided by the total number of
libraries across all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
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by the total number of libraries across all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding IGSFP. cDNA sequences and cDNA
library/tissue
information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto
CA).
VIII. Extension of IGSFP Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
lnc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mgz+, (NH~)ZSO4,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE
enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the
following parameters
for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 60°C, 1 min;
Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step
6: 68 °C, 5 min; Step 7: storage
at 4°C. In the alternative, the parameters for primer pair T7 and 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 p1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 p,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 S ~cl to 10 ~1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
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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 relegated 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 IGSFP Encoding
Polynucleotides
Common DNA sequence variants known as single nucleotide polymorphisms (SNPs)
were
identified in SEQ ID N0:13-24 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.
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,
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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, lnc.) 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
to Hispanic population comprised 324 individuals (162 male, 162 female), all
Mexican Hispanic. The
Asian population comprised 126 individuals (64 male, 62 female) with a
reported parental breakdown
of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
Allele
frequencies were first analyzed in the Caucasian population; in some cases
those SNPs which showed
no allelic variance in this population were not further tested in the other
three populations.
X. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:13-24 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
~,~ 3zp] adenosine- triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases: Ase
I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
XI. Microarrays
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The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, su ra.), 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), su ra).
Suggested substrates include silicon, silica, glass slides, glass chips, and
silicon wafers. Alternatively, a
procedure analogous to a dot or slot blot may also be used to arrange and link
elements to the surface
of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
A typical array may
be produced using available methods and machines well known to those of
ordinary skill in the art and
may contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science
l0 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/pl oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/p,l RNase inhibitor, 500 ~M dATP, 500 p.M dGTP, 500
p,M dTTP, 40 p.M
dCTP, 40 p,M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of O.SM sodium
hydroxide and
incubated for 20 minutes at 85°C to the stop the reaction and degrade
the RNA. Samples are purified
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using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and resuspended
in 14 u1 5X SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5 fig.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 p,1 of the array
element DNA, at an average
concentration of 100 ng/pl, is loaded into the open capillary printing element
by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2%
SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 ~l of sample mixture consisting of 0.2 ~g
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of 140
p,1 of 5X SSC in a corner of the chamber. The chamber containing the arrays is
incubated for about
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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
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
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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 N0:19 showed differential expression in toxicology studies
as
determined by microarray analysis. The expression of SEQ ID N0:19 was
decreased by at least two
fold in a human C3A liver cell line treated with various drugs (e.g.,
steroids, steroid hormones) relative
to untreated C3A cells. The human C3A cell line is a clonal derivative of
HepG2/C3 (hepatoma cell
line, isolated from a 15-year-old male with liver tumor), which was selected
for strong contact
inhibition of growth. The C3A cell line is well established as an in vitro
model of the mature human
l0 liver (Mickelson et al. (1995) Hepatology 22:866-875; Nageridra et al.
(1997) Am J Physiol 272:G408-
G416). Effects upon liver metabolism are important to understanding the
pharmacodynamics of a
drug. Therefore, SEQ 1D N0:19 is useful for understanding the pharmacodynamics
of a drug.
XII. Complementary Polynucleotides
Sequences complementary to the IGSFP-encoding sequences, or any parts thereof,
are used
to detect, decrease, or inhibit expression of naturally occurring IGSFP.
Although use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of IGSFP. 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 IGSFP-encoding
transcript.
XIII. Expression of IGSFP
Expression and purification of IGSFP is achieved using bacterial or virus-
based expression
systems. For expression of IGSFP in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the TS or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express IGSFP upon induction with isopropyl beta-
D-
thiogalactopyranoside (1PTG). Expression of IGSFP in eukaryotic cells is
achieved by infecting insect
or mammalian cell lines with recombinant Autographica californica nuclear
polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding IGSFP by either homologous recombination or
bacterial-mediated
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transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera fru 'per 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, IGSFP is synthesized as a fusion protein with,
e.g., glutathione S-
transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
l0 enzyme from Schistosoma iaponicum, enables the purification of fusion
proteins on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Pharmacia
Biotech). Following purification, the GST moiety can be proteolytically
cleaved from IGSFP at
specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity purification
using commercially available monoclonal and polyclonal anti-FLAG antibodies
(Eastman Kodak). 6-
His, a stretch of six consecutive histidine residues, enables purification on
metal-chelate resins
(QIAGEN). Methods for protein expression and purification are discussed in
Ausubel ( 1995, supra,
ch. 10 and 16). Purified IGSFP obtained by these methods can be used directly
in the assays shown
in Examples XVII and XVI)1 where applicable.
XIV. Functional Assays
IGSFP function is assessed by expressing the sequences encoding IGSFP 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 /.cg of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ~g of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
81
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
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
brornodeoxyuridine 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 IGSFP on gene expression can be assessed using highly
purified populations
of cells transfected with sequences encoding IGSFP and either CD64 or CD64-
GFP. CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind to
conserved regions of human
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success
NY). mRNA can be purified from the cells using methods well known by those of
skill in the art.
Expression of mRNA encoding IGSFP and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XV. Production of IGSFP Specific Antibodies
IGSFP 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 IGSFP amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide and anti-IGSFP activity by, for example, binding the peptide or
IGSFP to a substrate,
blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat
anti-rabbit IgG.
82
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WO 02/072794 PCT/US02/09052
XVI. Purification of Naturally Occurring IGSFP Using Specific Antibodies
Naturally occurring or recombinant IGSFP is substantially purified by
immunoaffinity
chromatography using antibodies specific for IGSFP. An immunoaffinity column
is constructed by
covalently coupling anti-IGSFP 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 IGSFP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of IGSFP (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/IGSFP 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 IGSFP is collected.
XVII. Identification of Molecules Which Interact with IGSFP
IGSFP, or biologically active fragments thereof, are labeled with l2sI 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 IGSFP, washed,
and any wells with labeled IGSFP complex are assayed. Data obtained using
different concentrations
of IGSFP are used to calculate values for the number, affinity, and
association of IGSFP with the
candidate molecules.
Alternatively, molecules interacting with IGSFP 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).
IGSFP may also be used in the PATHCALLING process (CuraGen Corp., New Haven
CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
XVIII. Demonstration of IGSFP Activity
An assay for IGSFP activity measures the ability of IGSFP to recognize and
precipitate
antigens from serum. This activity can be measured by the quantitative
precipitin reaction. (Golub, E.
S. et al. (1987) ImmunoloQV: A Synthesis, Sinauer Associates, Sunderland, MA,
pages 113-115.)
IGSFP is isotopically labeled using methods known in the art. Various serum
concentrations are
added to constant amounts of labeled IGSFP. IGSFP-antigen complexes
precipitate out of solution
and are collected by centrifugation. The amount of precipitable IGSFP-antigen
complex is
proportional to the amount of radioisotope detected in the precipitate. The
amount of precipitable
83
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
IGSFP-antigen complex is plotted against the serum concentration. For various
serum concentrations,
a characteristic precipitin curve is obtained, in which the amount of
precipitable IGSFP-antigen
complex initially increases proportionately with increasing serum
concentration, peaks at the
equivalence point, and then decreases proporkionately with further increases
in serum concentration.
Thus, the amount of precipitable IGSFP-antigen complex is a measure of IGSFP
activity which is
characterized by sensitivity to both limiting and excess quantities of
antigen.
Alternatively, an assay for IGSFP activity measures the expression of IGSFP on
the cell
surface. cDNA encoding IGSFP 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).
Immunoprecipitations
are performed using IGSFP-specific antibodies, and immunoprecipitated samples
are analyzed using
SDS-PAGE and imununoblotting techniques. The ratio of labeled
immunoprecipitant to unlabeled
immunoprecipitant is proportional to the amount of IGSFP expressed on the cell
surface.
Alternatively, an assay for IGSFP activity measures the amount of cell
aggregation induced by
overexpression of IGSFP. In this assay, cultured cells such as NIH3T3 are
transfected with cDNA
encoding IGSFP 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 (CLONTECI~, is useful for identifying stable
transfectants. The amount of cell
agglutination, or clumping, associated with transfected cells is compared with
that associated with
untransfected cells. The amount of cell agglutination is a direct measure of
IGSFP activity.
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the invention.
Although the invention has been described in connection with certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which are obvious
to those skilled in molecular biology or related fields are intended to be
within the scope of the
following claims.
84
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
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Table 5
PolynucleotideIncyte ProjectRepresentative Library
SEQ ID:
ID NO:
13 ~ 3855123CB BRAHNONOS
1
14 4547188CB COLXTDTO1
1
15 ' 3939883CB SKINBITO1
1
16 3163819CB TLYMTXT04
1
17 ~ 8518269CBTLYJTXFO1 '
1
18 ( 1592646CBEOSIHET02
1
19 7500191CB1 BRAIFEROS
20 7500099CB LUNGDIN02
1
21 17682434CB BRABDIK02
1
22 2202389CB SPLNFET02
1
23 7503597CB BRAHNONOS
1
24 7503603CB COLXTDTO1
1
101
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WO 02/072794 PCT/US02/09052
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CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
<110> INCYTE GENOMICS, INC.
YUE, Henry
XU, Yuming
THANGAVELU, Kavitha
WARREN, Bridget A.
TANG, Y. Tom
DUGGAN, Brendan M.
TRAN, Uyen K.
BAUGHN, Mariah R.
HONCHELL, Cynthia D.
BURFORD, Neil
FORSYTHE, Ian J.
YANG, Junming
MASON, Patricia M.
<120> IMMUNOLGLOBULIN SUPERFAMILY PROTEINS
<130> PF-0925 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/275,249; 60/316,810; 60/323,977; 60/348,447;
60/343,880
<151> 2001-03-12; 2001-08-31; 2001-09-21; 2001-10-26;
2001-11-02
<160> 24
<170> PERL Program
<210> 1
<211> 442
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 3855123CD1
<400> 1
Met Thr Thr Glu Pro Gln Ser Leu Leu Val Asp Leu Gly Ser Asp
1 5 10 15
Ala Ile Phe Ser Cys Ala Trp Thr Gly Asn Pro Ser Leu Thr Ile
20 25 30
Val Trp Met Lys Arg Gly Ser Gly Val Val Leu Ser Asn Glu Lys
35 40 45
Thr Leu Thr Leu Lys Ser Val Arg Gln Glu Asp Ala Gly Lys Tyr
50 55 60
Val Cys Arg Ala Val Val Pro Arg Val Gly Ala Gly Glu Arg Glu
65 70 75
Val Thr Leu Thr Val Asn Gly Pro Pro Ile Ile Ser Ser Thr Gln
80 85 90
Thr Gln His Ala Leu His Gly Glu Lys Gly Gln Ile Lys Cys Phe
95 100 105
Ile Arg Ser Thr Pro Pro Pro Asp Arg Ile Ala Trp Ser Trp Lys
110 115 120
1/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
Glu Asn Val Leu Glu Ser Gly Thr Ser Gly Arg Tyr Thr Val Glu
125 130 135
Thr Ile Ser Thr Glu Glu Gly Val Ile Ser Thr Leu Thr Ile Ser
140 145 150
Asn Ile Val Arg Ala Asp Phe Gln Thr Ile Tyr Asn Cys Thr Ala
155 160 165
Trp Asn Ser Phe Gly Ser Asp Thr Glu Ile Ile Arg Leu Lys Glu
170 175 .180
Gln Gly Ser Glu Met Lys Ser Gly Ala Gly Leu Glu Ala Glu Ser
185 190 195
Val Pro Met Ala Val Ile Ile Gly Val Ala Val Gly Ala Gly Val
200 205 210
Ala Phe Leu Val Leu Met Ala Thr Ile Val Ala Phe Cys Cys Ala
215 220 225
Arg Ser Gln Arg Asn Leu Lys Gly Val Val Ser Ala Lys Asn Asp
230 235 240
Ile Arg Val Glu Ile Val His Lys Glu Pro Ala Ser Gly Arg Glu
245 250 255
Gly Glu Glu His Ser Thr Ile Lys Gln Leu Met Met Asp Arg Gly
260 265 270
Glu Phe Gln Gln Asp Ser Val Leu Lys Gln Leu Glu Val Leu Lys
275 280 285
Glu Glu Glu Lys Glu Phe Gln Asn Leu Lys Asp Pro Thr Asn Gly
290 295 300
Tyr Tyr Ser Val Asn Thr Phe Lys Glu His His Ser Thr Pro Thr
305 310 315
Ile Ser Leu Ser Ser Cys Gln Pro Asp Leu Arg Pro Ala Gly Lys
320 325 330
Gln Arg Val Pro Thr Gly Met Ser Phe Thr Asn Ile Tyr Ser Thr
335 340 345
Leu Ser Gly Gln Gly Arg Leu Tyr Asp Tyr Gly Gln Arg Phe Val
350 355 360
Leu Gly Met Gly Ser Ser Ser Ile Glu Leu Cys Glu Arg Glu Phe
365 370 375
Gln Arg Gly Ser Leu Ser Asp Ser Ser Ser Phe Leu Asp Thr Gln
380 385 390
Cys Asp Ser Ser Val Ser Ser Ser Gly Lys Gln Asp Gly Tyr Val
395 400 405
Gln Phe Asp Lys Ala Ser Lys Ala Ser Ala Ser Ser Ser His His
410 415 420
Ser Gln Ser Ser Ser Gln Asn Ser Asp Pro Ser Arg Pro Leu Gln
425 430 435
Arg Arg Met Gln Thr His Val
440
<210> 2
<211> 577
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4547188CD1
<400> 2
Met Asp Gly Glu Ala Thr Val Lys Pro Gly Glu Gln Lys Glu Val
2/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
1 5 10 15
Val Arg Arg Gly Arg Glu Val Asp Tyr Ser Arg Leu Ile Ala Gly
20 25 30
Thr Leu Pro Gln Ser His Val Thr Ser Arg Arg Ala Gly Trp Lys
35 40 45
Met Pro Leu Phe Leu Ile Leu Cys Leu Leu Gln Gly Ser Ser Phe
50 55 60
Ala Leu Pro Gln Lys Arg Pro His Pro Arg Trp Leu Trp Glu Gly
65 70 75
Ser Leu Pro Ser Arg Thr His Leu Arg Ala Met Gly Thr Leu Arg
80 85 90
Pro Ser Ser Pro Leu Cys Trp Arg Glu Glu Ser Ser Phe Ala Ala
95 100 105
Pro Asn Ser Leu Lys Gly Ser Arg Leu Val Ser Gly Glu Pro Gly
110 115 120
Gly Ala Val Thr Ile Gln Cys His Tyr Ala Pro Ser Ser Val Asn
125 130 135
Arg His Gln Arg Lys Tyr Trp Cys Cys Leu Gly Pro Pro Arg Trp
140 145 150
Ile Cys Gln Thr Ile Val Ser Thr Asn Gln Tyr Thr His His Arg
155 160 165
Tyr Arg Asp Arg Val Ala Leu Thr Asp Phe Pro Gln Arg Gly Leu
170 175 180
Phe Val Val Arg Leu Ser Gln Leu Ser Pro Asp Asp Ile Gly Cys
185 190 195
Tyr Leu Cys Gly Ile Gly Ser Glu Asn Asn Met Leu Phe Leu Ser
200 205 210
Met Asn Leu Thr Ile Ser Ala Gly Pro Ala Ser Thr Leu Pro Thr
215 220 ~ 225
Ala Thr Pro Ala Ala Gly Glu Leu Thr Met Arg Ser Tyr Gly Thr
230 235 240
Ala Ser Pro Val Ala Asn Arg Trp Thr Pro Gly Thr Thr Gln Thr
245 250 255
Leu Gly Gln Gly Thr Ala Trp Asp Thr Val Ala Ser Thr Pro Gly
260 265 270
Thr Ser Lys Thr Thr Ala Ser Ala Glu Gly Arg Arg Thr Pro Gly
275 280 285
Ala Thr Arg Pro Ala Ala Pro Gly Thr Gly Ser Trp Ala Glu Gly
290 295 300
Ser Val Lys Ala Pro Ala Pro Ile Pro Glu Ser Pro Pro Ser Lys
305 310 315
Ser Arg Ser Met Ser Asn Thr Thr Glu Gly Val Trp Glu Gly Thr
320 325 330
Arg Ser Ser Val Thr Asn Arg Ala Arg Ala Ser Lys Asp Arg Arg
335 340 345
Glu Met Thr Thr Thr Lys Ala Asp Arg Pro Arg Glu Asp Ile Glu
350 355 360
Gly Val Arg Ile Ala Leu Asp Ala Ala Lys Lys Val Leu Gly Thr
365 370 375
Ile Gly Pro Pro Ala Leu Val Ser Glu Thr Leu Ala Trp Glu Ile
380 385 390
Leu Pro Gln Ala Thr Pro Val Ser Lys Gln Gln Ser Gln Gly Ser
395 400 405
Ile Gly Glu Thr Thr Pro Ala Ala Gly Met Trp Thr Leu Gly Thr
410 415 420
Pro Ala Ala Asp Val Trp Ile Leu Gly Thr Pro Ala Ala Asp Val
3/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
425 430 435
Trp Thr Ser Met Glu Ala Ala Ser Gly Glu Gly Ser Ala Ala Gly
440 445 450
Asp Leu Asp Ala Ala Thr Gly Asp Arg Gly Pro Gln Ala Thr Leu
455 460 465
Ser Gln Thr Pro Ala Val Gly Pro Trp Gly Pro Pro Gly Lys Glu
470 475 480
Ser Ser Val Lys Arg Thr Phe Pro Glu Asp Glu Ser Ser Ser Arg
485 490 495
Thr Leu Ala Pro Val Ser Thr Met Leu Ala Leu Phe Met Leu Met
500 505 510
Ala Leu Val Leu Leu Gln Arg Lys Leu Trp Arg Arg Arg Thr Ser
515 520 525
Gln Glu Ala Glu Arg Val Thr Leu Ile Gln Met Thr His Phe Leu
530 535 540
Glu Val Asn Pro Gln Ala Asp Gln Leu Pro His Val Glu Arg Lys
545 550 555
Met Leu Gln Asp Asp Ser Leu Pro Ala Gly Ala Ser Leu Thr Ala
560 565 570
Pro Glu Arg Asn Pro Gly Pro
575
<210> 3
<211> 385
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3939883CD1
<400> 3
Met Gln Thr Ser Ser Lys Pro Ser Asp Phe Leu Asn Leu Ala Lys
1 5 10 15
Lys Lys Arg Lys Phe Ser Glu Leu Leu Thr Thr Val Val Leu Leu
20 25 30
Cys Leu Leu Thr Pro Ser Trp Thr Ser Thr Gly Arg Met Trp Ser
35 40 '45
His Leu Asn Arg Leu Leu Phe Trp Ser Ile Phe Ser Ser Val Thr
50 55 60
Cys Arg Lys Ala Val Leu Asp Cys Glu Ala Met Lys Thr Asn Glu
65 70 75
Phe Pro Ser Pro Cys Leu Asp Ser Lys Thr Lys Val Val Met Lys
80 85 90
Gly Gln Asn Val Ser Met Phe Cys Ser His Lys Asn Lys Ser Leu
95 100 105
Gln Ile Thr Tyr Ser Leu Phe Arg Arg Lys Thr His Leu Gly Thr
110 115 120
Gln Asp Gly Lys Gly Glu Pro Ala Ile Phe Asn Leu Ser Ile Thr
125 130 135
Glu Ala His Glu Ser Gly Pro Tyr Lys Cys Lys Ala Gln Val Thr
140 145 150
Ser Cys Ser Lys Tyr Ser Arg Asp Phe Ser Phe Thr Ile Val Asp
155 160 165
Pro Val Thr Ser Pro Val Leu Asn Ile Met Val Ile Gln Thr Glu
170 175 180
4/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
Thr Asp Arg His Ile Thr Leu His Cys Leu Ser Val Asn Gly Ser
185 190 195
Leu Pro Ile Asn Tyr Thr Phe Phe Glu Asn His Val Ala Ile Ser
200 205 210
Pro Ala Ile Ser Lys Tyr Asp Arg Glu Pro Ala Glu Phe Asn Leu
215 220 225
Thr Lys Lys Asn Pro Gly Glu Glu Glu Glu Tyr Arg Cys Glu Ala
230 235 240
Lys Asn Arg Leu Pro Asn Tyr Ala Thr Tyr Ser His Pro Val Thr
245 250 255
Met Pro Ser Thr Gly Gly Asp Ser Cys Pro Phe Cys Leu Lys Leu
260 265 270
Leu Leu Pro Gly Leu Leu Leu Leu Leu Val Val Ile Ile Leu Ile
275 280 285
Leu Ala Phe Trp Val Leu Pro Lys Tyr Lys Thr Arg Lys Ala Met
290 295 300
Arg Asn Asn Val Pro Arg Asp Arg Gly Asp Thr Ala Met Glu Val
305 310 315
Gly Ile Tyr Ala Asn Ile Leu Glu Lys Gln Ala Lys Glu Glu Ser
320 325 330
Val Pro Glu Val Gly Ser Arg Pro Cys Val Ser Thr Ala Gln Asp
335 340 345
Glu Ala Lys His Ser Gln Glu Leu Gln Tyr Ala Thr Pro Val Phe
350 355 360
Gln Glu Val Ala Pro Arg Glu Gln Glu Ala Cys Asp Ser Tyr Lys
365 370 375
Ser Gly Tyr Vah Tyr Ser Glu Leu Asn Phe
380 385
<210> 4
<211> 221
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3163819CD1
<400> 4
Met Leu Trp Leu Phe Gln Ser Leu Leu Phe Val Phe Cys Phe Gly
1 5 10 15
Pro Gly Gln Leu Arg Asn Ile Gln Val Thr Asn His Ser Gln Leu
20. 25 30
Phe Gln Asn Met Thr Cys Glu Leu His Leu Thr Cys Ser Val Glu
35 40 45
Asp Ala Asp Asp Asn Val Ser Phe Arg Trp Glu Ala Leu Gly Asn
50 55 60
Thr Leu Ser Ser Gln Pro Asn Leu Thr Val Ser Trp Asp Pro Arg
65 70 75
Ile Ser Ser Glu Gln Asp Tyr Thr Cys Ile Ala Glu Asn Ala Val
80 85 90
Ser Asn Leu Ser Phe Ser Val Ser Ala Gln Lys Leu Cys Glu Asp
95 100 105
Val Lys Ile Gln Tyr Thr Asp Thr Lys Met Ile Leu Phe Met Val
110 115 120
Ser Gly Ile Cys Ile Val Phe Gly Phe Ile Ile Leu Leu Leu Leu
5/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
125 130 135
Val Leu Arg Lys Arg Arg Asp Ser Leu Ser Leu Ser Thr Gln Arg
140 145 150
Thr Gln Gly Pro Ala Glu Ser Ala Arg Asn Leu Glu Tyr Val Ser
155 160 165
Val Ser Pro Thr Asn Asn Thr Val Tyr Ala Ser Val Thr His Ser
170 175 180
Asn Arg Glu Thr Glu Ile Trp Thr Pro Arg Glu Asn Asp Thr Ile
185 190 195
Thr Ile Tyr Ser Thr Ile Asn His Ser Lys Glu Ser Lys Pro Thr
200 205 210
Phe Ser Arg Ala Thr Ala Leu Asp Asn Val Val
215 220
<210> 5
<211> 332
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 8518269CD1
<400> 5
Met Leu Trp Leu Phe Gln Ser Leu Leu Phe Val Phe Cys Phe Gly
1 5 10 15
Pro Gly Asn Val Val Ser Gln Ser Ser Leu Thr Pro Leu Met Val
20 25 30
Asn Gly Ile Leu Gly Glu Ser Val Thr Leu Pro Leu Glu Phe Pro
35 40 45
Ala Gly Glu Lys Val Asn Phe Ile Thr Trp Leu Phe Asn Glu Thr
50 55 60
Ser Leu Ala Phe Ile Val Pro His Glu Thr Lys Ser Pro Glu Ile
65 70 75
His Val Thr Asn Pro Lys Gln Gly Lys Arg Leu Asn Phe Thr Gln
80 85 90
Ser Tyr Ser Leu Gln Leu Ser Asn Leu Lys Met Glu Asp Thr Gly
95 100 105
Ser Tyr Arg Ala Gln Ile Ser Thr Lys Thr Ser Ala Lys Leu Ser
110 115 120
Ser Tyr Thr Leu Arg Ile Leu Arg Gln Leu Arg Asn Ile Gln Val
125 130 135
Thr Asn His Ser Gln Leu Phe Gln Asn Met Thr Cys Glu Leu His
140 145 150
Leu Thr Cys Ser Val Glu Asp Ala Asp Asp Asn Val Ser Phe Arg
155 160 165
Trp Glu Ala Leu Gly Asn Thr Leu Ser Ser Gln Pro Asn Leu Thr
170 175 180
Val Ser Trp Asp Pro Arg Ile Ser Ser Glu Gln Asp Tyr Thr Cys
185 190 195
Ile Ala Glu Asn Ala Val Ser Asn Leu Ser Phe Ser Val Ser Ala
200 205 210
Gln Lys Leu Cys Glu Asp Val Lys Ile Gln Tyr Thr Asp Thr Lys
215 220 225
Met Ile Leu Phe Met Val Ser Gly Ile Cys Ile Val Phe Gly Phe
230 235 240
6/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
Ile Ile Leu Leu Leu Leu Val Leu Arg Lys Arg Arg Asp Ser Leu
245 250 255
Ser Leu Ser Thr Gln Arg Thr Gln Gly Pro Ala Glu Ser Ala Arg
260 265 270
Asn Leu Glu Tyr Val Ser Val Ser Pro Thr Asn Asn Thr Val Tyr
275 280 285
Ala Ser Val Thr His Ser Asn Arg Glu Thr Glu Ile Trp Thr Pro
290 295 300
Arg Glu Asn Asp Thr Ile Thr Ile Tyr Ser Thr Ile Asn His Ser
305 310 315
Lys Glu Ser Lys Pro Thr Phe Ser Arg Ala Thr Ala Leu Asp Asn
320 325 330
Val Val
<210> 6
<211> 288
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 1592646CD1
<400> 6
Met Leu Pro His Phe Leu Gly Gly Glu Arg Val Arg Pro Ser Pro
1 5 10 15
Gly Ser Ser Ser Ser Gly Tyr Leu Pro Thr Met Ala Leu Val Leu
20 25 30
Ile Leu Gln Leu Leu Thr Leu Trp Pro Leu Cys His Thr Asp Ile
35 40 45
Thr Pro Ser Val Pro Pro Ala Ser Tyr His Pro Lys Pro Trp Leu
50 55 60
Gly Ala Gln Pro Ala Thr Val Val Thr Pro Gly Val Asn Val Thr
65 70 75
Leu Arg Cys Arg Ala Pro Gln Pro Ala Trp Arg Phe Gly Leu Phe
80 85 90
Lys Pro Gly Glu Ile Ala Pro Leu Leu Phe Arg Asp Val Ser Ser
95 100 105
Glu Leu Ala Glu Phe Phe Leu Glu Glu Val Thr Pro Ala Gln Gly
110 115 120
Gly Ser Tyr Arg Cys Cys Tyr Arg Arg Pro Asp Trp Gly Pro Gly
125 130 135
Val Trp Ser Gln Pro Ser Asp Val Leu Glu Leu Leu Val Thr Glu
140 145 150
Glu Leu Pro Arg Pro Ser Leu Val Ala Leu Pro Gly Pro Val Val
155 160 165
Gly Pro Gly Ala Asn Val Ser Leu Arg Cys Ala Gly Arg Leu Arg
170 175 180
Asn Met Ser Phe Val Leu Tyr Arg Glu Gly Val Ala Ala Pro Leu
185 190 195
Gln Tyr Arg His Ser Ala Gln Pro Trp Ala Asp Phe Thr Leu Leu
200 205 210
Gly Ala Arg Ala Pro Gly Thr Tyr Ser Cys Tyr Tyr His Thr Pro
215 220 225
Ser Ala Pro Tyr Val Leu Ser Gln Arg Ser Glu Val Leu Val Ile
7/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
230 235 240
Ser Trp Glu Asp Ser Gly Ser Ser Asp Tyr Thr Arg Gly Asn Leu
245 250 255
Val Arg Leu Gly Leu Ala Gly Leu Val Leu Ile Ser Leu Gly Ala
260 265 270
Leu Val Thr Phe Asp Trp Arg Ser Gln Asn Arg Ala Pro Ala Gly
275 280 285
Ile Arg Pro
<210> 7
<211> 1450
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500191CD1
<400> 7
Met Ala Ala Glu Arg Gly Ala Arg Arg Leu Leu Ser Thr Pro Ser
1 5 10 15
Phe Trp Leu Tyr Cys Leu Leu Leu Leu Gly Arg Arg Ala Pro Gly
20 25 30
Ala Ala Ala Ala Arg Ser Gly Ser Ala Pro Gln Ser Pro Gly Ala
35 40 45
Ser Ile Arg Thr Phe Thr Pro Phe Tyr Phe Leu Val Glu Pro Val
50 55 60
Asp Thr Leu Ser Val Arg Gly Ser Ser Val Ile Leu Asn Cys Ser
65 70 75
Ala Tyr Ser Glu Pro Ser Pro Lys Ile Glu Trp Lys Lys Asp Gly
80 85 90
Thr Phe Leu Asn Leu Val Ser Asp Asp Arg Arg Gln Leu Leu Pro
95 100 105
Asp Gly Ser Leu Phe Ile Ser Asn Val Val His Ser Lys His Asn
110 115 120
Lys Pro Asp Glu Gly Tyr Tyr Gln Cys Val Ala Thr Val Glu Ser
125 130 135
Leu Gly Thr Ile Ile Ser Arg Thr Ala Lys Leu Ile Val Ala Gly
140 145 150
Leu Pro Arg Phe Thr Ser Gln Pro Glu Pro Ser Ser Val Tyr Ala
155 160 165
Gly Asn Asn Ala Ile Leu Asn Cys Glu Val Asn Ala Asp Leu Val
170 175 180
Pro Phe Val Arg Trp Glu Gln Asn Arg Gln Pro Leu Leu Leu Asp
185 190 195
Asp Arg Val Ile Lys Leu Pro Ser Gly Met Leu Val Ile Ser Asn
200 205 210
Ala Thr Glu Gly Asp Gly Gly Leu Tyr Arg Cys Val Val Glu Ser
215 220 225
Gly Gly Pro Pro Lys Tyr Ser Asp Glu Val Glu Leu Lys Val Leu
230 235 240
Pro Asp Pro Glu Val Ile Ser Asp Leu Val Phe Leu Lys Gln Pro
245 250 255
Ser Pro Leu Val Arg Val Ile Gly Gln Asp Val Val Leu Pro Cys
260 265 270
8/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
Val Ala Ser Gly Leu Pro Thr Pro Thr Ile Lys Trp Met Lys Asn
275 280 285
Glu Glu Ala Leu Asp Thr Glu Ser Ser Glu Arg Leu Val Leu Leu
290 295 300
Ala Gly Gly Ser Leu Glu Ile Ser Asp Val Thr Glu Asp Asp Ala
305 310 315
Gly Thr Tyr Phe Cys Ile Ala Asp Asn Gly Asn Glu Thr Ile Glu
320 325 330
Ala Gln Ala Glu Leu Thr Val Gln Ala Gln Pro Glu Phe Leu Lys
335 340 345
Gln Pro Thr Asn Ile Tyr Ala His Glu Ser Met Asp Ile Val Phe
350 355 360
Glu Cys Glu Val Thr Gly Lys Pro Thr Pro Thr Val Lys Trp Val
365 370 375
Lys Asn Gly Asp Met Val Ile Pro Ser Asp Tyr Phe Lys Ile Val
380 385 390
Lys Glu His Asn Leu Gln Val Leu Gly Leu Val Lys Ser Asp Glu
395 400 405
Gly Phe Tyr Gln Cys Ile Ala Glu Asn Asp Val Gly Asn Ala Gln
410 415 420
Ala Gly Ala Gln Leu Ile Ile Leu Glu His Ala Pro Ala Thr Thr
425 430 435
Gly Pro Leu Pro Ser Ala Pro Arg Asp Val Val Ala Ser Leu Val
440 445 450
Ser Thr Arg Phe Ile Lys Leu Thr Trp Arg Thr Pro Ala Ser Asp
455 460 465
Pro His Gly Asp Asn Leu Thr Tyr Ser Val Phe Tyr Thr Lys Glu
470 475 480
Gly Ile Ala Arg Glu Arg Val Glu Asn Thr Ser His Pro Gly Glu
485 490 495
Met Gln Val Thr Ile Gln Asn Leu Met Pro Ala Thr Val Tyr Ile
500 505 510
Phe Arg Val Met Ala Gln Asn Lys His Gly Ser Gly Glu Ser Ser
515 520 525
Ala Pro Leu Arg Val Glu Thr Gln.Pro Glu Val Gln Leu Pro Gly
530 535 540
Pro Ala Pro Asn Leu Arg Ala Tyr Ala Ala Ser Pro Thr Ser Ile
545 550 555
Thr Val Thr Trp Glu Thr Pro Val Ser Gly Asn Gly Glu Ile Gln
560 565 570
Asn Tyr Lys Leu Tyr Tyr Met Glu Lys Gly Thr Asp Lys Glu Gln
575 580 585
Asp Val Asp Val Ser Ser His Ser Tyr Thr Ile Asn Gly Leu Lys
590 595 600
Lys Tyr Thr Glu Tyr Ser Phe Arg Val Val Ala Tyr Asn Lys His
605 610 615
Gly Pro Gly Val Ser Thr Pro Asp Val Ala Val Arg Thr Leu Ser
620 625 630
Asp Val Pro Ser Ala Ala Pro Gln Asn Leu Ser Leu Glu Val Arg
635 640 645
Asn Ser Lys Ser Ile Met Ile His Trp Gln Pro Pro Ala Pro Ala
650 655 660
Thr Gln Asn Gly Gln Ile Thr Gly Tyr Lys Ile Arg Tyr Arg Lys
665 670 675
Ala Ser Arg Lys Ser Asp Val Thr Glu Thr Leu Val Ser Gly Thr
680 685 690
9/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
Gln Leu Ser Gln Leu Ile Glu Gly Leu Asp Arg Gly Thr Glu Tyr
695 700 705
Asn Phe Arg Val Ala Ala Leu Thr Ile Asn Gly Thr Gly Pro Ala
710 715 720
Thr Asp Trp Leu Ser Ala Glu Thr Phe Glu Ser Asp Leu Asp Glu
725 730 735
Thr Arg Val Pro Glu Val Pro Ser Ser Leu His Val Arg Pro Leu
740 745 750
Val Thr Ser Ile Val Val Ser Trp Thr Pro Pro Glu Asn Gln Asn
755 760 765
Ile Val Val Arg Gly Tyr Ala Ile Gly Tyr Gly Ile Gly Ser Pro
770 775 780
His Ala Gln Thr Ile Lys Val Asp Tyr Lys Gln Arg Tyr Tyr Thr
785 790 795
Ile Glu Asn Leu Asp Pro Ser Ser His Tyr Val Ile Thr Leu Lys
800 805 810
Ala Phe Asn Asn Val Gly Glu Gly Ile Pro Leu Tyr Glu Ser Ala
815 820 825
Val Thr Arg Pro His Thr Asp Thr Ser Glu Val Asp Leu Phe Val
830 835 840
Ile Asn Ala Pro Tyr Thr Pro Val Pro Asp Pro Thr Pro Met Met
845 850 855
Pro Pro Val Gly Val Gln Ala Ser Ile Leu Ser His Asp Thr Ile
860 865 870
Arg Ile Thr Trp Ala Asp Asn Ser Leu Pro Lys His Gln Lys Ile
875 880 885
Thr Asp Ser Arg Tyr Tyr Thr Val Arg Trp Lys Thr Asn Ile Pro
890 895 900
Ala Asn Thr Lys Tyr Lys Asn Ala Asn Ala Thr Thr Leu Ser Tyr
905 910 915
Leu Val Thr Gly Leu Lys Pro Asn Thr Leu Tyr Glu Phe Ser Val
920 925 930
Met Val Thr Lys Gly Arg Arg Ser Ser Thr Trp Ser Met Thr Ala
935 940 945
His Gly Thr Thr Phe Glu Leu Val Pro Thr Ser Pro Pro Lys Asp
950 955 960
Val Thr Val Val Ser Lys Glu Gly Lys Pro Lys Thr Ile Ile Val
965 970 975
Asn Trp Gln Pro Pro Ser Glu Ala Asn Gly Lys Ile Thr Gly Tyr
980 985 990
Ile Ile Tyr Tyr Ser Thr Asp Val Asn Ala Glu Ile His Asp Trp
995 1000 1005
Val Ile Glu Pro Val Val Gly Asn Arg Leu Thr His Gln Ile Gln
1010 1015 1020
Glu Leu Thr Leu Asp Thr Pro Tyr Tyr Phe Lys Ile Gln Ala Arg
1025 1030 1035
Asn Ser Lys Gly Met Gly Pro Met Ser Glu Ala Val Gln Phe Arg
1040 1045 1050
Thr Pro Lys Ala Ser Gly Ser Gly Gly Lys Gly Ser Arg Leu Pro
1055 1060 1065
Asp Leu Gly Ser Asp Tyr Lys Pro Pro Met Ser Gly Ser Asn Ser
1070 1075 1080
Pro His Gly Ser Pro Thr Ser Pro Leu Asp Ser Asn Met Leu Leu
1085 1090 1095
Val Ile Ile Val Ser Val Gly Val Ile Thr Ile Val Val Val Val
1100 1105 1110
10/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
Ile Ile Ala Val Phe Cys Thr Arg Arg Thr Thr Ser His Gln Lys
1115 1120 1125
Lys Lys Arg Ala Ala Cys Lys Ser Val Asn Gly Ser His Lys Tyr
1130 1135 1140
Lys Gly Asn Ser Lys Asp Val Lys Pro Pro Asp Leu Trp Ile His
1145 1150 1155
His Glu Arg Leu Glu Leu Lys Pro Ile Asp Lys Ser Pro Asp Pro
1160 1165 1170
Asn Pro Ile Met Thr Asp Thr Pro Ile Pro Arg Asn Ser Gln Asp
1175 1180 1185
Ile Thr Pro Val Asp Asn Ser Met Asp Ser Asn Ile His Gln Arg
1190 1195 1200
Arg Asn Ser Tyr Arg Gly His Glu Ser Glu Asp Ser Met Ser Thr
1205 1210 1215
Leu Ala Gly Arg Arg Gly Met Arg Pro Lys Met Met Met Pro Phe
1220 1225 1230
Asp Ser Gln Pro Pro Gln Pro Val Ile Ser Ala His Pro Ile His
1235 1240 1245
Ser Leu Asp Asn Pro His His His Phe His Ser Ser Ser Leu Ala
1250 1255 1260
Ser Pro Ala Arg Ser His Leu Tyr His Pro Gly Ser Pro Trp Pro
1265 1270 1275
Ile Gly Thr Ser Met Ser Leu Ser Asp Arg Ala Asn Ser Thr Glu
1280 1285 1290
Ser Val Arg Asn Thr Pro Ser Thr Asp Thr Met Pro Ala Ser Ser
1295 1300 1305
Ser Gln Thr Cys Cys Thr Asp His Gln Asp Pro Glu Gly Ala Thr
1310 1315 1320
Ser Ser Ser Tyr Leu Ala Ser Ser Gln Glu Glu Asp Ser Gly Gln
1325 1330 1335
Ser Leu Pro Thr Ala His Val Arg Pro Ser His Pro Leu Lys Ser
1340 1345 1350
Phe Ala Val Pro Ala Ile Pro Pro Pro Gly Pro Pro Thr Tyr Asp
1355 1360 1365
Pro Ala Leu Pro Ser Thr Pro Leu Leu Ser Gln Gln Ala Leu Asn
1370 1375 1380
His His Ile His Ser Val Lys Thr Ala Ser Ile Gly Thr Leu Gly
1385 1390 1395
Arg Ser Arg Pro Pro Met Pro Val Val Val Pro Ser Ala Pro Glu
1400 1405 1410
Val Gln Glu Thr Thr Arg Met Leu Glu Asp Ser Glu Ser Ser Tyr
1415 1420 1425
Glu Pro Asp Glu Leu Thr Lys Glu Met Ala His Leu Glu Gly Leu
1430 1435 1440
Met Lys Asp Leu Asn Ala Ile Thr Thr Ala
1445 1450
<210> 8
<211> 551
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500099CD1
11/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
<400> 8
Met Val Ala Pro Lys Ser His Thr Asp Asp Trp Ala Pro Gly Pro
1 5 10 ~ 15
Phe Ser Ser Lys Pro Gln Arg Ser Gln Leu Gln Ile Phe Ser Ser
20 25 30
Val Leu Gln Thr Ser Leu Leu Phe Leu Leu Met Gly Leu Arg Ala
35 40 45
Ser Gly Lys Asp Ser Ala Pro Thr Val Val Ser Gly Ile Leu Gly
50 55 60
Gly Ser Val Thr Leu Pro Leu Asn Ile Ser Val Asp Thr Glu Ile
65 70 75
Glu Asn Val Ile Trp Ile Gly Pro Lys Asn Ala Leu Ala Phe Ala
80 85 90
Arg Pro Lys Glu Asn Val Thr Ile Met Val Lys Ser Tyr Leu Gly
95 100 105
Arg Leu Asp Ile Thr Lys Trp Ser Tyr Ser Leu Cys Ile Ser Asn
110 115 120
Leu Thr Leu Asn Asp Ala Gly Ser Tyr Lys Ala Gln Ile Asn Gln
125 130 135
Arg Asn Phe Glu Val Thr Thr Glu Glu Glu Phe Thr Leu Phe Val
140 145 150
Tyr Glu Gln Leu Gln Glu Pro Gln Val Thr Met Lys Ser Val Lys
155 160 165
Val Ser Glu Asn Phe Ser Cys Asn Ile Thr Leu Met Cys Ser Val
170 175 180
Lys Gly Ala Glu Lys Ser Val Leu Tyr Ser Trp Thr Pro Arg Glu
185 190 195
Pro His Ala Ser Glu Ser Asn Gly Gly Ser Ile Leu Thr Val Ser
200 205 210
Arg Thr Pro Cys Asp Pro Asp Leu Pro Tyr Ile Cys Thr Ala Gln
215 220 225
Asn Pro Val Ser Gln Arg Ser Ser Leu Pro Val His Val Gly Gln
230 235 240
Phe Cys Thr Asp Pro Gly Ala Ser Arg Gly Gly Thr Thr Gly Glu
245 250 255
Thr Val Val Gly Val Leu Gly Glu Pro Val Thr Leu Pro Leu Ala
260 265 270
Leu Pro Ala Cys Arg Asp Thr Glu Lys Val Val Trp Leu Phe Asn
275 280 285
Thr Ser Ile Ile Ser Lys Glu Arg Glu Glu Ala Ala Thr Ala Asp
290 295 300
Pro Leu Ile Lys Ser Arg Asp Pro Tyr Lys Asn Arg Val Trp Val
305 310 315
Ser Ser Gln Asp Cys Ser Leu Lys Ile Ser Gln Leu Lys Ile Glu
320 325 330
Asp Ala Gly Pro Tyr His Ala Tyr Val Cys Ser Glu Ala Ser Ser
335 340 345
Val Thr Ser Met Thr His Val Thr Leu Leu Ile Tyr Arg Pro Glu
350 355 360
Arg Asn Thr Lys Leu Trp Ile Gly Leu Phe Leu Met Val Cys Leu
365 370 375
Leu Cys Val Gly Ile Phe Ser Trp Cys Ile Trp Lys Arg Lys Gly
380 385 390
Arg Cys Ser Val Pro Ala Phe Cys Ser Ser Gln Ala Glu Ala Pro
395 400 405
Ala Asp Thr Pro Gly Tyr Glu Lys Leu Asp Thr Pro Leu Arg Pro
12/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
410 415 420
Ala Arg Gln Gln Pro Thr Pro Thr Ser Asp Ser Ser Ser Asp Ser
425 430 435
Asn Leu Thr Thr Glu Glu Asp Glu Asp Arg Pro Glu Val His Lys
440 445 450
Pro Ile Ser Gly Arg Tyr Glu Val Phe Asp Gln Val Thr Gln Glu
455 460 465
Gly Ala Gly His Asp Pro Ala Pro Glu Gly Gln Ala Asp Tyr Asp
470 475 480
Pro Val Thr Pro Tyr Val Thr Glu Val Glu Ser Val Val Gly Glu
485 490 495
Asn Thr Met Tyr Ala Gln Val Phe Asn Leu Gln Gly Lys Thr Pro
500 505 510
Val Ser Gln Lys Glu Glu Ser Ser Ala Thr Ile Tyr Cys Ser Ile
515 520 525
Arg Lys Pro Gln Val Val Pro Pro Pro Gln Gln Asn Asp Leu Glu
530 535 540
Ile Pro Glu Ser Pro Thr Tyr Glu Asn Phe Thr
545 550
<210> 9
<211> 336
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7682434CD1
<400> 9
Met Pro Pro Pro Ala Pro Gly Ala Arg Leu Arg Leu Leu Ala Ala
1 5 10 15
Ala Ala Leu Ala Gly Leu Ala Val Ile Ser Arg Gly Leu Leu Ser
20 25 30
Gln Ser Leu Glu Phe Asn Ser Pro Ala Asp Asn Tyr Thr Val Cys
35 40 45
Glu Gly Asp Asn Ala Thr Leu Ser Cys Phe Ile Asp Glu His Val
50 55 60
Thr Arg Val Ala Trp Leu Asn Arg Ser Asn Ile Leu Tyr Ala Gly
65 70 75
Asn Asp Arg Trp Thr Ser Asp Pro Arg Val Arg Leu Leu Ile Asn
80 85 90
Thr Pro Glu Glu Phe Ser Ile Leu Ile Thr Glu Val Gly Leu Gly
95 100 105
Asp Glu Gly Leu Tyr Thr Cys Ser Phe Gln Thr Arg His Gln Pro
110 115 120
Tyr Thr Thr Gln Val Tyr Leu Ile Val His Val Pro Ala Arg Ile
125 130 135
Val Asn Ile Ser Ser Pro Val Thr Val Asn Glu Gly Gly Asn Val
140 145 150
Asn Leu Leu Cys Leu Ala Val Gly Arg Pro Glu Pro Thr Val Thr
155 160 165
Trp Arg Gln Leu Arg Asp Gly Phe Thr Ser Glu Gly Glu Ile Leu
170 175 180
Glu Ile Ser Asp Ile Gln Arg Gly Gln Ala Gly Glu Tyr Glu Cys
185 190 195
13/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
Val Thr His Asn Gly Val Asn Ser Ala Pro Asp Ser Arg Arg Val
200 205 210
Leu Val Thr Val Asn Tyr Pro Pro Thr Ile Thr Asp Val Thr Ser
215 220 225
Ala Arg Thr Ala Leu Gly Arg Ala Ala Leu Leu Arg Cys Glu Ala
230 235 240
Met Ala Val Pro Pro Ala Asp Phe Gln Trp Tyr Lys Asp Asp Arg
245 250 255
Leu Leu Ser Ser Gly Thr Ala Glu Gly Leu Lys Val Gln Thr Glu
260 265 270
Arg Thr Arg Ser Met Leu Leu Phe Ala Asn Val Ser Ala Arg His
275 280 285
Tyr Gly Asn Tyr Thr Cys Arg Ala Ala Asn Arg Leu Gly Ala Ser
290 295 300
Ser Ala Ser Met Arg Leu Leu Arg Pro Gly Ser Leu Glu Asn Ser
305 310 315
Ala Pro Arg Pro Pro Gly Leu Leu Ala Leu Leu Ser Ala Leu Gly
320 325 330
Trp Leu Trp Trp Arg Met
335
<210> 10
<211> 241
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 2202389CD1
<400> 10
Met Lys Thr Leu Pro Ala Met Leu Gly Thr Gly Lys Leu Phe Trp
1 5 10 15
Val Phe Phe Leu Ile Pro Tyr Leu Asp Ile Trp Asn Ile His Gly
20 25 30
Lys Glu Ser Cys Asp Val Gln Leu Tyr Ile Lys Arg Gln Ser Glu
35 40 45
His Ser Ile Leu Ala Gly Asp Pro Phe Glu Leu Glu Cys Pro Val
50 55 60
Lys Tyr Cys Ala Asn Arg Pro His Val Thr Trp Cys Lys Leu Asn
65 70 75
Gly Thr Thr Cys Val Lys Leu Glu Asp Arg Gln Thr Ser Trp Lys
80 85 90
Glu Glu Lys Asn Ile Ser Phe Phe Ile Leu His Phe Glu Pro Val
95 100 105
Leu Pro Asn Asp Asn Gly Ser Tyr Arg Cys Ser Ala Asn Phe Gln
110 115 120
Ser Asn Leu Ile Glu Ser His Ser Thr Thr Leu Tyr Val Thr Gly
125 130 135
Lys Gln Asn Glu Leu Ser Asp Thr Ala Gly Arg Glu Ile Asn Leu
140 145 150
Val Asp Ala His Leu Lys Ser Glu Gln Thr Glu Ala Ser Thr Arg
155 160 165
Gln Asn Ser Gln Val Leu Leu Ser Glu Thr Gly Ile Tyr Asp Asn
170 175 180
Asp Pro Asp Leu Cys Phe Arg Met Gln Glu Gly Ser Glu Val Tyr
14/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
185 190 195
Ser Asn Pro Cys Leu Glu Glu Asn Lys Pro Gly Ile Val Tyr Ala
200 205 210
Ser Leu Asn His Ser Val Ile Gly Leu Asn Ser Arg Leu Ala Arg
215 220 225
Asn Val Lys Glu Ala Pro Thr Glu Tyr Ala Ser Ile Cys Val Arg
230 235 240
Ser
<210> 11
<211> 766
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503597CD1
<400> 11
Met Lys Pro Phe Gln Leu Asp Leu Leu Phe Val Cys Phe Phe Leu
1 5 10 15
Phe Ser Gln Glu Leu Gly Leu Gln Lys Arg Gly Cys Cys Leu Val
20 25 30
Leu Gly Tyr Met Ala Lys Asp Lys Phe Arg Arg Met Asn Glu Gly
35 40 45
Gln Val Tyr Ser Phe Ser Gln Gln Pro Gln Asp Gln Val Val Val
50 55 60
Ser Gly Gln Pro Val Thr Leu Leu Cys Ala Ile Pro Glu Tyr Asp
65 70 75
Gly Phe Val Leu Trp Ile Lys Asp Gly Leu Ala Leu Gly Val Gly
80 85 90
Arg Asp Leu Ser Ser Tyr Pro Gln Tyr Leu Val Val Gly Asn His
95 100 105
Leu Ser Gly Glu His His Leu Lys Ile Leu Arg Ala Glu Leu Gln
110 115 120
Asp Asp Ala Val Tyr Glu Cys Gln Ala Ile Gln Ala Ala Ile Arg
125 130 135
Ser Arg Pro Ala Arg Leu Thr Val Leu Val Pro Pro Asp Asp Pro
140 145 150
Val Ile Leu Gly Gly Pro Val Ile Ser Leu Arg Ala Gly Asp Pro
155 160 165
Leu Asn Leu Thr Cys His Ala Asp Asn Ala Lys Pro Ala Ala Ser
170 175 180
Ile Ile Trp Leu Arg Lys Gly Glu Val Ile Asn Gly Ala Thr Tyr
185 190 195
Ser Lys Thr Leu Leu Arg Asp Gly Lys Arg Glu Ser Ile Val Ser
200 205 210
Thr Leu Phe Ile Ser Pro Gly Asp Val Glu Asn Gly Gln Ser Ile
215 220 225
Val Cys Arg Ala Thr Asn Lys Ala Ile Pro Gly Gly Lys Glu Thr
230 235 240
Ser Val Thr Ile Asp Ile Gln His Pro Pro Leu Val Asn Leu Ser
245 250 255
Val Glu Pro Gln Pro Val Leu Glu Asp Asn Val Val Thr Phe His
260 265 270
15/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
Cys Ser Ala Lys Ala Asn Pro Ala Val Thr Gln Tyr Arg Trp Ala
275 280 285
Lys Arg Gly Gln Ile Ile Lys Glu Ala Ser Gly Glu Val Tyr Arg
290 295 300
Thr Thr Val Asp Tyr Thr Tyr Phe Ser Glu Pro Val Ser Cys Glu
305 310 315
Val Thr Asn Ala Leu Gly Ser Thr Asn Leu Ser Arg Thr Val Asp
320 325 330
Val Tyr Phe Gly Pro Arg Met Thr Thr Glu Pro Gln Ser Leu Leu
335 340 345
Val Asp Leu Gly Ser Asp Ala Ile Phe Ser Cys Ala Trp Thr Gly
350 355 360
Asn Pro Ser Leu Thr Ile Val Trp Met Lys Arg Gly Ser Gly Val
365 370 375
Val Leu Ser Asn Glu Lys Thr Leu Thr Leu Lys Ser Val Arg Gln
380 385 390
Glu Asp Ala Gly Lys Tyr Val Cys Arg Ala Val Val Pro Arg Val
395 400 405
Gly Ala Gly Glu Arg Glu Val Thr Leu Thr Val Asn Gly Pro Pro
410 415 420
Ile Ile Ser Ser Thr Gln Thr Gln His Ala Leu His Gly Glu Lys
425 430 435
Gly Gln Ile Lys Cys Phe Ile Arg Ser Thr Pro Pro Pro Asp Arg
440 445 450
Ile Ala Trp Ser Trp Lys Glu Asn Val Leu Glu Ser Gly Thr Ser
455 460 465
Gly Arg Tyr Thr Val Glu Thr Ile Ser Thr Glu Glu Gly Val Ile
470 475 480
Ser Thr Leu Thr Ile Ser Asn Ile Val Arg Ala Asp Phe Gln Thr
485 490 495
Ile Tyr Asn Cys Thr Ala Trp Asn Ser Phe Gly Ser Asp Thr Glu
500 505 510
Ile Ile Arg Leu Lys Glu Gln Glu Ser Val Pro Met Ala Val Ile
515 520 525
Ile Gly Val Ala Val Gly Ala Gly Val Ala Phe Leu Val Leu Met,
530 535 540
Ala Thr Ile Val Ala Phe Cys Cys Ala Arg Ser Gln Arg Asn Leu
545 550 555
Lys Gly Val Val Ser Ala Lys Asn Asp Ile Arg Val Glu Ile Val
560 565 570
His Lys Glu Pro Ala Ser Gly Arg Glu Gly Glu Glu His Ser Thr
575 580 585
Ile Lys Gln Leu Met Met Asp Arg Gly Glu Phe Gln Gln Asp Ser
590 595 600
Val Leu Lys Gln Leu Glu Val Leu Lys Glu Glu Glu Lys Glu Phe
605 610 615
Gln Asn Leu Lys Asp Pro Thr Asn Gly Tyr Tyr Ser Val Asn Thr
620 625 630
Phe Lys Glu His His Ser Thr Pro Thr Ile Ser Leu Ser Ser Cys
635 640 645
Gln Pro Asp Leu Arg Pro Ala Gly Lys Gln Arg Val Pro Thr Gly
650 655 660
Met Ser Phe Thr Asn Ile Tyr Ser Thr Leu Ser Gly Gln Gly Arg
665 670 675
Leu Tyr Asp Tyr Gly Gln Arg Phe Val Leu Gly Met Gly Ser Ser
680 685 690
16/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
Ser Ile Glu Leu Cys Glu Arg Glu Phe Gln Arg Gly Ser Leu Ser
695 700 705
Asp Ser Ser Ser Phe Leu Asp Thr Gln Cys Asp Ser Ser Val Ser
710 715 720
Ser Ser Gly Lys Gln Asp Gly Tyr Val Gln Phe Asp Lys Ala Ser
725 730 735
Lys Ala Ser Ala Ser Ser Ser His His Ser Gln Ser Ser Ser Gln
740 745 750
Asn Ser Asp Pro Ser Arg Pro Leu Gln Arg Arg Met Gln Thr His
755 760 765
Val
<210> 12
<211> 88
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503603CD1
<400> 12
Met Asp Gly Glu Ala Thr Val Lys Pro Gly Glu Gln Lys Glu Val
1 5 10 15
Val Arg Arg Gly Arg Glu Val Asp Tyr Ser Arg Leu Ile Ala Gly
20 25 30
Thr Leu Pro Gln Ser His Val Leu Leu Ser Pro Phe His Lys Lys
35 40 45
Asp Pro Ile Arg Asp Gly Cys Gly Arg Ala Leu Ser Pro Pro Gly
50 55 60
Pro Ile Ser Gly Pro Trp Glu His Ser Gly Leu Pro Arg Pro Ser
65 70 75
Ala Gly Gly Arg Arg Ala Pro Leu Gln Leu Gln Ile His
80 85
<210> 13
<211> 2691
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3855123CB1
<400> 13
ctccactggt caacccttct cggtggagcc acagccaagt gctggaggac atacgtcgtc 60
actttccact gctcttgcaa aggccaaccc agctgtcacc cagtacaggt ggccaatgcg 120
gggccagatc atcaaggagg catctggaga ggtgtacagg accacagtgg actacacgta 180
cttctcagag cccgtctcct gtgaggtgac caacgcctgg gcagcaccaa cctcagccgc 240
acggttgacg tctactttgg gccccggatg accacagaac cccaatcctt gctcgtggat 300
ctgggctctg atgccatctt cagctgcgcc tggaccggca acccatccct gaccatcgtc 360
tggatgaagc ggggctccgg agtggtcctg agcaatgaga agaccctgac cctcaaatcc 420
gtgcgccagg aggacgcggg caagtacgtg tgccgggctg tggtgccccg tgtgggagcc 480
ggggagagag aggtgaccct gaccgtcaat ggacccccca tcatctccag cacccagacc 540
cagcacgccc tccacggcga gaagggccag atcaagtgct tcatccggag cacgccgccg 600
17/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
ccggaccgca tcgcctggtc ctggaaggag aacgttctgg agtcgggcac atcggggcgc 660
tatacggtgg agaccatcag caccgaggag ggcgtcatct ccaccctgac catcagcaac 720
atcgtgcggg ccgacttcca gaccatctac aactgcacgg cctggaacag cttcggctcc 780
gacactgaga tcatccggct caaggagcaa ggttcggaaa tgaagtcggg agccgggctg 840
gaagcagagt ctgtgccgat ggccgtcatc attggggtgg ccgtaggagc tggtgtggcc 900
ttcctcgtcc ttatggcaac catcgtggcg ttctgctgtg cccgttccca gagaaatctc 960
aaaggtgttg tgtcagccaa aaatgatatc cgagtggaaa ttgtccacaa ggaaccagcc 1020
tctggtcggg agggtgagga gcactccacc atcaagcagc tgatgatgga ccggggtgaa 1080
ttccagcaag actcagtcct gaaacagctg gaggtcctca aagaagagga gaaagagttt 1140
cagaacctga aggaccccac caatggctac tacagcgtca acaccttcaa agagcaccac 1200
tcaaccccga ccatctccct ctccagctgc cagcccgacc tgcgtcctgc gggcaagcag 1260
cgtgtgccca caggcatgtc cttcaccaac atctacagca ccctgagcgg ccagggccgc 1320
ctctacgact acgggcagcg gtttgtgctg ggcatgggca gctcgtccat cgagctttgt 1380
gagcgggagt tccagagagg ctccctcagc gacagcagct ccttcctgga cacgcagtgt 1440
gacagcagcg tcagcagcag cggcaagcag gatggctatg tgcagttcga caaggccagc 1500
aaggcttctg cttcctcctc ccaccactcc cagtcctcgt cccagaactc tgaccccagt 1560
cgacccctgc agcggcggat gcagactcac gtctaaggat cacacaccgc gggtggggac 1620
gggccaggga agaggtcagg gcacgttctg gttgtccagg gacgaggggt actttgcaga 1680
ggacaccaga attggccact tccaggacag cctcccagcg cctctgccac tgccttcctt 1740
cgaagctctg atcaagcaca aatctgggtc cccaggtgct gtgtgccaga ggtgggcggg 1800
tggggagaca gacagaggct gcggctgagt gcgctgtgct tagtgctgga cacccgtgtc 1860
cccggccctt tcctggaggc ccctctacca cctgctctgc ccacaggcac aagtggcagc 1920
tataactctg ctttcatgaa actgcggtcc actctctggt ctctctgtgg gctctacccc 1980
tcactgacca caagctctac ctacccctgt gcctgtgctc ccatacagcc ctggggagaa 2040
ggggatgacg tcttcccagc actgagctgc cccagaaacc ccggctcccc actgctgctc 2100
atagcccata ccctggaggc tgacaagcca gaaatggcct tggctaaagg agcctctctc 2160
tcaccaggct ggccgggagc ccacccccaa tttgtttggt gttttgtgtc catactcttg 2220
cagttctgtc cttggacttg atgccgctga actctgcggt gggaccggtc ccgtcagagc 2280
ctggtgtact ggggggaggg agggaggagg gagcctgtgc tgacggagca cctcgccggg 2340
tgtgcccctc ctgggctgtg tgaccccagc ctccccaccc acctcctgct ttgtgtactc 2400
ctcccctccc cctcagcaca atcggagttc atataagaag tgcgggagct tctctggtca 2460
gggttctctg aacacttatg gagagagtgc ttcctgggaa gtgtggcgtt tgaaggggct 2520
ggagggcagg tctttaagat ggcgagactg cccttctcag ctgataaaca caagaacggc 2580
gatcctgtct tcagtaaggc tccacgagaa gagaggaagt atatctacac ctcaaccctc 2640
ctagtcacca cctgaaataa atgttaggga cactacaaaa aaaaaaaaaa a 2691
<210> 14
<211> 2518
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4547188CB1
<400> 14
ggaaggatat ggatcaatgt tttctttttt gaagctactg ttaccactcc tggaaaagtt 60
cttcaggaat aagtgacagt aagaatgaca agggattagg actggcttcc tcttataaat 120
aataaaatcc aaagagaagt gacttgagtc tccaggttta aagaagagca actagaagtc 180
gtccaaacac ctgcatctca taaggagaag aaaagtccac ctggatcttg tttctggact 240
gagatggatg gagaggccac agtgaagcct ggagaacaaa aggaagtggt gaggagagga 300
agagaagtgg actactccag gctcattgct ggcactttac cacaatctca cgtcaccagc 360
aggagggcag gatggaaaat gcccctcttc ctcatactgt gcctgctaca aggttcttct 420
ttcgcccttc cacaaaaaag accccatccg agatggctgt gggagggctc tctcccctcc 480
aggacccatc tccgggccat gggaacactc aggccttcct cgcccctctg ctggcgggag 540
gagagctcct ttgcagctcc aaattcattg aagggctcaa ggctggtgtc aggggagcct 600
18/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
ggaggagctg tcaccatcca gtgccattat gccccctcat ctgtcaacag gcaccagagg 660
aagtactggt gctgtctggg gcccccaaga tggatctgcc agaccattgt gtccaccaac 720
cagtatactc accatcgcta tcgtgaccgt gtggccctca cagactttcc acagagaggc 780
ttgtttgtgg tgaggctgtc ccaactgtcc ccggatgaca tcggatgcta cctctgcggc 840
attggaagtg aaaacaacat gctgttctta agcatgaatc tgaccatctc tgcaggtccc 900
gccagcaccc tccccacagc cactccagct gctggggagc tcaccatgag atcctatgga 960
acagcgtctc cagtggccaa cagatggacc ccaggaacca cccagacctt aggacagggg 1020
acagcatggg acacagttgc ttccactcca ggaaccagca agactacagc ttcagctgag 1080
ggaagacgaa ccccaggagc aaccaggcca gcagctccag ggacaggcag ctgggcagag 1140
ggttctgtca aagcacctgc tccgattcca gagagtccac cttcaaagag cagaagcatg 1200
tccaatacaa cagaaggtgt ttgggagggc accagaagct cggtgacaaa cagggctaga 1260
gccagcaagg acaggaggga gatgacaact accaaggctg ataggccaag ggaggacata 1320
gagggggtca ggatagctct tgatgcagcc aaaaaggtcc taggaaccat tgggccacca 1380
gctctggtct cagaaacttt ggcctgggaa atcctcccac aagcaacgcc agtttctaag 1440
caacaatctc agggttccat tggagaaaca actccagctg caggcatgtg gaccttggga 1500
actccagctg cagatgtgtg gatcttggga actccagctg cagatgtgtg gaccagcatg 1560
gaggcagcat ctggggaagg aagcgctgca ggggacctag atgctgccac tggagacaga 1620
ggtccccaag caacactgag ccagaccccg gcagtaggac cctggggacc ccctggcaag 1680
gagtcctccg tgaagcgtac ttttccagaa gatgaaagca gctctcggac cctggctcct 1740
gtctctacca tgctggccct gtttatgctt atggctctgg ttctattgca aaggaagctc 1800
tggagaagga ggacctctca ggaggcagaa agggtcacct taattcagat gacacatttt 1860
ctggaagtga acccccaagc agaccagctg ccccatgtgg aaagaaagat gctccaggat 1920
gactctcttc ctgctggggc cagcctgact gccccagaga gaaatccagg accctgaggg 1980
acagagagat gaactgctca gttaccatgg gagaaggacc aagatcaaag gccttcagga 2040
ccccagcctc tttccatcat ccttcctcca cctgtgggaa gagaagctga tgcagccggt 2100
gctccaccca tggaagaaag gctggctgtc cttgggccca agaaagtcaa gcattatcca 2160
cgtccaaagg tgacaagatg actcaaagga gacttcaaga acagtgtatg aaacactgga 2220
agaggtcacc taggaaaagc atgaaatttc cattcctgaa tgtttgcaaa tagaagaggc 2280
ttccaatcag tgtggaaagt gacaaatccc ctatcaacac tcccagccct tgctgggggc 2340
tccttttctg actactgtta gcactcagcc tcccattcac atgtattata tttaagtgta 2400
ccagccttgc cttctcaagt agattctaag ctcctttaag gcagtaattg cattttatct 2460
gtctcatgat gcccccagag aacttccaac tcagtagacc ccaataatac ctgtgtgc 2518
<210> 15
<211> 1522
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3939883CB1
<400> 15
aaaccagtat tatgcaaacc tcatccaaac cctctgattt ccttaacttg gctaagaaaa 60
agaggaagtt ctccgagtta ctcaccactg tggttctact atgccttctg accccgtctt 120
ggacttcaac tgggagaatg tggagccatt tgaacaggct cctcttctgg agcatatttt 180
cttctgtcac ttgtagaaaa gctgtattgg attgtgaggc aatgaaaaca aatgaattcc 240
cttctccatg tttggactca aagactaagg tggttatgaa gggtcaaaat gtatctatgt 300
tttgttccca taagaacaaa tcactgcaga tcacctattc attgtttcga cgtaagacac 360
acctgggaac ccaggatgga aaaggtgaac ctgcgatttt taacctaagc atcacagaag 420
cccatgaatc aggcccctac aaatgcaaag cccaagttac cagctgttca aaatacagtc 480
gtgacttcag cttcacgatt gtcgacccgg tgacttcccc agtgctgaac attatggtca 540
ttcaaacaga aacagaccga catataacat tacattgcct ctcagtcaat ggctcgctgc 600
ccatcaatta cactttcttt gaaaaccatg ttgccatatc accagctatt tccaagtatg 660
acagggagcc tgctgaattt aacttaacca agaagaatcc tggagaagag gaagagtata 720
ggtgtgaagc taaaaacaga ttgcctaact atgcaacata cagtcaccct gtcaccatgc 780
19/28
ttccagcaag actcagtcct gaaacagctg gaggtcctca aagaagagga
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
cctcaacagg cggagacagc tgtcctttct gtctgaagct actacttcca gggttattac 840
tgttgctggt ggtgataatc ctaattctgg ctttttgggt actgcccaaa tacaaaacaa 900
gaaaagctat gagaaataat gtgcccaggg accgtggaga cacagccatg gaagttggaa 960
tctatgcaaa tatccttgaa aaacaagcaa aggaggaatc tgtgccagaa gtgggatcca 1020
ggccgtgtgt ttccacagcc caagatgagg ccaaacactc ccaggagcta cagtatgcca 1080
cccccgtgtt ccaggaggtg gcaccaagag agcaagaagc ctgtgattct tataaatctg 1140
gatatgtcta ttctgaactc aacttctgaa atttacagaa acaaactaca tctcaggatg 1200
gagtctcact ctgttgccca ggctggagtt cagtggcgcg atcttggctc acttcaatct 1260
ccatcttccc agttcaagcg attctcatgc ctcgacctcc cgagtagctg ggattgcagg 1320
tgcccgctac cacgcccagc taatttttgt atttttagta gagatggggt ttcactatgg 1380
tggccaggct ggtcttgaac tcctgacctc agatgatctg cctgcctcgg cctcccaaag 1440
tgctggaact acaggcctga gccaccgtgc ccggccctga atcgctttag taagtaaagg 1500
gtctccaaga ataaaaaaaa as 1522
<210> 16
<211> 1084
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3163819CB1
<400> 16
ggaaagcatg ttgtggctgt tccaatcgct cctgtttgtc ttctgctttg gcccaggaca 60
actgaggaac atacaagtta ccaatcacag tcagctattt cagaatatga cctgtgagct 120
ccatctgact tgctctgtgg aggatgcaga tgacaatgtc tcattcagat gggaggcctt 180
gggaaacaca ctttcaagtc agccaaacct cactgtctcc tgggacccca ggatttccag 240
tgaacaggac tacacctgca tagcagagaa tgctgtcagt aatttatcct tctctgtctc 300
tgcccagaag ctttgcgaag atgttaaaat tcaatataca gataccaaaa tgattctgtt 360
tatggtttct gggatatgca tagtcttcgg tttcatcata ctgctgttac ttgttttgag 420
gaaaagaaga gattccctat ctttgtctac tcagcgaaca cagggccccg cagagtccgc 480
aaggaaccta gagtatgttt cagtgtctcc aacgaacaac actgtgtatg cttcagtcac 540
tcattcaaac agggaaacag aaatctggac acctagagaa aatgatacta tcacaattta 600
ctccacaatt aatcattcca aagagagtaa acccactttt tccagggcaa ctgcccttga 660
caatgtcgtg taagttgctg aaaggcctca gaggaattcg ggaatgacac gtcttctgat 720
cccatgagac agaacaaaga acaggaagct tggttcctgt tgttcctggc aacagaattt 780
gaatatctag gataggatga tcacctccag tccttcggac ttaaacctgc ctacctgagt 840
caaacaccta aggataacat catttccagc atgtggttca aataatattt tccaatccac 900
ttcaggccaa aacatgctaa agataacaca ccagcacatt gactctctct ttgataacta 960
agcaaatgga attatggttg acagagagtt tatgatccag aagacaacca cttctctcct 1020
tttagaaagc agcaggattg acttattgag aaataatgca gtgtgttggt tacatgtgta 1080
gtct 1084
<210> 17
<211> 1463
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 8518269CB1
<400> 17
caaaaacatt gactgcctca aggtctcaag caccagtctt caccgcggaa agcatgttgt 60
ggctgttcca atcgctcctg tttgtcttct gctttggccc agggaatgta gtttcacaaa 120
20/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
gcagcttaac cccattgatg gtgaacggga ttctggggga gtcagtaact cttcccctgg 180
agtttcctgc aggagagaag gtcaacttca tcacttggct tttcaatgaa acatctcttg 240
ccttcatagt accccatgaa accaaaagtc cagaaatcca cgtgactaat ccgaaacagg 300
gaaagcgact gaacttcacc cagtcctact ccctgcaact cagcaacctg aagatggaag 360
acacaggctc ttacagagcc cagatatcca caaagacctc tgcaaagctg tccagttaca 420
ctctgaggat attaagacaa ctgaggaaca tacaagttac caatcacagt cagctatttc 480
agaatatgac ctgtgagctc catctgactt gctctgtgga ggatgcagat gacaatgtct 540
cattcagatg ggaggccttg ggaaacacac tttcaagtca gccaaacctc actgtctcct 600
gggaccccag gatttccagt gaacaggact acacctgcat agcagagaat gctgtcagta 660
atttatcctt ctctgtctct gcccagaagc tttgcgaaga tgttaaaatt caatatacag 720
ataccaaaat gattctgttt atggtttctg ggatatgcat agtcttcggt ttcatcatac 780
tgctgttact tgttttgagg aaaagaagag attccctatc tttgtctact cagcgaacac 840
agggccccgc agagtccgca aggaacctag agtatgtttc agtgtctcca acgaacaaca 900
ctgtgtatgc ttcagtcact cattcaaaca gggaaacaga aatctggaca cctagagaaa 960
atgatactat cacaatttac tccacaatta atcattccaa agagagtaaa cccacttttt 1020
ccagggcaac tgcccttgac aatgtcgtgt aagttgctga aaggcctcag aggaattcgg 1080
gaatgacacg tcttctgatc ccatgagaca gaacaaagaa caggaagctt ggttcctgtt 1140
gttcctggca acagaatttg aatatctagg ataggatgat cacctccagt ccttcggact 1200
taaacctgcc tacctgagtc aaacacctaa ggataacatc atttccagca tgtggttcaa 1260
ataatatttt ccaatccact tcaggccaaa acatgctaaa gataacacac cagcacattg 1320
actctctctt tgataactaa gcaaatggaa ttatggttga cagagagttt atgatccaga 1380
agacaaccac ttctctcctt ttagaaagca gcaggattga cttattgaga aataatgcag 1440
tgtgttggtt acatgtgtag tct 1463
<210> 18
<211> 1557
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1592646CB1
<400> 18
agcggggcac tcgcgcagaa caaagatgga gccgtggagt gccatagggc tatgacacag 60
tcccccacag gcccccacct cgatactgtc ttccgtaaat gaggatctgg gtctggtttt 120
ctgatgttgc ctcatttcct gggaggggag agggtgcgac caagccctgg ctccagctct 180
agcgggtatc tgcccaccat ggccctggtg ctgatcctcc agctgctgac cctctggcct 240
ctgtgtcaca cagacatcac tccgtctgtc cccccagctt cataccaccc taagccatgg 300
ctgggagctc agccggctac agttgtgacc cctggggtca acgtgacctt gagatgccgg 360
gcaccccaac ccgcttggag atttggactt ttcaagcctg gagagatcgc tccccttctc 420
ttccgggatg tgtcctccga gctggcagaa ttctttctgg aggaggtgac tccagcccaa 480
gggggaagtt accgctgctg ctaccgaagg ccagactggg ggccgggtgt ctggtcccag 540
cccagcgatg tcctggagct gctggtgaca gaggagctgc cgcggccgtc gctggtggcg 600
ctgcccgggc cggtggtggg tcctggcgcc aacgtgagcc tgcgctgcgc gggccgcctg 660
cggaacatga gcttcgtgct gtaccgcgag ggcgtggcgg ccccgctgca gtaccgccac 720
tccgcgcagc cctgggccga cttcacgctg ctgggcgccc gcgcccccgg cacctacagc 780
tgctactatc acacgccctc cgcgccctac gtgctgtcgc agcgcagcga ggtgctggtc 840
atcagctggg aagactctgg ctcctccgac tacacccggg ggaacctagt ccgcctgggg 900
ctggccgggc tggtcctcat ctccctgggc gcgctggtca cttttgactg gcgcagtcag 960
aaccgcgctc ctgctggtat ccgcccctga gccccaggag cactgcagcc cgagacttcc 1020
aacctgagtg gcggagaagc tgggaccctg ggctggactg tcctttcctg cagccccaca 1080
gtcctgctgg ctgagctccg cggaacggtc cttagacccc gctgtgccct gtgctgtagc 1140
ttctttccag gcctttccca aggagtagct gaaaggaaga cgcgattagt ggttaagact 1200
tccaagccag aagacagagg gttcgaatcc cagcactgcc gtctactcac tgtagtagta 1260
gcagctacag aaaggtagta gtgagacgtg aagccagctg gacttcctgg gttgaatggg 1320
21/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
gacctggaga acttttctgt cttacaagag gattgtaaaa tggaccaatc agcactctgt 1380
aagatggacc aatcagcgct ctgtaaaatg gaccaatcag caggacatgg gcggggacaa 1440
taagggaata aaagctggcg agcgcggcac cccaccagag tctgcttcca cgctgtggga 1500
gctttgttct cttgctctac acaataaatc ttgctgctgc taaaaaaaaa aaaaagg 1557
<210> 19
<211> 5553
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500191CB1
<400> 19
tgcggccgcg ggagccgagc ttgcagcgag ggaccggctg aggcgcgcgg gagggaagga 60
ggcaagggct ccgcggcgct gtcgccgccg ctgccgctca ctctcgggga agagatggcg 120
gcggagcggg gagcccggcg actcctcagc accccctcct tctggctcta ctgcctgctg 180
ctgctcgggc gccgggcgcc gggcgccgcg gccgccagga gcggctccgc gccgcagtcc 240
ccaggagcca gcattcgaac gttcactcca ttttattttc tggtggagcc ggtggataca 300
ctctcagtta gaggctcttc tgttatatta aactgttcag catattctga gccttctcca 360
aaaattgaat ggaaaaaaga tggaactttt ttaaacttag tatcagatga tcgacgccag 420
cttctcccgg atggatcttt atttatcagc aatgtggtgc attccaaaca caataaacct 480
gatgaaggtt attatcagtg tgtggccact gttgagagtc ttggaactat tatcagtaga 540
acagcgaagc tcatagtagc aggtcttcca agatttacca gccaaccaga accttcctca 600
gtttatgctg ggaacaatgc aattctgaat tgtgaagtta atgcagattt ggtcccattt 660
gtgaggtggg aacagaacag acaacccctt cttctggatg atagagttat caaacttcca 720
agtggaatgc tggttatcag caatgcaact gaaggagatg gcgggcttta tcgctgcgta 780
gtggaaagtg gtgggccacc aaagtatagt gatgaagttg aattgaaggt tcttccagat 840
cctgaggtga tatcagactt ggtatttttg aaacagcctt ctcccttagt cagagtcatt 900
ggtcaggatg tagtgttgcc atgtgttgct tcaggacttc ctactccaac cattaaatgg 960
atgaaaaatg aggaggcact tgacacagaa agctctgaaa gattggtatt gctggcaggt 1020
ggtagcctgg agatcagtga tgttactgag gatgatgctg ggacttattt ttgtatagct 1080
gataatggaa atgagacaat tgaagctcaa gcagagctta cagtgcaagc tcaacctgaa 1140
ttcctgaagc agcctactaa tatatatgct cacgaatcta tggatattgt atttgaatgt 1200
gaagtgactg gaaaaccaac tccaactgtg aagtgggtca aaaatgggga tatggttatc 1260
ccaagtgatt attttaagat tgtaaaggaa cataatcttc aagttttggg tctggtgaaa 1320
tcagatgaag ggttctatca gtgcattgct gaaaatgatg ttggaaatgc acaagctgga 1380
gcccaactga taatccttga acatgcacca gccacaacgg gaccactgcc ttcagctcct 1440
cgggatgtcg tggcctccct ggtctctacc cgcttcatca aattgacgtg gcggacacct 1500
gcatcagatc ctcacggaga caaccttacc tactctgtgt tctacaccaa ggaagggatt 1560
gctagggaac gtgttgagaa taccagtcac ccaggagaga tgcaagtaac cattcaaaac 1620
ctaatgccag cgaccgtgta catctttaga gttatggctc aaaataagca tggctcagga 1680
gagagttcag ctccactgcg agtagaaaca caacctgagg ttcagctccc tggcccagca 1740
cctaaccttc gtgcatatgc agcttcgcct acctccatca ctgttacgtg ggaaacacca 1800
gtgtctggca atggggaaat tcagaattat aaattgtact acatggaaaa ggggactgat 1860
aaagaacagg atgttgatgt ttcaagtcac tcttacacca ttaatgggtt gaaaaaatat 1920
acagagtata gtttccgagt ggtggcctac aataaacatg gtcctggagt ttccacacca 1980
gatgttgctg ttcgaacatt gtcagatgtt cccagtgctg ctcctcagaa tctgtccttg 2040
gaagtgagaa attcaaagag tattatgatt cactggcagc cacctgctcc agccacacaa 2100
aatgggcaga ttactggcta caagattcgc taccgaaagg cctcccgaaa gagtgatgtc 2160
actgagacct tggtaagcgg gacacagctg tctcagctga ttgaaggtct tgatcggggg 2220
actgagtata atttccgagt ggctgctcta acaatcaatg gtacaggccc ggcaactgac 2280
tggctgtctg ctgaaacttt tgaaagtgac ctagatgaaa ctcgtgttcc tgaagtgcct 2340
agctctcttc acgtacgccc gctcgttact agcatcgtag tgagctggac tcctccagag 2400
aatcagaaca ttgtggtcag aggttacgcc attggttatg gcattggcag ccctcatgcc 2460
22/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
cagaccatca aagtggacta taaacagcgc tattacacca ttgaaaatct ggatcccagc 2520
tctcactatg tgattaccct gaaagcattt aataacgtgg gtgaaggcat ccccctgtat 2580
gagagtgctg tgaccaggcc tcacacagac acttctgaag ttgatttatt tgttattaat 2640
gctccataca ctccagtgcc agatcccact cccatgatgc caccagtggg agttcaggct 2700
tccattctga gtcatgacac catcaggatt acgtgggcag acaactcgct gcccaagcac 2760
cagaagatta cagactcccg atactacacc gtccgatgga aaaccaacat cccagcaaac 2820
accaagtaca agaatgcaaa tgcaaccact ttgagttatt tggtgactgg tttaaagccg 2880
aatacactct atgaattctc tgtgatggtg accaaaggtc gaagatcaag tacatggagt 2940
atgacagccc atgggaccac ctttgaatta gttccgactt ctccacccaa ggatgtgact 3000
gttgtgagta aagaggggaa acctaagacc ataattgtga attggcagcc tccctctgaa 3060
gccaatggca aaattacagg ttacatcata tattacagta cagatgtgaa tgcagagata 3120
catgactggg ttattgagcc tgttgtggga aacagactga ctcaccagat acaagagtta 3180
actcttgaca caccatacta cttcaaaatc caggcacgga actcaaaggg catgggaccc 3240
atgtctgaag ctgtccaatt cagaacacct aaagcctcag ggtctggagg gaaaggaagc 3300
cggctgccag acctaggatc cgactacaaa cctccaatga gcggcagtaa cagccctcat 3360
gggagcccca cctctcctct ggacagtaat atgctgctgg tcataattgt ttctgttggc 3420
gtcatcacca tcgtggtggt tgtgattatc gctgtctttt gtacccgtcg taccacctct 3480
caccagaaaa agaaacgagc tgcctgcaaa tcagtgaatg gctctcataa gtacaaaggg 3540
aattccaaag atgtgaaacc tccagatctc tggatccatc atgagagact ggagctgaaa 3600
cccattgata agtctccaga cccaaacccc atcatgactg atactccaat tcctcgcaac 3660
tctcaagata tcacaccagt tgacaactcc atggacagca atatccatca aaggcgaaat 3720
tcatacagag ggcatgagtc agaggacagc atgtctacac tggctggaag gcgaggaatg 3780
agaccaaaaa tgatgatgcc ctttgactcc cagccacccc agcctgtgat tagtgcccat 3840
cccatccatt ccctcgataa ccctcaccat catttccact ccagcagcct cgcttctcca 3900
gctcgcagtc atctctacca cccgggcagc ccatggccca ttggcacatc catgtccctt 3960
tcagacaggg ccaattccac agaatccgtt cgaaataccc ccagcactga caccatgcca 4020
gcctcttcgt ctcaaacatg ctgcactgat caccaggacc ctgaaggtgc taccagctcc 4080
tcttacttgg ccagctccca agaggaagat tcaggccaga gtcttcccac tgcccatgtt 4140
cgcccttccc acccattgaa gagcttcgcc gtgccagcaa tcccgcctcc aggacctccc 4200
acctatgatc ctgcattgcc aagcacacca ttactgtccc agcaagctct gaaccatcac 4260
attcactcag tgaagacagc ctccatcggg actctaggaa ggagccggcc tcctatgcca 4320
gtggttgttc ccagtgcccc tgaagtgcag gagaccacaa ggatgttgga agactccgag 4380
agtagctatg aaccagatga gctgaccaaa gagatggccc acctggaagg actaatgaag 4440
gacctaaacg ctatcacaac agcatgacga ccttcaccag gacctgactt caaacctgag 4500
tctggaagtc ttggaactta acccttgaaa acaaggaatt gtacagagta cgagaggaca 4560
gcacttgaga acacagaatg agccagcaga ctggccagcg cctctgtgta gggctggctc 4620
caggcatggc cacctgcctt cccctggtca gcctggaaga agcctgtgtc gaggcagctt 4680
ccctttgcct gctgatattc tgcaggactg ggcaccatgg gccaaaattt tgtgtccagg 4740
gaagaggcga gaagtgcaac ctgcatttca ctttgtggtc aggccgtgtc tttgtgctgt 4800
gactgcatca cctttatgga gtgtagacat tggcatttat gtacaatttt atttgtgtct 4860
tattttattt taccttcaaa aacaaaaacg ccatccaaaa ccaaggaagt ccttggtgtt 4920
ctccacaagt ggttgacatt tgactgcttg ttccaattat gtatggaaag tctttgacag 4980
tgtgggtcgt tcctggggtt ggcttgtttt ttggtttcat ttttattttt taattctgag 5040
tcattgcatc ctctaccagc tgttaatcca tcactctgag ggggaggaaa tgttgcattg 5100
ctgtttgtaa gcttttttta ttattttttt attataatta ttaaaggcct gactctttcc 5160
tctcatcact gtgagattac agatctattt gaattgaatg aaatgtaaca ttgaaaagac 5220
ttgtttgttg ctttctgtgc agtttcagta ttggggcggg tggggggctg ggggttggta 5280
ataggaaatg gaggggctgc tgaggtcctg tgaatgtttc tgtcattgta ctttcttcca 5340
gaagcctgca gagaatggaa gcatcttctt tattgtcctt tcctggcatg tccatcctta 5400
ttgtcactac gttgcaactg gagtttgatt tggatctggt tttaaaattc ttctgtgcaa 5460
tagatgggtt tgaggattta gcggccctga tgtcttggtc atagcctggt aagaatgtcc 5520
atgctgagga gccacatgtt gtatttctaa ctg 5553
<210> 20
<211> 1849
<212> DNA
23/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7500099CB1
<400> 20
aatagatcat catggtggca ccaaagagtc acacagatga ctgggctcct gggcctttct 60
ccagtaagcc acagaggagt cagctgcaaa tattctcttc tgttctacag acctctctcc 120
tcttcctgct catgggacta agagcctctg gaaaggactc agccccaaca gtggtgtcag 180
ggatcctagg gggttccgtg actctccccc taaacatctc agtagacaca gagattgaga 240
acgtcatctg gattggtccc aaaaatgctc ttgctttcgc acgtcccaaa gaaaatgtaa 300
ccattatggt caaaagctac ctgggccgac tagacatcac caagtggagt tactccctgt 360
gcatcagcaa tctgactctg aatgatgcag gatcctacaa agcccagata aaccaaagga 420
attttgaagt caccactgag gaggaattca ccctgttcgt ctatgagcag ctgcaggagc 480
cccaagtcac catgaagtct gtgaaggtgt ctgagaactt ctcctgtaac atcactctaa 540
tgtgctccgt gaagggggca gagaaaagtg ttctgtacag ctggacccca agggaacccc 600
atgcttctga gtccaatgga ggctccattc ttaccgtctc ccgaacacca tgtgacccag 660
acctgccata catctgcaca gcccagaacc ccgtcagcca gagaagctcc ctccctgtcc 720
atgttgggca gttctgtaca gatccaggag cctccagagg aggaacaacg ggggagactg 780
tggtaggggt cctgggagag ccagtcaccc tgccacttgc actcccagcc tgccgggaca 840
cagagaaggt tgtctggttg tttaacacat ccatcattag caaagagagg gaagaagcag 900
caacggcaga tccactcatt aaatccaggg atccttacaa gaacagggtg tgggtctcca 960
gccaggactg ctccctgaag atcagccagc tgaagataga ggacgccggc ccctaccatg 1020
cctacgtgtg ctcagaggcc tccagcgtca ccagcatgac acatgtcacc ctgctcatct 1080
accgacctga gagaaacaca aagctttgga ttgggttgtt cctgatggtt tgccttctgt 1140
gcgttgggat cttcagctgg tgcatttgga agcgaaaagg acggtgttca gtcccagcct 1200
tctgttccag ccaagctgag gccccagcgg atacaccagg atatgagaag ctggacactc 1260
ccctcaggcc tgccaggcaa cagcctacac ccacctcaga cagcagctct gacagcaacc 1320
tcacaactga ggaggatgag gacaggcctg aggtgcacaa gcccatcagt ggaagatatg 1380
aggtatttga ccaggtcact caggagggcg ctggacatga cccagcccct gagggccaag 1440
cagactatga tcccgtcact ccatatgtca cggaagttga gtctgtggtt ggagagaaca 1500
ccatgtatgc acaagtgttc aacttacagg gaaagacccc agtttctcag aaggaagaga 1560
gctcagccac aatctactgc tccatacgga aacctcaggt ggtgccacca ccacaacaga 1620
atgatcttga gattcctgaa agtcctacct atgaaaattt cacctgaaag gaaaagcagc 1680
tgctgcctct ctcctgggac cgtggggttg gaaagtcagc tggacctcat ggggcctggg 1740
gctcgcagac agaagcacct cagaatttcc ttcagtgcct cagagatgcc tggatgtggc 1800
ccctccccct ccttctcacc cttaaggact cccaaaccca ttaatagtt 1849
<210> 21
<211> 1427
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7682434CB1
<400> 21
cgccgcctct gccgcgatgc ccccccctgc gcccggggcc cggctccggc ttctcgccgc 60
cgccgccctg gccggcttgg ccgtcatcag ccgagggctg ctctcccaga gcctggagtt 120
caactctcct gccgacaact acacagtgtg tgaaggtgac aacgccaccc tcagctgctt 180
catcgacgag cacgtgaccc gcgtggcctg gctgaaccgc tccaacatcc tgtatgccgg 240
caatgaccgc tggaccagcg acccgcgggt gcggctgctc atcaacaccc ccgaggagtt 300
ctccatcctc atcaccgagg tggggctcgg cgacgagggc ctctacacct gctccttcca 360
gacccgccac cagccgtaca ccactcaggt ctacctcatt gtccacgtcc ctgcccgcat 420
24/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
tgtgaacatc tcgtcgcctg tgacggtgaa tgaggggggc aatgtgaacc tgctttgcct 480
ggccgtgggg cggccagagc ccacggtcac ctggagacag ctccgagacg gcttcacctc 540
ggagggagag atcctggaga tctctgacat ccagcggggc caggccgggg agtatgagtg 600
cgtgactcac aacggggtta actcggcgcc cgacagccgc cgcgtgctgg tcacagtcaa 660
ctatcctccg accatcacgg acgtgaccag cgcccgcacc gcgctgggcc gggccgccct 720
cctgcgctgc gaagccatgg cggttccccc cgcggatttc cagtggtaca aggatgacag 780
actgctgagc agcggcacgg ccgaaggcct gaaggtgcag acggagcgca cccgctcgat 840
gcttctcttt gccaacgtga gcgcccggca ttacggcaac tatacgtgtc gcgccgccaa 900
ccgactggga gcgtccagcg cctccatgcg gctcctgcgc ccaggatccc tggagaactc 960
agccccgagg cccccagggc tcctggccct cctctccgcc ctgggctggc tgtggtggag 1020
aatgtaggcg caacccagtg gagctcacct ccccctgcag ggggcctcag gccaagagtg 1080
agagaaacgg gggagcaaga gccgtgggtc tcgtgggggc agaagagctc tcggccacca 1140
aggaagaaga gagaggagaa gaggaggagg cagaggaaga aagatcttca gagaacccat 1200
cactgtgagg gataacgcaa aattatgcat ctttctacag ccattctcgc cacccgttca 1260
cgtttccgat tgtgacccac tcccgccacc ccatacccct ctctcttagc tcaggctgtc 1320
aactggcttg tgtgggtgtg ggtgtgtgag tgtgagcctg catgcatgtg taggtgtctg 1380
tgtctctgtt tgtgtgtgtg tgggggggtg ggctggggga agggact 1427
<210> 22
<211> 1014
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2202389CB1
<400> 22
cacagatgcc actggggtag gtaaactgac ccaactctgc agcactcaga agacgaagca 60
aagccttcta cttgagcagt ttttccatca ctgatatgtg caggaaatga agacattgcc 120
tgccatgctt ggaactggga aattattttg ggtcttcttc ttaatcccat atctggacat 180
ctggaacatc catgggaaag aatcatgtga tgtacagctt tatataaaga gacaatctga 240
acactccatc ttagcaggag atccctttga actagaatgc cctgtgaaat actgtgctaa 300
caggcctcat gtgacttggt gcaagctcaa tggaacaaca tgtgtaaaac ttgaagatag 360
acaaacaagt tggaaggaag agaagaacat ttcatttttc attctacatt ttgaaccagt 420
gcttcctaat gacaatgggt cataccgctg ttctgcaaat tttcagtcta atctcattga 480
aagccactca acaactcttt atgtgacagg aaagcaaaat gaactctctg acacagcagg 540
aagggaaatt aacctggttg atgctcacct taagagtgag caaacagaag caagcaccag 600
gcaaaattcc caagtactgc tatcagaaac tggaatttat gataatgacc ctgacctttg 660
tttcaggatg caggaagggt ctgaagttta ttctaatcca tgcctggaag aaaacaaacc 720
aggcattgtt tatgcttccc tgaaccattc tgtcattgga ctgaactcaa gactggcaag 780
aaatgtaaaa gaagcaccaa cagaatatgc atccatatgt gtgaggagtt aagtctgttt 840
ctgactccaa cagggaccac tgaatgatca gcatgttgac atcattgtct gggctcaaca 900
ggatgtcaaa taatatttct caatttgaga atttttactt tagaaatgtt catgttagtg 960
cttgggtctt aagggtccat aggataaatg attaaaattt ctctcagaaa ctta 1014
<210> 23
<211> 3695
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503597CB1
<400> 23
25/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
cccgcctgag gaagccgtgt gcctgggatg ccaagagcca gagaatggat cttctccgag 60
tggggacatt gctgacaatc ccggcttccc gaggcggcta agaacaggca gtttgtgtcg 120
gctggctgca gatacccaga ggcacaaaga gaccgaagcc acccggaggg acccacggac 180
ggacagatgg taggcgcgaa cccgagagga ccggcggagg ctgagcaccg agagccgcca 240
aggaagagaa actaaccaca gccaagttac cccgccggct ttccttcgct gcactaagga 300
atgaaaccct tccagctcga tctgctcttc gtctgcttct tcctcttcag tcaagagctg 360
ggcctccaga agagaggatg ctgtctggtg ctgggctaca tggccaagga caagtttcgg 420
agaatgaatg aaggccaagt ctattccttc agccagcagc cccaggacca ggtggtggtg 480
tcgggacagc cagtgacgct actttgcgcc atccccgaat acgatggctt cgttctgtgg 540
atcaaggacg gcttggctct gggtgtgggc agggacctct caagttaccc acagtacctg 600
gtggtaggga accacctgtc aggggagcac cacctgaaga tcctgagggc agagctgcaa 660
gacgatgcgg tgtacgagtg ccaggccatc caggccgcca tccgctcccg ccccgcacgc 720
ctcacagtcc tggtgccgcc tgatgacccc gtcatcctgg ggggccctgt gatcagcctg 780
cgtgcggggg accctctcaa cctcacctgc cacgcagaca atgccaagcc tgcagcctcc 840
atcatctggt tgcgaaaggg agaggtcatc aatggggcca cctactccaa gaccctgctt 900
cgggacggca agcgggagag catcgtcagc accctcttca tctcccctgg tgacgtggag 960
aatggccaga gcatcgtgtg tcgtgccacc aacaaagcca tccccggagg aaaggagacg 1020
tcggtcacca t.tgacatcca gcaccctcca ctggtcaacc tctcggtgga gccacagcca 1080
gtgctggagg acaacgtcgt cactttccac tgctctgcaa aggccaaccc agctgtcacc 1140
cagtacaggt gggccaagcg gggccagatc atcaaggagg catctggaga ggtgtacagg 1200
accacagtgg actacacgta cttctcagag cccgtctcct gtgaggtgac caacgccctg 1260
ggcagcacca acctcagccg cacggttgac gtctactttg ggccccggat gaccacagaa 1320
ccccaatcct tgctcgtgga tctgggctct gatgccatct tcagctgcgc ctggaccggc 1380
aacccatccc tgaccatcgt ctggatgaag cggggctccg gagtggtcct gagcaatgag 1440
aagaccctga ccctcaaatc cgtgcgccag gaggacgcgg gcaagtacgt gtgccgggct 1500
gtggtgcccc gtgtgggagc cggggagaga gaggtgaccc tgaccgtcaa tggacccccc 1560
atcatctcca gcacccagac ccagcacgcc ctccacggcg agaagggcca gatcaagtgc 1620
ttcatccgga gcacgccgcc gccggaccgc atcgcctggt cctggaagga gaacgttctg 1680
gagtcgggca catcggggcg ctatacggtg gagaccatca gcaccgagga gggcgtcatc 1740
tccaccctga ccatcagcaa catcgtgcgg gccgacttcc agaccatcta caactgcacg 1800
gcctggaaca gcttcggctc cgacactgag atcatccggc tcaaggagca agagtctgtg 1860
ccgatggccg tcatcattgg ggtggccgta ggagctggtg tggccttcct cgtccttatg 1920
gcaaccatcg tggcgttctg ctgtgcccgt tcccagagaa atctcaaagg tgttgtgtca 1980
gccaaaaatg atatccgagt ggaaattgtc cacaaggaac cagcctctgg tcgggagggt 2040
gaggagcact ccaccatcaa gcagctgatg atggaccggg gtgaattcca gcaagactca 2100
gtcctgaaac agctggaggt cctcaaagaa gaggagaaag agtttcagaa cctgaaggac 2160
cccaccaatg gctactacag cgtcaacacc ttcaaagagc accactcaac cccgaccatc 2220
tccctctcca gctgccagcc cgacctgcgt cctgcgggca agcagcgtgt gcccacaggc 2280
atgtccttca ccaacatcta cagcaccctg agcggccagg gccgcctcta cgactacggg 2340
cagcggtttg tgctgggcat gggcagctcg tccatcgagc tttgtgagcg ggagttccag 2400
agaggctccc tcagcgacag cagctccttc ctggacacgc agtgtgacag cagcgtcagc 2460
agcagcggca agcaggatgg ctatgtgcag ttcgacaagg ccagcaaggc ttctgcttcc 2520
tcctcccacc actcccagtc ctcgtcccag aactctgacc ccagtcgacc cctgcagcgg 2580
cggatgcaga ctcacgtcta aggatcacac accgcgggtg gggacgggcc agggaagagg 2640
tcagggcacg ttctggttgt ccagggacga ggggtacttt gcagaggaca ccagaattgg 2700
ccacttccag gacagcctcc cagcgcctct gccactgcct tccttcgaag ctctgatcaa 2760
gcacaaatct gggtccccag gtgctgtgtg ccagaggtgg gcgggtgggg agacagacag 2820
aggctgcggc tgagtgcgct gtgcttagtg ctggacaccc gtgtccccgg ccctttcctg 2880
gaggcccctc taccacctgc tctgcccaca ggcacaagtg gcagctataa ctctgctttc 2940
atgaaactgc ggtccactct ctggtctctc tgtgggctct acccctcgct gaccagaagc 3000
tctacctacc cctgtgcctg tgctcccata cagccctggg gagaagggga tgacgtcttc 3060
ccagcactga gctgccccag aaaccccggc tccccactgc tgctcatagc ccataccctg 3120
gaggctgaca agccagaaat ggccttggct aaaggagcct ctctctcacc aggctggccg 3180
ggagcccacc cccaatttgt ttggtgtttt gtgtccatac tcttgcagtt ctgtccttgg 3240
acttgatgcc gctgaactct gcggtgggac cggtccggtc agagcctggt gtactggggg 3300
gagggaggga ggagggagcc tgtgctgacg gagcacctcg ccgggtgtgc ccctcctggg 3360
26/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
ctgtgtgacc ccagcctccc cacccacctc ctgctttgtg tactcctccc ctccccctca 3420
gcacaatcgg agttcatata agaagtgcgg gagcttctct ggtcagggtt ctctgaacac 3480
ttatggagag agtgcttcct gggaagtgtg gcgtttgaag gggctggagg gcaggtcttt 3540
aagatggcga gactgccctt ctcagctgat aaacacaaga acggcgatcc tgtcttcagt 3600
aaggctccac gagaagagag gaagtatatc tacacctcaa ccctcctagt caccacctga 3660
aataaatgtt agggacacta ctccaaaaaa aaaaa 3695
<210> 24
<211> 2403
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7503603CB1
<400> 24
caggaataag tgacagtaag aatgacaagg gattaggact ggcttcctct tataaataat 60
aaaatccaaa gagaagtgac ttgagtctcc aggtttaaag gagagcaact agaagtcgtc 120
caaacacctg catctcataa ggagaagaaa agtccacctg gatcttgttt ctggactgag 180
atggatggag aggccacagt gaagcctgga gaacaaaagg aagtggtgag gagaggaaga 240
gaagtggact actccaggct cattgctggc actttaccac aatctcacgt tcttctttcg 300
cccttccaca aaaaagaccc catccgagat ggctgtggga gggctctctc ccctccagga 360
cccatctccg ggccatggga acactcaggc cttcctcgcc cctctgctgg cgggaggaga 420
gctcctttgc agctccaaat tcattgaagg gctcaaggct ggtgtcaggg gagcctggag 480
gagctgtcac catccagtgc cattatgccc cctcatctgt caacaggcac cagaggaagt 540
actggtgccg tctggggccc ccaagatgga tctgccagac cattgtgtcc accaaccagt 600
atactcacca tcgctatcgt gaccgtgtgg ccctcacaga ctttccacag agaggcttgt 660
ttgtggtgag gctgtcccaa ctgtccccgg atgacatcgg atgctacctc tgcggcattg 720
gaagtgaaaa caacatgctg ttcttaagca tgaatctgac catctctgca ggtcccgcca 780
gcaccctccc cacagccact ccagctgctg gggagctcac catgagatcc tatggaacag 840
cgtctccagt ggccaacaga tggaccccag gaaccaccca gaccttagga caggggacag 900
catgggacac agttgcttcc actccaggaa ccagcaagac tacagcttca gctgagggaa 960
gacgaacccc aggagcaacc aggccagcag ctccagggac aggcagctgg gcagagggtt 1020
ctgtcaaagc acctgctccg attccagaga gtccaccttc aaagagcaga agcatgtcca 1080
atacaacaga aggtgtttgg gagggcacca gaagctcggt gacaaacagg gctagagcca 1140
gcaaggacag gagggagatg acaactacca aggctgatag gccaagggag gacatagagg 1200
gggtcaggat agctcttgat gcagccaaaa aggtcctagg aaccattggg ccaccagctc 1260
tggtctcaga aactttggcc tgggaaatcc tcccacaagc aacgccagtt tctaagcaac 1320
aatctcaggg ttccattgga gaaacaactc cagctgcagg catgtggacc ttgggaactc 1380
cagctgcaga tgtgtggatc ttgggaactc cagctgcaga tgtgtggacc agcatggagg 1440
cagcatctgg ggaaggaagc gctgcagggg acctagatgc tgccactgga gacagaggtc 1500
cccaagcaac actgagccag accccggcag taggaccctg gggaccccct ggcaaggagt 1560
cctccgtgaa gcgtactttt ccagaagatg aaagcagctc tcggaccctg gctcctgtct 1620
ctaccatgct ggccctgttt atgcttatgg ctctggttct attgcaaagg aagctctgga 1680
gaaggaggac ctctcaggag gcagaaaggg tcaccttaat tcagatgaca cattttctgg 1740
aagtgaaccc ccaagcagac cagctgcccc atgtggaaag aaagatgctc caggatgact 1800
ctcttcctgc tggggccagc ctgactgccc cagagagaga aatccaggac cctgagggac 1860
agagagatga actgctcagt taccatggga gaaggaccaa gatcaaaggc cttcaggacc 1920
ccagcctctt tccatcatcc ttcctccacc tgtgggaaga gaagctgatg cagccggtgc 1980
tccacccatg gaagaaaggc tggctgtcct tgggcccaag aaagtcaagc attatccacg 2040
tccaaaggtg acaagatgac tcaaaggaga cttcaagaac agtgtatgaa acactggaag 2100
aggtcaccta ggaaaagcat gaaatttcca ttcctgaatg tttgaaaata gaagaggctt 2160
ccaatcagtg tggaaagtga caaatcccct atcaacactc ccagcccttg ctgggggctc 2220
cttttctgac tactgttagc actcagcctc ccattcacat gtattatatt taagtgtacc 2280
agccttgcct tctcaagtag attctaagct cctttaaggc agtaattgca ttttatctgt 2340
27/28
CA 02440618 2003-09-11
WO 02/072794 PCT/US02/09052
ctcatgatgc ccccagagaa cttccaactc agtaggaacc catttaatac ctgtgtctga 2400
ttg 2403
28/28
