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RNA METABOLISM PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of RNA
metabolism proteins
and to the use of these sequences in the diagnosis, treatment, and prevention
of nervous system,
autoimmune/inflammatory, cell proliferative, and developmental disorders, and
in the assessment of the
effects of exogenous compounds on the expression of nucleic acid and amino
acid sequences of RNA
metabolism proteins.
BACKGROUND OF THE INVENTION
Ribonucleic acid (RNA) is a linear single-stranded polymer of four
nucleotides, ATP, CTP,
UTP, and GTP. In most organisms, RNA is transcribed as a copy of
deoxyribonucleic acid (DNA),
the genetic material of the organism. In retroviruses RNA rather than DNA
serves as the genetic
material. RNA copies of the genetic material encode proteins or serve various
structural, catalytic, or
regulatory roles in organisms. RNA is classified according to its cellular
localization and function.
Messenger RNAs (mRNAs) encode polypeptides. Ribosomal RNAs (rRNAs) are
assembled, along
with ribosomal proteins, into ribosomes, which are cytoplasmic particles that
translate mRNA into
polypeptides. Transfer RNAs (tRNAs) are cytosolic adaptor molecules that
function in mRNA
translation by recognizing both an mRNA colon and the amino acid that matches
that colon.
Heterogeneous nuclear RNAs (hnRNAs) include mRNA precursors and other nuclear
RNAs of
various sizes. Small nuclear RNAs (snRNAs) are a part of the nuclear
spliceosome complex that
removes intervening, non-coding sequences (introns) and rejoins exons in pre-
mRNAs.
Proteins are associated with RNA during its transcription from DNA, RNA
processing, and
translation of mRNA into protein. Proteins are also associated with RNA as it
is used for structural,
catalytic, and regulatory purposes.
RNA Processing
Various proteins are necessary for processing of transcribed RNAs in the
nucleus. Pre-mRNA
processing steps include capping at the 5' end with methylguanosine,
polyadenylating the 3' end, and
splicing to remove introns. The spliceosomal complex is comprised of five
small nuclear
ribonucleoprotein particles (snRNPs) designated U1, U2, U4, U5, and U6. Each
snRNP contains a
single species of snRNA and about ten proteins. The RNA components of some
snRNPs recognize
and base-pair with intron consensus sequences. The protein components mediate
spliceosome
assembly and the splicing reaction.
An early step in pre-mRNA cleavage involves the cleavage factor Im (CF Im).
The human CF
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Im protein aids in the recruitment and assembly of processing factors that
make up the 3' end
processing complex (Ruegsegger, U. et aI (1998) MoI. Cell. 1:243-253). The
marine formin binding
proteins (FBP's) FBP11 and FBP12 are components of pxe-mRNA splicing complexes
that facilitate
the bridging of 5' and 3' ends of the intron. These proteins function through
bridging interactions
invloving U1 and U2 snRNPs. Autoantibodies to snRNP proteins are found in the
blood of patients
with systemic lupus erythematosus (Stryer, L. (1995) Biochemistry W.H. Freeman
and Company,
New York NY, p. 863).
Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified that
have roles in
splicing, exporting of the mature RNAs to the cytoplasm, and mRNA translation
(Biamonti, G. et al.
(1998) Clin. Exp. Rheumatol. 16:317-326). Some examples of hnRNPs include the
yeast proteins
Hrplp, involved in cleavage and polyadenylation at the 3' end of the RNA;
Cbp80p, involved in
capping the 5' end of the RNA; and Npl3p, a homolog of mammalian hnRNP A1,
involved in export
of mRNA from the nucleus (Shen, E.C. et al. (1998) Genes Dev. 12:679-691).
HnRNPs have been
shown to be important targets of the autoimmune response in rheumatic diseases
(Biamonti, supra).
Many snRNP and hnRNP proteins are characterized by an RNA recognition motif
(RRM).
(Reviewed in Birney, E. et al. (1993) Nucleic Acids Res. 21:5803-5816.) The
RRM is about 80 amino
acids in length and forms four j3-strands and two a-helices arranged in an
a/~3 sandwich. The RRM
contains a core RNP-1 octapeptide motif along with surrounding conserved
sequences. In addition to
snRNP proteins, examples of RNA-binding proteins which contain the above
motifs include
heteronuclear ribonucleoproteins which stabilize nascent RNA and factors which
regulate alternative
splicing. Alternative splicing factors include developmentally regulated
proteins, specific examples of
which have been identified in lower eukaryotes such as Drosophila melanoaaster
and Caenorhabditis
e1_ e~ans. These proteins play key roles in developmental processes such as
pattern formation and sex
determination, respectively. (See, for example, Hodgkin, J. et al. (1994)
Development 120:3681-3689.)
RNA Stability and Degradation
RNA helicases alter and regulate RNA conformation and secondary structure by
using energy
derived from ATP hydrolysis to destabilize and unwind RNA duplexes. The most
well-characterized
and ubiquitous family of RNA helicases is the DEAD-box family, so named for
the conserved B-type
ATP-binding motif which is diagnostic of proteins in this family. Over 40 DEAD-
box helicases have
been identified in organisms as diverse as bacteria, insects, yeast,
amphibians, mammals, and plants.
DEAD-box helicases function in diverse processes such as translation
initiation, splicing, ribosome
assembly, and RNA editing, transport, and stability. Some DEAD-box helicases
play tissue- and stage-
specific roles in spermatogenesis and embxyogenesis. All DEAD-box helicases
contain several
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conserved sequence motifs spread out over about 420 amino acids. These motifs
include an A-type
ATP binding motif, the DEAD-box/B-type ATP-binding motif, a
serinelarginine/threonine tripeptide of
unknown function, and a C-terminal glycine-rich motif with a possible role in
substrate binding and
unwinding. In addition, alignment of divergent DEAD-box helicase sequences has
shown that 37 amino
acid residues are identical among these sequences, suggesting that
conservation of these residues is
important for helicase function. (Reviewed in Linder, P. et al. (1989) Nature
337:121-122.)
Overexpression of the DEAD-box 1 protein (DDXl) may play a role in the
progression of
neuroblastoma (Nb) and retinoblastoma (Rb) tumors. These observations suggest
that DDX1 may
promote or enhance tumor progression by altering the normal secondary
structure and expression levels
of RNA in cancer cells. Other DEAD-box helicases have been implicated either
directly or indirectly in
ultraviolet light-induced tumors, B-cell lymphoma, and myeloid malignancies.
(Reviewed in Godbout,
R. et al. (1998) J. Biol. Chem. 273:21161-21168.)
Ribonucleases (RNases) catalyze the hydrolysis of phosphodiester bonds in RNA
chains, thus
cleaving the RNA. For example, RNase P is a ribonucleoprotein enzyme which
cleaves the 5' end of
pre-tRNAs as part of their maturation process. RNase H digests the RNA strand
of an RNA/DNA
hybrid. Such hybrids occur in cells invaded by retroviruses, and RNase H is an
important enzyme in
the retroviral replication cycle. RNase H domains are often found as a domain
associated with reverse
transcriptases. RNase activity in serum and cell extracts is elevated in a
variety of cancers and
infectious diseases (Schein, C.H. (1997) Nat. Biotechnol. 15:529-536).
Regulation of RNase activity
is being investigated as a means to control tumor angiogenesis, allergic
reactions, viral infection and
replication, and fungal infections.
Degradation of mRNAs having premature termination or nonsense codons is
accomplished
through a surveillance mechanism that has been termed nonsense-mediated mRNA
decay (NMD).
This mechanism helps eliminate flawed mRNAs that might code for nonfunctional
or deleterious
polypeptides. Various NMD components are linked to both yeast and human RNA
metabolism
disorders (Hentze, M. and Kulozik, A. (1999) Cell 96:307-310).
TRANSLATION
Ribosomal RNAs (rRNAs) are assembled, along with ribosomal proteins, into
ribosomes,
which are cytoplasmic particles that translate messenger RNA (mRNA) into
polypeptides. The
eukaryotic ribosome is composed of a 60S (large) subunit and a 40S (small)
subunit, which together
form the 80S ribosome. In addition to the 18S, 28S, SS, and 5.85 rRNAs,
ribosomes contain from 50
to over 80 different ribosomal proteins, depending on the organism. Ribosomal
proteins are classified
according to which subunit they belong (i.e., L, if associated with the large
60S large subunit or S if
associated with the small 40S subunit). E. coli ribosomes have been the most
thoroughly studied and
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contain 50 proteins, many of which are conserved in all life foams. The
structures of nine ribosomal
proteins have been solved to less than 3.0/~ resolution (i.e., S5, S6, 517,
L1, L6, L9, L12, L14, L30),
revealing common motifs, such as (3-a-~i protein folds in addition to acidic
and basic RNA-binding
motifs positioned between (3-strands. Most ribosomal proteins are believed to
contact rRNA directly
(reviewed in Liljas, A. and Garber, M. (1995) Curr. Opin. Struct. Biol. 5:721-
727, see also Woodson,
S.A. and Leontis, N.B. (1998) Curr. Opin. Struct. Biol. 8:294-300;
Ramakrishnan, V. and White,
S.W. (1998) Trends Biochem. Sci. 23:208-212.)
Ribosomal proteins may undergo post-translational modifications or interact
with other
ribosome-associated proteins to regulate translation. For example, the highly
homologous 40S
ribosomal protein S6 kinases (S6K1 and S6K2) play a key role in the regulation
of cell growth by
controlling the biosynthesis of translational components which make up the
protein synthetic
apparatus (including the ribosomal proteins). In the case of S6K1, at least
eight phosphorylation sites
are believed to mediate kinase activation in a hierarchical fashion (Dufner
and Thomas (1999) Exp.
Cell. Res. 253:100-109). Some of the ribosomal proteins, including L1, also
function as translational
repressors by binding to polycistronic mRNAs encoding ribosomal proteins
(reviewed in Liljas, A.
supra and Garber, M. supra).
Recent evidence suggests that a number of ribosomal proteins have secondary
functions
independent of their involvement in protein biosynthesis. These proteins
function as regulators of cell
proliferation and, in some instances, as inducers of cell death. For example,
the expression of human
ribosomal protein Ll3a has been shown to induce apoptosis by arresting cell
growth in the G2/M
phase of the cell cycle. Inhibition of expression of Ll3a induces apoptosis in
target cells, which
suggests that this protein is necessary, in the appropriate amount, for cell
survival. Similar results
have been obtained in yeast where inactivation of yeast homologues of Ll3a,
rp22 aiid rp23, results in
severe growth retardation and death. A closely related ribosomal protein, L7,
arrests cells in G1 and
also induces apoptosis. Thus, it appears that a subset of ribosomal proteins
may function as cell cycle
checkpoints and compose a new family of cell proliferation regulators.
Mapping of individual ribosomal proteins on the surface of intact ribosomes is
accomplished
using 3D immunocryoelectronmicroscopy, whereby antibodies raised against
specific ribosomal
proteins are visualized. Progress has been made toward the mapping of L1, L7,
and L12 while the
structure of the intact ribosome has been solved to only 20-2S A resolution
and inconsistencies exist
among different crude structures (Frank, J. (1997) Curr. Opin. Struct. Biol.
7:266-272).
Three distinct sites have been identified on the ribosome. The aminoacyl-tRNA
acceptor site
(A site) receives charged tRNAs (with the exception of the initiator-tRNA).
The peptidyl-tRNA site
(P site) binds the nascent polypeptide as the amino acid from the A site is
added to the elongating
chain. Deacylated tRNAs bind in the exit site (E site) prior to their release
from the ribosome. The
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structure of the ribosome is reviewed in Stryer, L. (1995) Biochemistry W.H.
Freeman and Company,
New York NY pp. 888-9081; Lodish, H. et al. (1995) Molecular Cell Biolo~y
Scientific American
Books, New York NY pp. 119-138; and Lewin, B (1997) Genes VI Oxford University
Press, Inc. New
York, NY).
tRNA Char~in~
Correct translation of the genetic code depends upon each amino acid forming a
linkage with the
appropriate transfer RNA (tRNA). The aminoacyl-tRNA synthetases (aaRSs) are
essential proteins
found in all living organisms. The aaRSs are responsible for the activation
and correct attachment of an
amino acid with its cognate tRNA, as the first step in protein biosynthesis.
Prokaryotic organisms have
at least twenty different types of aaRSs, one for each different amino acid,
while eukaryotes usually
have two aaRSs, a cytosolic form and a mitochondrial form, for each different
amino acid. The 20
aaRS enzymes can be divided into two structural classes. Class I enzymes add
amino acids to the 2'
hydroxyl at the 3' end of tRNAs while Class II enzymes add amino acids to the
3' hydroxyl at the 3' end
of tRNAs. Each class is characterized by a distinctive topology of the
catalytic domain. Class I
enzymes contain a catalytic domain based on the nucleotide-binding 'Rossman
fold'. In particular, a
consensus tetrapeptide motif is highly conserved (Prosite Document PDOC00161,
Aminoacyl-transfer
RNA synthetases class-I signature). Class II enzymes contain a central
catalytic domain, which consists
of a seven-stranded antiparallel 13-sheet domain, as well as N- and C-
terminal regulatory domains.
Class II enzymes are separated into two groups based on the heterodimeric or
homodimeric structure of
the enzyme; the latter group is further subdivided by the structure of the N-
and C-terminal regulatory
domains (Hartlein, M. and Cusack, S. (1995) J. Mol. Evol. 40:519-530). The
different aaRSs are
believed to be the result of divergent evolution, likely following gene
duplication events. Notably, amino
acids such as Gln, were among the last to appear in nature and evolutionary
studies suggest that Gln-
RSs appeared first in eukaryotes and were later horizontally transferred to
prokaryotes (Lamour, V. et
al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:8670-74 and Siatecka, M. et al.
(1998) Eur. J. Biochem.
256:80-7). The importance of Gln RS and Gln-tRNAG'" are discussed below.
In addition to their function in protein synthesis, specific aminoacyl tRNA
synthetases also play
roles in cellular fidelity, RNA splicing, RNA trafficking, apoptosis, and
transcriptional and translational
regulation. For example, human tyrosyl-tRNA synthetase can be proteolytically
cleaved into two
fragments with distinct cytokine activities. The carboxy-terminal domain
exhibits monocyte and
leukocyte chemotaxis activity as well as stimulating production of
myeloperoxidase, tumor necrosis
factor-a, and tissue factor. The N-terminal domain binds to the interleukin-8
type A receptor and
functions as an interleukin-8-like cytokine. Human tyrosyl-tRNA synthetase is
secreted from
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apoptotic tumor cells and may accelerate apoptosis (Wakasugi, K. and Schimmel,
P. (1999) Science
284:147-151). Mitochondrial Neurospora crassaTyrRS and S. cerevisiaeLeuRS are
essential factors
for certain group I intron splicing activities, and human mitochondrial LeuRS
can substitute for the
yeast LeuRS in a yeast null strain. Certain bacterial aaRSs are involved in
regulating their own
transcription or translation (Martinis, supra). Several aaRSs are able to
synthesize diadenosine
oligophosphates, a class of signalling molecules with roles in cell
proliferation, differentiation, and
apoptosis (Kisselev, L.L et al. (1998) FEBS Lett. 427:157-163; Vartanian, A.
et al. (1999) FEBS Lett.
456:175-180).
Under optimal conditions, polypeptide synthesis proceeds at a rate of
approximately 40 amino
acid residues per second. The rate of misincorporation during translation in
on the order of 10~ and is
primarily the result of aminoacyl-t-RNAs being charged with the incorrect
amino acid. Incorrectly
charged tRNA are tonic to cells as they result in the incorporation of
incorrect amino acid residues
into an elongating polypeptide. The rate of translation is presumed to be a
compromise between the
optimal rate of elongation and the need for translational fidelity.
Mathematical calculations predict
that 10-4 is indeed the maximum acceptable error rate for protein synthesis in
a biological system
(reviewed in Stryer, L. supra and Watson, J. et al. (1987) The
Benjamin/Cummings Publishing Co.,
Inc. Menlo Park, CA). A particularly error prone aminoacyl-tRNA charging
event, the charging of
tRNAG'n with Gln. A mechanism exist for the correction of this mischarging
event which likely has its
origins in evolution. Gln was among the last of the 20 naturally occurring
amino acids used
polypeptide synthesis to appear in nature. Gram positive eubacteria,
cyanobacteria, Archeae, and
eukaryotic organelles posses a noncanonical pathway for the synthesis of Gln-
tRNAG'n based on the
transformation of Glu-tRNA~'° (synthesized by Glu-tRNA synthetase,
GluRS) using the enzyme Glu-
tRNA~''1 amidotransferase (Glu-AdT). The reactions involved in the
transamidation pathway are as
follows (Curnow, A.W, et al. (1997) Nucleic Acids Symposium 36:2-4):
GluRS
( 1 ) tRNA~'n + GIu + ATP -~ GIu-tRNA~'~ + AMP + PPi
Glu-AdT
(2) Glu-tRNAG'n + Gln + ATP --~ GIn-tRNA~'" + Glu + ADP + P
A similar enzyme, Asp-tRNA'~" amidotransferase, exists in Archaea, which
transforms Asp-
tRNA'~" to Asn-tRNA'~°. Formylase, the enzyme that transforms Met-
tRNA~et to fMet-tRNA~e' in
eubacteria, is likely to be a related enzyme. A hydrolytic activity has also
been identified that destroys
mischarged Val-tRNAne (Schimmel, P. et al. (1998) FASEB J. 12:1599-1609). I
likely scenario for
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the evolution of Glu-AdT in primitive life forms is the absence a specific
glutaminyl-tRNA synthetase
(GlnRS), requiring an alternative pathway for the synthesis of Gln-tRNAG'". In
fact, deletion of the
Glu-AdT operon in Gram positive bacteria is lethal (Curnow, A.W. et al. (1997)
Proc. Natl. Acad. Sci.
U.S.A. 94:11819-11826). The existence of GluRS activity in other organisms has
been inferred by the
high degree of conservation in translation machinery in nature; however, GluRS
has not been
identified in all organisms, including Homo sapiens. Such an enzyme would be
responsible for
ensuring translational fidelity and reducing the synthesis of defective
polypeptides.
Autoantibodies against aminoacyl-tRNAs are generated by patients with
autoimmune diseases
such as rheumatic arthritis, dermatomyositis and polymyositis, and correlate
strongly with
complicating interstitial lung disease (1LD) (Freist, W. et aI. (1999) Biol.
Chem. 380:623-646; Freist,
W. et al. (1996) Biol. Chem. Hoppe Seyler 377:343-356). These antibodies
appear to be generated in
response to viral infection, and coxsackie virus has been used to induce
experimental viral myositis in
animals.
Comparison of aaRS structures between humans and pathogens has been useful in
the design
IS of novel antibiotics (Schimmel, supra). Genetically engineered aaRSs have
been utilized to allow site-
specific incorporation of unnatural amino acids into proteins in vivo (Liu,
D.R. et al. (1997) Proc.
Natl. Acad. Sci. USA 94:10092-10097).
Translation Initiation
Initiation of translation can be divided into three stages, 'The first stage
brings an initiator
transfer RNA (Met-tRNAf) together with the 40S ribosomal subunit to form the
43S preinitiation
complex. The second stage binds the 43S preinitiation complex to the mRNA,
followed by migration
of the complex to the correct AUG initiation codon. The third stage brings the
60S ribosomal subunit
to the 40S subunit to generate an 80S ribosome at the inititation codon.
Regulation of translation
primarily involves the first and second stage in the initiation process (V.M.
Pain (1996) Eur. J.
Biochem. 236:747-771).
Several initiation factors, many of which contain multiple subunits, are
involved in bringing
an initiator tRNA and 40S ribosomal subunit together. One eukaryotic
initiation factor (EIF) EIFSA
is an 18-kD protein containing the unique amino acid residue, hypusine (N
epsilon-(4-amino-2-
hydroxybutyl)lysine) (Rinaudo, M, et al. (1993) Gene 137:303-307). eIF2, a
guanine nucleotide
binding protein, recruits the initiator tRNA to the 40S ribosomal subunit.
Only when eIF2 is bound to
GTP does it associate with the initiator tRNA. elF2B, a guanine nucleotide
exchange protein, is
responsible for converting eIF2 from the GDP-bound inactive form to the GTP-
bound active form.
Two other factors, eIFlA and eIF3 bind and stabilize the 40S subunit by
interacting with 18S
ribosomal RNA and specific ribosomal structural proteins. eIF3 is also
involved in association of the
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40S ribosomal subunit with mRNA. The Met-tRNA f, eIFlA, elF3, and 40S
ribosomal subunit
together make up the 43S preinitiation complex (Pain, s_ upra).
Additional factors are required for binding of the 43S preinitiation complex
to an mRNA
molecule, and the process is regulated at several levels. eIF4F is a complex
consisting of three
proteins: eIF4E, eIF4A, and elF4G. eIF4E recognizes and binds to the mRNA 5'-
terminal m'GTP
cap, elF4A is a bidirectional RNA-dependent helicase, and eIF4G is a
scaffolding polypeptide, elF4G
has three binding domains. The N-terminal third of eIF4G interacts with eIF4E,
the central third
interacts with elF4A, and the C-terminal third interacts with eIF3 bound to
the 43S preinitiation
complex. Thus, elF4G acts as a bridge between the 40S ribosomal subunit and
the mRNA (M.W.
Hentze (1997) Science 275:500-501).
The ability of eIF4F to initiate binding of the 43S preinitiation complex is
regulated by
structural features of the mRNA. The mRNA molecule has an untranslated region
(UTR) between the
5' cap and the AUG start codon. In some mRNAs this region forms secondary
structures that impede
binding of the 43S preinitiation complex. The helicase activity of eIF4A is
thought to function in
removing this secondary structure to facilitate binding of the 43S
preinitiation complex (Pain, s_upra).
Translation Elongation
Elongation is the process whereby additional amino acids are joined to the
initiator
methionine to form the complete polypeptide chain. The elongation factors EF1
a, EF1 (3 y, and EF2
are involved in elongating the polypeptide chain following initiation. EFl a
is a GTP-binding protein.
In EF1 a's GTP-bound form, it brings an aminoacyl-tRNA to the ribosome's A
site. The amino acid
attached to the newly arrived aminoacyl-tRNA forms a peptide bond with the
initiatior methionine.
The GTP on EF1 a is hydrolyzed to GDP, and EF1 a-GDP dissociates from the
ribosome. EFl (3 y
binds EF1 a -GDP and induces the dissociation of GDP from EF1 a, allowing EF1
a to bind GTP and a
new cycle to begin.
As subsequent aminoacyl-tRNAs are brought to the ribosome, EF-G, another GTP-
binding
protein, catalyzes the translocation of tRNAs from the A site to the P site
and finally to the E site of
the ribosome. This allows the processivity of translation.
Translation Termination
The release factor eRF carries out termination of translation. eRF recognizes
stop codons in
the mRNA, leading to the release of the polypeptide chain from the ribosome.
The discovery of new RNA metabolism 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 nervous system, autoimmune/inflammatory, cell proliferative, and
developmental disorders,
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and in the assessment of the effects of exogenous compounds on the expression
of nucleic acid and
amino acid sequences of RNA metabolism proteins.
nervous system disorders, autoimmunelinflammatory disorders, and cell
proliferative disorders
including cancer
SUMMARY OF THE INVENTION
The invention features purified polypeptides, RNA metabolism proteins,
referred to collectively
as "RMEP" and individually as "RMEP-1," "RMEP-2," "RMEP-3," "RMEP-4," "RMEP-
5," "RMEP-
6," "RMEP-7," "RMEP-8," "RMEP-9," "RMEP-10," "RMEP-11," "RMEP-12," "RMEP-13,"
"RMEP-14," "RMEP-15," "RMEP-16," "RMEP-17," "RMEP-18," "RMEP-19," "RMEP-20,"
"RMEP-21," "RMEP-22," "RMEP-23," "RMEP-24," "RMEP-25," "RMEP-26," "RMEP-27,"
"RMEP-28," "RMEP-29," "RMEP-30," "RMEP-31," "RMEP-32," "RMEP-33," "RMEP-34,"
"RMEP-35," "RMEP-36," "RMEP-37," "RMEP-38," "RMEP-39," "RMEP-40," "RMEP-41,"
"RMEP-42," "RMEP-43," "RMEP-44," "RMEP-45," "RMEP-46," and "RMEP-47." In one
aspect,
the invention provides an isolated polypeptide selected from the group
consisting of a) a polypeptide
comprising an amino acid sequence selected from the group consisting of SEQ ID
N0:1-47, b) a
naturally occurring polypeptide comprising an amino acid sequence at least 90%
identical to an amino
acid sequence selected from the group consisting of SEQ ID NO:1-47, c) a
biologically active fragment
of a polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:1-47,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-47. In one alternative, the invention provides
an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO:1-47,
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 N0:1-47, b) a naturally occurring polypeptide comprising
an amino acid sequence
at least 90% identical to an amino acid sequence selected from the group
consisting of SEQ ID N0:1-
47, c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-47, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID NO:1-47. In
one alternative, the
polynucleotide encodes a polypeptide selected from the group consisting of SEQ
ID NO:1-47. In
another alternative, the polynucleotide is selected from the group consisting
of SEQ ID N0:48-94:
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group consisting
of a) a polypeptide comprising an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-47, b) a naturally occurring polypeptide comprising an amino acid
sequence at least 90% identical
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to an amino acid sequence selected from the group consisting of SEQ ID N0:1-
47, c) a biologically
active fragment of a polypeptide having an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-47, and d) an immunogenic fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ ID N0:1-47. 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 N0:1-47, b) a naturally occurring polypeptide comprising an amino acid
sequence at least 90%
identical to an amino acid sequence selected from the group consisting of SEQ
ID N0:1-47, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-47, and d) an immunogenic fragment of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ ID N0:1-47. 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 ID N0:1-47, b) a naturally occurring
polypeptide comprising
an amino acid sequence at least 90% identical to an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-47, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID N0:1-47, and d) an
immunogenic fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-47.
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 ID
N0:48-94, b) a naturally occurring polynucleotide comprising a polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:48-94, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). In one alternative,
the polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting of a)
a polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:48-94, b) a naturally occurring polynucleotide comprising a polynucleotide
sequence at least 90%
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identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:48-94, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) hybridizing the
sample with a probe comprising at least 20 contiguous nucleotides comprising a
sequence
complementary to said target polynucleotide in the sample, and which probe
specifically hybridizes to
said target polynucleotide, under conditions whereby a hybridization complex
is formed between said
probe and said target polynucleotide or fragments thereof, and b) detecting
the presence or absence of
said hybridization complex, and optionally, if present, the amount thereof. In
one alternative, the probe
comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample, said
target polynucleotide having a sequence of a polynucleotide selected from the
group consisting of a) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:48-94, b) a naturally occurring polynucleotide comprising a polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:48-94, 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 polymerise chain reaction
amplification, and b)
detecting the presence or absence of said amplified target polynucleotide ox
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:l-47, b) a naturally occurring polypeptide
comprising an-amino
acid sequence at least 90% identical to an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-47, c) a biologically active fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-47, and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-47, and
a pharmaceutically acceptable excipient Tn one embodiment, the composition
comprises an amino acid
sequence selected from the group consisting of SEQ ID NO:1-47. The invention
additionally provides a
method of treating a disease or condition associated with decreased expression
of functional RMEP,
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 N0:1-47, b) a naturally
occurring polypeptide
comprising an amino acid sequence at least 90% identical to an amino acid
sequence selected from the
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group consisting of SEQ ID N0:1-47, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID N0:1-47, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ ID
N0:1-47. 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 RMEP, comprising
administering to a patient in
need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as an
antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID N0:1-47, b) a
naturally occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID N0:1-47, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-47, and
d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-47. 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
RMEP, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID N0:1-47, b) a naturally
occurring polypeptide
cmoprising an amino acid sequence at least 90% identical to an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-47, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID NO:I-47, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ ID
N0:1-47. The method comprises a) combining the polypeptide with at least one
test compound under
suitable conditions, and b) detecting binding of the polypeptide to the test
compound, thereby
identifying a compound that specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
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acid sequence selected from the group consisting of SEQ ID NO:1-47, b) a
naturally occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID N0:1-47, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-47, and
d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-47. The method comprises a) combining the
polypeptide with at least one
test compound under conditions permissive for the activity of the polypeptide,
b) assessing the activity
of the polypeptide in the presence of the test compound, and c) comparing the
activity of the
polypeptide in the presence of the test compound with the activity of the
polypeptide in the absence of
the test compound, wherein a change in the activity of the polypeptide in the
presence of the test
compound is indicative of a compound that modulates the activity of the
polypeptide.
The invention further provides a method for screening a compound for
effectiveness in altering
expression of a target polynucleotide, wherein said target polynucleotide
comprises a sequence
selected from the group consisting of SEQ ID N0:48-94, the method comprising
a) exposing a sample
comprising the target polynucleotide to a compound, and b) detecting altered
expression of the target
polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound; b)
hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:48-94, ii) a
naturally occurring polynucleotide comprising a polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:48-94,
iii) a
polynucleotide having a sequence complementary to i), iv) a polynucleotide
complementary to the
polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization
occurs under conditions
whereby a specific hybridization complex is formed between said probe and a
target polynucleotide in
the biological sample, said target polynucleotide selected from the group
consisting of i) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:48-94, ii) a naturally occurring polynucleotide comprising a polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:48-94, 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
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biological sample, wherein a difference in the amount of hybridization complex
in the treated
biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability score for the match
between each
polypeptide and its GenBank homolog is also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including predicted
motifs and domains, along with the methods, algorithms, and searchable
databases used for analysis of
the polypeptides.
Table 4 lists the cDNA fragments which were used to assemble polynucleotide
sequences of the
invention, along with selected fragments of the polynucleotide sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will be
limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same meanings
as commonly understood by one of ordinary skill in the art to which this
invention belongs. Although
any machines, materials, and methods similar or equivalent to those described
herein can be used to
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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
eonnection 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
"RMEP" refers to the amino acid sequences of substantially purified RMEP
obtained from any
species, particularly a mammalian species, including bovine, ovine, porcine,
marine, equine, and human,
and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
RMEP. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of RMEP either by
directly interacting with
RMEP or by acting on components of the biological pathway in which RMEP
participates.
An "allelic variant" is an alternative form of the gene encoding RMEP. 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 RMEP include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as RMEP or a
polypeptide with at least one functional characteristic of RMEP. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding RMEP, and improper or unexpected hybridization to
allelic variants, with a
locus other than the normal chromosomal locus for the polynucleotide sequence
encoding RMEP. 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 RMEP. 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 RMEP is retained. For example, negatively charged
amino acids may
include aspartic acid and glutamic acid, and positively charged amino acids
may include lysine and
arginine. Amino acids with uncharged polar side chains having similar
hydrophilicity values may
include: asparagine and glutamine; and serine and threonine. Amino acids with
uncharged side chains
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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 polymerise chain reaction (PCR)
technologies well known
in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity of
RMEP. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of RMEP either by
directly interacting with RMEP or by acting on components of the biological
pathway in which RMEP
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 RMEP polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used to,
immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the
translation of RNA, or
synthesized chemically, and can be conjugated to a carrier protein if desired.
Commonly used carriers
that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and keyhole limpet
hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that makes
contact with a particular antibody. When a protein or a fragment of a protein
is used to immunize a
host animal, numerous regions of the protein may induce the production of
antibodies which bind
specifically to antigenic determinants (particular regions or three-
dimensional structures on the protein).
An antigenic determinant may compete with the intact antigen (i.e., the
immunogen used to elicit the
immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified sugar
groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having modified
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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 RMEP, or of
any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid sequences
that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising a
given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding RMEP or fragments of
RMEP may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be associated
with a stabilizing agent such as a carbohydrate. In hybridizations, the probe
may be deployed in an
aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium
dodecyl sulfate; SDS), and other
components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(Applied Biosystems,
Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which
has been assembled from
one or more overlapping cDNA, EST, or genomic DNA fragments using a computer
program for
fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison
WI) or Phrap
(University of Washington, Seattle WA). Some sequences have been both extended
and assembled to
produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to Ieast
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
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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 Tle, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of the
side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide. Chemical
modifications of a polynucleotide can include, for example, replacement of
hydrogen by an alkyl, acyl,
hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide
which retains at least one
biological or immunological function of the natural molecule. A derivative
polypeptide is one modified
by glycosylation, pegylation, or any similar process that retains at least one
biological or immunological
function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide 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.
A "fragment" is a unique portion of RMEP or the polynucleotide encoding RMEP
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
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to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10, 15,
16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25 % or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:48-94 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:48-94, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:48-94 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:48-94 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:48-94 and the region of SEQ ID N0:48-94 to which the fragment
corresponds are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID N0:1-47 is encoded by a fragment of SEQ ID N0:48-94. A
fragment
of SEQ ID NO: l-47 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:1-47. For example, a fragment of SEQ ID NO:1-47 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID N0:1-47.
The precise length of
a fragment of SEQ ID N0:1-47 and the region of SEQ ID N0:1-47 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
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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
Iiiggins, D.G.
and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992)
CABIOS 8:189-191.
For pairwise alignments of polynucleotide sequences, the default parameters
are set as follows:
Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted"
residue weight table is
selected as the default. Percent identity is reported by CLUSTAL V as the
"percent similarity" between
aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms is
provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment Search
Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which
is available from several
sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be aceessed 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
Pefialty for misrraatch: -2
Opera Gap: S arid Extension Gap: 2 penalties
Gap x drop-off. 50
Expect: 10
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example, as
defined by a particular SEQ ID number, or may be measured over a shorter
length, for example, over
the length of a fragment taken from a larger, defined sequence, for instance,
a fragment of at least 20, at
least 30, at least 40, at least 50, at least 70, at least 100, or at least 200
contiguous nucleotides. Such
lengths are exemplary only, and it is understood that any fragment length
supported by the sequences
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
shown herein, in the tables, figures, or Sequence Listing, may be used to
describe a length over which
percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes in
a nucleic acid sequence can be made using this degeneracy to produce multiple
nucleic acid sequences
that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default parameters
of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e
sequence alignment
program (described and referenced above). For pairwise alignments of
polypeptide sequences using
CLUSTAL V, the default parameters are set as follows: I~tuple=1, gap
penalty=3, window=5, and
"diagonals saved"=S. The PAM250 matrix is selected as the default residue
weight table. As with
polynucleotide alignments, the percent identity is reported by CLUSTAL V as
the "percent similarity"
between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12
(April-21-2000) with blastp set at default parameters. Such default parameters
may be, for example:
Matrix: BLOSUM62
Open Gap: 11 arzd Extension Gap: 1 pefaalties
Gap x drop-off.' SO
Expect: 10
Word Size: 3
Filter: ofa
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 S0,
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
21
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WO 01/83524 PCT/USO1/13862
describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely resembles
a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the stringency
of the hybridization process, with more stringent conditions allowing less non-
specific binding, i.e.,
binding between pairs of nucleic acid strands that are not perfectly matched.
Permissive conditions for
1 S 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 ~ ~ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about S°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 SO% of the target
sequence hybridizes to a perfectly matched probe. An equation for calculating
Tm and conditions for
nucleic acid hybridization are well known and can be found in Sambrook, J. et
al. (1989) Molecular
Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Press,
Plainview NY; specifically
see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1 % SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used. SSC concentration may
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
sheared and denatured salmon sperm DNA at about 100-200 ~g/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
22
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WO 01/83524 PCT/USO1/13862
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 RMEP
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
RMEP 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 RMEP. For example,
modulation may
cause an increase or a decrease in protein activity, binding characteristics,
or any other biological,
functional, or immunological properties of RMEP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
23
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WO 01/83524 PCT/USO1/13862
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an RMEP 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 RMEP.
"Probe" refers to nucleic acid sequences encoding RMEP, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule. Typical
labels include radioactive isotopes, ligands, chemiluminescent agents, and
enzymes. "Primers" are short
nucleic acids, usually DNA oligonucleotides, which may be annealed to a target
polynucleotide by
complementary base-pairing. The primer may then be extended along the target
DNA strand by a DNA
polymerase enzyme. Primer pairs can be used for amplification (and
identification) of a nucleic acid
sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers may
be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
24
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas South
West Medical Center, Dallas TX) is capable of choosing specific primers from
megabase sequences and
is thus useful fox designing primers on a genome-wide scope. The Primer3
primer selection program
(available to the public from the Whitehead InstituteJMIT 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 pxobes to identify fully or
partially complementary
polynucleotides in a sample of nucleic acids. Methods of oligonucleotide
selection are not limited to
those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques such
as those described in Sambrook, supra. The term recombinant includes nucleic
acids that have been
altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for
example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions (UTRs).
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Regulatory elements interact with host or viral proteins which control
transcription, translation, or RNA
stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose instead
of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing RMEP,
nucleic acids encoding RMEP, or fragments thereof may comprise a bodily fluid;
an extract from a cell,
chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA,
RNA, or cDNA, in
solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recognized by the
binding molecule. For
example, if an antibody is specific for epitope "A," the presence of a
polypeptide comprising the epitope
A, or the presence of free unlabeled A, in a reaction containing free labeled
A and the antibody will
reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides by
different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" 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
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WO 01/83524 PCT/USO1/13862
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 paxt 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. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention into
such organisms are widely known and provided in references such as Sambrook et
al. (1989), su ra,
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having at
least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of the
nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
set at default parameters. Such a pair of nucleic acids may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at
least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% or greater sequence
identity over a certain defined length. A variant may be described as, for
example, an "allelic" (as
defined above), "splice," "species," or "polymorphic" variant. A splice
variant may have significant
identity to a reference molecule, but will generally have a greater or lesser
number of polynucleotides
due to alternative splicing of exons during mRNA processing. The corresponding
polypeptide may
possess additional functional domains or lack domains that are present in the
reference molecule.
Species variants axe 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
27
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WO 01/83524 PCT/USO1/13862
species. Polymorphic variants also may encompass "single nucleotide
polymorphisms" (SNPs) in which
the polynucleotide sequence varies by one nucleotide base. The presence of
SNPs may be indicative of,
for example, a certain population, a disease state, or a propensity for a
disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having at
least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of the
polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
set at default parameters. Such a pair of polypeptides may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, at least 91 %, at least 92%, at
least 93%, at least 94%, at
least 95 %, at least 96%, at least 97%, at least 98%, or at least 99% or
greater sequence identity over a
certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human RNA metabolism proteins
(RMEP), the
polynucleotides encoding RMEP, and the use of these compositions for the
diagnosis, treatment, or
prevention of nervous system, autoimmunelinf7ammatory, cell proliferative, and
developmental
disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is denoted by
both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and
an Incyte polypeptide
sequence number (Incyte Polypeptide ID) as shown. Each polynucleotide sequence
is denoted by both a
polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and
an Incyte
polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the
invention. Column 3
shows the GenBank identification number (Genbank ID NO:) of the nearest
GenBank homolog. Column
4 shows the probability score for the match between each polypeptide and its
GenBank homolog.
Colwnn 5 shows the annotation of the GenBank homolog along with relevant
citations where applicable,
all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns l and 2
show the polypeptide sequence identification number (SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention. Column 3
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CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
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 Wn.
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structurelfanction 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 RNA metabolism
proteins. SEQ ID N0:46 is
29% identical to Glu-tRNAG'~ amidotransferase, subunit A, of Neisseria menin
i~ t~ (GenBank
ID g7226601) as determined by the Basic Local Alignment Search Tool (BLAST,
see Table 2). The
BLAST probability score is 1.3e-37, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ ID N0:46 also contains amidase
signature sequences
as determined by searching for statistically significant matches in the hidden
Markov model (HMM)-
based PFAM database of conserved protein family domains (see Table 3). Data
from BLIMPS and
PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:46
contains amidase
signature sequences, features of polypeptides involved in transamination
reactions. These data provide
evidence that SEQ ID N0:46 is related to the Glu-tRNAG'n amidotransferases
found in prokaryotes
and some cellular organelles but, until the instant invention, not in humans.
SEQ ID N0:47 is 97%
identical to the 60S acidic ribosomal protein of Zea mays (GenBank ID g790508)
as determined by the
Basic Local Alignment Search Tool (BLAST, see Table 2). The BLAST probability
score is 5.4e-51.
SEQ ID N0:47 also contains a 60S acidic ribosomal protein 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 analyses
provide further
corroborative evidence that SEQ ID N0:47 is a phosphorylated (hence likely to
be acidic) ribosomal
protein. SEQ ID NO:1-45 were analyzed and annotated in a similar manner. The
algorithms and
parameters for the analysis of SEQ ID NO;1-47 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Columns 1 and 2 list the
polynucleotide sequence
identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte
polynucleotide
consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide
of the invention.
Column 3 shows the length of each polynucleotide sequence in basepairs. Column
4 lists fragments of
the polynucleotide sequences which are useful, for example, in hybridization
or amplification
technologies that identify SEQ ID N0:48-94 or that distinguish between SEQ ID
N0:48-94 and
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WO 01/83524 PCT/USO1/13862
related polynucleotide sequences. Column 5 shows identification numbers
corresponding to cDNA
sequences, coding sequences (exons) predicted from genomic DNA, and/or
sequence assemblages
comprised of both cDNA and genomic DNA. These sequences were used to assemble
the full length
polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the
nucleotide start (5')
and stop (3') positions of the cDNA sequences in column 5 relative to their
respective full length
sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
642017H1 is the
identification number of an Incyte cDNA sequence, and BRSTNOT03 is the cDNA
library from which
it is derived. Incyte cDNAs for which cDNA libraries are not indicated were
derived from pooled
cDNA libraries (e.g., 70822015V1). Alternatively, the identification numbers
in column 5 may refer to
GenBank cDNAs or ESTs (e.g., g1136841) which contributed to the assembly of
the full length
polynucleotide sequences. Alternatively, the identification numbers in column
5 may refer to coding
regions predicted by Genscan analysis of genomic DNA. The Genscan-predicted
coding sequences may
have been edited prior to assembly. (See Example IV.) Alternatively, the
identification numbers in
column 5 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together by an
"exon stitching" algorithm. (See Example V.) Alternatively, the identification
numbers in column 5
may refer to assemblages of both cDNA and Genscan-predicted exons brought
together by an "exon-
stretching" algorithm. (See Example V.) In some cases, Incyte cDNA coverage
redundant with the
sequence coverage shown in column 5 was obtained to confirm the final
consensus polynucleotide
sequence, but the relevant Incyte cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide sequences
which were assembled using Incyte cDNA sequences. The representative cDNA
library is the Incyte
cDNA library which is most frequently represented by the Incyte cDNA sequences
which were used to
assemble and confirm the above polynucleotide sequences. The tissues and
vectors which were used to
construct the cDNA libraries shown in Table 5 are described in Table 6.
The invention also encompasses RMEP variants. A preferred RMEP 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 RMEP amino acid sequence, and which contains at least one
functional or structural
characteristic of RMEP.
The invention also encompasses polynucleotides which encode RMEP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected from
the group consisting of SEQ ID N0:48-94, which encodes RMEP. The
polynucleotide sequences of
SEQ ID N0:48-94, as presented in the Sequence Listing, embrace the equivalent
RNA sequences,
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
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
RMEP. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at least
about 85%, or even at least about 95% polynucleotide sequence identity to the
polynucleotide sequence
encoding RMEP. A particular aspect of the invention encompasses a variant of a
polynucleotide
sequence comprising a sequence selected from the group consisting of SEQ ID
N0:48-94 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 N0:48-94.
Any one of the polynucleotide variants described above can encode an amino
acid sequence which
contains at least one functional or sti~zctural characteristic of RMEP.
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 RMEP, 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
RMEP, and all such variations are to be considered as being specifically
disclosed.
Although nucleotide sequences which encode RMEP and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring RMEP under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding RMEP or its
derivatives possessing a substantially different codon usage, e.g., inclusion
of non-naturally occurring
codons. Codons may be selected to increase the rate at which expression of the
peptide occurs in a
particular prokaryotic or eukaxyotic host in accordance with the frequency
with which particular codons
are utilized by the host. Other reasons for substantially altering the
nucleotide sequence encoding
RMEP 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 RMEP
and
RMEP derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the synthetic
sequence may be inserted into any of the many available expression vectors and
cell systems using
reagents well known in the art. Moreover, synthetic chemistry may be used to
introduce mutations into
a sequence encoding RMEP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of hybridizing
31
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WO 01/83524 PCT/USO1/13862
to the claimed polynucleotide sequences, and, in particular, to those shown in
SEQ ID N0:48-94 and
fragments thereof under various conditions of stringency. (See, e.g., Wahl,
G.M. and S.L. Berger
(1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol.
152:507-511.)
Hybridization conditions, including annealing and wash conditions, are
described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of the
embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment of DNA
polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied Biosystems),
thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or
combinations of
polymerases and proofreading exonucleases such as those found in the ELONGASE
amplification
system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation
is automated with
machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV),
PTC200 thermal
cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler
(Applied Biosystems).
Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing
system (Applied
Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics,
Sunnyvale CA),
or other systems known in the art. The resulting sequences are analyzed using
a variety of algorithms
which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short
Protocols in Molecular
Biolo~y, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995)
Molecular Biology and
Biotechnolo~y, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding RMEP may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested pximers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another
method, inverse PCR, uses primers that extend in divergent directions to
amplify unknown sequence
from a circularized template. The template is derived from restriction
fragments comprising a known
genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988)
Nucleic Acids Res.
16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent to
known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al.
(1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction
enzyme digestions and
ligations may be used to insert an engineered double-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences are
known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res.
19:3055-3060). Additionally,
one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo
Alto CA) to
walk genomic DNA. This procedure avoids the need to screen libraries and is
useful in finding
32
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WO 01/83524 PCT/USO1/13862
intron/exon junctions. For all PCR-based methods, primers may be designed
using commercially
available software, such as OLIGO 4.06 primer analysis software (National
Biosciences, Plymouth
MN) or another appropriate program, to be about 22 to 30 nucleotides in
length, to have a GC content
of about 50% or more, and to anneal to the template at temperatures of about
68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T) library
does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence into 5'
non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze the
size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode RMEP may be cloned in recombinant DNA molecules that direct expression
of RMEP, 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 RMEP.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter RMEP-encoding sequences for a variety of
purposes including, but not
limited to, modification of the cloning, processing, and/or expression of the
gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction sites,
alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
33
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
improve the biological properties of RMEP, 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 RMEP 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,
RMEP itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide
synthesis can be performed using various solution-phase or solid-phase
techniques. (See, e.g., Creighton,
T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York
NY, pp. 55-60; and
Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be
achieved using the ABI
431A peptide synthesizer (Applied Biosystems). Additionally, the amino acid
sequence of RMEP, 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, su ra, pp. 28-53.)
In order to express a biologically active RMEP, the nucleotide sequences
encoding RMEP 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 RMEP. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
RMEP. Such signals
34
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding RMEP 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 RMEP and appropriate transcriptional and
translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. (1995)
Current Protocols in Molecular Biolo~v, John Wiley & Sons, New York NY, ch. 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding RMEP. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw
Hill, New
York NY, pp. 191-196; Logan, J, and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and
Harrington, J,J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di Nicola,
M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc.
Natl. Acad. Sci. USA
90(13):6340-6344; 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
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
upon the use intended for polynucleotide sequences encoding RMEP. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding RMEP can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1 plasmid
(Life Technologies). Ligation of sequences encoding RMEP 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 RMEP are needed, e.g. for the
production of antibodies,
vectors which direct high level expression of RMEP 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 RMEP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomvces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et
al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of RMEP. Transcription of
sequences encoding
RMEP may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV
used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO
J. 6:307-311).
Alternatively, plant promoters such as the small subunit of RUBISCO or heat
shock promoters may be
used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Brogue, R. et
al. (1984) Science
224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-
105.) These constructs can
be introduced into plant cells by direct DNA transformation or pathogen-
mediated transfection. (See,
e.g., The McGraw Hill Yearbook of Science and Technolo~v (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 RMEP
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 RMEP 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-
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CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
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 anuno 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
RMEP in cell lines is preferred. For example, sequences encoding RMEP can be
transformed into cell
lines using expression vectors which may contain viral origins of replication
and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include,
but are not limited to, the herpes simplex virus thymidine kinase and adenine
phosphoribosyltransferase
genes, for use in tk and apr cells, respectively. (See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232;
Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or
herbicide resistance can be
used as the basis for selection. For example, dlafr 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 a1. (1980)
Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J.
Mol. Biol. 150:1-14.)
Additional selectable genes have been described, e.g., trpB and hisD, which
alter cellular requirements
for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins
(GFP; Clontech),13
glucuronidase and its substrate 13-glucuronide, or luciferase and its
substrate luciferin may be used.
These markers can be used not only to identify transformants, but also to
quantify the amount of
transient or stable protein expression attributable to a specific vector
system. (See, e.g., Rhodes, C.A.
(1995) Methods Mol. Biol. 55:121-131.)
Although the presencelabsence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
conf'Irmed. For example, if the
sequence encoding RMEP is inserted within a marker gene sequence, transformed
cells containing
sequences encoding RMEP can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding RMEP under the
control of a single
37
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
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 RMEP
and that express
RMEP may be identified by a variety of procedures known to those of skill in
the art. These procedures
include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR
amplification, and
protein bioassay or immunoassay techniques which include membrane, solution,
or chip based
technologies for the detection and/or quantification of nucleic acid or
protein sequences.
Immunological methods for detecting and measuring the expression of RMEP 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 RMEP is preferred, but a
competitive binding assay may be
employed. These and other assays are well known in the art. (See, e.g.,
Hampton, R. et al. (1990)
Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. IV;
Coligan, J.E, et al.
(1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-
Interscience, New York
NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled hybridization
or PCR probes for detecting sequences related to polynucleotides encoding RMEP
include oligolabeling,
nick translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the
sequences encoding RMEP, or any fragments thereof, may be cloned into a vector
for the production of
an mRNA probe. Such vectors are known in the art, are commercially available,
and may be used to
synthesize RNA probes in vitro by addition of an appropriate RNA polymerase
such as T7, T3, or SP6
and labeled nucleotides. These procedures may be conducted using a variety of
commercially available
kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison
WI), and US
Biochemical. Suitable reporter molecules or labels which may be used for ease
of detection include
radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents,
as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding RMEP 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 RMEP may be designed to contain signal sequences
which direct secretion
of RMEP through a prokaryotic or eukaryotic cell membrane.
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CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
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, andlor
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 RMEP may be ligated to a heterologous sequence resulting in
translation of a fusion
protein in any of the aforementioned host systems. For example, a chimeric
RMEP protein containing a
heterologous moiety that can be recognized by a commercially available
antibody may facilitate the
screening of peptide libraries for inhibitors of RMEP 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-nayc, 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 RMEP encoding sequence and the heterologous protein
sequence, so that RMEP
may be cleaved away from the heterologous moiety following purification.
Methods for fusion protein
expression and purification are discussed in Ausubel (1995, supra, ch. 10). A
variety of commercially
available kits may also be used to facilitate expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled RMEP 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.
RMEP of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to RMEP. At least one and up to a plurality of test
compounds may be screened
for specific binding to RMEP. Examples of test compounds include antibodies,
oligonucleotides,
39
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
RMEP, e. g., a ligand or fragment thereof, a natural substrate, a structural
or functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which RMEP
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 RMEP,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing RMEP or cell membrane fractions which contain RMEP are
then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either RMEP 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
RMEP, either in solution
or affixed to a solid support, and detecting the binding of RMEP to the
compound. Alternatively, the
assay may detect or measure binding of a test compound in the presence of a
labeled competitor.
Additionally, the assay may be carried out using cell-free preparations,
chemical libraries, or natural
product mixtures, and the test compounds) may be free in solution or affixed
to a solid support.
RMEP of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of RMEP. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for RMEP
activity, wherein RMEP is combined with at least one test compound, and the
activity of RMEP in the
presence of a test compound is compared with the activity of RMEP in the
absence of the test
compound. A change in the activity of RMEP in the presence of the test
compound is indicative of a
compound that modulates the activity of RMEP. Alternatively, a test compound
is combined with an in
vitro or cell-free system comprising RMEP under conditions suitable for RMEP
activity, and the assay
is performed. In either of these assays, a test compound which modulates the
activity of RMEP may do
so indirectly and need not come in direct contact with the test compound. At
least one and up to a
plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding RMEP or their mammalian
homologs may be
"knocked out" in an animal model system using homologous recombination in
embryonic stem (ES)
cells. Such techniques are well known in the art and are useful for the
generation of animal models of
human disease. (See, e.g., U.S. Patent Number 5,175,383 and U.S. Patent Number
5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse embryo
CA 02407435 2002-10-24
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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
S system to knockout a gene of interest in a tissue- or developmental stage-
specific manner (Marth, J.D.
(1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids
Res. 25:4323-4330).
Transformed ES cells are identified and microinjected into mouse cell
blastocysts such as those from the
C57BL16 mouse strain. The blastocysts are surgically transferred to
pseudopregnant dams, and the
resulting chimeric progeny are genotyped and bred to produce heterozygous or
homozygous strains.
Transgenic animals thus generated may be tested with potential therapeutic or
toxic agents.
Polynucleotides encoding RMEP 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 RMEP can also be used to create "knockin" humanized
animals (pigs)
or transgenic animals (mice or rats) to model human disease. With knockin
technology, a region of a
polynucleotide encoding RMEP 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 RMEP, e.g., by secreting RMEP 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 RMEP and RNA metabolism proteins. In addition, the expression of
RMEP is closely
associated with diseased, proliferative, tumorous, and nervous tissues,
adrenal tissue, brain tumor
tissue, fetal colon tissue, adult colon tissue, prostate epithelial tissue,
lymph node cancer tissue,
ovarian tissue, pancreatic tissue, and fetal spleen tissue, as well as with
diseases of the lung, and
physiological conditions that result in anoxia. Therefore, RMEP appears to
play a role in nervous
system, autoimmune/inflammatory, cell proliferative, and developmental
disorders, as well as
neoplasms involving lung-specific tissues. In the treatment of disorders
associated with increased
RMEP expression or activity, it is desirable to decrease the expression or
activity of RMEP. In the
treatment of disorders associated with decreased RMEP expression or activity,
it is desirable to
41
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increase the expression or activity of RMEP.
Therefore, in one embodiment, RMEP 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 RMEP. Examples of such disorders include, but are not limited to, a nervous
system disorder such
as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms,
Alzheimer's disease, Pick's
disease, Huntington's disease, dementia, Parkinson's disease and other
extrapyramidal disorders,
amyotrophic lateral sclerosis and other motor neuron disorders, progressive
neural muscular atrophy,
retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other
demyelinating diseases, bacterial
and viral meningitis, brain abscess, subdural empyema, epidural abscess,
suppurative intracranial
thrombophlebitis, myelitis and radiculitis, viral central nervous system
disease; prion diseases including
kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome;
fatal familial insomnia,
nutritional and metabolic diseases of the nervous system, neurofibromatosis,
tuberous sclerosis,
cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental
retardation and other
developmental disorder of the central nervous system, cerebral palsy, a
neuroskeletal disorder, an
autonomic nervous system disorder, a cranial nerve disorder, a spinal cord
disease, muscular dystrophy
and other neuromuscular disorder, a peripheral nervous system disorder,
dermatomyositis and
polymyositis; inherited, metabolic, endocrine, and toxic myopathy; myasthenia
gravis, periodic
paralysis; a mental disorder including mood, anxiety, and schizophrenic
disorders; seasonal affective
disorder (SAD); akathesia, amnesia, catatonia, diabetic neuropathy, tardive
dyskinesia, dystonias,
paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; an
autoimmune/inflammatory
disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease,
adult respiratory
distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
polyenodocrinopathy-
candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact
dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
episodic lymphopenia
with 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 cell
proliferative disorder such as
actinic keratosis, arteriosclerosis, athexosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue
disease (MCTD), myelofibrosis, .paroxysmal nocturnal hemoglobinuria,
polycythemia vera, psoriasis,
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WO 01/83524 PCT/USO1/13862
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, and a developmental disorder such as renal
tubular acidosis, anemia,
Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular
dystrophy, epilepsy,
gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary
abnormalities, and mental
retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary
mucoepithelial dysplasia,
hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth
disease and
neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as
Syndenham's chorea and
cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital
glaucoma, cataract, and
sensorineural hearing loss.
In another embodiment, a vector capable of expressing RMEP 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 RMEP including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
RMEP 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 RMEP including,
but not limited to, those
provided above.
In still another embodiment, an agonist which modulates the activity of RMEP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or activity
of RMEP including, but not limited to, those listed above.
In a further embodiment, an antagonist of RMEP may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of RMEP.
Examples of such
disorders include, but are not limited to, those nervous system,
autoimmunelintlammatory, cell
proliferatave, and developmental described above. In one aspect, an antibody
which specitrcally binds
RMEP 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 RMEP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding RMEP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of RMEP including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made by
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WO 01/83524 PCT/USO1/13862
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 RMEP may be produced using methods which are generally known
in the art.
In particular, purified RMEP may be used to produce antibodies or to screen
libraries of pharmaceutical
agents to identify those which specifically bind RMEP. Antibodies to RMEP may
also be generated
using methods that are well known in the art. Such antibodies may include, but
are not limited to,
polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments,
and fragments produced
by a Fab expression library. Neutralizing antibodies (i.e., those which
inhibit dimer formation) are
generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans, and
others may be immunized by injection with RMEP 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 RMEP
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 RMEP 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 RMEP may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not limited
to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-
hybridoma technique.
(See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D, et al.
(1985) J. Immunol. Methods
81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030;
and Cole, S.P. et al.
(1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate 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
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CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
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 RMEP-
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 RMEP may also be
generated. For
example, such fragments include, but are not limited to, F(ab~2 fragments
produced by pepsin digestion
of the antibody molecule and Fab fragments generated by reducing the disulfide
bridges of the F(ab~2
fragments. Alternatively, Fab expression libraries may be constructed to allow
rapid and easy
identification of monoclonal Fab fragments with the desired specificity. (See,
e.g., Huse, W.D. et al.
(1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
RMEP and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two
non-interfering RMEP epitopes is generally used, but a competitive binding
assay may also be employed
(Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for RMEP. Affinity is
expressed as an association
constant, I~, which is defined as the molar concentration of RMEP-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The Ka determined
for a preparation of polyclonal antibodies, which are heterogeneous in their
affinities for multiple RMEP
epitopes, represents the average affinity, or avidity, of the antibodies for
RMEP. The I~ determined for
a preparation of monoclonal antibodies, which are monospecific fox a
particular RMEP epitope,
represents a true measure of affinity. High-affinity antibody preparations
with I~ ranging from about
I09 to 1012 Llmole are preferred for use in immunoassays in which the RMEP-
antibody complex must
withstand rigorous manipulations. Low-affinity antibody preparations with I~
ranging from about 106
to 10' L/mole are preferred for use in immunopurification and similar
procedures which ultimately
require dissociation of RMEP, preferably in active form, from the antibody
(Catty, D. (1988)
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; Liddell,
J.E. and A. Cryer
(1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York
NY).
The titer and avidity ofpolyclonal 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
antibodylml, preferably 5-10 mg
specific antibodylml, is generally employed in procedures requiring
precipitation of RMEP-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and
Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding RMEP, 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 RMEP.
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 RMEP.
(See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Cli. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morns, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding RMEP may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-linked
inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe
combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
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CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
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 aI. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D. (1988) Nature
335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93:11395-
11399), hepatitis B or C
virus (HBV, HCV); fungal parasites, such as Candida albicans and
Paracoccidioides brasiliensis; and
protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi). In
the case where a
genetic deficiency in RMEP expression or regulation causes disease, the
expression of RMEP 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 RMEP
are treated by constructing mammalian expression vectors encoding RMEP and
introducing these
vectors by mechanical means into RMEP-deficient cells. Mechanical transfer
technologies for use with
cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii) ballistic gold
particle delivery, (iii) liposome-mediated transfection, (iv) receptor-
mediated gene transfer, and (v) the
use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev.
Biochem. 62:191-217;
Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon (1998) Curr.
Opin. Biotechnol. 9:445-
450).
Expression vectors that may be effective for the expression of RMEP include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen,
Carlsbad CA),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON,
PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). RMEP 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 Blau, H.M. supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous gene
encoding RMEP from a normal individual.
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WO 01/83524 PCT/USO1/13862
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 RMEP expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding RMEP 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.5. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses a
method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ T-cells), and the
return of transduced cells to a patient are procedures well known to persons
skilled in the art of gene
therapy and have been well documented (Ranga, U. et aI. (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 RMEP to cells which have one or more genetic
abnormalities with respect to
the expression of RMEP. The construction and packaging of adenovirus-based
vectors are well known
to those with ordinary skill in the art. Replication defective adenovirus
vectors have proven to be
versatile for importing genes encoding immunoregulatory proteins into intact
islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
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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 RMEP to target cells which have one or more genetic
abnormalities with
respect to the expression of RMEP. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing RMEP to cells of the central nervous
system, for which HSV has a
tropism. The construction and packaging of herpes-based vectors are well known
to those with ordinary
skill in the art. A replication-competent herpes simplex virus (HSV) type 1-
based vector has been used
to deliver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp.
Eye Res. 169:385-395).
The construction of a HSV-1 virus vector has also been disclosed in detail in
U.S. Patent Number
5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which
is hereby incorporated
by reference. U.S. Patent Number 5,804,413 teaches the use of recombinant HSV
d92 which consists
of a genome containing at least one exogenous gene to be transferred to a cell
under the control of the
appropriate promoter for purposes including human gene therapy. Also taught by
this patent are the
construction and use of recombinant HSV strains deleted for ICP4, ICP27 and
ICP22. For HSV
vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et
al. (1994) Dev. Biol.
163:152-161, hereby incorporated by reference. The manipulation of cloned
herpesvirus sequences, the
generation of recombinant virus following the transfection of multiple
plasmids containing different
segments of the large herpesvirus genomes, the growth and propagation of
herpesvirus, and the infection
of cells with herpesvirus are techniques well known to those of ordinary skill
in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding RMEP to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based on
the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol. 9:464-
469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA, resulting
in the overproduction of capsid proteins relative to the viral proteins with
enzymatic activity (e.g.,
protease and polymerase). Similarly, inserting the coding sequence for RMEP
into the alphavirus
genome in place of the capsid-coding region results in the production of a
large number of RMEP-
coding RNAs and the synthesis of high levels of RMEP 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
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(Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of
alphaviruses will allow the
introduction of RMEP into a variety of cell types. The specific transduction
of a subset of cells in a
population may require the sorting of cells prior to transduction. The methods
of manipulating
infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and
performing alphavirus infections, are well known to those with ordinary skill
in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions -10
and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can
be achieved using triple helix base-pairing methodology. Triple helix pairing
is useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in
Huber, B.E. and B.I. Carr,
Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-
177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA by
preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding RMEP.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared by
any method known in the art for the synthesis of nucleic acid molecules. These
include techniques for
chemically synthesizing oligonucleotides such as solid phase phosphoramidite
chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA sequences
encoding RMEP. 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
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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 RMEP. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming
oligonucleotides,
transcription factors and other polypeptide transcriptional regulators, and
non-macromolecular
chemical entities which are capable of interacting with specific
polynucleotide sequences. Effective
compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
polynucleotide expression. Thus, in the treatment of disorders associated with
increased RMEP
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding RMEP may be therapeutically useful, and in the treament of disorders
associated with
decreased RMEP expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding RMEP 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 RMEP 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
RMEP 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 RMEP. 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
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can be carried out, for example, using a Schizosaccharom~ces 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 (Clarks, M.L, et al. (2000) Biochem.
Biophys. Res. Commun.
268:8-13). A particular embodiment of the present invention involves screening
a combinatorial
library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides,
peptide nucleic acids, and
modified oligonucleotides) for antisense activity against a specific
polynucleotide sequence (Bruise,
T.W. et al. (1997) U.S. Patent No. 5,686,242; Bntice, T.W. et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable for
use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells taken
from the patient and clonally propagated for autologous transplant back into
that same patient. Delivery
by transfection, by liposome injections, or by polycationic amino polymers may
be achieved using
methods which are well known in the art. (See, e.g., Goldman, C.K. et al.
(1997) Nat. Biotechnol.
15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of such
therapy, including, for example, mammals such as humans, dogs, cats, cows,
horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition which
generally comprises an active ingredient formulated with a pharmaceutically
acceptable excipient.
Excipients may include, for example, sugars, starches, ceIluloses, gums, and
proteins. Various
formulations are commonly known and are thoroughly discussed in the latest
edition of Remington's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of RMEP,
antibodies to RMEP, and mimetics, agonists, antagonists, or inhibitors of
RMEP.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the case
of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-acting
formulations is well-known in the art. In the case of macromolecules (e.g.
larger peptides and proteins),
recent developments in the field of pulmonary delivery via the alveolar region
of the lung have enabled
the practical delivery of drugs such as insulin to blood circulation (see,
e.g., Patton, J.S. et al., U.S.
Patent No. 5,997,848). Pulmonary delivery has the advantage of administration
without needle
injection, and obviates the need for potentially toxic penetration enhancers.
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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 RMEP or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, RMEP or a fragment thereof may be joined to a
short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, monkeys,
or pigs. An animal model may also be used to determine the appropriate
concentration range and route
of administration. Such information can then be used to determine useful doses
and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example RMEP
or fragments thereof, antibodies of RMEP, and agonists, antagonists or
inhibitors of RMEP, 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%o 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.
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Normal dosage amounts may vary from about 0.1 ~cg 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 RMEP may be used for
the diagnosis
of disorders characterized by expression of RMEP, or in assays to monitor
patients being treated with
RMEP or agonists, antagonists, or inhibitors of RMEP. Antibodies useful for
diagnostic purposes may
be prepared in the same manner as described above for therapeutics. Diagnostic
assays for RMEP
include methods which utilize the antibody and a label to detect RMEP in human
body fluids or in
extracts of cells or tissues. The antibodies may be used with or without
modification, and may be
labeled by covalent or non-covalent attachment of a reporter molecule. A wide
variety of reporter
molecules, several of which are described above, are known in the art and may
be used.
A variety of protocols for measuring RMEP, including ELISAs, RIAs, and FACS,
are known in
the art and provide a basis for diagnosing altered or abnormal levels of RMEP
expression. Normal or
standard values for RMEP expression are established by combining body fluids
or cell extracts taken
from normal mammalian subjects, for example, human subjects, with antibodies
to RMEP under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of RMEP
expressed in subject,
control, and disease samples from biopsied tissues are compared with the
standard values. Deviation
between standard and subject values establishes the parameters for diagnosing
disease.
In another embodiment of the invention, the polynucleotides encoding RMEP 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 RMEP may
be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
RMEP, and to monitor regulation of RMEP levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding RMEP or closely related
molecules may be used to
identify nucleic acid sequences which encode RMEP. 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 RMEP, allelic
variants, or related
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sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the RMEP encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:48-94 or from
genomic sequences including promoters, enhancers, and introns of the RMEP
gene.
Means for producing specific hybridization probes for DNAs encoding RMEP
include the
cloning of polynucleotide sequences encoding RMEP or RMEP derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a variety
of reporter groups, for example, by radionuclides such as 32P or 355, or by
enzymatic labels, such as
alkaline phosphatase coupled to the probe via avidin/biotin coupling systems,
and the like.
Polynucleotide sequences encoding RMEP may be used for the diagnosis of
disorders associated
with expression of RMEP. Examples of such disorders include, but are not
limited to, a nervous system
disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral
neoplasms, Alzheimer's
disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease
and other extrapyramidal
disorders, amyotrophic lateral sclerosis and other motor neuron disorders,
progressive neural muscular
atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and
other demyelinating diseases,
bacterial and viral meningitis, brain abscess, subdural empyema, epidural
abscess, suppurative
intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous
system disease; prion
diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-
Scheinker syndrome; fatal
familial insomnia, nutritional and metabolic diseases of the nervous system,
neurofibromatosis, tuberous
sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal
syndrome, mental retardation and
other developmental disorder of the central nervous system, cerebral palsy, a
neuroskeletal disorder, an
autonomic nervous system disorder, a cranial nerve disorder, a spinal cord
disease, muscular dystrophy
and other neuromuscular disorder, a peripheral nervous system disorder,
dermatomyositis and
polymyositis; inherited, metabolic, endocrine, and toxic myopathy; myasthenia
gravis, periodic
paralysis; a mental disorder including mood, anxiety, and schizophrenic
disorders; seasonal affective
disorder (SAD); akathesia, amnesia, catatonia, diabetic neuropathy, tardive
dyskinesia, dystonias,
paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; an
autoimmune/inflammatory
disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease,
adult respiratory
distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
polyenodocrinopathy-
candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact
dermatitis, Crohn's
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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 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, and a developmental disorder such as renal
tubular acidosis, anemia,
Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular
dystrophy, epilepsy,
gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary
abnormalities, and
mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome,
hereditary mucoepithelial
dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-
Marie-Tooth disease and
neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as
Syndenham's chorea and
cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital
glaucoma, cataract, and
sensorineural hearing loss. The polynucleotide sequences encoding RMEP 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 RMEP expression. Such qualitative or quantitative methods are well
known in the art.
In a particular aspect, the nucleotide sequences encoding RMEP may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding RMEP may be labeled by standard methods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a suitable
incubation period, the sample is washed and the signal is quantified and
compared with a standard
value. If the amount of signal in the patient sample is significantly altered
in comparison to a control
sample then the presence of altered levels of nucleotide sequences encoding
RMEP 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
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treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of RMEP,
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 RMEP, 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 RMEP
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 RMEP,
or a fragment of a polynucleotide complementary to the polynucleotide encoding
RMEP, 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 RMEP may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic disease in
humans. Methods of SNP detection include, but are not limited to, single-
stranded conformation
polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP,
oligonucleotide primers
derived from the polynucleotide sequences encoding RMEP are used to amplify
DNA using the
polymerase chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
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tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the secondary
and tertiary structures of PCR products in single-stranded form, and these
differences are detectable
using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such as
DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of
individual overlapping
DNA fragments which assemble into a common consensus sequence. These computer-
based methods
filter out sequence variations due to laboratory preparation of DNA and
sequencing errors using
statistical models and automated analyses of DNA sequence chromatograms. In
the alternative, SNPs
may be detected and characterized by mass spectrometry using, for example, the
high throughput
MASSARRAY system (Sequenom, Inc., San Diego CA).
Methods which may also be used to quantify the expression of RMEP include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standaxd curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C. et
al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple
samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used to
develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic profile.
In another embodiment, RMEP, fragments of RMEP, or antibodies specific for
RMEP may be
used as elements on a microaxray. 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
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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 SeiEaamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present invention
or their complements comprise a subset of a plurality of elements on a
microarray. The resultant
transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed molecular
fingerprints or toxicant signatures, which are indicative of mechanisms of
action and toxicity (Nuwaysir,
E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L. Anderson
(2000) Toxicol. Lett. 112-
113:467-471, expressly incorporated by reference herein). If a test compound
has a signature similar to
that of a compound with known toxicity, it is likely to share those toxic
properties. These fingerprints
or signatures are most useful and refined when they contain expression
information from a large number
of genes and gene families. Ideally, a genome-wide measurement of expression
provides the highest
quality signature. Even genes whose expression is not altered by any tested
compounds are important as
well, as the levels of expression of these genes are used to normalize the
rest of the expression data. The
normalization procedure is useful for comparison of expression data after
treatment with different
compounds. While the assignment of 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
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present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global pattern
of protein expression in a particular tissue or cell type. Each protein
component of a proteome can be
subjected individually to further analysis. Proteome expression patterns, or
profiles, are analyzed by
quantifying the number of expressed proteins and their relative abundance
under given conditions and at
a given time. A profile of a cell's proteome may thus be generated by
separating and analyzing the
polypeptides of a particular tissue or cell type. In one embodiment, the
separation is achieved using
two-dimensional gel electrophoresis, in which proteins from a sample are
separated by isoelectric
focusing in the first dimension, and then according to molecular weight by
sodium dodecyl sulfate slab
gel electrophoresis in the second dimension (Steiner and Anderson, supra). The
proteins are visualized
in the gel as discrete and uniquely positioned spots, typically by staining
the gel with an agent such as
Coomassie Blue or silver or fluorescent stains. The optical density of each
protein spot is generally
proportional to the level of the protein in the sample. The optical densities
of equivalently positioned
protein spots from different samples, for example, from biological samples
either treated or untreated
with a test compound or therapeutic agent, are compared to identify any
changes in protein spot density
related to the treatment. The proteins in the spots are partially sequenced
using, for example, standard
methods employing chemical or enzymatic cleavage followed by mass
spectrometry. The identity of the
prtotein 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 RMEP
to quantify the
levels of RMEP 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.
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Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures
may be useful in the
analysis of compounds which do not significantly affect the transcript image,
but which alter the
proteomic profile. In addition, the analysis of transcripts in body fluids is
difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated biological
sample are separated so that the amount of each protein can be quantified. The
amount of each protein
is compared to the amount of the corresponding protein in an untreated
biological sample. A difference
in the amount of protein between the two samples is indicative of a toxic
response to the test compound
in the treated sample. Individual proteins are identified by sequencing the
amino acid residues of the
individual proteins and comparing these partial sequences to the polypeptides
of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared with
the amount in an untreated biological sample. A difference in the amount of
protein between the two
samples is indicative of a toxic response to the test compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application 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 Microarravs: 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 RMEP
may be used to
generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a multi-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (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.
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7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop genetic
linkage maps, for example, which correlate the inheritance of a disease state
with the inheritance of a
particular chromosome region or restriction fragment length polymorphism
(RFLP). (See, for
example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA
83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic map
data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, 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
RMEP on a physical map
and a specific disorder, or a predisposition to a specific disorder, may help
define the region of DNA
associated with that disorder and thus may further positional cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse, may
reveal associated markers even if the exact chromosomal locus is not known.
This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely localized
by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia
to 11q22-23, any sequences
mapping to that area may represent associated or regulatory genes for further
investigation. (See, e.g.,
Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the
instant invention may
also be used to detect differences in the chromosomal location due to
translocation, inversion, etc.,
among normal, carrier, or affected individuals.
In another embodiment of the invention, RMEP, 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 RMEP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with RMEP, or
fragments thereof, and
washed. Bound RMEP is then detected by methods well known in the art. Purified
RMEP can also be
coated directly onto plates for use in the aforementioned drug screening
techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and immobilize
it on a solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
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antibodies capable of binding RMEP specifically compete with a test compound
for binding RMEP. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with RMEP.
In additional embodiments, the nucleotide sequences which encode RMEP may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are,
therefore, to be construed as merely illustrative, and not limitative of the
remainder of the disclosure
in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below,
including U.S. Ser. No. 60/201,875, U.S. Ser. No. 60/200,184, U.S. Ser. No.
60/202,090, U.S. Ser.
No. 60/210,232, and U.S. Ser. No. 60/220,553, are hereby expressly
incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database
(Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues
were homogenized and
lysed in guanidinium isothiocyanate, while others were homogenized and lysed
in phenol or in a suitable
mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic
solution of phenol and
guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl
cushions or extracted with
chloroform. RNA was precipitated from the lysates with either isopropanol or
sodium acetate and
ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA purity.
In some cases, RNA was treated with DNase. For most libraries, poly(A)+ RNA
was isolated using
oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles
(QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
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5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-1000
bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo Alto
CA), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli cells
including XL1-Blue, XL1-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
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in 384-
well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation such
as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200
thermal cycler (MJ
Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or
the MICROLAB
2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were
prepared using reagents
provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such
as the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides were
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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,
sera, 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, and hidden Markov model (HMM)-based protein family databases
such as PFAM.
(HMM is a probabilistic approach which analyzes consensus primary structures
of gene families. See,
for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The
queries were performed
using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA
sequences were
assembled to produce full length polynucleotide sequences. Alternatively,
GenBank cDNAs, GenBank
ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding
sequences (see Examples
IV and V) were used to extend Incyte cDNA assemblages to full length. Assembly
was performed using
programs based on Phred, Phrap, and Conned, and cDNA assemblages were screened
for open reading
frames using programs based on GeneMark, BLAST, and FASTA. The full length
polynucleotide
sequences were translated to derive the corresponding full length polypeptide
sequences. Alternatively,
a polypeptide of the invention may begin at any of the methionine residues of
the full length translated
polypeptide. Full length polypeptide sequences were subsequently analyzed by
querying against
databases such as the GenBank protein databases (genpept), SwissProt, BLOCKS,
PRINTS, DOMO,
PRODOM, Prosite, and hidden Markov model (HMM)-based protein family databases
such as PFAM.
Full length polynucleotide sequences are also analyzed using MACDNASIS PRO
software (Hitachi
Software Engineering, South San Francisco CA) and LASERGENE software
(DNASTAR).
Polynucleotide and polypeptide sequence alignments are generated using default
parameters specified by
the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence
alignment program
(DNASTAR), which also calculates the percent identity between aligned
sequences.
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
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which are incorporated by reference herein in their entirety, and the fourth
column presents, where
applicable, the scores, probability values, and other parameters used to
evaluate the strength of a match
between two sequences (the higher the score or the lower the probability
value, the greater the identity
between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide and
polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:48-94. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative RNA metabolism 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 RNA metabolism proteins, the encoded polypeptides were
analyzed by querying
against PFAM models for RNA metabolism proteins. Potential RNA metabolism
proteins were also
identified by homology to Incyte cDNA sequences that had been annotated as RNA
metabolism 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 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
conf'~rm 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" Seguences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification
program described in Example IV. Partial cDNAs assembled as described in
Example III were mapped
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to genomic DNA and parsed into clusters containing related cDNAs and Genscan
exon predictions from
one or more genomic sequences. Each cluster was analyzed using an algorithm
based on graph theory
and dynamic programming to integrate cDNA and genomic information, generating
possible splice
variants that were subsequently confirmed, edited, or extended to create a
full length sequence.
Sequence intervals in which the entire length of the interval was present on
more than one sequence in
the cluster were identified, and intervals thus identified were considered to
be equivalent by transitivity.
For example, if an interval was present on a cDNA and two genomic sequences,
then all three intervals
were considered to be equivalent. This process allows unrelated but
consecutive genomic sequences to
be brought together, bridged by cDNA sequence. Intervals thus identified were
then "stitched" together
by the stitching algorithm in the order that they appear along their parent
sequences to generate the
longest possible sequence, as well as sequence variants. Linkages between
intervals which proceed
along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic
sequence) were
given preference over linkages which change parent type (cDNA to genomic
sequence). The resultant
stitched sequences were translated and compared by BLAST analysis to the
genpept and gbpri public
databases. Incorrect exons predicted by Genscan were corrected by comparison
to the top BLAST hit
from genpept. Sequences were further extended with additional cDNA sequences,
or by inspection of
genomic DNA, when necessary.
"Stretched" Seguences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases using
the BLAST program. The nearest GenBank protein homolog was then compared by
BLAST analysis to
either Incyte cDNA sequences or GenScan exon predicted sequences described in
Example IV. A
chimeric protein was generated by using the resultant high-scoring segment
pairs (HSPs) to map the
translated sequences onto the GenBank protein homolog. Insertions or deletions
may occur in the
chimeric protein with respect to the original GenBank protein homolog. The
GenBank protein homolog,
the chimeric protein, or both were used as probes to search for homologous
genomic sequences from the
public human genome databases. Partial DNA sequences were therefore
"stretched" or extended by the
addition of homologous genomic sequences. The resultant stretched sequences
were examined to
determine whether it contained a complete gene.
VI. Chromosomal Mapping of RMEP Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:48-94 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
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SEQ ID N0:48-94 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances
are based on genetic markers mapped by Genethon which provide boundaries for
radiation hybrid
markers whose sequences were included in each of the clusters. Human genome
maps and other
resources available to the public, such as the NCBI "GeneMap'99" World Wide
Web site
(http://www.ncbi.nlm.nih.gov/genemap~, can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
In this manner, SEQ ID N0:53 was mapped to chromosome 1 within the interval
from 159.6
to 164.1 centiMorgans. SEQ ID N0:61 was mapped to chromosome 8 within the
interval from 30.70
to 60.00 centiMorgans. SEQ ID N0:69 was mapped to chromosome 10 within the
interval from
158.30 centiMorgans to the q terminus. SEQ ID N0:70 was mapped to chromosome 1
within the
interval from 63.90 to 74.80 centiMorgans. SEQ ID N0:71 was mapped to
chromosome 1 within the
interval from 159.60 to 164.10 centiMorgans. SEQ ID N0:73 was mapped to
chromosome 11 within
the interval from 34.30 to 37.00 centiMorgans. SEQ ID N0:75 was mapped to
chromosome 2 within
the interval from 107.10 to 118.00 centiMorgans. SEQ ID N0:76 was mapped to
chromosome 7
within the interval from 7.80 to 10.60 centiMorgans. SEQ ID NO:79 was mapped
to chromosome 22
within the interval from 22.20 to 40.20 centiMorgans. SEQ ID N0:81 was mapped
to chromosome 4
within the interval from the p terminus to 6.70 centiMorgans. SEQ ID NO: 84
was mapped to
chromosome 5 within the interval from 156.0 to 157.6 centiMorgans. SEQ ID NO:
88 was mapped to
chromosome 11 within the interval from 117.9 to 123.5 centiMorgans. SEQ ID
NO:91 was mapped to
chromosome 5 within the interval from 152.3 to 155.5 centiMorgans.
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)
sera, ch. 4 and 16.)
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Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is much
faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer search
can be modified to determine whether any particular match is categorized as
exact or similar. The basis
of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the length
of the sequence match. The product score is a normalized value between 0 and
100, and is calculated as
follows: the BLAST score is multiplied by the percent nucleotide identity and
the product is divided by
(5 times the length of the shorter of the two sequences). The BLAST score is
calculated by assigning a
score of +5 for every base that matches in a high-scoring segment pair (HSP),
and -4 for every
mismatch. Two sequences may share more than one HSP (separated by gaps). If
there is more than one
HSP, then the pair with the highest BLAST score is used to calculate the
product score. The product
score represents a balance between fractional overlap and quality in a BLAST
alignment. For example,
a product score of 100 is produced only for 100% identity over the entire
length of the shorter of the
two sequences being compared. A product score of 70 is produced either by 100%
identity and 70%
overlap at one end, or by 88% identity and 100% overlap at the other. A
product score of 50 is
produced either by 100% identity and 50% overlap at one end, or 79% identity
and 100% overlap.
Alternatively, polynucleotide sequences encoding RMEP are analyzed with
respect to the tissue
sources from which they were derived. For example, some full length sequences
are assembled, at least
in part, with overlapping Incyte cDNA sequences (see Example III). Each cDNA
sequence is derived
from a cDNA library constructed from a human tissue. Each human tissue is
classified into one of the
following organ/tissue categories: cardiovascular system; connective tissue;
digestive system;
embryonic structures; endocrine system; exocrine glands; genitalia, female;
genitalia, male; germ cells;
heroic and immune system; liver; musculoskeletal system; nervous system;
pancreas; respiratory system;
sense organs; skin; stomatognathic system; unclassified/mixed; or urinary
tract. The number of libraries
in each category is counted and divided by the total number of libraries
across all categories. Similarly,
each human tissue is classified into one of the following disease/condition
categories: cancer, cell line,
developmental, inflammation, neurological, trauma, cardiovascular, pooled, and
other, and the number
of libraries in each category is counted and divided by the total number of
libraries across all categories.
The resulting percentages reflect the tissue- and disease-specific expression
of cDNA encoding RMEP.
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cDNA sequences and cDNA library/tissue information are found in the LIFESEQ
GOLD database
(Incyte Genomics, Palo Alto CA).
VIII. Extension of RMEP Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target sequence
at temperatures of about 68 °C to about 72°C. Any stretch of
nucleotides which would result in hairpin
structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one extension
was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)yS 04,
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 andPCI 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 ~1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~l of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 /d to 10 /c1 aliquot of the reaction mixture was
analyzed by electrophoresis
on a 1 % agarose gel to determine which reactions were successful in extending
the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to relegation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentrateon (0.6 to 0.8%) agarose
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gels, fragments were excised, and agar digested with Agar ACE (Promega).
Extended clones were
religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector
(Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site overhangs,
and transfected into competent E. coli cells. Transformed cells were selected
on antibiotic-containing
media, and individual colonies were picked and cultured overnight at 37
°C in 384-well plates in LB/2x
carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham
Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following
parameters: Step 1:
94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min;
Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4
repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C.
DNA was quantified by PICOGREEN
reagent (Molecular Probes) as described above. Samples with low DNA recoveries
were reamplified
using the same conditions as described above. Samples were diluted with 20%
dimethysulfoxide (1:2,
v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the
DYENAMIC
DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator
cycle sequencing
ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5' regulatory sequences using the above procedure along
with oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:48-94 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 ~Ci of [y-
32P] adenosine
triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase
(DuPont NEN, Boston
MA). The labeled oligonucleotides are substantially purified using a SEPHADEX
G-25 superfine size
exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot
containing 10' counts per
minute of the labeled probe is used in a typical membrane-based hybridization
analysis of human
genomic DNA digested with one of the following endonucleases: Ase I, Bgl 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.
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Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
X. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, sera.), mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), supra). Suggested
substrates include silicon, silica, glass slides, glass chips, and silicon
wafers. Alternatively, a procedure
analogous to a dot or slot blot may also be used to arrange and link elements
to the surface of a
substrate using thermal, UV, chemical, or mechanical bonding procedures. A
typical array may be
produced using available methods and machines well known to those of ordinary
skill in the art and may
contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470;
Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson
(1998) Nat. Biotechnol.
16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The array
elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described in
detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the 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/~.il oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/iil RNase inhibitor, 500 ~M dATP, 500 ~M dGTP, 500
~M dTTP, 40 ~M
dCTP, 40 ~M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
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
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with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of O.SM sodium
hydroxide and
incubated for 20 minutes at 85 ° C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and resuspended
in 14 iil SX SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element is
amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
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 US
Patent No. 5,807,522, incorporated herein by reference. 1 irl of the array
element DNA, at an average
concentration of 100 ng/iil, 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 ~~1 of sample mixture consisting of 0.2 ~g
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65 ° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 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
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140 ~~1 of SX SSC in a corner of the chamber. The chamber containing the
arrays is incubated for
about 6.5 hours at 60° C. The arrays are washed for 10 min at 45
° C in a first wash buffer (1X SSC,
0.1 % SDS), three times for 10 minutes each at 45 ° C in a second wash
buffer (0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that location
to be correlated with a weight ratio of hybridizing species of 1:100,000. When
two samples from
different sources (e.g., representing test and control cells), each labeled
with a different fluorophore,
are hybridized to a single array for the purpose of identifying genes that are
differentially expressed,
the calibration is done by labeling samples of the calibrating cDNA with the
two fluorophores and
adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping emission
spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
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to obtain a numerical value corresponding to the average intensity of the
signal. The software used for
signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
XI. Complementary Polynucleotides
Sequences complementary to the RMEP-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring RMEP. 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 RMEP. 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 RMEP-encoding transcript.
XII. Expression of RMEP
Expression and purification of RMEP is achieved using bacterial or virus-based
expression
systems. For expression of RMEP in bacteria, cDNA is subcloned into an
appropriate vector containing
an antibiotic resistance gene and an inducible promoter that directs high
levels of cDNA transcription.
Examples of such promoters include, but are not limited to, the trp-lac (tac)
hybrid promoter and the T5
or T7 bacteriophage promoter in conjunction with the lac operator regulatory
element. Recombinant
vectors are transformed into suitable bacterial hosts, e.g., BL21 (DE3).
Antibiotic resistant bacteria
express RMEP upon induction with isopropyl beta-D-thiogalactopyranoside
(IPTG). Expression of
RMEP in eukaryotic cells is achieved by infecting insect or mammalian cell
lines with recombinant
Auto~raphica californica nuclear polyhedrosis virus (AcMNPV), commonly known
as baculovirus. The
nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding
RMEP by either
homologous recombination or bacterial-mediated transposition involving
transfer plasmid intermediates.
Viral infectivity is maintained and the strong polyhedrin promoter drives high
levels of cDNA
transcription. Recombinant baculovirus is used to infect Spodoptera frugiperda
(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, RMEP is synthesized as a fusion protein with,
e.g., glutathione S-
transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Schistosoma japonicum, enables the purification of fusion proteins
on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Pharmacia
Biotech). Following purification, the GST moiety can be proteolytically
cleaved from RMEP at
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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 RMEP obtained by these methods can be used directly in
the assays shown in
Examples XVI and XVII where applicable.
XIII. ~nctional Assays
RMEP function is assessed by expressing the sequences encoding RMEP 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 ,ug of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate the
apoptotic state of the cells and other cellular properties. FCM detects and
quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events include
changes in nuclear DNA content as measured by staining of DNA with propidium
iodide; changes in cell
size and granularity as measured by forward light scatter and 90 degree side
light scatter; down-
regulation of DNA synthesis as measured by decrease in bromodeoxyuridine
uptake; alterations in
expression of cell surface and intracellular proteins as measured by
reactivity with specific antibodies;
and alterations in plasma membrane composition as measured by the binding of
fluorescein-conjugated
Annexin V protein to the cell surface. Methods in flow cytometry are discussed
in Ormerod, M. G.
(1994) Flow Cytometry, Oxford, New York NY.
The influence of RMEP on gene expression can be assessed using highly purified
populations of
cells transfected with sequences encoding RMEP 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
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of mRNA encoding RMEP and other genes of interest can be analyzed by northern
analysis or
microarray techniques.
XIV. Production of RMEP Specific Antibodies
RMEP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the RMEP 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-I~LH
complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-RMEP
activity by, for example, binding the peptide or RMEP to a substrate, blocking
with 1 % BSA, reacting
with rabbit antisera, washing, and reacting with radio-iodinated goat anti-
rabbit IgG.
XV. Purification of Naturally Occurring RMEP Using Specific Antibodies
Naturally occurring or recombinant RMEP is substantially purified by
immunoaffinity
chromatography using antibodies specific for RMEP. An immunoafhnity column is
constructed by
covalently coupling anti-RMEP 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 RMEP are passed over the immunoafFnity column, and the column
is washed
under conditions that allow the preferential absorbance of RMEP (e.g., high
ionic strength buffers in the
presence of detergent). The column is eluted under conditions that disrupt
antibody/RMEP 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 RMEP is collected.
XVI. Identification of Molecules Which Interact with RMEP
RMEP, or biologically active fragments thereof, are labeled with lzsl 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 RMEP, washed, and
any wells with labeled RMEP complex are assayed. Data obtained using different
concentrations of
77
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
RMEP are used to calculate values for the number, affinity, and association of
RMEP with the
candidate molecules.
Alternatively, molecules interacting with RMEP 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).
RMEP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S. Patent
No. 6,057,101 ).
XVII. Demonstration of RMEP Activity
RMEP activity is demonstrated by a polyacrylamide gel mobility-shift assay. In
preparation
for this assay, RMEP is expressed by transforming a mammalian cell line such
as COS7, HeLa or
CHO with a eukaryotic expression vector containing RMEP cDNA. The cells are
incubated for 48-72
hours after transformation under conditions appropriate for the cell line to
allow expression and
accumulation of RMEP. Extracts containing solubilized proteins can be prepared
from cells
expressing RMEP by methods well known in the art. Portions of the extract
containing RMEP are
added to [32P]-labeled RNA. Radioactive RNA can be synthesized in vitro by
techniques well known
in the art. The mixtures are incubated at 25 °C in the presence of
RNase inhibitors under buffered
conditions for 5-10 minutes. After incubation, the samples are analyzed by
polyacrylamide gel
electrophoresis followed by autoradiography. The presence of a band on the
autoradiogram indicates
the formation of a complex between RMEP and the radioactive transcript. A band
of similar mobility
will not be present in samples prepared using control extracts prepared from
untransformed cells.
In the alternative, ribosomal protein function of RMEP is assessed by
expressing the
sequences encoding ribosomal proteins 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 Corporation), both of which contain the
cytomegalovirus
promoter (P~~). Between 5-10 ~g of recombinant vector are transfected into a
human cell line,
preferably of endothelial or hematopoietic origin, using either liposome
formulations or
electroporation. 1-2 ~g of an additional plasmid containing sequences encoding
a marker protein are
cotransfected.
Transient 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
78
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
used to identify transfected cells expressing GFP or CD64-GFP and to evaluate
the apoptotic state of
the cells and other cellular properties.
FCM detects and quantifies the uptake of fluorescent molecules that diagnose
events
preceding or coincident with cell death. These events include changes in
nuclear DNA content as
measured by staining of DNA with propidium iodide; changes in cell size and
granularity as measured
by forward light scatter and 90 degree side light scatter; down-regulation of
DNA synthesis as
measured by decrease in bromodeoxyuridine uptake; alterations in expression of
cell surface and
intracellular proteins as measured by reactivity with specific antibodies; and
alterations in plasma
membrane composition as measured by the binding of fluorescein-conjugated
Annexin V protein to
the cell surface. Methods in flow cytometry are discussed in Ormerod MG (1994)
Flow ~tometry,
Oxford University Press, New York NY.
The influence of ribosomal proteins on gene expression can be assessed using
highly purified
populations of cells transfected with sequences encoding a ribosomal protein
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, Inc., 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 a ribosomal
protein and other genes
of interest can be analyzed by northern analysis or microarray techniques.
In the alternative, RMEP activity is measured as the aminoaeylation of a
substrate tRNA in
the presence of [l4C]cysteine. RMEP is incubated with tRNA~ys and
[14C]cysteine (or appropriate
tRNA and amino acid substrates) in a buffered solution. [14C]-labeled product
is separated from free
[14C]-amino acid by chromatography, and the incorporated [laC] is quantified
by scintillation counter.
The amount of [14C] deteeted is proportional to the activity of RMEP in this
assay.
In the alternative, RMEP activity is measured by incubating a sample
containing RMEP in a
solution containing 1 mM ATP, 5 mM Hepes-KOH (pH 7.0), 2.5 mM KCl, 1.5 mM
magnesium
chloride, and 0.5 mM DTT along with misacylated [14C]-Glu-tRNAGln (e.g., 1
~iM) and a similar
concentration of unlabeled L-glutamine. Following the quenching of the
reaction with 3 M sodium
acetate (pH 5.0), the mixture is extracted with an equal volume of water-
saturated phenol, and the
aqueous and organic phases are separated by centrifugation at 15,000 x g at
room temperature for 1
min. The aqueous phase is removed and precipitated with 3 volumes of ethanol
at -70°C for 15 min.
The precipitated aminoacyl-tRNAs are recovered by centrifugation at 15,000 x g
at 4°C forl5 min.
The pellet is resuspended in of 25 mM KOH, deacylated at 65°C for 10
min., neutralized with 0.1 M
HCl (to final pH 6-7), and dried under vacuum. The dried pellet is resuspended
in water and spotted
onto a cellulose TLC plate. The plate is developed in either
isopropanol/fornuc acid/water or
79
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
ammonialwater/chloroform/methanol, The image is subjected to densitometric
analysis and the
relative amounts of Glu and Gln are calculated based on the Rf values and
relative intensities of the
spots. RMEP activity is calculated based on the amount of Gln resulting from
the transformation of
Glu while acylated as Glu-tRNAG'" (adapted from Curnow, A.W. et al. (1997)
Proc. Natt. Acad. Sci.
94:11819-26).
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.
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
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CA 02407435 2002-10-24
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CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<110> INCYTE GENOMICS, INC.
LAL, Preeti
YUE, Henry
TANG, Y. Tom
LU, Dyung Aina M.
AZIMZAI, Yalda
AU-YOUNG, Janice
HILLMAN, Jennifer L.
BAUGHN, Mariah R.
YAO, Monique G.
BURFORD, Neil
BATRA, Sajeev
POLICKY, Jennifer J.
<120> RNA METABOLISM PROTEINS
<130> PF-0771 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/200,184; 60/201,875; 60/202,090; 60/210,232; 60/220,553
<151> 2000-04-28; 2000-05-04; 2000-05-04; 2000-06-06; 2000-07-25
<160> 94
<170> PERL Program
<210> 1
<211> 245
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1622129CD1
<400> 1
Met Ala Gly Leu Glu Leu Leu Ser Asp Gln Gly Tyr Arg Val Asp
1 5 10 15
Gly Arg Arg Ala Gly Glu Leu Arg Lys Ile Gln Ala Arg Met Gly
20 25 30
Val Phe Ala Gln Ala Asp Gly Ser Ala Tyr Ile Glu Gln Gly Asn
35 40 45
Thr Lys Ala Leu Ala Val Val Tyr Gly Pro His Glu Ile Arg Gly
50 55 60
Ser Arg Ala Arg Ala Leu Pro Asp Arg Ala Leu Val Asn Cys Gln
65 70 75
Tyr Ser Ser AIa Thr Phe Ser Thr Gly Glu Arg Lys Arg Arg Pro
80 85 90
His Gly Asp Arg Lys Ser Cys Glu Met Gly Leu Gln Leu Arg Gln
95 100 105
Thr Phe Glu Ala Ala Ile Leu Thr Gln Leu His Pro Arg Ser Gln
110 115 120
Ile Asp Ile Tyr Val Gln Val Leu Gln Ala Asp Gly Gly Thr Tyr
125 130 135
Ala Ala Cys Val Asn Ala Ala Thr Leu Ala Val Leu Asp Ala Gly
140 145 150
Ile Pro Met Arg Asp Phe Val Cys Ala Cys Ser Ala Gly Phe Val
155 160 165
Asp Gly Thr Ala Leu Ala Asp Leu Ser His Val Glu Glu Ala Ala
170 175 180
Gly Gly Pro Gln Leu Ala Leu Ala Leu Leu Pro Ala Ser Gly G1n
185 190 195
Ile Ala Leu Leu Glu Met Asp Ala Arg Leu His Glu Asp His Leu
200 205 210
Glu Arg Val Leu Glu Ala Ala Ala Gln Ala Ala Arg Asp Val His
215 220 225
1164
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Thr Leu Leu Asp Arg Val Val Arg Gln His Va1 Arg Glu Ala Ser
230 235 240
Ile Leu Leu Gly Asp
245
<210> 2
<211> 118
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1820078CD1
<400> 2
Met Thr Asp Thr Ala Glu Ala Val Pro Lys Phe Glu Glu Met Phe
1 5 10 15
Ala Ser Arg Phe Thr Glu Asn Asp Lys Glu Tyr Gln Glu Tyr Leu
20 25 30
Lys Arg Pro Pro Glu Ser Pro Pro Tle Val Glu Glu Trp Asn Ser
35 40 45
Arg Ala Gly Gly Asn Gln Arg Asn Arg Gly Asn Arg Leu Gln Asp
50 55 60
Asn Arg Gln Phe Arg Gly Arg Asp Asn Arg Trp Gly Trp Pro Ser
65 70 75
Asp Asn Arg Ser Asn Gln Trp His Gly Arg Ser Trp Gly Asn Asn
80 85 90
Tyr Pro Gln His Arg Gln Glu Pro Tyr Tyr Pro Gln Gln Tyr Gly
95 100 105
His Tyr Gly Tyr Asn Gln Arg Pro Pro Tyr Gly Tyr Tyr
110 115
<210> 3
<211> 179
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1527017CD1
<400> 3
Met Phe Gly Ser Ser Arg Arg Leu Ser Ser Ser Lys Leu Leu Gln
1 5 10 15
Gln Gly Lys Thr Ser Ser Val Phe Glu Asp Pro Val Ile Ser Lys
20 25 30
Phe Thr Asn Met Met Met Ile Gly Gly Asn Lys Val Leu Ala Arg
35 40 45
Ser Leu Met Ile Gln Thr Leu Glu Ala Val Lys Arg Lys Gln Phe
50 55 60
Glu Lys Tyr His Ala Ala Ser Ala G1u Glu Gln Ala Thr Ile Glu
65 70 75
Arg Asn Pro Tyr Thr Ile Phe His Gln Ala Leu Lys Asn Cys Glu
80 85 90
Pro Met Ile Gly Leu Val Pro Ile Leu Lys Gly G1y Arg Phe Tyr
95 100 105
Gln Val Pro Val Pro Leu Pro Asp Arg Arg Arg Arg Phe Leu Ala
110 115 120
Met Lys Trp Met Ile Thr Glu Cys Arg Asp Lys Lys His Gln Arg
125 130 135
Thr Leu Met Pro Glu Lys Leu Ser His Lys Leu Leu Glu Ala Phe
140 145 150
His Asn Gln Gly Pro Val Ile Lys Arg Lys His Asp Leu His Lys
155 160 165
Met Ala Glu Ala Asn Arg Ala Leu Ala His Tyr Arg Trp Trp
170 175
2/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<210> 4
<211> 101
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1647264CD1
<400> 4
Met Glu Arg Pro Asp Lys Ala Ala Leu Asn Ala Leu Gln Pro Pro
1 5 10 15
Glu Phe Arg Asn Glu Ser Ser Leu Ala Ser Thr Leu Lys Thr Leu
20 25 30
Leu Phe Phe Thr Ala Leu Met Ile Thr Val Pro Ile Gly Leu Tyr
35 40 45
Phe Thr Thr Lys Ser Tyr Ile Phe Glu Gly Ala Leu Gly Met Ser
50 55 60
Asn Arg Asp Ser Tyr Phe Tyr Ala Ala Ile Val Ala Val Val Ala
65 70 75
Val His Val Val Leu Ala Leu Phe Val Tyr Val Ala Trp Asn Glu
80 85 90
Gly Ser Arg G1n Trp Arg Glu Gly Lys Gln Asp
95 100
<210> 5
<211> 145
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1721989CD1
<400> 5
Met Ala Phe Phe Thr Gly Leu Trp Gly Pro Phe Thr Cys Val Ser
1 5 10 15
Arg Val Leu Ser His His Cys Phe Ser Thr Thr Gly Ser Leu Ser
20 25 30
Ala Ile Gln Lys Met Thr Arg Val Arg Val Val Asp Asn Ser Ala
35 40 45
Leu Gly Asn Ser Pro Tyr His Arg Ala Pro Arg Cys Ile His Val
50 55 60
Tyr Lys Lys Asn G1y Val Gly Lys Val Gly Asp Gln Ile Leu Leu
65 70 75
Ala Ile Lys Gly Gln Lys Lys Lys Ala Leu Ile Val Gly His Cys
80 85 90
Met Pro Gly Pro Arg Met Thr Pro Arg Phe Asp Ser Asn Asn Val
95 100 105
Val Leu Ile Glu Asp Asn Gly Asn Pro Val Gly Thr Arg Ile Lys
110 115 120
Thr Pro Ile Pro Thr Ser Leu Arg Lys Arg Glu Gly Glu Tyr Ser
12 5 13 0 13 5
Lys Val Leu Ala Ile Ala Gln Asn Phe Val
140 145
<210> 6
<211> 249
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1730581CD1
<400> 6
Met Ala Ala Gln Ser Ala Pro Lys Val Val Leu Lys Ser Thr Thr
3/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
l 5 10 15
Lys Met Ser Leu Asn Glu Arg Phe Thr Asn Met Leu Lys Asn Lys
20 25 30
Gln Pro Thr Pro Val Asn Ile Arg Ala Ser Met Gln Gln Gln Gln
35 40 45
Gln Leu Ala Ser Ala Arg Asn Arg Arg Leu Ala Gln Gln Met Glu
50 55 60
Asn Arg Pro Ser Val Gln Ala Ala Leu Lys Leu Lys Gln Lys Ser
65 70 75
Leu Lys Gln Arg Leu Gly Lys Ser Asn Ile Gln Ala Arg Leu Gly
80 85 90
Arg Pro Ile Gly Ala Leu Ala Arg Gly Ala Ile Gly Gly Arg Gly
95 100 105
Leu Pro Ile Ile Gln Arg Gly Leu Pro Arg Gly Gly Leu Arg Gly
110 115 120
Gly Arg Ala Thr Arg Thr Leu Leu Arg G1y Gly Met Ser Leu Arg
125 130 135
Gly Gln Asn Leu Leu Arg Gly Gly Arg Ala Val Ala Pro Arg Met
140 145 150
Gly Leu Arg Arg Gly Gly Val Arg Gly Arg Gly Gly Pro Gly Arg
155 160 165
Gly Gly Leu Gly Arg Gly Ala Met Gly Arg Gly Gly Ile Gly Gly
170 175 180
Arg Gly Arg Gly Met Ile Gly Arg Gly Arg Gly Gly Phe Gly Gly
185 190 195
Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Ala Leu Ala Arg Pro
200 205 210
Val Leu Thr Lys Glu G1n Leu Asp Asn Gln Leu Asp Ala Tyr Met
215 220 225
Ser Lys Thr Lys Gly His Leu Asp Ala Glu Leu Asp Ala Tyr Met
230 ' 235 240
Ala Gln Thr Asp Pro Glu Thr Asn Asp
245
<210> 7
<211> 265
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1740714CD1
<400> 7
Met Arg Arg Ala Glu Leu Ala Gly Leu Lys Thr Met Ala Trp Val
1 5 10 15
Pro Ala Glu Ser Ala Val Glu Glu Leu Met Pro Arg Leu Leu Pro
20 25 30
Val Glu Pro Cys Asp Leu Thr Glu Gly Phe Asp Pro Ser Val Pro
35 40 45
Pro Arg Thr Pro Gln Glu Tyr Leu Arg Arg Val Gln Ile Glu Ala
50 55 60
Ala Gln Cys Pro Asp Val Val Val Ala Gln Ile Asp Pro Lys Lys
65 70 75
Leu Lys Arg Lys Gln Ser Val Asn Ile Ser Leu Ser Gly Cys Gln
80 85 90
Pro Ala Pro Glu Gly Tyr Ser Pro Thr Leu Gln Trp Gln Gln Gln
95 100 105
Gln Val Ala Gln Phe Ser Thr Val Arg Gln Asn Val Asn Lys His
110 115 120
Arg Ser His Trp Lys Ser Gln Gln Leu Asp Ser Asn Val Thr Met
125 13 0 135
Pro Lys Ser Glu Asp Glu Glu Gly Trp Lys Lys Phe Cys Leu Gly
140 145 150
Glu Lys Leu Cys Ala Asp Gly Ala Val Gly Pro Ala Thr Asn Glu
155 160 165
Ser Pro Gly Ile Asp Tyr Val Gln Ala Thr Val Thr Ser Val Leu
4/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
170 175 180
Glu Tyr Leu Ser Asn Trp Phe Gly Glu Arg Asp Phe Thr Pro Glu
185 190 195
Leu Gly Arg Trp Leu Tyr Ala Leu Leu Ala Cys Leu Glu Lys Pro
200 205 210
Leu Leu Pro Glu Ala His Ser Leu Ile Arg Gln Leu Ala Arg Arg
215 220 225
Cys Ser Glu Val Arg Leu Leu Val Asp Ser Lys Asp Asp Glu Arg
230 235 240
Val Pro Ala Leu Asn Leu Leu Ile Cys Leu Val Ser Arg Tyr Phe
245 250 255
Asp Gln Arg Asp Leu Ala Asp Glu Pro Ser
260 265
<210> 8
<211> 306
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1850596CD1
<400> 8
Met Ser Leu Lys Leu Gln Ala Ser Asn Val Thr Asn Lys Asn Asp
1 5 10 15
Pro Lys Ser Ile Asn Ser Arg Val Phe Ile Gly Asn Leu Asn Thr
20 25 30
Ala Leu Val Lys Lys Ser Asp Val Glu Thr Ile Phe Ser Lys Tyr
35 40 45
Gly Arg Val Ala Gly Cys Ser Val His Lys Gly Tyr A1a Phe Val
50 55 60
Gln Tyr Ser Asn Glu Arg His Ala Arg A1a A1a Val Leu Gly Glu
65 70 75
Asn Gly Arg Val Leu Ala Gly Gln Thr Leu Asp Ile Asn Met Ala
80 85 90
Gly Glu Pro Lys Pro Asp Arg Pro Lys Gly Leu Lys Arg Ala Ala
95 100 105
Ser Ala Ile Tyr Ser Gly Tyr Ile Phe Asp Tyr Asp Tyr Tyr Arg
110 115 120
Asp Asp Phe Tyr Asp Arg Leu Phe Asp Tyr Arg Gly Arg Leu Ser
125 130 135
Pro Val Pro Val Pro Arg Ala Val Pro Val Lys Arg Pro Arg Val
140 145 150
Thr Val Pro Leu Val Arg Arg Val Lys Thr Asn Val Pro Val Lys
155 160 165
Leu Phe A1a Arg Ser Thr Ala Val Thr Thr Ser Ser Ala Lys Ile
170 175 180
Lys Leu Lys Ser Ser Glu Leu Gln Ala I1e Lys Thr Glu Leu Thr
185 190 195
Gln Ile Lys Ser Asn Ile Asp Ala Leu Leu Ser Arg Leu Glu Gln
200 205 210
Ile Ala Ala Glu Gln Lys Ala Asn Pro Asp Gly Lys Lys Lys Gly
215 220 225
Asp Gly Gly Gly Ala Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly
230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Ser Ser Arg Pro
245 250 255
Pro Ala Pro Gln Glu Asn Thr Thr Ser Glu Ala Gly Leu Pro Gln
260 265 270
Gly Glu Ala Arg Thr Arg Asp Asp Gly Asp Glu Glu Gly Leu Leu
275 280 285
Thr His Ser Glu Glu Glu Leu Glu His Ser Gln Asp Thr Asp Ala
290 295 300
Asp Asp Gly Ala Leu Gln
305
5/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<210> 9
<211> 332
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1856109CD1
<400> 9
Met Ala Ser Gly Leu Val Arg Leu Leu Gln Gln Gly His Arg Cys
1 5 10 15
Leu Leu Ala Pro Val Ala Pro Lys Leu Val Pro Pro Val Arg Gly
20 25 30
Val Lys Lys Gly Phe Arg Ala Ala Phe Arg Phe Gln Lys Glu Leu
35 40 45
Glu Arg Gln Arg Leu Leu Arg Cys Pro Pro Pro Pro Val Arg Arg
50 55 60
Ser Glu Lys Pro Asn Trp Asp Tyr His Ala Glu Ile Gln Ala Phe
65 70 75
Gly His Arg Leu Gln Glu Asn Phe Ser Leu Asp Leu Leu Lys Thr
80 85 90
Ala Phe Val Asn Ser Cys Tyr Ile Lys Ser Glu Glu Ala Lys Arg
95 100 105
Gln Gln Leu Gly Ile Glu Lys Glu Ala Val Leu Leu Asn Leu Lys
110 115 120
Ser Asn Gln Glu Leu Ser Glu Gln Gly Thr Ser Phe Ser Gln Thr
125 130 135
Cys Leu Thr Gln Phe Leu Glu Asp Glu Tyr Pro Asp Met Pro Thr
140 145 150
Glu Gly Ile Lys Asn Leu Val Asp Phe Leu Thr Gly Glu Glu Val
155 160 165
Val Cys His Val Ala Arg Asn Leu Ala Val Glu Gln Leu Thr Leu
170 175 180
Ser Glu Glu Phe Pro Val Pro Pro Ala Val Leu Gln Gln Thr Phe
185 190 195
Phe Ala Val Ile Gly Ala Leu Leu Gln Ser Ser Gly Pro Glu Arg
200 205 210
Thr Ala Leu Phe Ile Arg Asp Phe Leu Ile Thr Gln Met Thr G1y
215 220 225
Lys Glu Leu Phe Glu Met Trp Lys Ile Ile Asn Pro Met Gly Leu
230 235 240
Leu Val Glu Glu Leu Lys Lys Arg Asn Val Ser Ala Pro Glu Ser
245 250 255
Arg Leu Thr Arg Gln Ser Gly Gly Thr Thr A1a Leu Pro Leu Tyr
260 265 270
Phe Val Gly Leu Tyr Cys Asp Lys Lys Leu Ile Ala Glu Gly Pro
275 280 285
Gly Glu Thr Val Leu Va1 Ala Glu Glu Glu Ala Ala Arg Val Ala
290 295 300
Leu Arg Lys Leu Tyr Gly Phe Thr Glu Asn Arg Arg Pro Trp Asn
305 310 315
Tyr Ser Lys Pro Lys Glu Thr Leu Arg Ala Glu Lys Ser Ile Thr
320 325 330
Ala Ser
<210> 10
<211> 279
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1921719CD1
<400> 10
6/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Met Ala Ala Pro Val Arg Arg Thr Leu Leu Gly Val Ala Gly Gly
1 5 10 15
Trp Arg Arg Phe Glu Arg Leu Trp Ala Gly Ser Leu Ser Ser Arg
20 25 30
Ser Leu Ala Leu Ala Ala Ala Pro Ser Ser Asn Gly Ser Pro Trp
35 40 45
Arg Leu Leu Gly Ala Leu Cys Leu Gln Arg Pro Pro Val Val Ser
50 55 60
Lys Pro Leu Thr Pro Leu Gln Glu G1u Met Ala Ser Leu Leu Gln
65 70 75
Gln Ile Glu Ile Glu Arg Ser Leu Tyr Ser Asp His Glu Leu Arg
80 85 90
Ala Leu Asp Glu Asn Gln Arg Leu Ala Lys Lys Lys Ala Asp Leu
95 100 105
His Asp Glu Glu Asp Glu Gln Asp Ile Leu Leu Ala Gln Asp Leu
110 115 120
Glu Asp Met Trp Glu Gln Lys Phe Leu Gln Phe Lys Leu Gly Ala
125 13 0 135
Arg Ile Thr Glu A1a Asp Glu Lys Asn Asp Arg Thr Ser Leu Asn
140 145 150
Arg Lys Leu Asp Arg Asn Leu Val Leu Leu Val Arg Glu Lys Phe
155 160 165
Gly Asp Gln Asp Val Trp Ile Leu Pro Gln Ala Glu Trp Gln Pro
170 175 180
Gly Glu Thr Leu Arg Gly Thr Ala Glu Arg Thr Leu Ala Thr Leu
185 190 195
Ser Glu Asn Asn Met Glu Ala Lys Phe Leu Gly Asn Ala Pro Cys
200 205 210
Gly His Tyr Thr Phe Lys Phe Pro Gln Ala Met Arg Thr Glu Ser
215 220 225
Asn Leu Gly Ala Lys Val Phe Phe Phe Lys Ala Leu Leu Leu Thr
230 235 240
Gly Asp Phe Ser Gln Ala Gly Asn Lys Gly His His Val Trp Val
245 250 255
Thr Lys Asp G1u Leu Gly Asp Tyr Leu Lys Pro Lys Tyr Leu Ala
260 265 270
Gln Val Arg Arg Phe Val Ser Asp Leu
275
<210> 11
<211> 239
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2099829CD1
<400> 11
Met Pro Lys Ser Lys Arg Asp Lys Lys Val Ser Leu Thr Lys Thr
1 5 10 15
Ala Lys Lys Gly Leu G1u Leu Lys G1n Asn Leu Ile Glu Glu Leu
20 25 30
Arg Lys Cys Val Asp Thr Tyr Lys Tyr Leu Phe Ile Phe Ser Val
35 40 45
Ala Asn Met Arg Asn Ser Lys Leu Lys Asp Ile Arg Asn Ala Trp
50 55 60
Lys His Ser Arg Met Phe Phe Gly Lys Asn Lys Val Met Met Val
65 70 75
Ala Leu Gly Arg Ser Pro Ser Asp Glu Tyr Lys Asp Asn Leu His
80 85 90
Gln Val Ser Lys Arg Leu Arg Gly Glu Val Gly Leu Leu Phe Thr
95 100 105
Asn Arg Thr Lys Glu Glu Val Asn Glu Trp Phe Thr Lys Tyr Thr
110 115 120
Glu Met Asp Tyr Ala Arg Ala Gly Asn Lys Ala Ala Phe Thr Val
125 130 135
7/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Ser Leu Asp Pro Gly Pro Leu Glu Gln Phe Pro His Ser Met Glu
140 145 150
Pro Gln Leu Arg Gln Leu Gly Leu Pro Thr Ala Leu Lys Arg Gly
155 160 165
Val Val Thr Leu Leu Ser Asp Tyr Glu Val Cys Lys Glu Gly Asp
170 175 180
Val Leu Thr Pro Glu Gln Ala Arg Val Leu Lys Leu Phe Gly Tyr
185 190 195
Glu Met Ala Glu Phe Lys Val Thr Ile Lys Tyr Met Trp Asp Ser
200 205 210
Gln Ser Gly Arg Phe Gln Gln Met Gly Asp Asp Leu Pro Glu Ser
215 220 225
Ala Ser Glu Ser Thr Glu Glu Ser Asp Ser Glu Asp Asp Asp
230 235
<210> 12
<211> 291
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2416915CD1
<400> 12
Met Asp Phe Glu Asn Leu Phe Ser Lys Pro Pro Asn Pro Ala Leu
1 5 10 15
Gly Lys Thr Ala Thr Asp Ser Asp Glu Arg Ile Asp Asp Glu Ile
20 25 30
Asp Thr Glu Val Glu Glu Thr Gln Glu Glu Lys I1e Lys Leu Glu
35 40 45
Cys Glu Gln Ile Pro Lys Lys Phe Arg His Ser Ala Ile Ser Pro
50 55 60
Lys Ser Ser Leu His Arg Lys Ser Arg Ser Lys Asp Tyr Asp Val
65 70 75
Tyr Ser Asp Asn Asp Ile Cys Ser Gln Glu Ser Glu Asp Asn Phe
80 85 90
Ala Lys Glu Leu Gln Gln Tyr Ile Gln Ala Arg Glu Met Ala Asn
95 100 105
Ala Ala G1n Pro G1u Glu Ser Thr Lys Lys Glu Gly Val Lys Asp
110 115 120
Thr Pro Gln Ala Ala Lys Gln Lys Asn Lys Asn Leu Lys Ala G1y
125 130 135
His Lys Asn Gly Lys Gln Lys Lys Met Lys Arg Lys Trp Pro G1y
140 145 150
Pro Gly Asn Lys Gly Ser Asn Ala Leu Leu Arg Asn Ser Gly Ser
155 160 165
Gln Glu Glu Asp Gly Lys Pro Lys Glu Lys Gln Gln His Leu Ser
170 175 180
Gln Ala Phe Ile Asn Gln His Thr Val Glu Arg Lys Gly Lys Gln
185 190 195
Ile Cys Lys Tyr Phe Leu Glu Arg Lys Cys I1e Lys Gly Asp Gln
200 205 210
Cys Lys Phe Asp His Asp Ala Glu Ile G1u Lys Lys Lys Glu Met
215 220 225
Cys Lys Phe Tyr Val Gln Gly Tyr Cys Thr Arg Gly Glu Asn Cys
230 235 240
Leu Tyr Leu His Asn Glu Tyr Pro Cys Lys Phe Tyr His Thr Gly
245 250 255
Thr Lys Cys Tyr Gln Gly Glu Tyr Cys Lys Phe Ser His Ala Pro
260 265 270
Leu Thr Pro Glu Thr Gln Glu Leu Leu Ala Lys Val Leu Asp Thr
275 280 285
Glu Lys Lys Ser Cys Lys
290
<210> 13
8/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<211> 451
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2472784CD1
<400> 13
Met Ala Gly Ala Gly Pro Ala Pro Gly Leu Pro Gly Ala Gly Gly
1 5 10 15
Pro Val Val Pro Gly Pro Gly Ala Gly Ile Pro Gly Lys Ser Gly
20 25 30
Glu Glu Arg Leu Lys Glu Met Glu Ala Glu Met Ala Leu Phe Glu
35 40 45
Gln Glu Val Leu Gly Ala Pro Val Pro Gly Ile Pro Thr Ala Val
50 55 60
Pro Ala Val Pro Thr Val Pro Thr Val Pro Thr Val Glu Ala Met
65 70 75
Gln Val Pro Ala Ala Pro Val Ile Arg Pro Ile Ile A1a Thr Asn
80 85 90
Thr Tyr Gln Gln Va1 Gln Gln Thr Leu Glu A1a Arg Ala Ala Ala
95 100 105
Ala Ala Thr Val Val Pro Pro Met Val Gly Gly Pro Pro Phe Val
110 115 120
Gly Pro Val Gly Phe Gly Pro Gly Asp Arg Ser His Leu Asp Ser
125 130 135
Pro Glu Ala Arg Glu Ala Met Phe Leu Arg Arg Ala A1a Ala Val
140 145 150
Pro Arg Pro Met Ala Leu Pro Pro Pro His Gln Ala Leu Va1 Gly
155 160 165
Pro Pro Leu Pro Gly Pro Pro Gly Pro Pro Met Met Leu Pro Pro
170 175 180
Met Ala Arg Ala Pro Gly Pro Pro Leu G1y Ser Met Ala Ala Leu
185 190 195
Arg Pro Pro Leu Glu Glu Pro Ala Ala Pro Arg Glu Leu Gly Leu
200 205 210
Gly Leu Gly Leu Gly Leu Lys Glu Lys Glu Glu Ala Val Val A1a
215 220 225
Ala A1a Ala Gly Leu Glu Glu Ala Ser A1a Ala Val Ala Val Gly
230 235 240
Ala Gly Gly Ala Pro Ala Gly Pro Ala Val Ile Gly Pro Ser Leu
245 250 255
Pro Leu A1a Leu Ala Met Pro Leu Pro Glu Pro Glu Pro Leu Pro
260 265 270
Leu Pro Leu Glu Val Val Arg Gly Leu Leu Pro Pro Leu Arg Ile
275 280 285
Pro Glu Leu Leu Ser Leu Arg Pro Arg Pro Arg Pro Pro Arg Pro
290 295 300
Glu Pro Pro Pro Gly Leu Met Ala Leu Glu Val Pro Glu Pro Leu
305 310 315
Gly Glu Asp Lys Lys Lys Gly Lys Pro Glu Lys Leu Lys Arg Cys
320 325 330
Ile Arg Thr Ala Ala G1y Ser Ser Trp Glu Asp Pro Ser Leu Leu
335 340 345
Glu Trp Asp Ala Asp Asp Phe Arg Ile Phe Cys Gly Asp Leu Gly
350 355 360
Asn Glu Val Asn Asp Asp Ile Leu Ala Arg Ala Phe Ser Arg Phe
365 370 375
Pro Ser Phe Leu Lys Ala Lys Val Ile Arg Asp Lys Arg Thr Gly
380 385 390
Lys Thr Lys Gly Tyr Gly Phe Val Ser Phe Lys Asp Pro Ser Asp
395 400 405
Tyr Val Arg Ala Met Arg Glu Met Asn Gly Lys Tyr Val Gly Ser
410 415 420
Arg Pro Ile Lys Leu Arg Lys Ser Met Trp Lys Asp Arg Asn Leu
425 430 435
9/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Asp Val Val Arg Lys Lys Gln Lys Glu Lys Lys Lys Leu Gly Leu
440 445 450
Arg
<210> 14
<211> 600
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2598981CD1
<400> 14
Met Pro Glu Ile Arg Val Thr Pro Leu Gly Ala Gly Gln Asp Val
1 5 10 15
Gly Arg Ser Cys Ile Leu Val Ser Ile Ala Gly Lys Asn Val Met
20 25 30
Leu Asp Cys Gly Met His Met Gly Phe Asn Asp Asp Arg Arg Phe
35 40 45
Pro Asp Phe Ser Tyr Ile Thr Gln Asn Gly Arg Leu Thr Asp Phe
50 55 60
Leu Asp Cys Val Ile Ile Ser His Phe His Leu Asp His Cys Gly
65 70 75
Ala Leu Pro Tyr Phe Ser Glu Met Val Gly Tyr Asp Gly Pro Ile
80 85 90
Tyr Met Thr His Pro Thr Gln Ala Ile Cys Pro Ile Leu Leu Glu
95 100 105
Asp Tyr Arg Lys Ile Ala Val Asp Lys Lys Gly Glu Ala Asn Phe
110 115 120
Phe Thr Ser Gln Met Ile Lys Asp Cys Met Lys Lys Val Val Ala
125 130 135
Val His Leu His Gln Thr Va1 Gln Val Asp Asp Glu Leu Glu Ile
140 145 150
Lys Ala Tyr Tyr Ala Gly His Val Leu Gly Ala Ala Met Phe Gln
155 160 165
Ile Lys Va1 Gly Ser Glu Ser Val Val Tyr Thr Gly Asp Tyr Asn
170 175 180
Met Thr Pro Asp Arg His Leu Gly Ala A1a Trp Ile Asp Lys Cys
185 190 195
Arg Pro Asn Leu Leu Ile Thr Glu Ser Thr Tyr Ala Thr Thr Ile
200 205 210
Arg Asp Ser Lys Arg Cys Arg Glu Arg Asp Phe Leu Lys Lys Val
215 220 225
His Glu Thr Val Glu Arg Gly Gly Lys Val Leu Ile Pro Val Phe
230 235 240
Ala Leu Gly Arg Ala Gln Glu Leu Cys Ile Leu Leu Glu Thr Phe
245 250 255
Trp Glu Arg Met Asn Leu Lys Val Pro Ile Tyr Phe Ser Thr G1y
260 265 270
Leu Thr Glu Lys Ala Asn His Tyr Tyr Lys Leu Phe Ile Pro Trp
275 280 285
Thr Asn Gln Lys Ile Arg Lys Thr Phe Val Gln Arg Asn Met Phe
290 295 300
G1u Phe Lys His Ile Lys Ala Phe Asp Arg Ala Phe Ala Asp Asn
305 310 315
Pro Gly Pro Met Val Val Phe Ala Thr Pro Gly Met Leu His Ala
320 325 330
Gly Gln Ser Leu Gln Ile Phe Arg Lys Trp Ala Gly Asn Glu Lys
335 340 345
Asn Met Val Ile Met Pro Gly Tyr Cys Val Gln Gly Thr Val Gly
350 355 360
His Lys I1e Leu Ser Gly Gln Arg Lys Leu Glu Met Glu Gly Arg
365 370 375
Gln Val Leu Glu Val Lys Met Gln Val Glu Tyr Met Ser Phe Ser
380 385 390
10/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Ala His Ala Asp Ala Lys G1y Ile Met Gln Leu Val Gly Gln Ala
395 400 405
Glu Pro Glu Ser Val Leu Leu Val His Gly Glu Ala Lys Lys Met
410 415 420
Glu Phe Leu Lys Gln Lys Ile Glu Gln G1u Leu Arg Val Asn Cys
425 430 435
Tyr Met Pro Ala Asn G1y Glu Thr Val Thr Leu Pro Thr Ser Pro
440 445 450
Ser Ile Pro Val Gly Ile Ser Leu Gly Leu Leu Lys Arg Glu Met
455 460 465
Ala Gln Gly Leu Leu Pro Glu Ala Lys Lys Pro Arg Leu Leu His
470 475 480
Gly Thr Leu Ile Met Lys Asp Ser Asn Phe Arg Leu Val Ser Ser
485 490 495
Glu Gln Ala Leu Lys Glu Leu Gly Leu Ala Glu His Gln Leu Arg
500 505 510
Phe Thr Cys Arg Val His Leu His Asp Thr Arg Lys Glu Gln Glu
515 520 525
Thr Ala Leu Arg Val Tyr Ser His Leu Lys Ser Val Leu Lys Asp
530 535 540
His Cys Val Gln His Leu Pro Asp Gly Ser Val Thr Val Glu Ser
545 550 555
Val Leu Leu Gln Ala Ala Ala Pro Ser Glu Asp Pro Gly Thr Lys
560 565 570
Val Leu Leu Val Ser Trp Thr Tyr Gln Asp Glu Glu Leu Gly Ser
575 580 585
Phe Leu Thr Ser Leu Leu Lys Lys Gly Leu Pro Gln Ala Pro Ser
590 595 600
<210> 15
<211> 217
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2738075CD1
<400> 15
Met Ser Gly Gly Leu A1a Pro Ser Lys Ser Thr Val Tyr Val Ser
1 5 l0 l5
Asn Leu Pro Phe Ser Leu Thr Asn Asn Asp Leu Tyr Arg Ile Phe
20 25 30
Ser Lys Tyr Gly Lys Va1 Val Lys Val Thr Ile Met Lys Asp Lys
35 40 45
Asp Thr Arg Lys Ser Lys Gly Val Ala Phe Ile Leu Phe Leu Asp
50 55 60
Lys Asp Ser Ala Gln Asn Cys Thr Arg Ala Ile Asn Asn Lys Gln
65 70 75
Leu Phe Gly Arg Val Ile Lys Ala Ser Ile Ala Ile Asp Asn Gly
80 85 90
Arg Ala Ala Glu Phe Ile Arg Arg Arg Asn Tyr Phe Asp Lys Ser
95 100 105
Lys Cys Tyr Glu Cys Gly Glu Ser Gly His Leu Ser Tyr Ala Cys
110 115 120
Pro Lys Asn Met Leu Gly Glu Arg Glu Pro Pro Lys Lys Lys Glu
125 13 0 135
Lys Lys Lys Lys Lys Lys Ala Pro Glu Pro Glu G1u Glu Ile Glu
140 145 150
Glu Va1 Glu Glu Ser Glu Asp Glu Gly Glu Asp Pro Ala Leu Asp
155 160 165
Ser Leu Ser Gln A1a Ile Ala Phe Gln Gln Ala Lys Ile Glu G1u
170 175 180
Glu G1n Lys Lys Trp Lys Pro Ser Ser Gly Val Pro Ser Thr Ser
185 190 195
Asp Asp Ser Arg Arg Pro Arg Ile Lys Lys Ser Thr Tyr Phe Ser
11/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
200 205 210
Asp Glu Glu Glu Leu Ser Asp
215
<210> 16
<211> 319
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2279049CD1
<400> 16
Met Lys Ile Glu Leu Ser Met Gln Pro Trp Asn Pro Gly Tyr Ser
1 5 10 15
Ser Glu Gly Ala Thr Ala Gln Glu Thr Tyr Thr Cys Pro Lys Met
20 25 30
Ile Glu Met Glu Gln Ala Glu Ala Gln Leu Ala Glu Leu Asp Leu
35 40 45
Leu Ala Ser Met Phe Pro Gly Glu Asn Glu Leu Ile Val Asn Asp
50 55 60
G1n Leu Ala Val Ala Glu Leu Lys Asp Cys Ile Glu Lys Lys Thr
65 70 75
Met Glu Gly Arg Ser Ser Lys Val Tyr Phe Thr Ile Asn Met Asn
80 85 90
Leu Asp Val Ser Asp G1u Lys Met Ala Met Phe Ser Leu Ala Cys
95 100 105
Ile Leu Pro Phe Lys Tyr Pro Ala Val Leu Pro Glu Ile Thr Val
110 115 120
Arg Ser Va1 Leu Leu Ser Arg Ser Gln Gln Thr Gln Leu Asn Thr
125 130 135
Asp Leu Thr Ala Phe Leu Gln Lys His Cys His Gly Asp Val Cys
140 145 150
Ile Leu Asn Ala Thr Glu Trp Val Arg G1u His Ala Ser Gly Tyr
155 160 165
Val Ser Arg Asp Thr Ser Ser Ser Pro Thr Thr Gly Ser Thr Val
170 175 180
Gln Ser Val Asp Leu Ile Phe Thr Arg Leu Trp Ile Tyr Ser His
185 ~ 190 195
His Ile Tyr Asn Lys Cys Lys Arg Lys Asn Ile Leu Glu Trp Ala
200 205 210
Lys Glu Leu Ser Leu Ser Gly Phe Ser Met Pro Gly Lys Pro Gly
215 220 225
Val Val Cys Val Glu Gly Pro Gln Ser Ala Cys Glu Glu Phe Trp
230 235 240
Ser Arg Leu Arg Lys Leu Asn Trp Lys Arg Ile Leu Ile Arg His
245 250 255
Arg Glu Asp Ile Pro Phe Asp Gly Thr Asn Asp Glu Thr Glu Arg
260 265 270
Gln Arg Lys Phe Ser Ile Phe Glu Glu Lys Va1 Phe Ser Val Asn
275 280 285
Gly Ala Arg Gly Asn His Met Asp Phe Gly Gln Leu Tyr Gln Phe
290 295 300
Leu Asn Thr Lys Gly Cys Gly Asp Val Phe Gln Met Phe Phe Gly
305 310 315
Val Glu Gly Gln
<210> 17
<211> 108
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2660904CD1
12/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<400> 17
Met Ser His His Ala Glu Ile Gln Arg Asp Ile Leu Glu Ser Cys
1 5 10 15
Asn His Val Arg Lys Lys Val Pro Val Thr Phe Val Gly Ala Gly
20 25 30
Gly Gln Asp Pro Glu Val Pro Glu Glu Leu Leu His Leu Leu Gln
35 40 45
Pro Gly Gln Arg Val Pro Gln Asp Val Gln His His Leu Leu Glu
50 55 60
Pro Arg Asp Arg Trp Ala His Leu Glu Val Leu Lys Lys Val Asp
65 70 75
Leu Leu Leu Gln Val Met Ala Ala Thr Gly Tyr Phe His Ala Ser
80 85 90
Leu Gln Arg Gly Glu Ile Met Arg Ser Pro Gly Pro Val Ala Arg
95 100 105
Asn Ser Pro
<210> 18
<211> 92
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3179424CD1
<400> 18
Met Ala Val Leu Ala Gly Ser Leu Leu Gly Pro Thr Ser Arg Ser
1 5 10 15
Ala Ala Leu Leu Gly G1y Arg Trp Leu Gln Pro Arg Ala Trp Leu
20 25 30
Gly Phe Pro Asp Ala Trp Gly Leu Pro Thr Pro Gln Gln Ala Arg
35 40 45
Gly Lys Ala Arg Gly Asn G1u Tyr Gln Pro Ser Asn Ile Lys Arg
50 55 60
Lys Asn Lys His Gly Trp Val Arg Arg Leu Ser Thr Pro A1a Gly
65 70 75
Val Gln Val Ile Leu Arg Arg Met Leu Lys Gly Arg Lys Ser Leu
80 85 90
Ser His
<210> 19
<211> 268
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2885096CD1
<400> 19
Met Ala Gly Gly Val Pro Gly Gln Pro Ala Gly Val Gly Leu Ala
1 5 10 15
Leu Ile Ala Thr Asp Ser Gln Glu Thr Arg Pro Gly Arg Ala Gly
20 25 30
Pro Gly Ser Gly Glu Ser Leu Ser Ala Ser His Leu Phe Ile Ser
35 40 45
Asp Phe Ala Tyr Cys Trp Glu Asn Phe Val Cys Asn Glu Gly Gln
50 55 60
Pro Phe Met Pro Trp Tyr Lys Phe Asp Asp Asn Tyr Ala Ser Leu
65 70 75
His Arg Thr Leu Lys Glu Ile Leu Arg Asn Pro Met Glu Ala Met
80 85 90
Tyr Pro His Ile Phe Tyr Phe His Phe Lys Asn Leu Leu Lys Ala
95 100 105
13/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Cys Gly Arg Asn Glu Ser Trp Leu Cys Phe Thr Met Glu Val Thr
1'10 115 12 0
Lys His His Ser Ala Val, Phe Arg Lys Lys Gly Val Phe Arg Asn
125 130 135
Gln Val Asp Pro Glu Thr His Cys His Ala Glu Arg Cys Phe Leu
140 145 150
Ser Trp Phe Cys Asp Asp Ile Leu Ser Pro Asn Thr Asn Tyr Glu
155 160 165
Val Thr Trp Tyr Thr Ser Trp Ser Pro Cys Pro Glu Cys Ala Gly
170 175 180
Glu Val A1a Glu Phe Leu Ala Arg His Ser Asn Val Asn Leu Thr
185 190 195
Ile Phe Thr Ala Arg Leu Cys Tyr Phe Trp Asp Thr Asp Tyr Gln
200 205 210
Glu Gly Leu Cys Ser Leu Ser Gln Glu Gly Ala Ser Val Lys Ile
215 220 225
Met Gly Tyr Lys Asp Phe Val Ser Cys Trp Lys Asn Phe Val Tyr
230 235 240
Ser Asp Asp Glu Pro Phe Lys Pro Trp Lys Gly Leu Gln Thr Asn
245 250 255
Phe Arg Leu Leu Lys Arg Arg Leu Arg Glu Ile Leu Gln
260 265
<210> 20
<211> 624
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2901076CD1
<400> 20
Met Asn Ser Gly G1y Gly Phe Gly Leu Gly Leu Gly Phe Gly Leu
1 5 10 15
Thr Pro Thr Ser Val Ile Gln Val Thr Asn~Leu Ser Ser Ala Val
20 25 30
Thr Ser Glu Gln Met Arg Thr Leu Phe Ser Phe Leu Gly Glu Ile
35 40 45
Glu Glu Leu Arg Leu Tyr Pro Pro Asp Asn Ala Pro Leu Ala Phe
50 55 60
Ser Ser Lys Val Cys Tyr Va1 Lys Phe Arg Asp Pro Ser Ser Val
65 70 75
Gly Val Ala Gln His Leu Thr Asn Thr Val Phe Tle Asp Arg Ala
80 85 90
Leu Ile Val Val Pro Cys Ala Glu Gly Lys Ile Pro Glu Glu Ser
95 100 105
Lys Ala Leu Ser Leu Leu Ala Pro A1a Pro Thr Met Thr Ser Leu
110 115 120
Met Pro Gly Ala Gly Leu Leu Pro I1e Pro Thr Pro Asn Pro Leu
125 130 135
Thr Thr Leu G1y Val Ser Leu Ser Ser Leu Gly Ala Ile Pro Ala
140 145 150
Ala Ala Leu Asp Pro Asn Ile Ala Thr Leu Gly Glu Ile Pro Gln
155 160 165
Pro Pro Leu Met Gly Asn Val Asp Pro Ser Lys Ile Asp Glu Ile
170 175 180
Arg Arg Thr Val Tyr Val Gly Asn Leu Asn Ser Gln Thr Thr Thr
185 190 195
Ala Asp Gln Leu Leu Glu Phe Phe Lys Gln Val Gly Glu Val Lys
200 205 210
Phe Val Arg Met Ala Gly Asp Glu Thr Gln Pro Thr Arg Phe Ala
215 220 225
Phe Val Glu Phe Ala Asp Gln Asn Ser VaI Pro Arg A1a Leu Ala
23 0 235 240
Phe Asn Gly Val Met Phe Gly Asp Arg Pro Leu Lys Ile Asn His
245 250 255
14/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Ser Asn Asn Ala Ile Val Lys Pro Pro Glu Met Thr Pro Gln Ala
260 265 270
Ala Ala Lys Glu Leu Glu Glu Val Met Lys Arg Val Arg G1u Ala
275 280 285
Gln Ser Phe Ile Ser Ala Ala Ile Glu Pro Glu Ser Gly Lys Ser
290 295 300
Asn Glu Arg Lys Gly Gly Arg Ser Arg Ser His Thr Arg Ser Lys
305 310 315
Ser Arg Ser Ser Ser Lys Ser His Ser Arg Arg Lys Arg Ser Gln
320 325 330
Ser Lys His Arg Ser Arg Ser His Asn Arg Ser Arg Ser Arg Gln
335 340 345
Lys Asp Arg Arg Arg Ser Lys Ser Pro His Lys Lys Arg Ser Lys
350 355 360
Ser Arg Glu Arg Arg Lys Ser Arg Ser Arg Ser His Ser Arg Asp
365 370 375
Lys Arg Lys Asp Thr Arg Glu Lys Ile Lys Glu Lys Glu Arg Va1
380 385 390
Lys Glu Lys Asp Arg Glu Lys Glu Arg Glu Arg Glu Lys Glu Arg
395 400 405
Glu Lys Glu Lys Glu Arg Gly Lys Asn Lys Asp Arg Asp Lys G1u
410 415 420
Arg G1u Lys Asp Arg Glu Lys Asp Lys Glu Lys Asp Arg Glu Arg
425 430 435
Glu Arg Glu Lys Glu His Glu Lys Asp Arg Asp Lys Glu Lys Glu
440 445 450
Lys Glu Gln Asp Lys Glu Lys Glu Arg Glu Lys Asp Arg Ser Lys
455 460 465
Glu Ile Asp Glu Lys Arg Lys Lys Asp Lys Lys Ser Arg Thr Pro
470 475 480
Pro Arg Ser Tyr Asn Ala Ser Arg Arg Ser Arg Ser Ser Ser Arg
485 490 495
Glu Arg Arg Arg Arg Arg Ser Arg Ser Ser Ser Arg Ser Pro Arg
500 505 510
Thr Ser Lys Thr T1e Lys Arg Lys Ser Ser Arg Ser Pro Ser Pro
515 520 525
Arg Ser Arg Asn Lys Lys Asp Lys Lys Arg Glu Lys Glu Arg Asp
530 535 540
His Ile Ser Glu Arg Arg Glu Arg Glu Arg Ser Thr Ser Met Arg
545 550 555
Lys Ser Ser Asn Asp Arg Asp Gly Lys Glu Lys Leu Glu Lys Asn
560 565 570
Ser Thr Ser Leu Lys Glu Lys Glu His Asn Lys Glu Pro Asp Ser
575 580 585
Ser Val Ser Lys Glu Val Asp Asp Lys Asp Ala Pro Arg Thr Glu
590 595 600
Glu Asn Lys Ile Gln His Asn Gly Asn Cys Gln Leu Asn Glu Glu
605 610 615
Asn Leu Ser Thr Lys Thr Glu Ala Val
620
<210> 21
<211> 419
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3074572CD1
<400> 21
Met Ala Glu Leu Pro Ser Ala Trp Gln Tyr Cys
Ala Val Arg Gly
1 5 10 15
Ala Pro Gly Gln Arg Ala Val Val Gln Phe Ser
Asp Ser Leu Asn
20 25 30
Gly Lys Gln Pro Gly Asn Met Phe Thr Leu Tyr
Leu Ser Arg G1u
35 40 45
15/64
CA 02407435 2002-10-24
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Asn Lys Asp Ser Thr Asn Pro Arg Lys Arg Asn Gln Arg Ile Leu
50 55 60
Ala Ala Glu Thr Asp Arg Leu Ser Tyr Val Gly Asn Asn Phe Gly
65 70 75
Thr Gly Ala Leu Lys Cys Asn Thr Leu Cys Arg His Phe Val Gly
80 85 90
I1e Leu Asn Lys Thr Ser Gly Gln Met Glu Val Tyr Asp Ala Glu
95 100 105
Leu Phe Asn Met Gln Pro Leu Phe Ser Asp Val Ser Val Glu Ser
110 115 120
Glu Leu Ala Leu Glu Ser Gln Thr Lys Thr Tyr Arg G1u Lys Met
125 130 135
Asp Ser Cys Ile Glu Ala Phe Gly Thr Thr Lys Gln Lys Arg Ala
140 145 150
Leu Asn Thr Arg Arg Met Asn Arg Val Gly Asn Glu Ser Leu Asn
155 160 165
Arg Ala Val Ala Lys Ala Ala Glu Thr Ile Ile Asp Thr Lys Gly
170 175 180
Val Thr Ala Leu Val Ser Asp Ala Ile His Asn Asp Leu Gln Asp
185 190 195
Asp Ser Leu Tyr Leu Pro Pro Cys Tyr Asp Asp Ala Ala Lys Pro
200 205 210
Glu Asp Val Tyr Lys Phe Glu Asp Leu Leu Ser Pro Ala Glu Tyr
215 220 225
Glu Ala Leu Gln Ser Pro Ser Glu Ala Phe Arg Asn Val Thr Ser
230 235 240
Glu Glu Ile Leu Lys Met Ile Glu Glu Asn Ser His Cys Thr Phe
245 250 255
Val Ile Glu Ala Leu Lys Ser Leu Pro Ser Asp Val Glu Ser Arg
260 265 270
Asp Arg Gln Ala Arg Cys Ile Trp Phe Leu Asp Thr Leu Ile Lys
275 280 285
Phe Arg Ala His Arg Val Val Lys Arg Lys Ser Ala Leu Gly Pro
290 295 300
Gly Val Pro His Ile Ile Asn Thr Lys Leu Leu Lys His Phe Thr
305 310 315
Cys Leu Thr Tyr Asn Asn Gly Arg Leu Arg Asn Leu I1e Ser Asp
320 325 330
Ser Met Lys Ala Lys Ile Thr Ala Tyr Val Ile Ile Leu Ala Leu
335 340 345
His Ile His Asp Phe Gln Ile Asp Leu Thr Val Leu Gln Arg Asp
350 355 360
Leu Lys Leu Ser Glu Lys Arg Met Met Glu Ile Ala Lys Ala Met
365 370 375
Arg Leu Lys I1e Ser Lys Arg Arg Val Ser Val Ala Ala Gly Ser
380 385 390
Glu Glu Asp His Lys Leu Gly Thr Leu Ser Leu Pro Leu Pro Pro
395 400 405
Ala Gln Thr Ser Asp Arg Leu Ala Lys Arg Arg Lys Ile Thr
410 415
<210> 22
<211> 743
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1437895CD1
<400> 22
Met Glu Glu Glu Gly Leu Glu Cys Pro Asn Ser Ser Ser Glu Lys
1 5 10 15
Arg Tyr Phe Pro Glu Ser Leu Asp Ser Ser Asp Gly Asp Glu Glu
20 25 30
G1u Val Leu Ala Cys Glu Asp Leu Glu Leu Asn Pro Phe Asp Gly
35 40 45
16/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Leu Pro Tyr Ser Ser Arg Tyr Tyr Lys Leu Leu Lys Glu Arg Glu
50 55 60
Asp Leu Pro Ile Trp Lys Glu Lys Tyr Ser Phe Met Glu Asn Leu
65 70 75
Leu Gln Asn Gln Ile Val Ile Val Ser Gly Asp Ala Lys Cys Gly
80 85 90
Lys Ser Ala Gln Val Pro Gln Trp Cys Ala Glu Tyr Cys Leu Ser
95 100 105
Ile His Tyr Gln His Gly Gly Val Ile Cys Thr Gln Val His Lys
110 115 120
Gln Thr Val Val Gln Leu Ala Leu Arg Val Ala Asp Glu Met Asp
125 130 135
Val Asn Ile Gly His Glu Val Gly Tyr Val Ile Pro Phe Glu Asn
140 145 150
Cys Cys Thr Asn Glu Thr Ile Leu Arg Tyr Cys Thr Asp Asp Met
155 160 l65
Leu Gln Arg Glu Met Met Ser Asn Pro Phe Leu Gly Ser Tyr Gly
170 175 180
Val Ile Ile Leu Asp Asp Ile His Glu Arg Ser Ile Ala Thr Asp
185 190 195
Val Leu Leu Gly Leu Leu Lys Asp Val Leu Leu Ala Arg Pro Glu
200 205 210
Leu Lys Leu Ile Ile Asn Ser Ser Pro His Leu Ile Ser Lys Leu
215 220 225
Asn Ser Tyr Tyr Gly Asn Val Pro Val Ile Glu Val Lys Asn Lys
230 235 240
His Pro Val Glu Val Val Tyr Leu Ser Glu Ala Gln Lys Asp Ser
245 250 255
Phe Glu Ser Ile Leu Arg Leu Ile Phe Glu Ile His His Ser Gly
260 265 270
Glu Lys Gly Asp Ile Val Val Phe Leu Ala Cys Glu G1n Asp Ile
275 280 285
Glu Lys Va1 Cys Glu Thr Val Tyr Gln Gly Ser Asn Leu Asn Pro
290 295 300
Asp Leu Gly Glu Leu Val Val Va1 Pro Leu Tyr Pro Lys Glu Lys
305 310 315
Cys Ser Leu Phe Lys Pro Leu Asp G1u Thr Glu Lys Arg Cys Gln
320 325 330
Val Tyr Gln Arg Arg Val Val Leu Thr Thr Ser Ser Gly Glu Phe
335 340 345
Leu Ile Trp Ser Asn Ser Val Arg Phe Val I1e Asp Val Gly Val
350 355 360
Glu Arg Arg Lys Val Tyr Asn Pro Arg Ile Arg Ala Asn Ser Leu
365 370 375
Val Met Gln Pro Ile Ser Gln Ser Gln Ala Glu Ile Arg Lys Gln
380 385 390
Ile Leu Gly Ser Ser Ser Ser Gly Lys Phe Phe Cys Leu Tyr Thr
395 400 405
Glu Glu Phe Ala Ser Lys Asp Met Thr Pro Leu Lys Pro Ala Glu
410 415 420
Met Gln G1u Ala Asn Leu Thr Ser Met Val Leu Phe Met Lys Arg
425 430 435
Ile Asp Ile Ala Gly Leu Gly His Cys Asp Phe Met Asn Arg Pro
440 445 450
Ala Pro Glu Ser Leu Met Gln Ala Leu Glu Asp Leu Asp Tyr Leu
455 460 465
Ala Ala Leu Asp Asn Asp Gly Asn Leu Ser Glu Phe Gly Ile Ile
470 475 480
Met Ser Glu Phe Pro Leu Asp Pro Gln Leu Ser Lys Ser Ile Leu
485 490 495
Ala Ser Cys Glu Phe Asp Cys Val Asp Glu Val Leu Thr Ile A1a
500 505 510
Ala Met Val Thr Ala Pro Asn Cys Phe Ser His Val Pro His Gly
515 520 525
Ala Glu Glu Ala Ala Leu Thr Cys Trp Lys Thr Phe Leu His Pro
530 535 540
Glu Gly Asp His Phe Thr Leu Ile Ser Ile Tyr Lys Ala Tyr Gln
17/64
CA 02407435 2002-10-24
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545 550 555
Asp Thr Thr Leu Asn Ser Ser Ser Glu Tyr Cys Val Glu Lys Trp
560 565 570
Cys Arg Asp Tyr Phe Leu Asn Cys Ser Ala Leu Arg Met Ala Asp
575 580 585
Val Ile Arg Ala Glu Leu Leu Glu Ile Ile Lys Arg Ile Glu Leu
590 595 600
Pro Tyr Ala Glu Pro Ala Phe Gly Ser Lys Glu Asn Thr Leu Asn
605 610 615
Ile Lys Lys Ala Leu Leu Ser Gly Tyr Phe Met Gln Ile Ala Arg
620 625 630
Asp Val Asp Gly Ser Gly Asn Tyr Leu Met Leu Thr His Lys Gln
635 640 645
Val Ala Gln Leu His Pro Leu Ser Gly Tyr Ser Ile Thr Lys Lys
650 655 660
Met Pro Glu Trp Val Leu Phe His Lys Phe Ser Ile Ser G1u Asn
665 670 675
Asn Tyr Ile Arg Ile Thr Ser Glu Ile Ser Pro Glu Leu Phe Met
680 685 690
Gln Leu Val Pro Gln Tyr Tyr Phe Ser Asn Leu Pro Pro Ser Glu
695 700 705
Ser Lys Asp Ile Leu Gln Gln Val Val Asp His Leu Ser Pro Val
710 715 720
Ser Thr Met Asn Lys Glu Gln Gln Met Cys Glu Thr Cys Pro Glu
725 730 735
Thr Glu Gln Arg Cys Thr Leu Gln
740
<210> 23
<211> 284
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1454656CD1
<400> 23
Met Arg Arg Pro Cys Asn Pro Val Arg Ala Ala Lys Arg Thr Ala
1 5 10 15
Ala Ala Ala Arg Ala Pro Arg Gly Leu Glu Val Thr Met Leu Arg
20 25 30
Val Ala Trp Arg Thr Leu Ser Leu Ile Arg Thr Arg Ala Val Thr
35 40 45
Gln Val Leu Val Pro Gly Leu Pro Gly Gly Gly Ser Ala Lys Phe
50 55 60
Pro Phe Asn Gln Trp Gly Leu Gln Pro Arg Ser Leu Leu Leu G1n
65 70 75
Ala Ala Arg Gly Tyr Val Va1 Arg Lys Pro Ala Gln Ser Arg Leu
80 85 90
Asp Asp Asp Pro Pro Pro Ser Thr Leu Leu Lys Asp Tyr Gln Asn
95 100 105
Val Pro Gly Ile Glu Lys Val Asp Asp Val Val Lys Arg Leu Leu
110 115 120
Ser Leu Glu Met Ala Asn Lys Lys Glu Met Leu Lys Ile Lys Gln
12 5 13 0 13 5
Glu Gln Phe Met Lys Lys Ile Val Ala Asn Pro Glu Asp Thr Arg
140 145 150
Ser Leu Glu Ala Arg Ile Ile Ala Leu Ser Val Lys Ile Arg Ser
155 160 165
Tyr Glu Glu His Leu Glu Lys His Arg Lys Asp Lys Ala His Lys
170 175 180
Arg Tyr Leu Leu Met Ser Ile Asp Gln Arg Lys Lys Met Leu Lys
185 190 195
Asn Leu Arg Asn Thr Asn Tyr Asp Val Phe Glu Lys Ile Cys Trp
200 205 210
Gly Leu Gly Ile Glu Tyr Thr Phe Pro Pro Leu Tyr Tyr Arg Arg
18/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
2l5 220 225
Ala His Arg Phe ValThrLys AlaLeu Cys ArgVal
Arg Lys Ile
230 235 240
Phe Gln Glu G1n LysLeuLys ArgArg Arg LeuLys
Thr Lys Ala
245 250 255
Ala Ala Ala Ala GlnLysGln LysArg Arg ProAsp
Ala Ala Asn
260 265 270
Ser Pro A1a Ala IleProLys LeuLys Asp Gln
Lys Thr Ser
275 280
<210> 24
<211> 248
<212> PRT
<213> Homo
Sapiens
<220>
<221> misc_feature
<223> Incyte No: 121130CD1
ID
<400> 24
Met Ala Ala Gln Ser Ala Pro Lys Val Val Leu Lys Ser Thr Thr
1 5 10 15
Lys Met Ser Leu Asn Glu Arg Phe Thr Asn Met Leu Lys Asn Lys
20 25 30
Gln Pro Thr Pro Val Asn Ile Arg Ala Ser Met G1n Gln Gln Gln
35 40 45
Gln Leu Ala Ser Ala Arg Asn Arg Arg Leu Ala Gln Gln Met Glu
50 55 60
Asn Arg Pro Ser Val Gln Ala Ala Leu Lys Leu Lys Gln Ser Leu
65 70 75
Lys Gln Arg Leu Gly Lys Ser Asn Ile Gln Ala Arg Leu Gly Arg
80 85 90
Pro Ile Gly Ala Leu Ala Arg Gly Ala I1e Gly Gly Arg Gly Leu
95 100 105
Pro Ile Ile Gln Arg Gly Leu Pro Arg Gly Gly Leu Arg Gly G1y
110 ll5 120
Arg Ala Thr Arg Thr Leu Leu Arg Gly Gly Met Ser Leu Arg Gly
125 130 135
Gln Asn Leu Leu Arg Gly Gly Arg Ala Val Ala Pro Arg Met Gly
140 145 150
Leu Arg Arg Gly G1y Val Arg Gly Arg G1y Gly Pro Gly Arg G1y
155 160 165
Gly Leu Gly Arg Gly Ala Met Gly Arg Gly Gly Ile Gly Gly Arg
170 175 180
Gly Arg Gly Met Ile Gly Arg Gly Arg Gly Gly Phe Gly Gly Arg
185 190 195
Gly Arg Gly Arg Gly Arg Gly Arg Gly Ala Leu Ala Arg Pro Val
200 205 210
Leu Thr Lys Glu Gln Leu Asp Asn Gln Leu Asp Ala Tyr Met Ser
215 220 225
Lys Thr Lys Gly His Leu Asp Ala Glu Leu Asp Ala Tyr Met Ala
230 235 240
Gln Thr Asp Pro Glu Thr Asn Asp
245
<2l0> 25
<211> 214
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1257715CD1
<400> 25
Met Arg Pro Gly Gly Phe Leu Gly Ala~Gly Gln Arg Leu Ser Arg
1 5 10 15
19/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Ala Met Ser Arg Cys Val Leu Glu Pro Arg Pro Pro Gly Lys Arg
20 25 30
Trp Met Val Ala Gly Leu Gly Asn Pro Gly Leu Pro Gly Thr Arg
35 40 45
His Ser Val Gly Met Ala Val Leu Gly Gln Leu Ala Arg Arg Leu
50 55 60
Gly Val Ala Glu Ser Trp Thr Arg Asp Arg His Cys Ala Ala Asp
65 70 75
Leu Ala Leu Ala Pro Leu Gly Asp Ala Gln Leu Val Leu Leu Arg
80 85 90
Pro Arg Arg Leu Met Asn Ala Asn Gly Arg Ser Val Ala Arg Ala
95 100 105
Ala Glu Leu Phe Gly Leu Thr Ala Glu Glu Val Tyr Leu Val His
110 115 120
Asp Glu Leu Asp Lys Pro Leu Gly Arg Leu Ala Leu Lys Leu Gly
12 5 13 0 13 5
Gly Ser Ala Arg Gly His Asn Gly Val Arg Ser Cys Ile Ser Cys
140 145 150
Leu Asn Ser Asn Ala Met Pro Arg Leu Arg Val Gly Ile Gly Arg
155 160 165
Pro Ala His Pro Glu Ala Val Gln Ala His Val Leu Gly Cys Phe
170 175 180
Ser Pro Ala Glu Gln Glu Leu Leu Pro Leu Leu Leu Asp Arg Ala
185 190 195
Thr Asp Leu Ile Leu Asp His Ile Arg Glu Arg Ser Gln Gly Pro
200 205 210
Ser Leu Gly Pro
<210> 26
<211> 184
<2l2> PRT
<2l3> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1342022CD1
<400> 26
Met Thr Thr Arg Pro Ala Phe Ile Leu His His Ser Asp Cys Phe
1 5 10 15
Ser Ser Arg Ser Ser Arg Ile Arg His Glu Gly Val Trp Arg Arg
20 25 30
Arg Ala Glu Met Ala Pro Arg Lys Gly Lys Glu Lys Lys Glu Glu
35 40 45
Gln Val Ile Ser Leu Gly.Pro Gln Val Ala Glu Gly Glu Asn Val
50 55 60
Phe Gly Va1 Cys His Ile Phe Ala Ser Phe Asn Asp Thr Phe Val
65 70 75
His Val Thr Asp Leu Ser Gly Lys Glu Thr Ile Cys Arg Val Thr
80 85 90
Gly G1y Met Lys Val Lys Ala Asp Arg Asp Glu Ser Ser Pro Tyr
95 100 105
Ala Ala Met Leu Ala Ala Gln Asp Val Ala Gln Arg Cys Lys Glu
110 115 120
Leu Gly Ile Thr Ala Leu His Ile Lys Leu Arg Ala Thr Gly Gly
125 130 135
Asn Arg Thr Lys Thr Pro Gly Pro Gly Ala Gln Ser Ala Leu Arg
140 145 150
Ala Leu Ala Arg Ser Gly Met Lys Ile Gly Arg Ile Glu Asp Val
155 160 165
Thr Pro Ile Pro Ser Asp Ser Thr Arg Arg Lys Gly Gly Arg Arg
170 175 180
Gly Arg Arg Leu
<210> 27
20/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<211> 371
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 194704CD1
<400> 27
Met Ser Ala Gln Ala Gln Met Arg Ala Leu Leu Asp Gln Leu Met
1 5 10 15
Gly Thr Ala Arg Asp Gly Asp Glu Thr Arg Gln Arg Val Lys Phe
20 25 30
Thr Asp Asp Arg Val Cys Lys Ser His Leu Leu Asp Cys Cys Pro
35 40 45
His Asp Ile Leu Ala Gly Thr Arg Met Asp Leu Gly Glu Cys Thr
50 55 60
Lys Ile His Asp Leu Ala Leu Arg Ala Asp Tyr Glu Ile Ala Ser
65 70 75
Lys G1u Arg Asp Leu Phe Phe Glu Leu Asp Ala Met Asp His Leu
80 85 90
Glu Ser Phe Ile Ala Glu Cys Asp Arg Arg Thr Glu Leu Ala Lys
95 100 105
Lys Arg Leu Ala Glu Thr Gln Glu Glu Ile Ser Ala Glu Val Ser
110 115 120
Ala Lys Ala Glu Lys Val His Glu Leu Asn Glu Glu Ile Gly Lys
125 13 0 135
Leu Leu Ala Lys Ala Glu Gln Leu Gly Ala Glu Gly Asn Val Asp
140 145 150
Glu Ser Gln Lys Ile Leu Met Glu Val Glu Lys Val Arg A1a Lys
155 160 165
Lys Lys Glu Ala Glu Glu Glu Tyr Arg Asn Ser Met Pro Ala Ser
170 175 180
Ser Phe Gln Gln Gln Lys Leu Arg Val Cys Glu Val Cys Ser Ala
185 190 195
Tyr Leu Gly Leu His Asp Asn Asp Arg Arg Leu A1a Asp His Phe
200 205 210
Gly Gly Lys Leu His Leu Gly Phe Ile Gln Ile Arg Glu Lys Leu
215 220 225
Asp Gln Leu Arg Lys Thr Val Ala Glu Lys Gln Glu Lys Arg Asn
230 235 240
Gln Asp Arg Leu Arg Arg Arg Glu Glu Arg Glu Arg Glu Glu Arg
245 250 255
Leu Ser Arg Arg Ser Gly Ser Arg Thr Arg Asp Arg Arg Arg Ser
260 265 270
Arg Ser Arg Asp Arg Arg Arg Arg Arg Ser Arg Ser Thr Ser Arg
275 280 285
Glu Arg Arg Lys Leu Ser Arg Ser Arg Ser Arg Asp Arg His Arg
290 295 300
Arg His Arg Ser Arg Ser Arg Ser His Ser Arg Gly His Arg Arg
305 310 315
Ala Ser Arg Asp Arg Ser Ala Lys Tyr Lys Phe Ser Arg Glu Arg
320 325 330
Ala Ser Arg Glu Glu Ser Trp Glu Ser Gly Arg Ser Glu Arg Gly
335 340 345
Pro Pro Asp Trp Arg Leu Glu Ser Ser Asn Gly Lys Met Ala Ser
350 355 360
Arg Arg Ser Glu Glu Lys Glu Ala Gly Glu Ile
365 370
<210> 28
<211> 396
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
21/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<223> Incyte ID No: 607270CD1
<400> 28
Met Ala Ala Pro Cys Val Ser Tyr Gly Gly Ala Val Ser Tyr Arg
1 5 10 15
Leu Leu Leu Trp Gly Arg Gly Ser Leu Ala Arg Lys Gln Gly Leu
20 25 30
Trp Lys Thr Ala Ala Pro Glu Leu Gln Thr Asn Val Arg Ser Gln
35 4D 45
Ile Leu Arg Leu Arg His Thr Ala Phe Val Ile Pro Lys Lys Asn
50 55 60
Val Pro Thr Ser Lys Arg Glu Thr Tyr Thr Glu Asp Phe Ile Lys
65 70 75
Lys Gln Ile Glu Glu Phe Asn Ile Gly Lys Arg His Leu Ala Asn
80 85 90
Met Met Gly Glu Asp Pro Glu Thr Phe Thr Gln Glu Asp Ile Asp
95 100 105
Arg Ala Ile Ala Tyr Leu Phe Pro Ser Gly Leu Phe Glu Lys Arg
110 115 120
Ala Arg Pro Val Met Lys His Pro Glu Gln Ile Phe Pro Arg Gln
125 130 135
Arg Ala Ile Gln Trp Gly Glu Asp Gly Arg Pro Phe His Tyr Leu
140 145 150
Phe Tyr Thr Gly Lys Gln Ser Tyr Tyr Ser Leu Met His Asp Val
155 160 165
Tyr Gly Met Leu Leu Asn Leu Glu Lys His Gln Ser His Leu~Gln
170 175 180
Ala Lys Ser Leu Leu Pro Glu Lys Thr Val Thr Arg Asp Val Ile
185 190 195
Gly Ser Arg Trp Leu Ile Lys Glu Glu Leu Glu Glu Met Leu Val
200 205 210
Glu Lys Leu Ser Asp Leu Asp Tyr Met Gln Phe Ile Arg Leu Leu
215 220 225
Glu Lys Leu Leu Thr Ser Gln Cys Gly Ala Ala Glu Glu Glu Phe
230 235 240
Val Gln Arg Phe Arg Arg Ser Val Thr Leu Glu Ser Lys Lys Gln
245 250 255
Leu Ile Glu Pro Val Gln Tyr Asp G1u G1n Gly Met Ala Phe Ser
260 265 270
Lys Ser Glu Gly Lys Arg Lys Thr Ala Lys Ala Glu Ala Ile Val
275 280 285
Tyr Lys His Gly Ser Gly Arg Ile Lys Val Asn Gly Ile Asp Tyr
290 295 300
Gln Leu Tyr Phe Pro Ile Thr Gln Asp Arg Glu Gln Leu Met Phe
305 310 315
Pro Phe His Phe Val Asp Arg Leu Gly Lys His Asp Val Thr Cys
320 325 330
Thr Val Ser Gly Gly Gly Arg Ser Ala Gln Ala G1y Ala Ile Arg
335 340 345
Leu Ala Met Ala Lys Ala Leu Cys Ser Phe Val Thr Glu Asp Glu
350 355 360
Val Glu Trp Met Arg Gln Ala Gly Leu Leu Thr Thr Asp Pro Arg
365 370 375
Val Arg Glu Arg Lys Lys Pro Gly Gln Glu Gly Ala Arg Arg Lys
380 385 390
Phe Thr Trp Lys Lys Arg
395
<210> 29
<211> 184
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 758546CD1
22/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<400> 29
Met Val Arg Lys Leu Lys Phe His Glu Gln Lys Leu Leu Lys Gln
1 5 10 15
Val Asp Phe Leu Asn Trp Glu Val Thr Asp His Asn Leu His Glu
20 25 30
Leu Arg Val Leu Arg Arg Tyr Arg Leu Gln Arg Arg Glu Asp Tyr
35 40 45
Thr Arg Tyr Asn Gln Leu Ser Arg Ala Val Arg Glu Leu Ala Arg
50 55 60
Arg Leu Arg Asp Leu Pro Glu Arg Asp Gln Phe Arg Val Arg Ala
65 70 75
Ser Ala Ala Leu Leu Asp Lys Leu Tyr Ala Leu G1y Leu Val Pro
80 85 90
Thr Arg Gly Ser Leu Glu Leu Cys Asp Phe Val Thr Ala Ser Ser
95 100 105
Phe Cys Arg Arg Arg Leu Pro Thr Val Leu Leu Lys Leu Arg Met
110 115 120
Ala Gln His Leu Gln Ala Ala Val Ala Phe Val Glu Gln Gly His
125 130 135
Val Arg Val Gly Pro Asp Val Val Thr Asp Pro Ala Phe Leu Val
140 145 150
Thr Arg Ser Met Glu Asp Phe Val Thr Trp Val Asp Ser Ser Lys
155 160 165
Ile Lys Arg His Val Leu Glu Tyr Asn Glu Glu Arg Asp Asp Phe
170 175 180
Asp Leu Glu Ala
<210> 30
<211> 282
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 866043CD1
<400> 30
Met Leu Leu Ser Thr Ser Met Asp Lys Thr Phe Lys Val Trp Asn
1 5 10 15
Ala Val Asp Ser Gly His Cys Leu Gln Thr Tyr Ser Leu His Thr
20 25 30
Glu Ala Val Arg Ala Ala Arg Trp Ala Pro Cys Gly Arg Arg Ile
35 40 45
Leu Ser Gly Gly Phe Asp Phe Ala Leu His Leu Thr Asp Leu Glu
50 55 60
Thr Gly Thr Gln Leu Phe Ser Gly Arg Ser Asp Phe Arg Ile Thr
65 70 75
Thr Leu Lys Phe His Pro Lys Asp His Asn Ile Phe Leu Cys Gly
80 85 90
Gly Phe Ser Ser Glu Met Lys Ala Trp Asp Ile Arg Thr Gly Lys
95 100 105
Val Met Arg Ser Tyr Lys Ala Thr Ile Gln G1n Thr Leu Asp Ile
110 115 120
Leu Phe Leu Arg Glu Gly Ser Glu Phe Leu Ser Ser Thr Asp A1a
125 130 135
Ser Thr Arg Asp Ser Ala Asp Arg Thr Ile Ile Ala Trp Asp Phe
140 145 150
Arg Thr Ser Ala Lys Ile Ser Asn Gln Ile Phe His Glu Arg Phe
155 160 165
Thr Cys Pro Ser Leu Ala Leu His Pro Arg Glu Pro Val Phe Leu
170 175 180
Ala Gln Thr Asn Gly Asn Tyr Leu Ala Leu Phe Ser Thr Val Trp
185 190 195
Pro Tyr Arg Met Ser Arg Arg Arg Arg Tyr Glu Gly His Lys Val
200 205 210
Glu Gly Tyr Ser Val Gly Cys Glu Cys Ser Pro Gly Gly Asp Leu
23/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
215 220 225
Leu Val Thr Gly Ser Ala Asp Gly Arg Val Leu Met Tyr Ser Phe
230 235 240
Arg Thr Ala Ser Arg Ala Cys Thr Leu Gln Gly His Thr Gln Ala
245 250 255
Cys Val Gly Thr Thr Tyr His Pro Val Leu Pro Ser Val Leu Ala
260 265 270
Thr Cys Ser Trp Gly Gly Asp Met Lys Ile Trp His
275 280
<210> 31
<211> 125
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 927065CD1
<400> 31
Met Pro Ala Pro Ala Ala Thr Tyr Glu Arg Val Val Tyr Lys Asn
1 5 10 15
Pro Ser Glu Tyr His Tyr Met Lys Val Cys Leu Glu Phe Gln Asp
20 25 30
Cys Gly Val Gly Leu Asn Ala Ala Gln Phe Lys Gln Leu Leu Ile
35 40 45
Ser Ala Val Lys Asp Leu Phe Gly Glu Val Asp Ala Ala Leu Pro
50 55 60
Leu Asp Ile Leu Thr Tyr G1u G1u Lys Thr Leu Ser Ala Ile Leu
65 70 75
Arg Tle Cys Ser Ser Gly Leu Val Lys Leu Trp Ser Ser Leu Thr
80 85 90
Leu Leu Arg Ile Pro Ile Lys Gly Lys Lys Cys Ala Phe Arg Val
95 100 105
T1e Gln Val Ser Pro Phe Leu Leu Ala Leu Ser Gly Asn Ser Arg
110 115 120
Glu Leu Val Leu Asp
125
<210> 32
<211> 365
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 938071CD1
<400> 32
Met Ala Pro Val Ser Gly Ser Arg Ser Pro Asp Arg Glu Ala Ser
1 5 10 15
Gly Ser Gly Gly Arg Arg Arg Ser Ser Ser Lys Ser Pro Lys Pro
20 25 30
Ser Lys Ser Ala Arg Ser Pro Arg Gly Arg Arg Ser Arg Ser His
35 40 45
Ser Cys Ser Arg Ser Gly Asp Arg Asn Gly Leu Thr His Gln Leu
50 55 60
Gly G1y Leu Ser Gln Gly Ser Arg Asn Gln Ser Tyr Arg Ser Arg
65 70 . 75
Ser Arg Ser Arg Ser Arg Glu Arg Pro Ser Ala Pro Arg G1y Ile
80 85 90
Pro Phe Ala Ser Ala Ser Ser Ser Val Tyr Tyr Gly Ser Tyr Ser
95 100 105
Arg Pro Tyr Gly Ser Asp Lys Pro Trp Pro Ser Leu Leu Asp Lys
110 115 120
Glu Arg Glu Glu Ser Leu Arg Gln Lys Arg Leu Ser Glu Arg Glu
125 130 135
24/64
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Arg I1e Gly Glu Leu Gly Ala Pro Glu Val Trp Gly Leu Ser Pro
140 145 150
Lys Asn Pro Glu Pro Asp Ser Asp Glu His Thr Pro Val Glu Asp
155 160 165
Glu Glu Pro Lys Lys Ser Thr Thr Ser Ala Ser Thr Ser Glu Glu
170 175 180
Glu Lys Lys Lys Lys Ser Ser Arg Ser Lys Glu Arg Ser Lys Lys
185 190 195
Arg Arg Lys Lys Lys Ser Ser Lys Arg Lys His Lys Lys Tyr Ser
200 205 210
Glu Asp Ser Asp Ser Asp Ser Asp Ser Glu Thr Asp Ser Ser Asp
215 220 225
Glu Asp Asn Lys Arg Arg A1a Lys Lys Ala Lys Lys Lys Glu Lys
230 235 240
Lys Lys Lys His Arg Ser Lys Lys Tyr Lys Lys Lys Arg Ser Lys
245 250 255
Lys Ser Arg Lys Glu Ser Ser Asp Ser Ser Ser Lys Glu Ser Gln
260 265 270
Glu Glu Phe Leu Glu Asn Pro Trp Lys Asp Arg Thr Lys Ala Glu
275 280 285
Glu Pro Ser Asp Leu Ile Gly Pro Glu Ala Pro Lys Thr Leu Thr
290 295 300
Ser G1n Asp Asp Lys Pro Leu Lys His Arg Arg Met Glu Ala Val
305 310 315
Arg Leu Arg Lys Glu Asn Gln Ile Tyr Ser Ala Asp Glu Lys Arg
320 325 330
Ala Leu Ala Ser Phe Asn Gln Glu Glu Arg Arg Lys Arg Glu Asn
335 340 345
Lys Ile Leu Ala Ser Phe Arg Glu Met Val Tyr Arg Lys Thr Lys
350 355 360
Gly Lys Asp Asp Lys
365
<210> 33
<211> 672
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3295984CD1
<400> 33
Met Arg Ser Ile Arg Ser Phe Ala Asn Asp Asp Arg His Val Met
1 5 10 15
Val Lys His Ser Thr Ile Tyr Pro Ser Pro Glu Glu Leu Glu Ala
20 25 30
Val Gln Asn Met Val Ser Thr Val Glu Cys Ala Leu Lys His Val
35 40 45
Ser Asp Trp Leu Asp Glu Thr Asn Lys Gly Thr Lys Thr G1u Gly
50 55 60
Glu Thr Glu Val Lys Lys Asp Glu Ala Gly Glu Asn Tyr Ser Lys
65 70 75
Asp Gln Gly Gly Arg Thr Leu Cys Gly Val Met Arg Tle Gly Leu
80 85 90
Val Ala Lys Gly Leu Leu Ile Lys Asp Asp Met Asp Leu Glu Leu
95 200 105
Val Leu Met Cys Lys Asp Lys Pro Thr Glu Thr Leu Leu Asn Thr
110 115 120
Val Lys Asp Asn Leu Pro Ile Gln Ile Gln Lys Leu Thr Glu Glu
125 130 135
Lys Tyr Gln Val Glu G1n Cys Val Asn Glu Ala Ser Ile Ile Ile
140 145 150
Arg Asn Thr Lys Glu Pro Thr Leu Thr Leu Lys Val Ile Leu Thr
155 160 165
Ser Pro Leu Ile Arg Asp Glu Leu Glu Lys Lys Asp Gly Glu Asn
170 175 180
25/64
CA 02407435 2002-10-24
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Val Ser Met Lys Asp Pro Pro Asp Leu Leu Asp Arg Gln Lys Cys
185 190 195
Leu Asn Ala Leu Ala Ser Leu Arg His Ala Lys Trp Phe Gln Ala
200 205 210
Arg Ala Asn Gly Leu Lys Ser Cys Val Ile Val Leu Arg Ile Leu
215 220 225
Arg Asp Leu Cys Asn Arg Val Pro Thr Trp Ala Pro Leu Lys Gly
230 235 240
Trp Pro Leu Glu Leu Ile Cys Glu Lys Ser Ile Gly Thr Cys Asn
245 250 255
Arg Pro Leu Gly Ala Gly Glu Ala Leu Arg Arg Val Met Glu Cys
260 265 270
Leu Ala Ser Gly Ile Leu Leu Pro Gly Gly Pro Gly Leu His Asp
275 280 285
Pro Cys Glu Arg Asp Pro Thr Asp Ala Leu Ser Tyr Met Thr Ile
290 295 300
Gln Gln Lys Glu Asp Ile Thr His Ser Ala Gln His Ala Leu Arg
305 310 315
Leu Ser Ala Phe Gly Gln Ile Tyr Lys Val Leu Glu Met Asp Pro
320 325 330
Leu Pro Ser Ser Lys Pro Phe Gln Lys Tyr Ser Trp Ser Val Thr
335 340 345
Asp Lys Glu Gly Ala Gly Ser Ser Ala Leu Lys Arg Pro Phe Glu
350 355 360
Asp Gly Leu Gly Asp Asp Lys Asp Pro Asn Lys Lys Met Lys Arg
365 370 375
Asn Leu Arg Lys Ile Leu Asp Ser Lys Ala Ile Asp Leu Met Asn
380 385 390
Ala Leu Met Arg Leu Asn Gln Ile Arg Pro Gly Leu Gln Tyr Lys
395 400 405
Leu Leu Ser Gln Ser Gly Pro Va1 His Ala Pro Val Phe Thr Met
410 415 420
Ser Val Asp Val Asp Gly Thr Thr Tyr G1u Ala Ser Gly Pro Ser
425 430 435
Lys Lys Thr Ala Lys Leu His Val Ala Val Lys Val Leu Gln Ala
440 445 450
Met Gly Tyr Pro Thr Gly Phe Asp Ala Asp Ile Glu Cys Met Ser
455 460 465
Ser Asp Glu Lys Ser Asp Asn Glu Ser Lys Asn Glu Thr Va1 Ser
470 475 480
Ser Asn Ser Ser Asn Asn Thr Gly Asn Ser Thr Thr G1u Thr Ser
485 490 495
Ser Thr Leu Glu Val Arg Thr Gln Gly Pro Ile Leu Thr Ala Ser
500 505 510
Gly Lys Asn Pro Val Met Glu Leu Asn Glu Lys Arg Arg Gly Leu
515 520 525
Lys Tyr Glu Leu Ile Ser Glu Thr Gly Gly Ser His Asp Lys Arg
530 535 540
Phe Val Met Glu Val Glu Val Asp Gly Gln Lys Phe Arg Gly Ala
545 550 555
Gly Pro Asn Lys Lys Val Ala Lys Ala Ser Ala Ala Leu Ala Ala
560 565 570
Leu Glu Lys Leu Phe Ser Gly Pro Asn Ala Ala Asn Asn Lys Lys
575 580 585
Lys Lys Ile Ile Pro Gln Ala Lys Gly Val Val Asn Thr Ala Val
590 595 600
Ser Ala Ala Val Gln Ala Val Arg Gly Arg Gly Arg Gly Thr Leu
605 610 615
Thr Arg Gly Ala Phe Val Gly Ala Thr Ala Ala Pro Gly Tyr Ile
620 625 630
Ala Pro Gly Tyr Gly Thr Pro Tyr Gly Tyr Ser Thr Ala Ala Pro
63 5 64 0 645
Ala Tyr Gly Leu Pro Lys Arg Met Val Leu Leu Pro Val Met Lys
650 655 660
Phe Pro Thr Tyr Pro Val Pro His Tyr Ser Phe Phe
665 670
26/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<210> 34
<211> 430
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4545237CD1
<400> 34
Met Ala Thr Ala Val Arg Ala Val Gly Cys Leu Pro Val Leu Cys
1 5 10 15
Ser Gly Thr Ala Gly His Leu Leu Gly Arg Gln Cys Ser Leu Asn
20 25 30
Thr Leu Pro Ala Ala Ser Tle Leu Ala Trp Lys Ser Va1 Leu Gly
35 40 45
Asn Gly His Leu Ser Ser Leu Gly Thr Arg Asp Thr His Pro Tyr
50 55 60
Ala Ser Leu Ser Arg Ala Leu Gln Thr G1n Cys Cys Ile Ser Ser
65 70 75
Pro Ser His Leu Met Ser Gln Gln Tyr Arg Pro Tyr Ser Phe Phe
80 85 90
Thr Lys Leu Thr Ala Asp Glu Leu Trp Lys Gly Ala Leu Ala Glu
95 100 105
Thr Gly Ala Gly Ala Lys Lys Gly Arg Gly Lys Arg Thr Lys Lys
110 115 120
Lys Lys Arg Lys Asp Leu Asn Arg Gly G1n Ile Ile Gly Glu Gly
125 130 135
Arg Tyr Gly Phe Leu Trp Pro Gly Leu Asn Va1 Pro Leu Met Lys
140 145 150
Asn Gly Ala Val Gln Thr Ile Ala Gln Arg Ser Lys Glu Glu Gln
155 160 165
Glu Lys Val Glu Ala Asp Met Ile Gln Gln Arg Glu Glu Trp Asp
170 175 180
Arg Lys Lys Lys Met Lys Val Lys Arg Glu Arg Gly Trp Ser Gly
185 190 195
Asn Ser Trp Gly Gly Ile Ser Leu Gly Pro Pro Asp Pro Gly Pro
200 205 210
Cys G1y Glu Thr Tyr Glu Asp Phe Asp Thr Arg Ile Leu Glu Val
215 220 225
Arg Asn Val Phe Thr Met Thr Ala Lys Glu Gly Arg Lys Lys Ser
230 235 240
Ile Arg Val Leu Val Ala Val Gly Asn G1y Lys Gly Ala Ala Gly
245 250 255
Phe Ser Ile Gly Lys Ala Thr Asp Arg Met Asp A1a Phe Arg Lys
260 265 270
Ala Lys Asn Arg Ala Val His His Leu His Tyr Ile Glu Arg Tyr
275 280 285
Glu Asp His Thr Ile Phe His Asp Ile Ser Leu Arg Phe Lys Arg
290 295 300
Thr His Ile Lys Met Lys Lys Gln Pro Lys Gly Tyr Gly Leu Arg
305 310 315
Cys His Arg Ala Ile I1e Thr Ile Cys Arg Leu Ile Gly Ile Lys
320 325 330
Asp Met Tyr Ala Lys Val Ser Gly Ser Ile Asn Met Leu Ser Leu
335 340 345
Thr Gln Gly Leu Phe Arg Gly Leu Ser Arg Gln Glu Thr His Gln
350 355 360
Gln Leu Ala Asp Lys Lys Gly Leu His Val Val Glu Ile Arg Glu
365 370 375
Glu Cys Gly Pro Leu Pro Ile Val Val Ala Ser Pro Arg Gly Pro
380 385 390
Leu Arg Lys Asp Pro Glu Pro Glu Asp Glu Val Pro Asp Val Lys
395 400 405
Leu Asp Trp Glu Asp Val Lys Thr Ala Gln Gly Met Lys Arg Ser
410 415 420
Val Trp Ser Asn Leu Lys Arg Ala Ala Thr
27/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
425 430
<210> 35
<211> 137
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4942964CD1
<400> 35
Met Ala Asp Ser Lys Ala Thr Ser Ala Val Thr Leu Arg Thr Arg
1 5 10 15
Lys Phe Met Thr Asn Arg Leu Leu Ala Arg Lys Gln Phe Va1 Leu
20 25 30
Glu Val Ile His Pro Gly Arg Ala Asn Val Ser Lys Ala Glu Leu
35 40 45
Lys Glu Arg Leu Ala Lys Ala Tyr Glu Val Lys Asp Pro Asn Thr
50 55 60
Ile Phe Val Phe Lys Phe Arg Thr His Phe Gly Gly Gly Lys Ser
65 70 75
Thr Gly Phe Gly Leu Ile Tyr Asp Asn Leu Glu Ala Ala Lys Lys
80 85 90
Phe Glu Pro Lys Tyr Arg Leu Ile Arg Asn Gly Leu Ala Thr Lys
95 100 105
Val Glu Lys Ser Arg Lys Gln Met Lys G1u Arg Lys Asn Arg Ala
110 115 120
Lys Lys Ile Arg Gly Val Lys Lys Thr Lys Ala Gly Asp Ala Lys
125 130 135
Lys Lys
<210> 36
<211> 380
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5702144CD1
<400> 36
Met Arg Ser Arg Val Leu Trp Gly Ala Ala Arg Trp Leu Trp Pro
1 5 10 15
Arg Arg Ala Val Gly Pro Ala Arg Arg Pro Leu Ser Ser Gly Ser
20 25 30
Pro Pro Leu Glu Glu Leu Phe Thr Arg Gly Gly Pro Leu Arg Thr
35 40 45
Phe Leu Glu Arg Gln Ala Gly Ser Glu Ala His Leu Lys Val Arg
50 55 60
Arg Pro Glu Leu Leu A1a Val Ile Lys Leu Leu Asn Glu Lys G1u
65 70 75
Gln Glu Leu Arg Glu Thr Glu His Leu Leu His Asp Glu Asn Glu
80 85 90
Asp Leu Arg Lys Leu Ala Glu Asn Glu Ile Thr Leu Cys Gln Lys
95 100 105
Glu Ile Thr Gln Leu Lys His Gln Ile Ile Leu Leu Leu Val Pro
110 115 120
Ser Glu Glu Thr Asp Glu Asn Asp Leu Ile Leu Glu Val Thr Ala
125 130 135
Gly Val Gly Gly Gln Glu Ala Met Leu Phe Thr Ser Glu Ile Phe
140 145 150
Asp Met Tyr Gln Gln Tyr Ala Ala Phe Lys Arg Trp His Phe Glu
155 160 165
Thr Leu Glu Tyr Phe Pro Ser Glu Leu Gly Gly Leu Arg His Ala
170 175 180
28/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Ser Ala Ser Ile Gly Gly Ser Glu Ala Tyr Arg His Met Lys Phe
185 190 195
Glu Gly Gly Val His Arg Val Gln Arg Val Pro Lys Thr Glu Lys
200 205 2l0
Gln Gly Arg Ile His Thr Ser Thr Met Thr Val Ala Ile Leu Pro
215 220 225
Gln Pro Thr Glu Ile Asn Leu Val Ile Asn Pro Lys Asp Leu Arg
230 235 240
Ile Asp Thr Lys Arg Ala Ser Gly Ala Gly Gly Gln His Val Asn
245 250 255
Thr Thr Asp Ser Ala Val Arg Ile Val His Leu Pro Thr Gly Val
260 265 270
Val Ser Glu Cys Gln Gln Glu Arg Ser Gln Leu Lys Asn Lys Glu
275 280 285
Leu Ala Met Thr Lys Leu Arg Ala Lys Leu Tyr Ser Met His Leu
290 295 300
Glu Glu Glu Ile Asn Lys Arg Gln Asn Ala Arg Lys Ile Gln Ile
305 310 315
Gly Ser Lys Gly Arg Ser Glu Lys Ile Arg Thr Tyr Asn Phe Pro
320 325 330
Gln Asn Arg Val Thr Asp His Arg Ile Asn Lys Thr Leu His Asp
335 340 345
Leu Glu Thr Phe Met Gln Gly Asp Tyr Leu Leu Asp Glu Leu Val
350 355 360
Gln Ser Leu Lys Glu Tyr Ala Asp Tyr Glu Ser Leu Val Glu Ile
365 370 375
Ile Ser Gln Lys Val
380
<210> 37
<2ll> 206
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5862945CD1
<400> 37
Met Ala Ala Ala Val Leu Gly G1n Leu Gly Ala Leu Trp Ile His
1 5 10 15
Asn Leu Arg Ser Arg Gly Lys Leu Ala Leu Gly Val Leu Pro Gln
20 25 30
Ser Tyr Ile His Thr Ser Ala Ser Leu Asp Ile Ser Arg Lys Trp
35 40 45
G1u Lys Lys Asn Lys Ile Val Tyr Pro Pro Gln Leu Pro Gly Glu
50 55 60
Pro Arg Arg Pro Ala Glu Ile Tyr His Cys Arg Arg Gln Ile Lys
65 70 75
Tyr Ser Lys Asp Lys Met Trp Tyr Leu Ala Lys Leu Ile Arg Gly
80 85 90
Met Ser Ile Asp Gln Ala Leu Ala Gln Leu Glu Phe Asn Asp Lys
95 100 105
Lys Gly Ala Lys Ile Ile Lys Glu Val Leu Leu Glu Ala Gln Asp
110 115 120
Met A1a Val Arg Asp His Asn Val Glu Phe Arg Ser Asn Leu Tyr
12 5 l3 0 13 5
Ile Ala Glu Ser Thr Ser Gly Arg Gly Gln Cys Leu Lys Arg Ile
140 145 150
Arg Tyr His Gly Arg Gly Arg Phe Gly Ile Met Glu Lys Val Tyr
155 160 165
Cys His Tyr Phe Val Lys Leu Val Glu Gly Pro Pro Pro Pro Pro
170 175 180
Glu Pro Pro Lys Thr Ala Val A1a His A1a Lys Glu Tyr Ile Gln
185 190 195
Gln Leu Arg Ser Arg Thr Ile Val His Thr Leu
200 205
29/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<210> 38
<211> 190
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6319547CD1
<400> 38
Met Glu Ala Glu Thr Lys Thr Leu Pro Leu Glu Asn Ala Ser Ile
1 5 10 15
Leu Ser Glu Gly Ser Leu Gln G1u Gly His Arg Leu Trp Ile Gly
20 25 30
Asn Leu Asp Pro Lys Ile Thr Glu Tyr His Leu Leu Lys Leu Leu
35 40 45
Gln Lys Phe Gly Lys Val Lys Gln Phe Asp Phe Leu Phe His Lys
50 55 60
Ser Gly A1a Leu Glu Gly Gln Pro Arg Gly Tyr Cys Phe Val Asn
65 70 75
Phe Glu Thr Lys Gln Glu Ala G1u Gln Ala Ile Gln cys Leu Asn
80 85 90
Gly Lys Leu Ala Leu Ser Lys Lys Leu Val Val Arg Trp Ala His
95 100 105
Ala Gln Val Lys Arg Tyr Asp His Asn Lys Asn Asp Lys Ile Leu
110 115 120
Pro Ile Ser Leu Glu Pro Ser Ser Ser Thr Glu Pro Thr Gln Ser
12 5 13 0 13 5
Asn Leu Ser Val Thr Ala Lys Ile Lys Ala Ile Glu Ala Lys Leu
140 145 150
Lys Met Met Ala Glu Asn Pro Asp Ala Glu Tyr Pro Ala Ala Pro
155 160 165
Val Tyr Ser Tyr Phe Lys Pro Pro Asp Lys Lys Arg Thr Thr Pro
170 175 180
Tyr Ser Arg Thr Ala Trp Lys Ser Arg Arg
185 190
<210> 39
<211> 434
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 000124CD1
<400> 39
Met Leu Arg Cys Leu Tyr His Trp His Arg Pro Val Leu Asn Arg
1 5 10 15
Arg Trp Ser Arg Leu Cys Leu Leu Lys Gln Tyr Leu Phe Thr Met
20 25 30
Lys Leu Gln Ser Pro Glu Phe Gln Ser Leu Phe Thr Glu Gly Leu
35 40 45
Lys Ser Leu Thr Glu Leu Phe Val Lys Glu Asn His Glu Leu Arg
50 55 60
Ile Ala Gly Gly Ala Val Arg Asp Leu Leu Asn Gly Val Lys Pro
65 70 75
Gln Asp Ile Asp Phe Ala Thr Thr Ala Thr Pro Thr Gln Met Lys
80 85 90
Glu Met Phe Gln Ser Ala Gly Ile Arg Met Ile Asn Asn Arg Gly
95 100 105
Glu Lys His Gly Thr Ile Thr Ala Arg Leu His Glu Glu Asn Phe
110 115 120
Glu Ile Thr Thr Leu Arg Ile Asp Val Thr Thr Asp GIy Arg His
125 130 135
Ala Glu Val Glu Phe Thr Thr Asp Trp Gln Lys Asp Ala Glu Arg
140 145 150
30/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Arg Asp Leu Thr Ile Asn Ser Met Phe Leu Gly Phe Asp Gly Thr
155 160 165
Leu Phe Asp Tyr Phe Asn Gly Tyr Glu Asp Leu Lys Asn Lys Lys
170 175 180
Val Arg Phe Val G1y His Ala Lys Gln Arg Ile Gln Glu Asp Tyr
185 190 195
Leu Arg Ile Leu Arg Tyr Phe Arg Phe Tyr Gly Arg Ile Val Asp
200 205 210
Lys Pro Gly Asp His Asp Pro Glu Thr Leu Glu Ala Ile Ala Glu
215 220 225
Asn Ala Lys Gly Leu Ala Gly Ile Ser Gly Glu Arg Ile Trp Val
230 235 240
Glu Leu Lys Lys Ile Leu Val Gly Asn His Val Asn His Leu Ile
245 250 255
His Leu Ile Tyr Asp Leu Asp Val Ala Pro Tyr Ile Gly Leu Pro
260 265 270
Ala Asn Ala Ser Leu Glu Glu Phe Asp Lys Val Ser Lys Asn Va1
275 280 285
Asp Gly Phe Ser Pro Lys Pro Val Thr Leu Leu Ala Ser Leu Phe
290 295 300
Lys Val Gln Asp Asp Val Thr Lys Leu Asp Leu Arg Leu Lys Ile
305 310 315
Ala Lys Glu Glu Lys Asn Leu Gly Leu Phe Ile Val Lys Asn Arg
320 325 330
Lys Asp Leu Ile Lys Ala Thr Asp Ser Ser Asp Pro Leu Lys Pro
335 340 345
Tyr Gln Asp Phe Ile Ile Asp Ser Arg Glu Pro Asp Ala Thr Thr
350 355 360
Arg Val Cys Glu Leu Leu Lys Tyr Gln Gly Glu His Cys Leu Leu
365 370 375
Lys Glu Met Gln Gln Trp Ser Ile Pro Pro Phe Pro Val Ser Gly
380 385 390
His Asp Ile Arg Lys Val Gly Ile Ser Ser Gly Lys Glu Ile Gly
395 400 405
Ala Leu Leu Gln Gln Leu Arg Glu Gln Trp Lys Lys Ser Gly Tyr
410 415 420
Gln Met Glu Lys Asp Glu Leu Leu Ser Tyr Ile Lys Lys Thr
425 430
<210> 40
<211> 339
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1659474CD1
<400> 40
Met Ala Ala Gly Cys Ser Glu Ala Pro Arg Pro Thr Ala Ala Ser
1 5 10 15
Asp Gly Ser Leu Val Gly Gln Ala Gly Val Leu Pro Cys Leu Glu
20 25 30
Leu Pro Thr Tyr Ala Ala Ala Cys Ala Leu Val Asn Ser Arg Tyr
35 40 45
Ser Cys Leu Val Ala Gly Pro His Gln Arg His Ile Ala Leu Ser
50 ~ 55 60
Pro Arg Tyr Leu Asn Arg Lys Arg Thr Gly Ile Arg Glu Gln Leu
65 70 75
Asp Ala Glu Leu Leu Arg Tyr Ser G1u Ser Leu Leu Gly Val Pro
80 85 90
Ile Ala Tyr Asp Asn Ile Lys Val Val Gly Glu Leu Gly Asp Ile
95 100 105
Tyr Asp Asp Gln Gly His Ile His Leu Asn Ile Glu Ala Asp Phe
110 115 120
Val Ile Phe Cys Pro Glu Pro Gly Gln Lys Leu Met Gly Ile Val
125 130 135
31/64
CA 02407435 2002-10-24
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Asn Lys Val Ser Ser Ser His Ile Gly Cys Leu Val His Gly Cys
140 145 150
Phe Asn Ala Ser Ile Pro Lys Pro Glu Gln Leu Ser Ala Glu Gln
155 160 165
Trp Gln Thr Met Glu Ile Asn Met Gly Asp Glu Leu Glu Phe Glu
170 175 180
Val Phe Arg Leu Asp Ser Asp Ala Ala Gly Val Phe Cys Tle Arg
185 190 195
Gly Lys Leu Asn Ile Thr Ser Leu Gln Phe Lys Arg Ser Glu Val
200 205 210
Ser Glu Glu Val Thr Glu Asn Gly Thr Glu Glu Ala Ala Lys Lys
215 220 225
Pro Lys Lys Lys Lys Lys Lys Lys Asp Pro Glu Thr Tyr Glu Val
230 235 240
Asp Ser Gly Thr Thr Lys Leu Ala Asp Asp Ala Asp Asp Thr Pro
245 250 255
Met Glu G1u Ser Ala Leu Gln Asn Thr Asn Asn Ala Asn Gly Ile
260 265 270
Trp Glu Glu Glu Pro Lys Lys Lys Lys Lys Lys Lys Lys His Gln
275 280 285
Glu Val Gln Asp Gln Asp Pro Val Phe Gln Gly Ser Asp Ser Ser
290 295 300
Gly Tyr Gln Ser Asp His Lys Lys Lys Lys Lys Glu Lys Lys Thr
305 310 315
Asn Ser Glu Glu Ala Glu Phe Thr Pro Pro Leu Lys Cys Ser Pro
320 325 330
Lys Arg Lys Gly Lys Ser Asn Phe Leu
335
<210> 41
<211> 599
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2267892CD1
<400> 41
Met Asp Val His Asp Leu Phe Arg Arg Leu Gly Ala Gly Ala Lys
1 5 10 15
Phe Asp Thr Arg Arg Phe Ser Ala Asp Ala Ala Arg Phe Gln Ile
20 25 30
Gly Lys Arg Lys Tyr Asp Phe Asp Ser Ser Glu Val Leu Gln Gly
35 40 45
Leu Asp Phe Phe Gly Asn Lys Lys Ser Val Pro Gly Val Cys Gly
50 55 60
Ala Ser Gln Thr His Gln Lys Pro G1n Asn Gly Glu Lys Lys Glu
65 70 75
Glu Ser Leu Thr Glu Arg Lys Arg Glu Gln Ser Lys Lys Lys Arg
80 85 90
Lys Thr Met Thr Ser Glu Ile Ala Ser Gln Glu Glu Gly Ala Thr
95 100 105
Ile Gln Trp Met Ser Ser Val Glu Ala Lys Ile Glu Asp Lys Lys
110 115 120
Val Gln Arg Glu Ser Lys Leu Thr Ser Gly Lys Leu Glu Asn Leu
125 130 135
Arg Lys Glu Lys Ile Asn Phe Leu Arg Asn Lys His Lys I1e His
140 145 150
Val Gln Gly Thr Asp Leu Pro Asp Pro Ile Ala Thr Phe Gln Gln
155 160 165
Leu Asp Gln Glu Tyr Lys Ile Asn Ser Arg Leu Leu G1n Asn Ile
170 175 180
Leu Asp Ala Gly Phe Gln Met Pro Thr Pro Ile Gln Met Gln Ala
185 190 195
Ile Pro Val Met Leu His Gly Arg Glu Leu Leu Ala Ser Ala Pro
200 205 210
32/64
CA 02407435 2002-10-24
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Thr Gly Ser Gly Lys Thr Leu Ala Phe Ser Ile Pro Ile Leu Met
215 220 225
Gln Leu Lys Gln Pro Ala Asn Lys Gly Phe Arg Ala Leu Ile Ile
230 235 240
Ser Pro Thr Arg Glu Leu Ala Ser Gln Ile His Arg Glu Leu Ile
245 250 255
Lys Ile Ser Glu Gly Thr Gly Phe Arg Ile His Met Ile His Lys
260 265 270
Ala A1a Val Ala Ala Lys Lys Phe Gly Pro Lys Ser Ser Lys Lys
275 280 285
Phe Asp Ile Leu Val Thr Thr Pro Asn Arg Leu Ile Tyr Leu Leu
290 295 300
Lys Gln Asp Pro Pro Gly Ile Asp Leu Ala Ser Val Glu Trp Leu
305 310 315
Val Val Asp Glu Ser Asp Lys Leu Phe Glu Asp Gly Lys Thr Gly
320 325 330
Phe Arg Asp Gln Leu Ala Ser I1e Phe Leu Ala Cys Thr Ser His
335 340 345
Lys Val Arg Arg Ala Met Phe.Ser Ala Thr Phe Ala Tyr Asp Val
350 355 360
Glu Gln Trp Cys Lys Leu Asn Leu Asp Asn Val Ile Ser Val Ser
365 370 375
Ile Gly Ala Arg Asn Ser Ala Val Glu Thr Val Glu Gln Glu Leu
380 385 390
Leu Phe Val Gly Ser G1u Thr Gly Lys Leu Leu Ala Met Arg Glu
395 400 405
Leu Val Lys Lys Gly Phe Asn Pro Pro Val Leu Val Phe Val Gln
410 415 420
Ser Ile Glu Arg Ala Lys Glu Leu Phe His Glu Leu Ile Tyr Glu
425 430 435
Gly Ile Asn Val Asp Val Ile His Ala Glu Arg Thr Gln Gln Gln
440 445 450
Arg Asp Asn Thr Val His Ser Phe Arg Ala Gly Lys Ile Trp Val
455 460 465
Leu Ile Cys Thr Ala Leu Leu Ala Arg Gly Ile Asp Phe Lys Gly
470. 475 480
Val Asn Leu Val Ile Asn Tyr Asp Phe Pro Thr Ser Ser Val Glu
485 490 495
Tyr Ile His Arg Ile Gly Arg Thr Gly Arg Ala Gly Asn Lys Gly
500 505 510
Lys Ala Ile Thr Phe Phe Thr Glu Asp Asp Lys Pro Leu Leu Arg
515 520 525
Ser Val Ala Asn Val Ile Gln Gln Ala Gly Cys Pro Val Pro Glu
530 535 540
Tyr Ile Lys Gly Phe Gln Lys Leu Leu Ser Lys Gln Lys Lys Lys
545 550 555
Met Ile Lys Lys Pro Leu Glu Arg Glu Ser Ile Ser Thr Thr Pro
560 565 570
Lys Cys Phe Leu Glu Lys A1a Lys Asp Lys Gln Lys Lys Val Thr
575 580 585
Gly Gln Asn Ser Lys Lys Lys Val Ala Leu Glu Asp Lys Ser
590 595
<210> 42
<211> 334
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2670307CD1
<400> 42
Met Ala Ala Gly Ser Gly Met Ala Lys Thr Trp G1u
Ser Gln Leu
1 5 10 15
Ala Asn Asn Gln Glu Ala Gln Ser Asp Glu Ile Tyr
Met Ile Lys
20 25 30
33/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Tyr Asp Lys Lys Gln Gln Gln Glu Ile Leu Ala Ala Lys Pro Gly
35 40 45
Leu Arg Ile His His Tyr Phe Lys Tyr Cys Lys Ile Ser Ala Leu
50 55 60
Ala Leu Leu Lys Met Val Met His Ala Arg Ser Gly Gly Asn Leu
65 70 75
Glu Val Met Gly Leu Met Leu Gly Lys Val Asp Gly Glu Thr Met
80 85 90
Ile Ile Met Asp Ser Phe Ala Leu Pro Val Glu Gly Thr Glu Thr
95 100 105
Arg Val Asn Ala Gln Ala Ala Ala Tyr Glu Tyr Met Ala Ala Tyr
110 115 120
Ile Glu Asn Ala Lys Gln Val Gly Arg Leu Glu Asn Ala Ile Gly
125 13 0 13 5
Trp Tyr His Ser His Pro Gly Tyr Gly Cys Trp Leu Ser Gly Ile
140 145 150
Asp Val Ser Thr Gln Met Leu Asn Gln Gln Phe G1n Glu Pro Phe
155 160 165
Val Ala Val Val Ile Asp Pro Thr Arg Thr Ile Ser Ala Gly Lys
170 175 280
Val Asn Leu Gly Ala Phe Arg Thr Tyr Pro Lys Gly Tyr Lys Pro
185 190 195
Pro Asp Glu Gly.Pro Ser Glu Tyr Gln Thr Ile Pro Leu Asn Lys
200 205 210
Ile Glu Asp Phe Gly Val His Cys Lys Gln Tyr Tyr Ala Leu Glu
215 220 225
Val Ser Tyr Phe Lys Ser Ser Leu Asp Arg Lys Leu Leu Glu Leu
230 235 240
Leu Trp Asn Lys Tyr Trp Val Asn Thr Leu Ser Ser Ser Ser Leu
245 250 255
Leu Thr Asn A1a Asp Tyr Thr Thr Gly Gln Va1 Phe Asp Leu Ser
260 265 270
Glu Lys Leu Glu Gln Ser Glu Ala Gln Leu Gly Arg Gly Ser Phe
275 280 285
Met Leu Gly Leu Glu Thr His Asp Arg Lys Ser Glu Asp Lys Leu
290 295 300
Ala Lys Ala Thr Arg Asp Ser Cys Lys Thr Thr Ile G1u Ala Ile
305 310 315
His Gly Leu Met Ser Gln Val Ile Lys Asp Lys Leu Phe Asn Gln
320 325 330
Ile Asn Ile Ser
<210> 43
<211> 448
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4524210CD1
<400> 43
Met Asn Lys Glu Ile Val Thr Ala Leu Gly Lys Gln Glu Ala Glu
1 5 10 15
Arg Lys Phe Glu Thr Leu Leu Lys His Leu Ser His Pro Pro Ser
20 25 30
Phe Thr Thr Val Arg Val Asn Thr His Leu Ala Ser Val Gln His
35 40 45
Val Lys Asn Leu Leu Leu Asp Glu Leu Gln Lys Gln Phe Asn Gly
50 55 60
Leu Ser Val Pro Ile Leu Gln His Pro Asp Leu Gln Asp Val Leu
65 70 75
Leu Ile Pro Val Ile Gly Pro Arg Lys Asn Ile Lys Lys Gln Gln
80 85 90
Cys Glu Ala Ile Val Gly Ala G1n Cys Gly Asn Ala Val Leu Arg
95 100 105
34/64
CA 02407435 2002-10-24
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Gly Ala His Val Tyr Ala Pro Gly Ile Val Ser Ala Ser Gln Phe
110 115 120
Met Lys Ala Gly Asp Val Ile Ser Val Tyr Ser Asp Ile Lys Gly
125 130 135
Lys Cys Lys Lys Gly Ala Lys Glu Phe Asp Gly Thr Lys Val Phe
140 145 150
Leu Gly Asn Gly Ile Ser Glu Leu Ser Arg Lys Glu Ile Phe Ser
155 160 165
Gly Leu Pro Glu Leu Lys Gly Met Gly Ile Arg Met Thr Glu Pro
170 175 180
Val Tyr Leu Ser Pro Ser Phe Asp Ser Val Leu Pro Arg Tyr Leu
185 190 195
Phe Leu Gln Asn Leu Pro Ser Ala Leu Val Ser His Val Leu Asn
200 205 210
Pro Gln Pro Gly Glu Lys Ile Leu Asp Leu Cys Ala Ala Pro Gly
215 220 225
Gly Lys Thr Thr His Ile Ala Ala Leu Met His Asp Gln Gly Glu
230 235 240
Val Ile Ala Leu Asp Lys Ile Phe Asn Lys Val Glu Lys Ile Lys
245 250 255
Gln Asn Ala Leu Leu Leu Gly Leu Asn Ser Ile Arg Ala Phe Cys
260 265 270
Phe Asp Gly Thr Lys A1a Val Lys Leu Asp Met Val Glu Asp Thr
275 280 285
Glu Gly Glu Pro Pro Phe Leu Pro Glu Ser Phe Asp Arg Ile Leu
290 295 300
Leu Asp Ala Pro Cys Ser Gly Met Gly Gln Arg Pro Asn Met Ala
305 310 315
Cys Thr Trp Ser Val Lys Glu Val Ala Ser Tyr Gln Pro Leu Gln
320 325 330
Arg Lys Leu Phe Thr Ala Ala_Val Gln Leu Leu Lys Pro Glu Gly
335 340 345
Val Leu Val Tyr Ser Thr Cys Thr Ile Thr Leu Ala Glu Asn Glu
350 355 360
Glu Gln Val Ala Trp Ala Leu Thr Lys Phe Pro Cys Leu G1n Leu
365 370 375
Gln Pro Gln Glu Pro Gln Ile Gly Gly Glu Gly Met Arg Gly Ala
380 385 390
Gly Leu Ser Cys Glu Gln Leu Lys Gln Leu Gln Arg Phe Asp Pro
395 400 405
Ser Ala Val Pro Leu Pro Asp Thr Asp Met Asp Ser Leu Arg G1u
410 415 420
Ala Arg Arg Glu Asp Met Leu Arg Leu Ala Asn Lys Asp Ser Ile
425 43 0 435
Gly Phe Phe I1e A1a Lys Phe Val Lys Cys Lys Ser Thr
440 445
<210> 44
<211> 420
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5584860CD1
<400> 44
Met Ala Thr Ser Leu Gly Ser Asn Thr Tyr Asn Arg Gln Asn Trp
1 5 10 15
Glu Asp Ala Asp Phe Pro Ile Leu Cys Gln Thr Cys Leu Gly Glu
20 25 30
Asn Pro Tyr Ile Arg Met Thr Lys Glu Lys Tyr Gly Lys Glu Cys
35 40 45
Lys Ile Cys Ala Arg Pro Phe Thr Val Phe Arg Trp Cys Pro Gly
50 55 60
Val Arg Met Arg Phe Lys Lys Thr Glu Val Cys Gln Thr Cys Ser
65 70 75
35/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
Lys Leu Lys Asn Val Cys Gln Thr Cys Leu Leu Asp Leu Glu Tyr
80 85 90
Gly Leu Pro Ile Gln Val Arg Asp Ala Gly Leu Ser Phe Lys Asp
95 100 105
Asp Met Pro Lys Ser Asp Val Asn Lys Glu Tyr Tyr Thr Gln Asn
110 115 120
Met Glu Arg Glu Ile Ser Asn Ser Asp Gly Thr Arg Pro Val Gly
125 130 135
Met Leu Gly Lys A1a Thr Ser Thr Ser Asp Met Leu Leu Lys Leu
140 145 150
Ala Arg Thr Thr Pro Tyr Tyr Lys Arg Asn Arg Pro His Ile Cys
155 160 165
Ser Phe Trp Val Lys Gly Glu Cys Lys Arg Gly Glu Glu Cys Pro
170 175 180
Tyr Arg His Glu Lys Pro Thr Asp Pro Asp Asp Pro Leu Ala Asp
185 190 195
Gln Asn Ile Lys Asp Arg Tyr Tyr Gly I1e Asn Asp Pro Val Ala
200 205 210
Asp Lys Leu Leu Lys Arg Ala Ser Thr Met Pro Arg Leu Asp Pro
215 220 225
Pro Glu Asp Lys Thr Ile Thr Thr Leu Tyr Val Gly Gly Leu Gly
230 235 240
Asp Thr Ile Thr Glu Thr Asp Leu Arg Asn His Phe Tyr Gln Phe
245 250 255
Gly Glu I1e Arg Thr Ile Thr Val Val Gln Arg Gln Gln Cys Ala
260 265 270
Phe Ile Gln Phe Ala Thr Arg Gln Ala Ala Glu Val Ala Ala Glu
275 280 285
Lys Ser Phe Asn Lys Leu Ile Val Asn Gly Arg Arg Leu Asn Val
290 295 300
Lys Trp Gly Arg Ser Gln Ala Ala Arg Gly Lys Glu Lys Glu Lys
305 310 315
Asp Gly Thr Thr Asp Ser Gly Ile Lys Leu Glu Pro Val Pro Gly
320 325 330
Leu Pro Gly Ala Leu Pro Pro Pro Pro Ala Ala Glu Glu Glu Ala
335 340 345
Ser Ala Asn Tyr Phe Asn Leu Pro Pro Ser Gly Pro Pro Ala Val
350 355 360
Val Asn Ile Ala Leu Pro Pro Pro Pro Gly I1e Ala Pro Pro Pro
365 370 375
Pro Pro G1y Phe Gly Pro His Met Phe His Pro Met G1y Pro Pro
380 385 390
Pro Pro Phe Met Arg Ala Pro Gly Pro Ile His Tyr Pro Ser Gln
395 400 405
Asp Pro Gln Arg Met Gly Ala His Ala Gly Lys His Ser Ser Pro
410 415 420
<210> 45
<211> 137
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5807892CD1
<400> 45
Met Val His Leu Thr Thr Leu Leu Cys Lys Ala Tyr Arg Gly Gly
1 5 10 15
His Leu Thr Ile Arg Leu Ala Leu Gly Gly Cys Thr Asn Arg Pro
20 25 30
Phe Tyr Arg Ile Va1 Ala Ala His Asn Lys Cys Pro Arg Asp Gly
35 40 45
Arg Phe Val Glu Gln Leu Gly Ser Tyr Asp Pro Leu Pro Asn Ser
50 55 60
His Gly Glu Lys Leu Val Ala Leu Asn Leu Asp Arg Ile Arg His
36/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
65 70 75
Trp Ile Gly Cys Gly Ala His Leu Ser Lys Pro Met Glu Lys Leu
80 85 90
Leu Gly Leu Ala Gly Phe Phe Pro Leu His Pro Met Met Ile Thr
95 100 105
Asn Ala Glu Arg Leu Arg Arg Lys Arg Ala Arg Glu Val Leu Leu
110 115 120
Ala Ser G1n Lys Thr Asp Ala Glu Ala Thr Asp Thr Glu Ala Thr
125 13 0 135
Glu Thr
<210> 46
<211> 556
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3210044CD1
<400> 46
Met Met Asn Leu Pro Phe Asn Arg Asp Ala Val Phe Tyr His Glu
1 5 10 15
Asp G1u Thr Asn Cys Leu Leu Leu Ile Met Ala Pro Ser Phe Thr
20 25 30
Ala Arg Ile Gln Leu Phe Leu Leu Arg Ala Leu Gly Phe Leu Ile
35 40 45
Gly Leu Val Gly Arg Ala Ala Leu Val Leu Gly Gly Pro Lys Phe
50 55 60
Ala Ser Lys Thr Pro Arg Pro Val Thr Glu Pro Leu Leu Leu Leu
65 70 75
Ser Gly Met Gln Leu Ala Lys Leu Ile Arg Gln Arg Lys Val Lys
80 85 90
Cys Ile Asp Val Val Gln Ala Tyr Ile Asn Arg Ile Lys Asp Val
95 100 105
Asn Pro Met Ile Asn Gly Ile Va1 Lys Tyr Arg Phe Glu Glu Ala
110 115 120
Met Lys Glu Ala His Ala Val Asp Gln Lys Leu Ala Glu Lys Gln
125 130 135
Glu Asp Glu A1a Thr Leu Glu Asn Lys Trp Pro Phe Leu Gly Val
140 145 150
Pro Leu Thr Val Lys Glu Ala Phe G1n Leu Gln Gly Met Pro Asn
155 160 165
Ser Ser Gly Leu Met Asn Arg Arg Asp Ala Ile Ala Lys Thr Asp
170 175 180
Ala Thr Val Val Ala Leu Leu Lys Gly Ala Gly Ala Ile Pro Leu
185 190 195
Gly Ile Thr Asn Cys Ser Glu Leu Cys Met Trp Tyr Glu Ser Ser
200 205 210
Asn Lys Ile Tyr Gly Arg Ser Asn Asn Pro Tyr Asp Leu Gln His
215 220 225
Ile Val Gly Gly Ser Ser Gly G1y Glu Gly Cys Thr Leu Ala Ala
230 235 240
Ala Cys Ser Val Ile Gly Val Gly Ser Asp Ile Gly Gly Ser Ile
245 250 255
Arg Met Pro Ala Phe Phe Asn Gly Ile Phe Gly His Lys Pro Ser
260 265 270
Pro Gly Val Val Pro Asn Lys Gly Gln Phe Pro Leu Ala Val Gly
275 280 285
Ala Gln Glu Leu Phe Leu Cys Thr Gly Pro Met Cys Arg Tyr A1a
290 295 300
Glu Asp Leu Ala Pro Met Leu Lys Val Met Ala Gly Pro Gly Ile
305 310 315
Lys Arg Leu Lys Leu Asp Thr Lys Val His Leu Lys Asp Leu Lys
320 325 330
Phe Tyr Trp Met Glu His Asp Gly Gly Ser Phe Leu Met Ser Lys
37/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
335 340 345
Val Asp Gln Asp Leu Ile Met Thr Gln Lys Lys Val Val Val His
350 355 360
Leu Glu Thr Ile Leu Gly Ala Ser Val Gln His Val Lys Leu Lys
365 370 375
Lys Met Lys Tyr Ser Phe Gln Leu Trp Ile Ala Met Met Ser Ala
380 385 390
Lys Gly His Asp Gly Lys Glu Pro Val Lys Phe Val Asp Leu Leu
395 400 405
Gly Asp His Gly Lys His Val Ser Pro Leu Trp Glu Leu Ile Lys
410 415 420
Trp Cys Leu Gly Leu Ser Val Tyr Thr Ile Pro Ser Ile Gly Leu
425 430 435
Ala Leu Leu Glu Glu Lys Leu Arg Tyr Ser Asn Glu Lys Tyr Gln
440 445 450
Lys Phe Lys Ala Val Glu Glu Ser Leu Arg Lys Glu Leu Val Asp
455 460 465
Met Leu Gly Asp Asp Gly Val Phe Leu Tyr Pro Ser His Pro Thr
470 475 480
Val Ala Pro Lys His His Val Pro Leu Thr Arg Pro Phe Asn Phe
485 490 495
Ala Tyr Thr Gly Val Phe Ser Ala Leu G1y Leu Pro Val Thr Gln
500 505 510
Cys Pro Leu Gly Leu Asn Ala Lys Gly Leu Pro Leu Gly Ile Gln
515 520 525
Val Val Ala Gly Pro Phe Asn Asp His Leu Thr Leu Ala Val Ala
530 535 540
Gln Tyr Leu Glu Lys Thr Phe Gly Gly Trp Val Cys Pro Gly Lys
545 550 555
Phe
<210> 47
<211> 111
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4942454CD1
<400> 47
Met Lys Phe Val Ala Ala Tyr Leu Leu Ala Val Leu Ala Gly Asn
l 5 10 15
Ser Ser Pro Ser Ala Glu Asp Leu Thr Ala Ile Leu Glu Ser Val
20 25 30
Gly Cys Glu Val Asp Asn Glu Lys Met Glu Leu Leu Leu Ser Gln
35 40 45
Leu Ser Gly Lys Asp Ile Thr Glu Leu Ile Ala Ala Gly Arg Glu
50 55 60
Lys Phe Ala Ser Val Pro Cys Gly Gly Gly Gly Val Ala Val Ala
65 70 75
Ala Ala Ala Pro Ala Ala Gly Gly Ala Pro Ala Ala Glu Ala Lys
80 85 90
Lys Glu Glu Lys Val Glu Glu Lys Glu Glu Ser Asp Asp Asp Met
95 100 105
Gly Phe Ser Leu Phe Asp
110
<210> 48
<211> 882
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1622129CB1
38/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<400> 48
cccacgcgtc cgcggagccg ccgggagctg tagttctccc gcggctcaga gaagtaggca 60
gagagcggac ctggcggccg ggcagcatgg cggggctgga gctcttgtcg gaccagggct 120
accgggtgga cgggcggcgc gccggggagc tgcgcaagat ccaggcgcgg atgggcgtgt 180
tcgcgcaggc tgacggctcg gcctacattg agcagggcaa caccaaggca ctggctgtgg 240
tctacggccc gcacgagatc cggggctccc gggctcgagc cctgccggac agggccctag 300
tgaactgtca atatagttca gcgaccttca gcacaggtga gcgcaagcga cggccacatg 360
gggaccgtaa gtcctgtgag atgggcctgc agctccgcca gactttcgaa gcagccatcc 420
tcacacagct gcacccacgc tcccagattg atatctatgt gcaggtgcta caggcagatg 480
gtgggaccta tgcagcttgt gtgaatgcag ccacgctggc agtgctggat gccgggatac 540
ccatgagaga ctttgtgtgt gcgtgctcag ctggcttcgt ggacggcaca gccctggcgg 600
acctcagcca tgtggaggaa gcagctggtg gcccccagct ggccctggcc ctgctgccag 660
cctcaggaca gattgcgctg cttgagatgg atgcccggct gcacgaggac cacctggagc 720
gggtgttgga ggctgctgcc caggctgccc gagatgtgca caccctctta gatcgagtgg 780
tccggcagca tgtgcgtgag gcctctatct tgctggggga ctgaccaccc agccacccat 840
gtccagaata aaaccctcct ctgcccacaa aaaaaaaaaa as 882
<210> 49
<211> 1220
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1820078CB1
<400> 49
ctcaacttta gcccgccgga agcggaagtc aggtggttgt cggattttag aggaaggcgc 60
tcggttacat tggagaactg gagtggtctg gagttccacg gtgtagtgga ccagaggcca 120
cctctcctgg gcttctcagt gtctcgccgg cggggttcgg cctgagctgg attgacatag 180
cccttggcgg atttaaacaa cctaaacatt aagcagtaca gctgcctcaa acctttggga 240
ttttcagaat gactgacact gccgaagctg ttccaaagtt tgaagagatg tttgctagta 300
gattcacaga aaatgacaag gagtatcagg aatacctgaa acgccctcct gagtctcctc 360
caattgttga ggaatggaat agcagagctg gtgggaacca aagaaacaga ggcaatcggt 420
tgcaagacaa cagacagttc agaggcaggg acaacagatg ggggtggcca agtgacaatc 480
gatccaatca gtggcatgga cgatcctggg gtaacaacta cccgcaacac agacaagaac 540
cttactatcc ccagcaatat ggacattatg gttacaacca gcggcctcct tacggttact 600
actgatagaa atgttggcag cttttagtaa aagcatttac tctgttacca tgagaaaagt 660
ttgggtgtct tctgttggtc atagttttac atctgatttt acagaatgga ttattgattt 720
tttggaagtt gagactttaa aaaaaataga tcttacttgc gaaatgcgat ggttgctggg 780
aatacctgaa actgtggatt atattgcttg acttctacct cagaatcttc tttgtttcat 840
gacttaatag tgctttaagt ttggtatatt atttgacctc taggaattct ttgttttaca 900
cagaaataaa aattttaaaa tagaaaatgc ttttactttg taaggtaaga gagtatccat 960
atgcttagat gtgctcgttt ctaaaattct agaggttgat ataatcagct catgaatgca 1020
cagctatgct ttttgtgata gattgtacat aacatcagca gttgaaaggt aaaacaattg 1080
cttttttttt tttttgcatt tgttaagtga ctatggtact ttgtgattcc ttaatctata 1140
gatgagtcag ctccacactt gagtctcttt ttagagggaa atcagtaata aagctgtaaa 1200
ataaggaagg aaaaaaaaaa 1220
<210> 50
<211> 2020
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1527017CB2
<220>
<221> unsure
<222> 1905, 1909, 1936, 1942, 1959, 1967, 1972, 1979, 1989, 1999, 2002
<223> a, t, c, g, or other
<400> 50
gagaggagtg agtgccgtca ccgagggccg cgccagactg cgacggatac agggagggca 60
agggtttcct tttggcgctt ccctttggac cccggagtga aaaactctaa cgtccagatc 120
agtggagaga aacgcagatt taggaccctg aggagtcttt ttcacccgtt tcccgtcact 180
39/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
cgctcaggcg cgccgagggc agtccttgtg gggtcctcgt ggccagccaa gatggttgcc 240
cccgcagtga aggttgcccg aggatggtcg ggcctggcgt tgggcgtgcg gcgggctgtc 300
ttgcagcttc caggggctaa ctcaggtgag atggagccgc tatagtcctg aattcaagga 360
tcccttgatt gacaaggaat attatcgcaa gccagtggag gagctaactg aggaggagaa 420
atatgttcgg gagctcaaga agactcagct catcaaagct gctccagcag gggaaaacaa 480
gttctgtgtt tgaagaccca gtcatcagta aattcaccaa catgatgatg ataggaggaa 540
acaaagtact ggccagatcc ctcatgattc agactctgga agctgtgaaa aggaagcagt 600
ttgagaagta ccatgccgct tctgcagagg aacaggcaac catcgaacgc aacccctaca 660
ccatcttcca tcaagcactg aaaaactgtg agcctatgat tgggctggta cccatcctca 720
agggaggccg tttctaccag gtccctgtac ccctacccga ccggcgtcgc cgcttcctag 780
ccatgaagtg gatgatcact gagtgccggg ataaaaagca ccagcggaca ctgatgccgg 840
agaagctgtc acacaagctg ctggaggctt tccataacca gggccccgtg atcaagagga 900
agcatgactt gcacaagatg gcagaggcca accgtgccct ggcccactac cgctggtggt 960
agagtctcca ggaggagccc agggccctct gccgcaagaa acagtgtgag ctactgccac 1020
gctgaaaact acctgtgggt taaggatgta gttcctttgt aagggtgggc aggcctcgta 1080
agaaagatgt agcagcatat tcactatccg ttaatccttc tttctttgag gctggaactt 1140
gctctctctg cccctatttc cttgtaaaga gggagcacat tgacttggga atttcctcca 1200
ggaaactcag ggctgttttc tcttccctta ggttggggcg gacctttgga catataaagg 1260
aagcagtttt agtatcagaa aagatttatt agaaaattct cacgctgaac tggtgtagca 1320
tgtggtgcag cattcagtga aactggctgg aggaaatagg cttgtttcca gagttgtcct 1380
tatacaaaat gtataaaaag cagtttctgg tgtgacttgt gctctgcctc caccccttga 1440
catcccaaaa tatcccacca gtggctatgc ttacccattt tacagatggg taaactgagg 1500
caccaaggta gtagttgcac taatggttac acagtgcagt ggctcttggg agttgccctt 1560
ctctgcctgg ccgtggtggg ttgtggtggg gaaaggggct cagggcagga ccacggcata 1620
agtgggaaac atctcaccag gagatgggaa agtctagaag ggaagacact caaagtctgg 1680
aagggaaaag tctttgggtg aggcagagac tccactgcca gctttagagg tgggtagaag 1740
aaaggccagt gctggtgagg aaaccctgat ctggaggcta gtcggagact tcgctgtagt 1800
atacttgtgg cactggcgtt gcttccagcc gttggccgtt gttctttccc aagcccgggc 1860
ccgcccccgg gaaacttcca aatgaatttt tcccaaggca aagcnagcna accttggggg 1920
cccaagggga agcttnaaag gncccaattt ttggggaanc caagggnaaa gncccattnc 1980
ccccggggnt ttaaggccnc cnaaaaagaa ggcttccctt 2020
<210> 51
<211> 637
<212> DNA
<213> Homo .sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1647264CB1
<400> 51
cggccagtgc aagctaaaat taaccctcac taaagggaat aagcttgcgg ccgccgagcc 60
cagctccgcc gccgagcgcc tgtgccggca ccgtacacca tggagcgccc ggataaggcg 120
gcgctgaacg cactgcagcc tcctgagttc agaaatgaaa gctcattagc atctacactg 180
aagacgctcc tgttcttcac agctttaatg atcactgttc ctattgggtt atatttcaca 240
actaaatctt acatatttga aggcgccctt gggatgtcca atagggacag ctatttttac 300
gctgctattg ttgcagtggt cgccgtccat gtggtgctgg ccctctttgt gtatgtggcc 360
tggaatgaag gctcacgaca gtggcgtgaa ggcaaacagg attaaagtga acatcacctt 420
tttatagcat taaattcatt ttttaaaatg ataaatgctg gagggggcca tctgatttga 480
ataaagttga aagaacatgt taaagtcagt cttaaggagt cacgtttgag tatgtaaatt 540
ttgatccttc taatatgttg ggtttgatat tcagttttac tgtatgaatc gattgcaatg 600
agaattggaa aagtagtaca agaatatgta attatta 637
<210> 52
<211> 717
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1721989CB1
<400> 52
ggctgggcct ggcgcgcagg cgctaggaag aggccgcgtg gggcgaaggc ggcgcttggc 60
tggtggggcc cgcggcggga ttttcccggg cggcgagagc ggatctatct tgggatccca 120
tggctttctt tactgggctc tggggcccct tcacctgtgt aagcagagtg ctgagccatc 180
40164
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
actgtttcag caccactggg agtctgagtg cgattcagaa gatgacgcgg gtacgagtgg 240
tggacaacag tgccctgggg aacagcccat accatcgggc tcctcgctgc atccatgtct 300
ataagaagaa tggagtgggc aaggtgggcg accagatact actggccatc aagggacaga 360
agaaaaaggc gctcattgtg gggcactgca tgcctggccc ccgaatgacc cccagatttg 420
actccaacaa cgtggtcctc attgaggaca acgggaaccc tgtggggaca cgaattaaga 480
cacccatccc caccagcctg cgcaagcggg aaggcgagta ttccaaggtg ctggccattg 540
ctcagaactt tgtgtgagtt gagcccaggc ctctggttgc aggactcgtg aatggagcag 600
ttctgagaac cacccttttg ctaagggagc ttgggagcca catggctgct cccttcacac 660
tgggtaacag tgtagtatcc tgtgagagaa taaatgtatt catttaaaaa aaaaaaa 717
<210> 53
<211> 2061
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1730581CB1
<400> 53
ggctcgcaca cacttcggca cgaggaaagg caggaaaggg caggccgggt gagcagacgg 60
atcggccgac tagacagcca accagcaaca acgaactgag ctcgcatact accgcttacg 120
catctaacca accgcccatc tagctaaccc gagcccctcc accgtcaact caggttcggc 180
cggtccccgg cccgcctgcc ggagccgtgg tggcagcccc gggaggagca ctggcgtctg 240
tttccttcga ttctcgggat tcgaagatgg ctgcacagtc agcgccgaaa gttgtgctaa 300
aaagcaccac caagatgtct ctaaatgagc gctttactaa tatgctgaag aacaaacagc 360
cgacgccagt gaatattcgg gcttcgatgc agcaacaaca gcagctagcc agtgccagaa 420
acagaagact ggcccagcag atggagaata gaccctctgt ccaggcagca ttaaaactta 480
agcagaagag cttaaagcag cgcctgggta agagtaacat ccaggcacgg ttaggccgac 540
ccataggggc cctggccagg ggagcaatcg gaggacgagg cctacccata atccagagag 600
gcttgcccag aggaggacta cgtgggggac gtgccaccag aaccctactt aggggcggga 660
tgtcactccg aggtcaaaac ctgctccgag gtggacgagc cgtagctccc cgaatgggct 720
taagaagagg tggtgttcga ggtcgtggag gtcctgggag agggggccta gggcgtggag 780
ctatgggtcg tggcggaatc ggtggtagag gtcggggtat gataggtcgg ggaagagggg 840
gctttggagg ccgaggccga ggccgtggac gagggagagg tgcccttgct cgccctgtat 900
tgaccaagga gcagctggac aaccaattgg atgcatatat gtcgaaaaca aaaggacacc 960
tggatgctga gttggatgcc tacatggcgc agacagatcc cgaaaccaat gattgaagcc 1020
tgcccatcct cccatgagag actcttgtta gtcaacacat ctgtaaataa ccttgagata 1080
acagatgaga agaaatctga ttgatgctgg atggacctat cacaataggc tgtggactta 1140
cttgccacca gcttgtgcat ttagtgtgtt ccttttactt tttgatactg tgttgtatga 1200
aacccttttg tcctttgatt tggttttttg tttttgtttt tttagggggg agggggggtt 1260
tcccctcctt tgcccagact tctctttgaa cacaaatgca ttagccttgt ggctagaaca 1320
ccctcttcct acctctgtct cccctcactt gtcatatgct ctgacatgct aacatttctt 1380
ttgttcatcc ctgttgcccc cacagaaaca tcccagaaaa accggtcagt gttccttcct 1440
ccctgatcct taggtttctg aaatagggtt ctgttacatc ctcttcgata gcctgtttaa 1500
aatgtttaga aggtctggag ctcaaaaatg cgttcttcca cattgataat ttagtaaact 1560
gagaacattg acatcactac agggcagcat aagaggttgc ttacatgtgg tagcagctct 1620
ggtttgattc aagttgctac catgtacatt gacagcacat ataccataac cagcgtgttg 1680
ggttgaattg cactttctac ctttgtatga gatttacaga ctttccttct gggtttgtat 1740
catgaccaga ggggtactat agggttggtt tatactgcaa tatagaggat cagaagccat 1800
ttgatttggt aggtgtgtca gaagggagaa tgatggcaga cgaactgctg gaagaggtca 1860
gaagatagcc atgctaaaat gcaattatat cctcatgttt atcccaaact aatcttggac 1920
ttttccactc attagctttg ttttgccctt gtttcccttg aaggtttaag ttcaaccata 1980
ttctgtcaac tgttcagttt cagtggaatc ttgtatttct ggttcattat aacaaactgt 2040
tcgcttaaaa aaaaaaaaaa a 2061
<210> 54
<211> 1307
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1740714CB1
<220>
<221> unsure
41/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<222> 1301
<223> a, t, c, g, or other
<400> 54
gcgctgtgac ctagaatggg cgcatgcgcc gagcggaact ggctggtttg aaaaccatgg 60
cgtgggtacc agcggagtcc gcagtggaag agttgatgcc tcggctattg ccggtagagc 120
cttgcgactt gacggaaggt ttcgatccct cggtaccccc gaggacgcct caggaatacc 180
tgaggcgggt ccagatcgaa gcagctcaat gtccagatgt tgtggtagct caaattgacc 240
caaagaagtt gaaaaggaag caaagtgtga atatttctct ttcaggatgc caacccgccc 300
ctgaaggtta ttccccaaca cttcaatggc aacagcaaca agtggcacag ttttcaactg 360
ttcgacagaa tgtgaacaaa catagaagtc actggaaatc acaacagttg gatagtaatg 420
tgacaatgcc aaaatctgaa gatgaagaag gctggaagaa attttgtctg ggtgaaaagt 480
tatgtgctga cggggctgtt ggaccagcca caaatgaaag tcctggaata gattatgtac 540
aagcaacagt aactagtgtc ttggaatatc tgagtaattg gtttggagaa agagacttta 600
ctccagaatt gggaagatgg ctttatgctt tattggcttg tcttgaaaag cctttgttac 660
ctgaggctca ttcactgatt cggcagcttg caagaaggtg ctctgaagtg aggctcttag 720
tggatagcaa agatgatgag agggttcctg ctttgaattt attaatctgc ttggttagca 780
ggtattttga ccaacgtgat ttagctgatg agccatcttg atgtagctga tctctcaggg 840
atagaagata tttctcatga aggcagccta actctgagga aaacaatgcc aattcaagta 900
cagatttcaa cacatcttca acactatgtg aagggttcac atcttaacct gtgcaattca 960
gattgatact cagaatatgg gttgatttga atatctgaaa tatcaatgga aaatcccact 1020
cagtttttga tgaacagttt gaacagtttt ctgtaatcaa gcagcttgca tagaaattgt 1080
atgatgaaat tttacatagg ttcttggtgc tgttttgttc tttttttgtt ttttgttgtt 1140
ttgttattta cttatataca tataaaattt tattgaaaat atgttttggt tactaaaatt 1200
ttgtttgact cctaacaaaa gacaatggat ggccttagca tcagaattaa aataatctgg 1260
attaaatggc aatgtgttca tagtcagcaa taaaattaaa natttta 1307
<210> 55
<211> 1357
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1850596CB1
<400> 55
ggggcgcgcg acggcgccag ctcggggcag cggaacccag agaagctgaa ggggcggtag 60
cggcggcgac ggcgacgacg acgactcccg cgcgtgtgcc cagcctcttc ccgccgcagc 120
cgcccttttc ctccctccct tacgtccccg agtgcggcag taccgcctcc ttcccagccg 180
cgcggcttcc tccagacctc tcggcgcggg tgagccctat tcccagaggc aggtggtgct 240
gaccctgtaa cccaaaggag gaaacagctg gctaagctca tcattgttac tggtgggcac 300
catgtccttg aagcttcagg caagcaatgt aaccaacaag aatgacccca agtccatcaa 360
ctctcgagtc ttcattggaa acctcaacac agctctggtg aagaaatcag atgtggagac 420
catcttctct aagtatggcc gtgtggccgg ctgttctgtg cacaagggct atgcctttgt 480
tcagtactcc aatgagcgcc atgcccgggc agctgtgctg ggagagaatg ggcgggtgct 540
ggccgggcag accctggaca tcaacatggc tggagagcct aagcctgaca gacccaaggg 600
gctaaagaga gcagcatctg ccatatacag tggctacatc tttgactatg attactaccg 660
ggacgacttc tacgacaggc tcttcgacta ccggggccgt ctgtcgcccg tgccagtgcc 720
cagggcggtc cctgtgaagc gaccccgggt cacagtccct ttggtccggc gtgtcaaaac 780
taacgtacct gtcaagctct ttgcccgctc cacagctgtc accaccagct cagccaagat 840
caagttaaag agcagtgagc tgcaggccat caagacggag ctgacacaga tcaagtccaa 900
tatcgatgcc ctgctgagcc gcttggagca gatcgctgcg gagcaaaagg ccaatccaga 960
tggcaagaag aagggtgatg gaggtggcgc cggcggcggc ggcggtggtg gtggcagcgg 1020
tggcggtggc agtggtggtg gcggtggcgg tggcagcagc cggccaccag ccccccaaga 1080
gaacacaact tctgaggcag gcctgcccca gggggaagca cggacccgag acgacggcga 1140
tgaggaaggg ctcctgacac acagcgagga agagctggaa cacagccagg acacagacgc 1200
ggatgatggg gccttgcagt aagcagcctg acaggagcaa tggccaccag caggtgaagg 1260
gcatcgctgc cccaggcctc aagccgggca cccaaccctg gatgccaccc cccagcgggt 1320
accagaggaa agctggcagc aggcgcctcc tccccca 1357
<210> 56
<211> 1749
<212> DNA
<213> Homo Sapiens
<220>
42164
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<221> misc_featuxe
<223> Incyte ID No: 1856109CB1
<400> 56
ctggcccgac tactttcgtt ccgtcttcca tcgttttctc tcgtgcaatg gcgtccgggc 60
tggtaagatt gctgcagcag ggacatcgct gcctcctggc tccagtcgcc cccaagctgg 120
tccctccggt tcggggagtg aagaagggat tccgcgccgc cttccgcttc cagaaggagt 180
tagagcggca gcgccttctg cggtgcccgc cgccgcccgt gcgccgttca gagaagccga 240
actgggatta ccatgcagaa atacaagctt ttggacatcg gttacaggaa aacttttcct 300
tagatcttct caaaactgca tttgttaata gctgctatat taaaagtgag gaggccaaac 360
gccaacaact tgggatagag aaagaagctg ttcttctgaa tcttaaaagt aatcaagaac 420
tatccgaaca agggacatct ttttcacaga cttgccttac acagtttctt gaagacgagt 480
acccagacat gcccactgaa ggcataaaaa atcttgttga ctttctcact ggtgaggaag 540
tcgtgtgtca cgtggctaga aacttggctg tggagcagtt aacactgagt gaagaattcc 600
cagtgccccc agctgtgtta cagcagactt tctttgcagt tattggagcc ctgttacaga 660
gcagtggacc tgagaggact gcacttttca tcagggactt cttaattact caaatgactg 720
gaaaagagct ctttgagatg tggaagataa taaatcccat ggggctattg gtagaagaac 780
tgaagaaaag gaatgtttca gctcctgaat caagacttac taggcagtct ggtggcacca 840
cagctttgcc tttgtatttt gttggcttat actgtgataa aaagttgatt gcagaaggac 900
ctggggaaac agtattggtt gcagaagaag aggctgctcg agtggccctt agaaaacttt 960
atggattcac agaaaataga cggccgtgga actattccaa gcccaaagaa accttgagag 1020
cagaaaagag catcactgcc agctagccgc catggatgca gcagcctgaa acttgagagc 2080
gaaagtgaga taaatgtcaa aggtgtttca agccagacat tttcacaatt gtgaagaaat 1140
agatgttttg tttctgtttt ttactgtgtt cccaaaatta aataaatgtt aaccaagtca 1200
cagtgttttt ggttttgttt ttctgaaatc ttggtttgat caaatctttt tttttttctc 1260
ttgagatgga gtcttactct gtcgcccagg ctggactgca gtggtgcgat ctcggctcac 1320
tgcaacctcc acctcacagg ttcaagcgat tctcgtggct cagcctccct agtagctggg 1380
attacaggca cacaccacca tacctggcta atttttgtat ttttggtaga gatggggttt 1440
caccaagttg gctagactag tcttgaactc ctgacctcag gtgatccacc cgccttggcc 1500
tcccaaagtg ctgggattac aggtgtgagc cactataccc gaccagatca aatctttttt 1560
tgacattttt gcaaaaaaat tttcctaatg ttcttgattt aattgtatag aatttgtata 1620
attaggtgta ttttatttgc ctctagcttt gaggtatcat aatttatgta tcttatgtga 1680
attttttgct gtaataccaa taaagttttt tttctccaca tgttaaaaaa aaaaaaaaaa 1740
aaaaaaaaa 1749
<210> 57
<211> 991
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1921719CB1
<400> 57
cgaaagatgg cggcgcccgt aaggcggacg ctgttagggg tggcgggggg ttggcggcgg 60
ttcgagaggc tctgggccgg cagtctaagc tctcgcagcc tggctcttgc agccgcaccc 120
tcaagcaacg gatccccatg gcgcttgttg ggcgcgttgt gcctgcagcg gccacctgta 180
gtctccaagc cgttgacccc attgcaggaa gagatggcgt ctctactgca gcagattgag 240
atagagagaa gcctgtattc agaccacgag cttcgtgctc tggatgaaaa ccagcgactg 300
gcaaagaaga aagctgacct tcatgatgaa gaagatgaac aggatatatt gctggcgcaa 360
gatttggaag atatgtggga gcagaaattt ctacagttca aacttggagc tcgcataaca 420
gaagctgatg aaaagaatga ccgaacatcc ctgaacagga agctagacag gaaccttgtc 480
ctgttagtca gagagaagtt tggagaccag gatgtttgga tactgcccca ggcagagtgg 540
cagcctgggg agacccttcg aggaacagct gaacgaaccc tggccacact ctcagaaaac 600
aacatggaag ccaagttcct aggaaatgca ccctgtgggc actacacatt caagttcccc 660
caggcaatgc ggacagagag taacctcgga gccaaggtgt tcttcttcaa agcactgcta 720
ttaactggag acttttccca ggctgggaat aagggccatc atgtgtgggt cactaaggat 780
gagctgggtg actatttgaa accaaaatac ctggcccaag ttaggaggtt tgtttcagac 840
ctctgatggg ccgagctgcc tgtggacggt gctcagacaa gtctgggatt agagcctcaa 900
ggacattgtg tgattgcctc acatttgcag gtaatatcaa gcagcaaact aaattctgag 960
aaataaacga gtctattact gaaaaaaaaa a 991
<210> 58
<211> 1188
<212> DNA
<213> Homo sapiens
43!64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<220>
<221> misc_feature
<223> Incyte ID No: 2099829CB1
<400> 58
ccgtcttccg ccgcacgtgg attcagcgcg atgcccaaat ccaagcgcga caagaaagtc 60
tccttaacca aaactgccaa gaaaggcttg gaattgaaac aaaacctgat agaagagctt 120
cggaaatgtg tggacaccta caagtacctt ttcatcttct ctgtggccaa catgaggaac 180
agcaagctga aggacatccg gaacgcctgg aagcacagcc ggatgttctt tggcaaaaac 240
aaggtgatga tggtggcctt gggtcggagc ccatctgatg aatacaaaga caacctgcac 300
caggtcagca aaaggttgag gggtgaggtg ggtctcctgt tcaccaaccg cacaaaggag 360
gaggtgaatg agtggttcac gaaatacaca gaaatggact acgcccgagc tggtaacaaa 420
gcagctttca ctgtgagcct ggatccaggg cccctggagc agttccccca ctccatggag 480
ccacagctca ggcagctggg cctgcccacc gccctcaaga gaggtgtggt gactctgctg 540
tctgactacg aggtgtgcaa ggagggcgat gtgctgaccc cagagcaggc tcgcgtcctg 600
aagctttttg ggtatgagat ggctgaattc aaggtgacca tcaaatacat gtgggattca 660
cagtcgggaa ggttccagca gatgggagac gacttgccag agagcgcatc tgagtccaca 720
gaagagtcag actcagaaga tgatgactga aagggactcg ggactgaagg tctcctggaa 780
gcttctgggt ctcactggac catcaggact gctgccgccc ctctggagag agcagctttt 840
tatttgtctg tagacaggga acatgatggg cactgacctc ctgtaaagaa taaaactgtg 900
ggccgggcgc ggtggctcac gcctggaatc ccagcacttt gggaagccga ggtgggcaga 960
tcataaggtc aggagattaa gaccatcctg gctaacacgg tgaaaccccg tctctactaa 1020
aaatagaaaa aaaaactagt tgggcatagt ggcatgtgcc tgtagtccca gctactcagg 1080
aggctgaggc aggagaatca cttgaacccg ggaggtggag gttgccgtga gttgagattg 1140
gaccactgct ctccagcctg ggcaacagag taaaactctg tcccaaaa 1188
<210> 59
<211> 1454
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2416915CB1
<400> 59
gttgtcactc tctcgggttg ttactctgta gcttcccggc tcgcgaaagg gaggacctgt 60
ctgggtcatg gattttgaga atcttttctc aaaacccccc aacccggccc tcggcaaaac 120
ggccacggac tctgacgaaa gaatcgatga tgaaatagat acagaagttg aagaaacaca 180
agaagagaaa attaaactgg agtgcgagca aattcccaaa aaatttagac actctgcaat 240
atcaccaaaa agttcgctgc atagaaaatc aagaagtaag gactatgatg tatatagtga 300
taatgatatc tgcagtcagg aatcagaaga taattttgcc aaagagcttc aacagtacat 360
acaagccaga gaaatggcaa atgctgctca acctgaagaa tctacaaaga aagaaggagt 420
aaaagatacc ccacaggctg ctaaacaaaa aaataaaaat cttaaagctg gtcacaagaa 480
tggcaaacag aagaaaatga agcgaaaatg gcctggccct ggaaacaaag gatcaaatgc~540
tttgctgagg aacagcggct cacaggaaga ggatggtaaa cctaaagaga agcagcagca 600
tttgagtcag gcattcatca accaacatac agtggaacgc aagggaaaac aaatttgtaa 660
atattttctt gaaaggaaat gtattaaggg agaccagtgt aaatttgatc atgatgcaga 720
gatagaaaaa aaaaaggaaa tgtgtaagtt ttatgtacaa ggatattgta ccagaggtga 780
aaactgtctg tatttgcata atgaatatcc ttgtaagttt taccatacag gaacaaaatg 840
ttatcaggga gaatactgca agttttctca tgctccactg actcctgaaa cacaagaatt 900
gttggctaaa gttttggata ctgaaaagaa gtcatgtaaa taaaatagac ataaaaaggt 960
agcaatgtac agataaagag tactttaacg cccatgcgtg ttcaagactg ttcaagactg 1020
gtgatttgga gtagtttaca agattcctca ttcagagtgc cctcttgtgt gactggggtg 1080
atgtgcagct tccataatgg atgggacaga gagctgggat ctaatgtaca agtgaagggc 1140
ttggtcttcc ctgagacatt ccagccattg gaataggaga ggagcatata tggcagaggt 1200
gatggctggt gggtaaatgt gatagtaaat tgtagaaacc tcttctgatt gattggattt 2260
ccttaataaa atcggaagca aggttaggct gagtgagggt gagtaaagag gtagaggagg 1320
tttgaggaga gagaactgct cggaagacat tggtagatgg accataaaaa cagagttagt 1380
tctctttatg acattaaata gttttacaac atatttttaa tggttcacaa tttcatttta 1440
gggcttaaaa taaa 1454
<210> 60
<211> 1588
<212> DNA
<213> Homo Sapiens
44/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<220>
<221> misc_feature
<223> Incyte ID No: 2472784CB1
<400> 60
cccggacgaa gggggagagt agacagcaga accagcggcg gcggctaagc agagactgta 60
gtagcggcga cagcgacgac ggcagcgatg gctggggcgg ggccagcccc gggactcccg 120
ggtgcaggag gacccgtggt cccgggtcct ggcgctggca tcccgggcaa aagcggcgag 180
gaacgcttga aggaaatgga ggcggagatg gccctgtttg agcaggaagt tctgggggct 240
ccagtacctg gaatcccaac tgctgtgcct gcggtgccca ctgtccccac ggtccccaca 300
gtagaagcga tgcaggtccc agcggctcct gtgatccgcc caattatcgc gaccaacaca 360
taccagcagg tccagcagac tctggaggcc cgagcagctg ctgcagccac agtagttcct 420
cccatggtgg gtggccctcc ttttgtaggc cctgttggct ttggccctgg tgatcggagt 480
cacctggaca gcccagaggc tcgagaagcc atgttcctgc ggcgggcagc agccgtcccc 540
cgccctatgg ccctaccgcc ccctcaccag gccctcgtgg gcccccctct gcctgggccc 600
cctggaccac ccatgatgct gccaccaatg gctcgggctc cagggccccc gctgggctcc 660
atggctgcac tgaggccccc tctggaagag ccagcagcac cccgagagct gggcctaggc 720
ctggggttgg gcctgaaaga gaaggaagag gcagtggtgg cggcggcggc tgggctggag 780
gaggctagcg cggctgtggc cgtgggggca ggaggtgccc cagctggccc tgcagtcatt 840
gggcccagcc tgccgctggc cctggccatg ccattgcccg agcctgagcc cctgcccctc 900
ccgttggagg tcgtccgcgg cctcctgccc ccgctgcgca ttcctgaact cctgtccctg 960
cgtcctcggc cccggccccc tcggccagag ccacccccag gcctcatggc tcttgaggtc 1020
ccagagcccc tgggtgaaga caagaagaag gggaagccag agaaattgaa acggtgcatt 1080
cgcacagcgg cagggagcag ctgggaggac cccagcctgc tggagtggga tgcagatgac 1140
ttccggatct tctgtgggga tctgggcaat gaggtgaacg atgacatctt ggcacgcgcc 1200
ttcagccgct tcccatcctt ccttaaggcc aaggtgatcc gtgacaagcg cacaggcaag 1260
accaagggct acggcttcgt cagcttcaag gaccccagcg actacgtgcg cgccatgcgt 1320
gagatgaatg ggaagtatgt gggctcgcgc cccatcaagc ttcgcaagag catgtggaag 1380
gaccggaatc tggacgtggt ccgcaagaag cagaaggaaa agaagaagct gggcctgaga 1440
tagggtctgt ggccaggcac ccgctcccac ctggccgggc gctggctcct ccctcagttc 1500
tctttggaaa acccccagct gtccacccat cccctgcccc aaaaccagtt tcaataaatt 1560
tacgttcatt tccaaaaaaa aaaaaaaa 1588
<210> 61
<211> 2111
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2598981CB1
<400> 61
cgcaggcggg cctcgcgggt ccgggagcgc ggcggagacg atgcctgaga tcagagtcac 60
gcccttgggg gccggccagg acgtgggccg aagctgcatc ctggtctcca ttgcgggcaa 120
gaatgtcatg ctggactgtg gaatgcacat gggcttcaat gacgaccgac gcttccctga 180
cttctcctac atcacccaga acggccgcct aacagacttc ctggactgtg tgatcattag 240
ccacttccac ctggaccact gcggggcact cccctacttc agcgagatgg tgggctacga 300
cgggcccatc tacatgactc accccaccca ggccatctgc cccatcttgc tggaggacta 360
ccgcaagatc gccgtagaca agaagggcga ggccaacttc ttcacctccc agatgatcaa 420
agactgcatg aagaaggtgg tggctgtcca cctccaccag acggtccagg tagatgatga 480
gctggagatc aaggcctact atgcaggcca cgtgctgggg gcagccatgt tccagattaa 540
agtgggctca gagtctgtgg tctacacggg tgattataac atgaccccag accgacactt 600
aggagctgcc tggattgaca agtgccgccc caacctgctc atcacagagt ccacgtacgc 660
cacgaccatc cgtgactcca agcgctgccg ggagcgagac ttcctgaaga aagtccacga 720
gaccgtggag cgtggtggga aggtgctgat acctgtgttc gcgctgggcc gcgcccagga 780
gctctgcatc ctcctggaga ccttctggga gcgcatgaac ctgaaggtgc ccatctactt 840
ctccacgggg ctgaccgaga aggccaacca ctactacaag ctgttcatcc cctggaccaa 900
ccagaagatc cgcaagactt ttgtgcagag gaacatgttt gagttcaagc acatcaaggc 960
cttcgaccgg gcttttgctg acaacccagg accgatggtt gtgtttgcca cgccaggaat 1020
gctgcacgct gggcagtccc tgcagatctt ccggaaatgg gccggaaacg aaaagaacat 1080
ggtcatcatg cccggctact gcgtgcaggg caccgtcggc cacaagatcc tcagcgggca 1140
gcggaagctc gagatggagg ggcggcaggt gctggaggtc aagatgcagg tggagtacat 1200
gtcattcagc gcacacgcgg acgccaaggg catcatgcag ctggtgggcc aggcagagcc 1260
ggagagcgtg ctgctggtgc atggcgaggc caagaagatg gagttcctga agcagaagat 1320
cgagcaggag ctccgggtca actgctacat gccggccaat ggcgagacgg tgacgctgcc 1380
cacaagcccc agcatccccg taggcatctc gctggggctg ctgaagcggg agatggcgca 1440
45/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
ggggctgctc cctgaggcca agaagcctcg gctcctgcac ggcaccctga tcatgaagga 1500
cagcaacttc cggctggtgt cctcagagca agccctcaaa gagctgggtc tggctgagca 1560
ccagctgcgc ttcacctgcc gcgtgcacct gcatgacaca cgcaaggagc aggagacggc 2620
attgcgcgtc tacagccacc tcaagagcgt cctgaaggac cactgtgtgc agcacctccc 1680
ggacggctct gtgactgtgg agtccgtcct cctccaggcc gccgcccctt ctgaggaccc 1740
aggcaccaag gtgctgctgg tctcctggac ctaccaggac gaggagctgg ggagcttcct 1800
cacatctctg ctgaagaagg gcctccccca ggcccccagc tgaggccggc aactcaccca 1860
gccgccacct ctgccctctc ccagctggac agaccctggg cctgcacttc aggactgtgg 1920
gtgccctggg tgaacagacc ctgcaggtcc catccctggg gacagaggcc ttgtgtcacc 1980
tgcctgccca ggcagctgtt tgcagctgaa gaaacaaact ggtctccagg ctgtcttgcc 2040
tttattcctg gttagggcag gtggtcctag acagcagttt ccagtaaaag ctgaacaaaa 2100
gaaaaaaaaa a 2111
<210> 62
<211> 1155
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2738075CB1
<220>
<221> unsure
<222> 1150-1155
<223> a, t, c, g, or other
<400> 62
cccacgcgtc cgcccacgcg tccgcccacg cgtccggccg ggaagaagca ccgtggctgc 60
tattatctgc tctccgcgcc tgacccctcc caggactcgt gatgccaagg ccgctgcgag 120
cggctacgaa gagtcggggt tgagccccag ctgagccgag ggctcgcact cttctggtct 180
cccaggccca acccacctga agaaatgagt ggtggattgg ctccaagtaa gagcacagtg 240
tatgtatcca acttgccttt ttccctgaca aacaatgact tgtaccggat attttccaag 300
tatggcaaag ttgtaaaggt taccatcatg aaagataaag ataccaggaa gagtaaaggg 360
gttgcattta ttttattttt ggataaagac tctgcacaaa actgtaccag ggcaataaac 420
aacaaacagt tatttggtag agtgataaaa gcaagcattg ctattgacaa tggaagagca 480
gctgagttca tccgaaggcg aaactacttt gataaatcta agtgttatga atgtggggaa 540
agtggacact taagttatgc ctgtccgaaa aatatgctcg gagaacgtga gcctccaaag 600
aagaaagaaa aaaagaaaaa aaagaaagct cctgaaccag aagaagaaat tgaggaagta 660
gaagaaagtg aagatgaagg ggaggatcct gctcttgaca gcctcagtca ggccatagca 720
ttccagcaag ccaaaattga agaagaacaa aaaaaatgga aacccagttc aggagtcccc 780
tcaacatcag atgattcaag acgcccaagg ataaagaaaa gcacatattt cagtgatgag 840
gaagaactta gtgattaaaa tcttgcccca gcacagtaat aaaaatcaag atttgttagt 900
aacaatcttg aagagctaat tttaataaaa ataagaaaaa ttaatactat catgttaata 960
ctattattgt catcccaaga aaaaagatat tttaaaaatt tatttgaaaa gttcattata 1020
agggctttat tcatgcctga tttgtttaca tgaggacttc tgaaattaat ccttaaaaca 1080
aacttcctga agaccgaaaa gttgaatgat ttattgttac ttatattaat aaacttttca 1140
agagaaaaan nnnnn 1155
<210> 63
<211> 1673
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2279049CB1
<400> 63
gttttgggtc gcagtatgct agaattttga ggctcccttc tgatgaaaat tgagctgtcc 60
atgcagccat ggaacccggg ttacagcagt gagggggcca cggctcaaga aacttacaca 120
tgtccaaaaa tgattgagat ggagcaggcg gaggcccagc ttgctgagtt agacctgcta 180
gccagtatgt tccctggtga gaatgagctc atagtgaatg accagctggc tgtagcagaa 240
ctgaaagatt gtattgaaaa gaagacaatg gaggggcgat cttcaaaagt ctactttact 300
atcaatatga acctggatgt atctgacgaa aaaatggcga tgttttctct ggcctgtatt 360
cttcccttta aatacccggc agttctgcct gaaattactg tcagatcagt attattgagt 420
agatcccagc agactcagct gaacacagat ctgactgcat tcctgcaaaa acattgtcat 480
46/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
ggagatgttt gtatactgaa tgccacagag tgggttagag aacacgcctc tggctatgtc 540
agcagagata cttcatcttc acccaccaca ggaagcacag tccagtcagt tgacctcatc 600
ttcacgagac tctggatcta cagccatcat atctataaca aatgcaaaag aaagaatatt 660
ctagagtggg caaaggagct ttccctgtct gggtttagca tgcctggaaa acctggtgtt 720
gtttgtgtgg aaggcccaca aagtgcctgt gaagaattct ggtcaagact cagaaaatta 780
aactggaaga gaattttaat tcgccatcga gaagacattc cttttgatgg tacaaatgat 840
gaaacggaaa gacaaaggaa attttccatt tttgaagaaa aagtgttcag tgttaatgga 900
gccaggggaa accacatgga ctttggtcag ctctatcagt tcttaaacac caaaggatgt 960
ggggatgttt tccagatgtt ctttggtgta gaaggacaat gacatcaaga gtagttgaaa 1020
gtatcttgcc actgttggcc ttttgatttt tttttcccac tttttcttga aagattaagt 1080
aattttattt tagttccatt ctagaatgtt ggggagtggg gcacaagaaa aaatagtata 1140
gctgaaatgc atctgttaaa aatgtcatga ttgaaagcag aactgagttt caaattacaa 1200
ccttaaaatt gttgttagat atttcttcac atatcagctg cccattttga aaaagaaatt 1260
atccataaag gtaatgttgg tgctccaatt tgccagccat tcccaacccc cttctccctt 1320
acctgccttc actaaagaac ccagaaaagc taattgctcc cctttcagcc tctgttgcaa 1380
ctaacaactc tcagtggcct caggacacag ctttggcctt gggaattctg ggaaaacttt 1440
tacttcctga ttaaagatac atatgcagct aggccacctc ctccccccct tactgccata 1500
aacaccaaag tgatgactgg agctggagga gttatttgaa ccacgacgaa gggccaagag 1560
aaccacgaag atgccagttg ccacattgtt gagctgctga cccaacacca gccattgcct 1620
gtctctaaac atcttatgaa ataaaaccag ttttgtttaa aaaaaaaaaa aaa 1673
<210> 64
<211> 584
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2660904CB1
<400> 64
aaaataaggc atttgggatg ccgatgttaa aaggagaagg taatcagaga agaaaaaagg 60
aagtcggtgg agagttctgc gaagattttg aagatttcta gagaagcagg gggttctgga 120
gaagggaata gacttggcca gatacccagg aagacttcct caacagatgt cccatcatgc 180
tgagattcaa cgcgacattt tagagtcatg caaccatgtg agaaaaaaag tcccagtaac 240
ctttgttggg gctggagggc aggatcccga ggtcccggag gagctgctcc acctcctcca 300
gccaggacag cgcgtgcctc aggacgtcca gcaccacctt ctggagcccc gagacaggtg 360
ggctcacctg gaggtgctga agaaggtcga cctccttctt caggtcatgg ctgcaacagg 420
atattttcat gcaagcctgc aaagaggtga gatcatgagg agcccaggcc ctgtggccag 480
aaatagcccc tgacgtgacc ttcgaggaac agcgttcttg actctgccac gaagcaggca 540
gcgccacgta ttggccgcct tgggaggact cagttttttt cttt 584
<210> 65
<211> 978
<212> DNA
<213> Homo Sapiens
<220>
<222> misc_feature
<223> Incyte ID No: 3179424CB1
<400> 65
ccggctacct gttggtgtgt atgcagagca tccctgtgcc ccgcggatat agactggcgc 60
gcctctgttg cgcaggcgca gaactacaac ttcagggttt tccccaacgg cctctttttt 120
gcacgttagg agaaactaca tttcccataa tcctttgttc cagggctgga gcggctctgg 180
gctccggaat cgcccgcagc cggtactgcg ggacccactg cggatatggc tgtcttggct 240
ggatccctgt tgggccccac gagtaggtcg gcagcgttgc tgggtggcag gtggctccag 300
ccccgggcct ggctggggtt cccagacgcc tggggcctcc ccaccccgca gcaggcccgg 360
ggcaaggctc gcgggaatga gtatcagccg agcaacatca aacgcaagaa caagcacggc 420
tgggtccggc gcctgagcac gccggccggc gtgcaggtca tccttcgccg aatgctcaag 480
ggccgcaagt cgctgagcca ttgaggatcg cgacgcagtc ggcgggaccc tcatggaagc 540
atcgccctcg cctcggacct tgcctggcgc tatttttgca gggagctggg gagcaggaac 600
gcctcggacc tgagtgctct ccatattgtg gggttgaagt ctggatggga gcttgccaag 660
tcccttttta ggctttttaa ttaggaagca tttcgaacct gcgcaacaga ccaaagaaca 720
gtacaaagaa catccgtgta cccagtaccc tgactaccga ctacctacaa cccgtccctg 780
ccccatcctg agttcttttg aagctgatct caggcatcgg attatttctt ctgtaaatat 840
ttcagaatgt atctctccaa gatgagagct cattaaaaga caattacaaa gcttatcaca 900
47/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
tccaaaagaa ttatcaataa ttttgaaata ttattaaacg tgtaataaat gttcaaagtt 960
ccacttgcaa aaaaaaaa 978
<210> 66
<211> 1055
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2885096CB1
<400> 66
cagcctcaga gatggatgga tctgcaatgc catggctggg ggtgttccag ggcagcctgc 60
aggggtgggg ctggcactga ttgcaactga cagccaggag accaggcctg ggagggcagg 120
cccagggtca ggggagagcc tgagtgcttc ccacctcttc atctcagact ttgcatactg 280
ctgggaaaac tttgtgtgca atgaaggtca gccattcatg ccttggtaca aattcgatga 240
caattatgca tccctgcacc gcacgctaaa ggagattctc agaaacccga tggaggcaat 300
gtacccacac atattctact tccactttaa aaacctactg aaagcctgtg gtcggaacga 360
aagctggctg tgcttcacca tggaagttac aaagcaccac tcagctgtct tccggaagaa 420
gggcgtcttc cgaaaccagg tggatcctga gacccattgt catgcagaaa ggtgcttcct 480
ctcttggttc tgtgacgaca tactgtctcc taacacaaac tacgaggtca cctggtacac 540
atcttggagc ccttgcccag agtgtgcagg ggaggtggcc gagttcctgg ccaggcacag 600
caacgtgaat ctcaccatct tcaccgcccg cctctgctac ttctgggata cagattacca 660
ggaggggctc tgcagcctga gtcaggaagg ggcctccgtg aagatcatgg gctacaaaga 720
ttttgtatct tgttggaaaa actttgtgta cagtgatgat gagccattca agccttggaa 780
gggactacaa accaactttc gacttctgaa aagaaggcta cgggagattc tccagtgagg 840
ggtctccctg ggcctcatgg tctgtctctt ctagcctcct gctcatgctg cacgggcctc 900
ccctccatcc tgcaccagct gtgcttttgc ctggtcatcc tgagcccctc ctggcctcag 960
ggccattcca tagtgccccc ctgcctcacc acctactctc cgctctccca ggttcttcct 1020
gcagaggcct ctttctgcct ccatggctat ccatc 1055
<210> 67
<211> 2220
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2901076CB1
<400> 67
cggctccgtc gctgacgcgt cgtagacgtt ggggagcggg aaggcaacgg cagcgggatc 60
gggatgaaca gcggcggcgg cttcggtttg ggcttaggct tcggcctcac ccccacgtcg 120
gtgattcagg tgacgaatct gtcgtcggcg gtgaccagcg agcagatgcg gacgcttttt 180
tccttcctag gagaaatcga ggagctgcgg ctctaccccc cggacaacgc acctcttgct 240
ttttcctcca aagtatgtta tgttaagttt cgtgatccat caagtgttgg cgtggcccag 300
catctaacta acacggtttt tattgacaga gctctgatag ttgttccttg tgcagaaggt 360
aaaatcccag aggaatccaa agccctctct ttattggctc ctgctccaac catgacaagt 420
ctgatgcctg gtgcaggatt gcttccaata ccgaccccaa atcctttgac tactcttggt 480
gtttcactta gcagtttggg agctatacca gcagcagcac tagaccccaa cattgcaaca 540
ettggagaga taccacagcc accacttatg ggaaacgtgg atccttccaa aatagatgaa 600
attaggagaa cggtttatgt tggaaatctg aattcccaga caacgacagc tgatcaacta 660
cttgaatttt ttaaacaagt tggagaagtg aagtttgtgc ggatggcagg tgatgagact 720
cagccaactc ggtttgcttt tgtggaattt gcagaccaaa attctgtacc aagggccctt 780
gcttttaatg gagttatgtt tggagacagg ccactgaaaa taaatcactc caacaatgca 840
atagtaaaac cccctgagat gacacctcag gctgcagcta aggagttaga agaagtaatg 900
aagcgagtac gagaagctca gtcatttatc tcagcagcta ttgaaccaga gtctggaaag 960
agcaatgaaa gaaaaggcgg tcgatctcgt tcccatactc gctcaaaatc caggtctagc 1020
tcaaaatccc attctagaag gaaaagatca caatcaaaac acaggagtag atcccataat 1080
agatcacgtt caagacagaa agacagacgt agatctaaga gcccacataa aaaacgctct 1140
aaatcaaggg agagacggaa gtcaaggagt cgttcgcatt cacgggacaa gagaaaagac 1200
actcgagaaa agatcaagga aaaggaaaga gtgaaagaga aagacaggga aaaggagaga 1260
gagagggaaa aggaacgtga aaaagaaaag gaacggggta aaaacaaaga ccgggacaag 1320
gaacgggaaa aggaccggga aaaagacaag gaaaaggaca gagagagaga acgggaaaaa 1380
gagcatgaga aggatcgaga caaagagaag gaaaaggaac aggacaaaga aaaggaacga 1440
gaaaaagaca gatccaaaga gatagatgaa aaaagaaaga aggataaaaa atccagaaca 1500
48/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
ccacccagga gttacaatgc atcgcgaaga tctcgtagtt ccagcaggga aaggcgtagg 1560
aggaggagca ggagttcttc cagatcgcca agaacatcaa aaaccataaa aaggaaatct 1620
tctagatctc cgtcccccag gagcagaaat aagaaggata aaaagagaga aaaagaaagg 1680
gaccacatca gtgaaagaag agagagagaa cgttcaacgt ctatgagaaa gagttctaat 1740
gatagagatg ggaaggagaa gttggagaag aacagtactt cacttaaaga gaaagagcac 1800
aataaagaac cagattcaag tgtgagcaaa gaagtagatg acaaggatgc accaaggact 1860
gaggaaaaca aaatacagca caatgggaat tgtcagctga atgaagaaaa cctctctacc 1920
aaaacagaag cagtatagga ccgacaagtg tacctctgca ctcaatgctg gaatcaaatc 1980
caaagctttt aattctctca acaagatgta aacaggaaag aaatctagtt gagcatgaag 2040
ataggatcta acagcttttc cagttgttag atgactttgt ggccatcttg ttattgagta 2100
agaaaataaa gcatggacat catgaaaata acagatgtta cccaaactca tcttctaaaa 2160
tctgtgcatt tccatggtgg ctgacacact tgtcatgtgg tctgttagtg tttgccaaga 2220
<210> 68
<211> 1890
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3074572CB1
<400> 68
ggcggtgccc ggccggggcc acgccttttc cggcccgcag cgcggcctgg gctcccgcgt 60
gtttaaaagt gcgcttgtgg ctgctgctgt cttaactcct gtgcttggcg gacagacagg 120
cgagatggcg gcggaggtgt tgccgagtgc gaggtggcag tattgtgggg cgcccgacgg 180
gagccagaga gctgtactgg tccagttctc caacgggaag ctacagagtc caggcaacat 240
gcgctttacc ttgtatgaga acaaagattc caccaacccc aggaagagga atcaacggat 300
cctggcagct gaaacagata ggctctccta tgtgggaaac aattttggga ctggagccct 360
caaatgcaac actttgtgca ggcactttgt gggaattttg aacaagacct ctggccaaat 420
ggaagtatat gatgctgaat tgttcaatat gcagccacta ttttcagatg tatcagttga 480
gagtgaactg gcgctagaga gtcagaccaa aacttacaga gaaaagatgg attcttgtat 540
tgaagccttt ggtaccacca aacagaagcg agctctgaac accaggagaa tgaacagagt 600
tggcaatgaa tctttgaatc gtgcagtggc taaagctgca gagactatca ttgatacgaa 660
gggtgtgact gctctggtca gcgatgctat ccacaatgac ttgcaagatg actccctcta 720
ccttcctccc tgctatgatg atgcagccaa gcctgaagac gtgtataaat ttgaagatct 780
tctttcccct gcggagtatg aagctcttca gagcccatct gaagctttca ggaacgtcac 840
gtcagaagaa atactgaaga tgattgagga gaacagccat tgcacctttg tcatagaagc 900
gttgaagtct ttgccatcag atgtggagag ccgagaccgc caggcccgat gcatatggtt 960
tctggatacc ctcatcaaat ttcgagctca tagggtagtt aagcggaaaa gtgctctggg 1020
acctggagtt ccccacatca tcaacaccaa actgctgaag cactttactt gcttgaccta 1080
caacaatggc agattacgga acttaatttc ggattctatg aaggcgaaga ttactgcata 2140
tgtgatcata cttgccttgc acatacatga cttccaaatt gacctgacag tgttacagag 1200
ggacttgaag ctcagtgaga aaaggatgat ggagatagcc aaagccatga ggctgaagat 2260
ctccaaaaga agggtgtctg tggccgccgg cagtgaagaa gatcacaaac tgggcaccct 1320
gtccctcccg ctgectccag cccagacctc agaccgectg gcaaagcgga ggaagattac 2380
ctagacgcat gctttccaga cagggcgttt tggctgcatc acagccactg gctggtccta 1440
ttcatttcca tttttatgta tgttttgaaa agaaaaggtc cggggatggt ggctcacacc 1500
tgaaatccca gcactttggg aggccgaggc aggaagatca ttgagctcag gagtttgaaa 1560
ccagtctgga caacataggg agaccccatc tctaccggag gaaaaaaaaa agagtcaggc 1620
ctggtggtgt gcgcctgtaa tcccagctac tcgggaggct gaggcaggac gattacttga 1680
gcttgggaaa tcaaggttgc agtgagctat gattgtgtgg ccacactcca tcctgggtca 1740
cagagtgaga ccttgtctca aaaaaagtaa cataaggaaa aaagaagcct tgctttagca 1800
caggtatgaa gccagaagcc agcatctcaa ctgtgcttgt cttatgcaga aatataaagc 1860
gatggccagg ttggacttca aaaaaaaaaa 1890
<210> 69
<211> 2893
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1437895CB1
<400> 69
aattgggctc accaggatcg tccaggataa tcttccaatc tcaagtgtgg tttattgaca 60
49164
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
atcatttaca atgccgaaga gtgctgtagt gagccagcac agtgggtaac acagcaacgg 120
agaacagatg caggtttgag gaatttaact tgctaaaacc ttgaactgaa gtcttagaga 180
ttggaacata cgggtttgta taaataggct tttaagccct gtttgcaatg ggttactgat 240
aggagaaact tgcttgtgga atgtcagctg cgtgagctca ctgtcagaca agatggaaga 300
agaagggctg gagtgtccaa actcttcctc tgaaaaacgc tattttcctg aatccctgga 360
ttccagcgat ggggatgagg aagaggtttt ggcctgtgag gatttggaac ttaacccctt 420
tgatggattg ccatattcat cacgttatta taaacttctg aaagaaagag aagatcttcc 480
tatatggaaa gaaaaatact cctttatgga gaacctgctt caaaatcaaa tcgtgattgt 540
ttcaggagat gctaaatgtg gtaagagcgc tcaggttcct cagtggtgtg ctgaatattg 600
tctttccatc cactaccagc acgggggcgt gatatgcaca caggtccaca agcagactgt 660
ggtccagctc gccctgcggg tggcggatga aatggatgtt aacattggtc atgaggttgg 720
ctacgtgatc cctttcgaga actgctgtac caacgaaaca atcctgaggt attgtactga 780
tgatatgctg caaagagaaa tgatgtccaa tccttttttg ggtagctatg gggtcatcat 840
cttagatgat attcatgaaa gaagcattgc aactgatgtg ttacttggac ttcttaaaga 900
tgttttacta gcaagaccag aactgaagct cataattaac tcctcacctc acctgatcag 960
caaactcaat tcttattatg gaaacgtgcc tgtcatagaa gtgaaaaata aacaccctgt 1020
ggaggttgtg taccttagtg aggctcaaaa ggattctttt gagtctattt tacgccttat 1080
ctttgaaatt caccactcgg gtgagaaagg tgacattgta gtctttctgg cctgtgaaca 1140
agatattgag aaagtctgtg aaactgtcta tcaaggatct aacctaaacc cagatcttgg 1200
agaactggtg gttgttcctt tgtatccaaa agagaaatgt tcattgttca agccactcga 1260
tgaaacagaa aaaagatgcc aagtttatca aagaagagtg gtgttaacta ctagctctgg 1320
agagtttttg atctggagca actcagtcag atttgttatc gatgtgggtg tggaaagaag 1380
aaaggtgtac aacccgagaa taagagcaaa ctcgctcgtc atgcagccca tcagccagag 1440
ccaggcagag atacgcaagc agattcttgg ctcatcttct tcaggaaaat ttttctgcct 1500
gtacactgaa gaatttgcct ccaaagacat gacgccactg aagccagcag aaatgcagga 1560
agccaaccta acaagcatgg tgctttttat gaagaggata gacattgcgg gcctaggcca 1620
ctgtgacttc atgaacagac cagcaccaga aagtttgatg caggcattgg aagacttaga 1680
ttatctggca gcactggata atgatggaaa tctttctgaa tttggaatca tcatgtcaga 1740
gtttcctctt gatccacaac tctcgaagtc tatcttagcg tcctgtgaat ttgactgtgt 1800
agatgaagtg ctaacaatcg cagccatggt aacagctcca aattgctttt cacatgtgcc 1860
acatggagct gaagaggctg ccttgacttg ttggaagaca tttttacatc ccgaaggaga 1920
tcactttacc ctcatcagca tttacaaggc ttaccaagac acaactctga attctagcag 1980
tgagtactgt gtggaaaagt ggtgtcgtga ttacttcctc aactgttcag cactcagaat 2040
ggcagatgtt attcgagctg aactcttaga aattatcaag cgaatcgagc ttccctatgc 2200
agaacctgct tttggctcca aggaaaacac tctaaacata aagaaagctc ttctgtccgg 2160
ttactttatg cagattgctc gggatgttga tggatcaggt aactacttaa tgctgacaca 2220
taagcaggtt gctcagctgc atcccctgtc tggttactca atcaccaaga agatgccaga 2280
gtgggtcctc ttccataaat tcagcatttc tgagaacaac tacatcagga ttacctcaga 2340
aatctctcct gaactattta tgcagctggt accacaatac tatttcagta atctgcctcc 2400
tagtgaaagt aaggacattc tacagcaagt agtggatcac ctatcccctg tgtcaacaat 2460
gaataaggaa cagcaaatgt gtgagacgtg ccctgaaact gaacagagat gcactctcca 2520
gtgactcccc agcaaacaca aggtgcagca gggtcccaaa ggtagctgga tggctgaact 2580
gctggatatg ggagatacat gacgcgaaga cggatttcac atccacagga cggtcttgaa 2640
gaaaataaca ctgtgtatat tattttaaaa taaaaaatag aagtttttat tgagttcttt 2700
aaattactac tccatgcttt tcttcttctt ggaaaagttt ttaaatcaac cactcataat 2760
ttgaccaaaa ttttaaaaaa ctggtatttt gtaaatgtgt cagagacaca tgggacagaa 2820
ccctactttt tgtagaggaa cttaatctga ataaagtctg agtttttcag taaaaaaaaa 2880
aaaaaaaaaa aag 2893
<210> 70
<212> 885
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_teature
<223> Incyte ID No: 1454656CB1
<400> 70
ccagcatgcg gcgcccatgt aacccggtcc gtgccgcaaa gcgaacggcg gccgcggcgc 60
gggccccgcg ggggttagag gtcaccatgc tgagggtcgc gtggaggacg ctgagtttga 120
ttcggacccg ggcagttacc caggtcctag tacccgggct gccgggcggt gggagcgcca 180
agtttccttt caaccagtgg ggcctgcagc ctcgaagtct cctcctccag gccgcgcgcg 240
gatatgtcgt ccggaaacca gcccagtcta ggctggatga tgacccacct ccttctacgc 300
tgctcaaaga ctaccagaat gtccctggaa ttgagaaggt tgatgatgtc gtgaaaagac 360
tcttgtcttt ggaaatggcc aacaagaagg agatgctaaa aatcaagcaa gaacagttta 420
tgaagaagat tgttgcaaac ccagaggaca ccagatccct ggaggctcga attattgcct 480
50/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
tgtctgtcaa gatccgcagt tatgaagaac acttggagaa acatcgaaag gacaaagccc 540
acaaacgcta tctgctaatg agcattgacc agaggaaaaa gatgctcaaa aacctccgta 600
acaccaacta tgatgtcttt gagaagatat gctgggggct gggaattgag tacaccttcc 660
cccctctgta ttaccgaaga gcccaccgcc gattcgtgac caagaaggct ctgtgcattc 720
gggttttcca ggagactcaa aagctgaaga agcgaagaag agccttaaag gctgcagcag 780
cagcccaaaa acaagcaaag cggaggaacc cagacagccc tgccaaagcc ataccaaaga 840
cactcaaaga cagccaataa attctgttca atcatttaaa aaaaa 885
<210> 71
<211> 1269
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 121130CB1
<400> 71
tcagacaagc actggacgtg gcggccattt tgttttggac accgagcagg agctggcggc 60
cgctgcagac gaaaggcagg aaagggcagg ccgggtgagc agacggatcg gccgactaga 120
cagccaacca gcaacaacga actgagctcg catactaccg cttacgcatc taaccaaccg 180
cccatctagc taacccgagc ccctccaccg tcaactcagg ttcggccggt ccccggcccg 240
cctgccggag ccgtggtggc agccccggga ggagcactgg cgtctgtttc cttcgattct 300
cgggattcga agatggctgc acagtcagcg ccgaaagttg tgctaaaaag caccaccaag 360
atgtctctaa atgagcgctt tactaatatg ctgaagaaca aacagccgac gccagtgaat 420
attcgggctt cgatgcagca acaacagcag ctagccagtg ccagaaacag aagactggcc 480
cagcagatgg agaatagacc ctctgtccag gcagcattaa aacttaagca gagcttaaag 540
cagcgcctgg gtaagagtaa catccaggca cggttaggcc gacccatagg ggccctggcc 600
aggggagcaa tcggaggacg aggcctaccc ataatccaga gaggcttgcc cagaggagga 660
ctacgtgggg gacgtgccac cagaacccta cttaggggcg ggatgtcact ccgaggtcaa 720
aacctgctcc gaggtggacg agccgtagct ccccgaatgg gcttaagaag aggtggtgtt 780
cgaggtcgtg gaggtcctgg gagagggggc ctagggcgtg gagctatggg tcgtggcgga 840
atcggtggta gaggtcgggg tatgataggt cggggaagag ggggctttgg aggccgaggc 900
cgaggccgtg gacgagggag aggtgccctt gctcgccctg tattgaccaa ggagcagctg 960
gacaaccaat tggatgcata tatgtcgaaa acaaaaggac acctggatgc tgagttggat 1020
gcctacatgg cgcagacaga tcccgaaacc aatgattgaa gcctgcccat cctcccatga 1080
gagactcttg ttagtcaaca catctgtaaa taaccttgag ataacagatg agaagaaatc 1140
tgattgatgc tggatggacc tatcacaata ggctgtggac ttacttgcca ccagcttgtg 1200
catttagtgt gttcctttta ctttttgata ctgtgttgta tgaaaccctt ttgtcctttg 1260
aaaaaaaaa 1269
<210> 72
<211> 1066
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1257715CB1
<400> 72
cggctcgagg tgaatggggg cagcatgagg ccgggcggct ttttgggcgc cggacagcgg 60
ctgagtagag ccatgagccg atgtgttttg gagcctcgcc ccccggggaa gcggtggatg 120
gtggctggcc tggggaatcc cggactgccc ggcacgcgac acagcgtggg catggcggtg 180
ctggggcagc tggcgcggcg gctgggtgtg gcggagagtt ggacgcgcga ccggcactgt 240
gccgccgacc tcgccctggc cccgctgggg gatgcccaac tggtcctgct ccggccacgg 300
cggcttatga acgccaacgg gcgcagcgtg gcccgggctg cggagctgtt tgggctgact 360
gccgaggaag tctacctggt gcatgatgag ctggacaagc ccctggggag actggctctg 420
aagctggggg gcagtgccag gggccacaat ggagtccgtt cctgcattag ctgcctcaac 480
tccaatgcaa tgccaaggct gcgggtgggt atcgggcgcc cggcgcaccc tgaggcggtt 540
caggcccatg tgctgggctg cttctcccct gctgagcagg agctgctgcc tctgttgctg 600
gatcgagcca ccgacctgat cttggaccac atccgtgagc gaagccaggg gccctcactg 660
gggccgtgac actagtggcc atggctgcct gcctgactgt agtgcccacc aacccagcca 720
ctgccacaga gctgccacgc cagccttggt atctactttt tatacaaatc tcctctagac 780
tgttccaggc tgcctgcgga ttaaagtggg ggtgactgtg actggaccag tccatttctg 840
gagtaggttc ttctctctgt gtcctacttg ggacgtaggg gaacttcagg aagactaaac 900
ttttcaagcc tttttagaga accaggggca cgcatctctc cttgggtggg ccatgggact 960
51164
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
gtgactcctg gtggggacac gcagccttct gaggtctcgt ggccacagtg gagctgagca 1020
tgaccagcag ttgctgcagc atctccttgt gccatggctg gaacgt 1066
<210> 73
<211> 639
<212> DNA
<213> Homo Sapiens
<220>
<222> misc_feature
<223> Incyte ID No: 1342022CB1
<400> 73
ggggagacac gtgcccttgg tactatgacc actagaccag cattcatatt acaccacagt 60
gactgcttct cgagccgctc gagccgaatt cggcacgagg gagtctggag acgacgtgca 120
gaaatggcac ctcgaaaggg gaaggaaaag aaggaagaac aggtcatcag cctcggacct 180
caggtggctg aaggagagaa tgtatttggt gtctgccata tctttgcatc cttcaatgac 240
acttttgtcc atgtcactga tctttctggc aaagaaacca tctgccgtgt gactggtggg 300
atgaaggtaa aggcagaccg agatgaatcc tcaccatatg ctgctatgtt ggctgcccag 360
gatgtggccc agaggtgcaa ggagctgggt atcaccgccc tacacatcaa actccgggcc 420
acaggaggaa ataggaccaa gacccctgga cctggggccc agtcggccct cagagccctt 480
gcccgctcgg gtatgaagat cgggcggatt gaggatgtca cccccatccc ctctgacagc 540
actcgcagga aggggggtcg ccgtggtcgc cgtctgtgaa caagattcct caaaatattt 600
tctgttaata aattgccttc atgtaaaaaa aaaaaaaaa 639
<210> 74
<211> 1420
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 194704CB1
<400> 74
ggccgacgcg accatcgttt gtcgacgccg ctgccaccgc ctgcctgaga gaagtcgtcg 60
cggccgaccc cgtcgcctcc gccggctacc atgtccgccc aggcgcagat gcgggccctg 120
ctggaccagc tcatgggcac ggctcgggac ggagacgaaa ccagacagag ggtcaagttt 180
acagatgacc gtgtctgcaa gagtcacctt ctggactgct gcccccatga catcctggct 240
gggacgcgca tggatttagg agaatgtacc aaaatccacg acttggccct ccgagcagat 300
tatgagattg caagtaaaga aagagacctg ttttttgaat tagatgcaat ggatcacttg 360
gagtccttta ttgctgaatg tgatcggaga actgagctcg ccaagaagcg gctggcagaa 420
acacaggagg aaatcagtgc ggaagtttct gcaaaggcag aaaaagtaca tgagttaaat 480
gaagaaatag gaaaactcct tgctaaagcc gaacagctag gggctgaagg taatgtggat 540
gaatcccaga'agattcttat ggaagtggaa aaagttcgtg cgaagaaaaa agaagctgag 600
gaagaataca gaaattccat gcctgcatcc agttttcagc agcaaaagct gcgtgtctgc 660
gaggtctgtt cagcctacct tggtctccat gacaatgacc gtcgcctggc agaccacttc 720
ggtggcaagt tacacttggg gttcattcag atccgagaga agcttgatca gttgaggaaa 780
actgtcgctg aaaagcagga gaagagaaat caggatcgct tgaggaggag agaggagagg 840
gaacgggagg agcgtctgag caggaggtcg ggatcaagaa ccagagatcg caggaggtca 900
cgctcccggg atcggcgtcg gaggcggtca agatctacct cccgagagcg acggaaattg 960
tcccggtccc ggtcccgaga tagacatcgg cgccaccgca gccgttcccg gagccacagc 1020
cggggacatc gtcgggcttc ccgggaccga agtgcgaaat acaagttctc cagagagcgg 1080
gcatccagag aggagtcctg ggagagcggg cggagcgagc gagggccccc ggactggagg 1140
cttgagagct ccaacgggaa gatggcttca cggaggtcag aagagaagga ggccggcgag 1200
atctgaaccc gtctcccggg tgctgtaaat agtctgataa acgttcacac agtctaaaat 1260
taccctttat atttgctgaa tacaactcat cttttgtagt ttaaaatttc tattgttttg 1320
gagctagctg tgagtttcta gaagtgtaca gagttgctcc tgtgttcccg ggtcatgttg 1380
agtaggaata aataaatctg atgctgccaa aaaaaaaaaa 1420
<210> 75
<211> 1457
<212> DNA
<213> Homo Sapiens
<220>
<221> mist feature
52/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<223> Incyte ID No: 607270CB2
<400> 75
gcgccattag cgcctgcgcc gtctctaggc cccgccccct cacccctccg gtcctggagc 60
tcccacagct aacatggcgg cgccctgtgt gtcctacggc ggagcagttt cgtaccggct 120
tcttctctgg ggtaggggta gcctcgcccg gaagcaaggc ctctggaaaa ccgcggcccc 180
tgagttgcaa acaaatgtca gatcccagat attaaggcta agacatactg catttgtaat 240
accaaagaaa aacgttceta cctcaaaacg tgaaacttac acagaggatt ttattaaaaa 300
gcagattgaa gagttcaaca taggaaagag acatttagcc aacatgatgg gagaagatcc 360
agaaactttc actcaagaag atattgacag agctattgct taccttttcc caagtggttt 420
gtttgagaaa cgagccaggc cagtaatgaa gcatcctgaa cagatttttc caagacaaag 480
agcaatccag tggggagaag atggccgtcc atttcactat ctcttctata ctggcaaaca 540
gtcatactat tcattaatgc atgatgtata tggaatgtta ctcaatttag aaaaacatca 600
aagtcacttg caagccaaaa gtctgctccc agaaaaaact gtaaccagag acgtgattgg 660
cagcagatgg ctgattaagg aggaactaga agaaatgtta gtggaaaaac tgtcagatct 720
agattatatg cagttcattc ggctgctaga aaagttattg acatcgcagt gtggtgctgc 780
tgaggaagaa tttgtgcaga ggtttcgaag aagtgtaact cttgaatcaa aaaaacagct 840
gattgaacct gtacagtatg atgagcaagg aatggccttt agcaaaagcg aaggtaaaag 900
aaagactgca aaagcagaag caattgttta taaacatgga agtggaagaa taaaagtaaa 960
tggaattgat taccagcttt acttcccgat cacacaggac agagaacagc tgatgttccc 1020
tttccacttt gttgaccggc tgggaaagca cgacgtgacc tgcacagtct cagggggcgg 1080
gaggtcagcg caggctggag caatacgact ggcaatggca aaagccttgt gcagctttgt 1140
caccgaggac gaggtcgagt ggatgagaca agctggacta cttactactg atccacgtgt 1200
gagggaacgg aagaagccag gccaagaggg agcccgcaga. aagtttacgt ggaagaaacg 1260
ctaagggttt gctcccagga aaggagagga agagctatat atatgtgccg acatgtggca 1320
gacacacagt aaataatggc tgaccagcat gagggcagta ctgtcagaaa tttctttgag 1380
ctgtgagatg gatttatttt taaatgctac tttgtaaagg tgacctttaa aaaataaaag 1440
gaaaataaag aaaaaaa 1457
<210> 76
<211> 1184
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 758546CB1
<400> 76
caggccgtcc aggtcttggg gcgccgcggc ggaaatcgcg cggatgccag aacgcgctct 60
cagcttcggg tcctgcggct gcggctgccg ccatcatggt gcggaagctt aagttccacg 120
agcagaagct gctgaagcag gtggacttcc tgaactggga ggtcaccgac cacaacctgc 180
acgagctgcg cgtgctgcgg cgttaccggc tgcagcggcg ggaggactac acgcgctaca 240
accagctgag ccgtgccgtg cgtgagctgg cgcggcgcct gcgcgacctg cccgaacgcg 300
accagttccg cgtgcgcgct tcggccgcgc tgctggacaa gctgtatgct ctcggcttgg 360
tgcccacgcg cggttcgctg gagctctgcg acttcgtcac ggcctcgtcc ttctgccgcc 420
gccgcctccc caccgtgctc ctcaagctgc gcatggcgca gcaccttcag gctgccgtgg 480
cctttgtgga gcaagggcac gtacgcgtgg gccctgacgt ggttaccgac cccgccttcc 540
ttgtcacgcg cagcatggag gactttgtca cttgggtgga ctcgtccaag atcaagcggc 600
acgtgctaga gtacaatgag gagcgcgatg acttcgatct ggaagcctag cggatctccc 660
actttgcatg gctgtctttt acagatggga aaactgaggc ctgatgctgg agattctatg 720
agggtgctct cctcaagggt atcagacggt cgtaggttct taagaatttg attcatcagt 780
ggcaggccat gcatagagcc acgggaggtg cgtccttgtt ttccaggaaa tgttcttaga 840
acttggacta ctgattatta attgactgtg ccttgggaaa cagtgggaag taacttggtg 900
cagcactggg gtattgttgg cttcttgtgt tggaaacttt gtaatgtaaa aggaaaaact 960
ggaaatcccc acgccctgtt tccctttatc gtcttgtggt tggactggtt caattcgttt 1020
aactcgaatt cttgctcctg gccgtggtta agctgtgtac agatgatgga gagtttggcc 1080
tcaagttttt ataaactgag cgagactagt gttcaggatc tcctcccttg tttaaatgtc 1140
aataaatgcc ccaactgctt tgtaagtgca aaaaaaaaaa aaaa 1184
<210> 77
<211> 1638
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
53/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<223> Incyte~ID No: 866043CB1
<400> 77
atcggggatc ttgccccagc cagaggetac agtggcccgg gaaggagcct caagtcacct 60
tccccatcaa agagccttct tgttcttctc tgtggacgag ccatgttcca gccagccaca 120
tgcccctggc agctgcccgc tttaagcaag taaaactctc caggaacttt cccaagtcat 180
ctttccgtgc tcaaagtgag tctgaaaccg tagtaaaaat ggcagctctt ttcagaagaa 240
aaaatgtgag gactgtgtgg taccctatac tcccagaaga ctaagacagc ggcaggcatt 300
aagcacggag acaggcaagg gtaaagacgt ggagccacag gggccccctg cagggcgtgc 360
cccagcccct ctctacgtgg gcccgggagt gtctgagttt attcagccgt atttgaatag 420
ccattataaa gaaaccacag ttccccggaa agtgcttttc cacctgagag gccacagggg 480
ccctgtcaac accattcagt ggtgtccagt cctttctaag agccacatgc ttctctccac 540
ttctatggat aaaactttca aggtatggaa cgccgtggac tccgggcact gcctgcagac 600
ctactccctg cacacagagg cagtgcgggc cgcccggtgg gctccctgtg gccggcgcat 660
cctcagtggt ggctttgact tcgcgctgca cctaacagac cttgaaacag gaacccagct 720
atttagtggt cgaagtgact ttagaatcac taccttgaaa ttccatccaa aagaccacaa 780
catcttttta tgtggaggct tcagctctga aatgaaagct tgggatataa ggactggcaa 840
ggtgatgaga agctacaagg cgaccatcca gcagaccttg gacatcctgt tcctccggga 900
aggctccgag ttcctgagca gcacagacgc ttccacccgg gactcagctg accgcaccat 960
tattgcctgg gatttccgga cctctgccaa aatctccaac cagattttcc acgagaggtt 1020
cacctgcccc agcctcgcct tgcacccgag agagcccgtg ttcctggcac agaccaatgg 1080
caactacctg gcccttttct ccactgtgtg gccctaccgg atgagcagac ggcggcgcta 1140
tgaagggcac aaggtggagg gctactcagt gggctgcgag tgctccccag gcggtgactt 1200
gctggtgacg ggcagcgccg atggccgggt cctgatgtac agcttccgca cagccagccg 1260
agcatgcaca ctgcaggggc acacacaggc ctgtgtcggc accacctatc accccgtgct 1320
gccctccgtc ctcgccacct gctcctgggg aggggacatg aagatctggc actgagcttt 1380
ttgtcactga accttcccga tgccagctgg gctcttggac tcccctcttc ctcaagggta 1440
gatgagagga acgagcacag aggttggctg tgggtcctgg gtaccacctt ctgagcctca 1500
gtttcctcat ctgtaaagtg gggagaaaag tctgtttgcc tcaggagtgt gaggactaca 1560
ctagtgaaag cgcctggcgg gcagccggcg atgcccaata aatgtgtgtt ttgctgtttg 1620
ttaagtgaaa aaaaaaaa 1638
<210> 78
<211> 701
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 927065CB1
<400> 78
tcacgcttcg tggggcggga cgaggagaag ccaaacgtaa agacaccagg agtttctcgg 60
gcccagctgt ggctgctgcc ggggagcccc aagccttggc gggtccttgc ggcgaatagg 120
agtctggtca ggcgtcaggc tagtccgacg aagagtgggt gtgatcagca ctggaaaaga 180
tgcctgcccc tgctgccaca tatgaaagag tagtttacaa aaacccttcc gagtaccact 240
acatgaaagt ctgcctagaa tttcaagatt gtggagttgg actgaatgct gcacagttca 300
aacagctgct tatttcggct gtgaaggacc tgtttgggga ggttgatgcc gccttacctt 360
tggacatcct aacctatgaa gagaagacct tgtcagccat cttgagaata tgtagcagtg 420
gtcttgtcaa attgtggagc tctttgaccc tgttaaggat ccctattaaa ggcaaaaaat 480
gtgctttccg ggtgattcag gtttctccat ttcttcttgc attatctggt aatagtaggg 540
aactagtatt ggattgaatg aatagtcttc cattttggaa acgttcatcc actctcatat 600
ttattttttg gtgccctgca tgtttgaaga ctgaaagcag gctaaaagct cttgatgaaa 660
tttgagggtg ctgaaagatg ttcccactaa tttccagcca t 701
<210> 79
<211> 1829
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 938071CB1
<400> 79
gggttttgca gaagtaccca gaactgtgtc caaggtttcc tcagatttgg gctgttccgc 60
agcggcaggt cccgggaacc aaggcaacag acatcttcct aggctcgcga gagcgccccc 120
54/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
ttgtcccacg gctgctgggg ccccccagta gccatggctc cggtgtccgg ctcacgcagc 180
ccggataggg aggcctcggg ctcgggggga agacgtcgca gttcgtcgaa gagtccgaag 240
cccagcaaat ctgcccgctc cccgcggggc cgccgctctc gctcgcactc ttgctctcgg 300
tccggggacc ggaatggact cacccatcag ctgggtggcc tcagccaagg ctcccgaaac 360
cagtcctacc gctcacgctc gcggtcgcgt tctagagagc ggccctctgc gccccggggc 420
atccccttcg cttctgcctc ctcgtcagtc tattacggca gctactcgcg cccctacggg 480
agcgacaagc cttggcctag cctcctcgac aaggagaggg aggagagcct gcggcagaag 540
agattaagtg agagagagag aattggagaa ttgggagctc ctgaagtatg gggactttct 600
ccaaagaatc ctgaaccaga ttctgatgaa catacaccag tggaggatga agagccaaag 660
aaaagcacta cttcagcttc tacttcagaa gaagaaaaaa agaagaagtc tagccgttca 720
aaagaaaggt ccaagaaaag gagaaagaaa aaatcatcga aaagaaaaca taagaagtat 780
tctgaagata gcgacagtga ctctgattct gaaacagact ccagtgatga agataacaaa 840
aggagagcaa agaaagccaa gaaaaaggaa aagaagaaga aacacagatc gaagaaatat 900
aagaaaaaga ggtctaagaa gagcagaaaa gagtccagtg attcaagctc taaagaatcc 960
caagaagagt ttctggaaaa tccctggaag gatcgaacaa aggctgaaga accatcagat 1020
ttaattggcc cagaggctcc aaaaacactt acctctcaag atgataaacc tttgaagcat 1080
cgccgaatgg aggctgtgcg actgcgaaaa gagaaccaga tctacagtgc tgatgagaag 1140
agagcccttg catcctttaa ccaagaagag agacgaaaga gagagaacaa gattctggcc 1200
agttttcgag aaatggttta cagaaagacc aaagggaagg atgacaaata aagattttct 1260
gattgtccag aagacatttt taacaacaaa aaagaaagtc tgggttccac acatacatag 1320
aaaaagatta ttatgttctg agaaagcttt acagtgctac tgtgccttct atttaattct 1380
ttcagtcctt caataaaaag ctgcttattg atataacttt agcaagttct ttgggttatt 1440
ttggattgac catagtaact ttctggttta aaaatccaaa ttatgggctg ggcacggttg 1500
ctcacgcctg tagtctcagc ctcctgaaat gctgggattg caggtgtgag caaccgtgcc 1560
tggccgtttt tgttaaggtt atttgatctg cattattatt acatgcctat gataaatttt 1620
taattacccc tgtgtataaa agggctttcc gattatccta ttgggaaaat gcccgcttgc 1680
cttatatttt taagtggttg tttttcaaaa gtgtttaaat aagggcggcc atatttcaaa 1740
gtattggaca aaaagttttt ttaattataa tttttggaga cgggggtctc ctctgttacc 1800
caggctagag ttcagttgac cgagatctt 1829
<210> 80
<211> 2541
<222> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3295984CB1
<400> 80
caagaaagag gggaaaggat cggaaaaaga agctaaaata ctatagaaaa ccatgagatc 60
tattcgatct tttgctaatg atgatcgcca tgttatggtg aaacattcaa caatctatcc 120
atctccggag gaacttgaag ctgttcagaa tatggtatct actgttgaat gtgctcttaa 180
acatgtctca gattggttgg atgaaacaaa taaaggcaca aaaacagagg gtgagacaga 240
agtgaagaaa gatgaggccg gagaaaacta ttccaaggat caaggtggtc ggacattgtg 300
tggtgtaatg aggattggcc tggttgcaaa aggcttgctg attaaagatg atatggactt 360
ggagctggtt ttaatgtgca aagacaaacc cacagagacc ctgttaaata cagtcaaaga 420
taatcttcct attcagattc agaaactcac agaagagaaa tatcaagtgg aacaatgtgt 480
aaatgaggca tctattataa ttcggaatac aaaagagccc acgctaactt tgaaggtgat 540
acttacctca cctctaatta gggacgaatt ggagaagaag gatggagaaa atgtttcgat 600
gaaagatcct ccggacttat tggacaggca gaaatgcctg aacgccttgg cgtctcttcg 660
acatgccaaa tggtttcagg caagggcaaa tggattaaaa tcatgtgtaa ttgtcctccg 720
cattctgcgt gatttgtgca acagagtccc cacatgggca ccattgaaag gatggccact 780
agaacttata tgtgaaaagt ctataggtac ttgtaataga cctttgggcg ctggggaggc 840
cttgagacga gtaatggagt gtttggcatc tggaatacta cttcctgggg gtcctggtct 900
tcatgatcct tgtgagcgag acccaacaga tgctctgagc tatatgacca tccagcaaaa 960
agaagatatt acccacagtg cacagcatgc actcagacta tcagcctttg gtcagattta 1020
caaagtgctg gagatggacc cccttccatc tagtaagcct tttcagaagt attcctggtc 1080
agttactgat aaagaaggtg ctgggtcttc agctctaaag aggccatttg aagatggatt 1140
aggggatgat aaagacccca acaagaagat gaaacgaaac ttaaggaaaa ttctggatag 1200
taaagcaata gaccttatga atgcactaat gaggctaaat cagatcaggc ctgggcttca 1260
gtataagctc ctatctcagt ctggccccgt tcatgcccca gtcttcacaa tgtctgtaga 1320
tgtggatggc acaacatatg aagcctcagg accatccaag aaaacagcaa aacttcacgt 1380
agcggtgaag gtattgcagg caatgggata tccaacaggc tttgatgcag atattgaatg 1440
tatgagttcc gatgaaaaat cagataatga aagtaaaaat gaaacagtgt cttcaaactc 1500
aagcaataat actggaaatt ctacaactga aacctccagt accttagagg taagaactca 1560
gggccctatc ctcacagcaa gtggcaaaaa ccctgtaatg gagctcaatg aaaaaagaag 1620
55/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
aggtctcaag tatgaactca tctcagagac tggtggaagc catgacaagc gctttgtaat 1680
ggaggtagaa gtagatggac agaaattcag aggcgcaggt ccaaataaga aagtggcaaa 1740
ggcgagtgca gctttagctg ccttggagaa actgttttct ggacccaatg cggcaaataa 1800
taagaaaaag aagattatcc ctcaggcaaa gggcgttgtg aatacagctg tgtctgcagc 1860
agtccaagct gttcggggca gaggaagagg aactctaaca aggggagctt ttgttggggc 1920
gacagctgct cctggctaca tagctccagg ctatggaaca ccatatggtt acagcacagc 1980
tgcccctgcc tatggtttac ccaagagaat ggttctgtta cccgttatga aatttccaac 2040
atatcctgtt ccccactact cattctttta gcaaatgaca gaagctaatt cctattgaac 2100
aacaatacag tacaacacag aatgttagag aaaaagcctt tttatcctgc tttctttgaa 2160
cacatacttg atcaaaatta tttgtaaaga acatctttcc tactttttga ttttaacaaa 2220
tgcaaattta gttctctaaa acttgaaaaa aaaaaaagaa accagttctg tgaaaacggt 2280
acctcatttc tggaaaataa cttataccag cccttctgtt ctagggaaat aaaagtctag 2340
cagttcaaag tttaagtttt aagagacgta tcagattatg taaaattaaa tttgtgaagg 2400
atgtatagag tctcaaacac tgatcacaaa taaactgctt tgttgtaaca cagagtactg 2460
cctggttcct gatgcagtca ctgattctta gttgattgat atgtatttgc cccagggcac 2520
tttaatttgg gctgtagtta t 2541
<210> 81
<211> 1647
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4545237CB1
<400> 81
gtccgcggtc ggccgggctc cgcctgcagt gtggcccgtc cggacagtcc ctcaccccgg 60
cctgcgctgc tgcgtggact cgggcctcag gaattccgct gcggcccaag gcttgccgtt 120
tgacgaggag cagtcgcggt aggcggtggg caaggctgcc ctgggcggag gccgaggcgc 180
ggctcggact ccagcatggc gaccgcggtg cgcgctgtgg gctgcctccc cgtgctgtgt 240
agcgggacgg caggtcattt attggggagg cagtgttccc taaacacctt accagcagct 300
tccattttgg catggaagag tgttctcggc aatggccatt tgtcatcact gggaaccaga 360
gacacccatc cctacgccag cttgagccgt gcactgcaga cacaatgctg tatttcttct 420
cccagtcacc tgatgagcca gcagtataga ccatatagtt tcttcactaa attgactgca 480
gatgagctgt ggaaaggcgc tttagcagag actggtgctg gagcaaaaaa aggaagaggc 540
aaaagaacta aaaagaagaa aagaaaggat ctgaacaggg gtcagatcat tggtgaaggg 600
cgttatggtt ttctatggcc cggactgaat gtccctctta tgaaaaatgg agcagtgcag 660
accattgccc aaagaagcaa ggaagagcag gagaaggtgg aggcagacat gatccagcag 720
agagaagagt gggaccgaaa gaagaagatg aaggttaaac gggagcgagg atggagtgga 780
aactcatggg gaggcatcag tcttggcccc cctgaccctg gtccctgtgg agaaacatat 840
gaggattttg ataccaggat acttgaggta agaaacgttt tcactatgac tgcgaaagag 900
ggaagaaaga aatcgatccg tgtcttggtg gctgtgggga acggaaaagg agctgcaggt 960
ttttctattg ggaaagctac tgatcggatg gatgctttca ggaaagcaaa gaacagagca 1020
gttcaccatt tgcattatat agaacgatat gaagaccata caatattcca tgatatttca 1080
ttaagattta aaaggacgca tatcaagatg aagaaacaac ccaaaggtta cggcctccgc 1140
tgccacaggg ccatcatcac catctgccgg ctcattggca tcaaagacat gtatgccaag 1200
gtctctgggt ccattaatat gctcagcctc acccagggcc tcttccgtgg gctctccaga 1260
caggaaaccc atcaacagct ggctgataag aagggcctcc atgttgtgga aatccgggag 1320
gaatgtggcc ctctgcccat tgtggttgcg tccccccggg ggcccttgag gaaggatcca 1380
gagccagaag atgaggttcc agacgtcaaa ctggactggg aagatgtgaa gactgcacag 1440
ggaatgaagc gctctgtgtg gtctaatttg aagagagccg ccacgtaacc tctctggcct 1500
tgtgcagcca gttcctgtgc tgccctgcac ctaggagaga ctcagcccct cacagcttgg 1560
gatgttacct tgccttttgt ttgttttgag ggaagtttaa tctttaaact ctttggaaat 1620
aaataattat agctttcaaa aaaaaaa 1647
<210> 82
<211> 735
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte TD No: 4942964CB1
<220>
<221> unsure
56/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<222> 721
<223> a, t, c, g, or other
<400> 82
ctcgttcctg tcgcgcagca cgacctccac ttccacatct cccccggcgt cggcgcggtc 60
agttgaacca tggcggactc caaggccacc tcggcggtca ccctccgcac ccgcaagttc 120
atgaccaacc gcctcctggc ccgcaagcaa ttcgtgcttg aggtgatcca ccccggccgc 180
gccaacgtct ccaaggcgga gttgaaggag aggcttgcca aggcgtacga ggtgaaggac 240
cccaacacca tctttgtctt caagttccgc acccacttcg gaggaggaaa gtccactggt 300
ttcggcctca tctacgacaa cctcgaggct gccaagaagt tcgagccgaa ataccgcctc 360
atcaggaatg gtcttgctac taaggttgag aagtcccgca agcaaatgaa ggagcggaag 420
aacagggcca agaagatccg tggtgtcaag aagaccaaag ctggtgacgc caagaagaag 480
taaacgttcg tttacatttg tattactgtt ctgggctctg ggtggtctag ctgcaatgtc 540
ataattatgg tcgtgttagg ttttgttcca cccttggcac tgaagtgatt ttttttgtaa 600
ttcctcggca ctgaagtgaa gttttgtctg aatattgcct cgtaacataa ttgcccggtc 660
cctgttctag ttgtggcgca gtctggtttg ttttgacatt tgtaatcgtg gttaatgtgg 720
ntggatcggt tcatg
735
<210> 83
<211> 2614
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5702144CB1
<400> 83
gtgcgctctc acccttatct ccaaattctg ggtgttgtcg cgagggctgc tgtgtccgga 60
acttccggtt ccggtcaggg tccgcgatct cggactaagg atgcggtccc gggttctgtg 120
gggcgctgcc cggtggctct ggccccgccg ggccgttggc ccagcccgcc ggcccctgag 180
ctccggtagc ccgccgctgg aggagctgtt cacccggggc gggcccttgc ggaccttcct 240
cgagcgccag gcggggtctg aagcccattt gaaggtcagg aggcccgagt tgctggcggt 300
gatcaaactg ctgaacgaga aggagcagga gctgcgggag actgagcact tgctgcacga 360
tgagaatgaa gatttaagga aacttgcaga gaatgaaatc actttgtgtc aaaaagaaat 420
aactcagctg aagcatcaga ttatcttact tttggttccc tcagaagaaa cagatgaaaa 480
tgatttgatc ctggaagtaa ctgcaggagt tggaggtcag gaggcaatgt tgtttacatc 540
agagatattt gatatgtatc agcaatatgc tgcatttaaa agatggcatt ttgaaaccct 600
ggaatatttt ccaagtgaac taggtggcct tagacatgca tctgccagca ttgggggttc 660
agaagcctat aggcacatga aatttgaagg aggtgttcac agagtacaaa gagtgccaaa 720
gacagaaaag caaggccgca tccatactag caccatgact gtagcaatat taccccagcc 780
tactgagatt aatctggtga ttaatccgaa agatttgaga attgacacta agcgagccag 840
tggagctggg gggcagcatg taaataccac ggacagtgct gtccggatag ttcatcttcc 900
aacaggtgtt gtttctgaat gtcaacaaga gagatctcag ctgaaaaata aagagctggc 960
tatgacaaag ttacgtgcaa aactgtacag catgcatcta gaagaagaaa taaataaaag 1020
acagaatgct agaaaaattc agattggaag taaaggaaga tcagagaaaa taagaacata 1080
taattttcca cagaaccggg tcacagatca cagaataaac aagacgctgc atgatcttga 1140
aacttttatg caaggagatt atctactgga tgaacttgta cagtcattga aggaatacgc 1200
cgattatgaa tctttagtag aaattatttc ccaaaaagtt taagttgatt tgttatttat 1260
agactttcgt agcttagaaa aattctacag tacatccaca tagggtgaaa gtacccttac 1320
tetcttgaaa aacgttgagt taacacagtt ggaggtaata tgcatattct gaagtcatag 1380
ataatttaca cagatctctc tcaatgcatt agcaaaaatc atacaatata cagatggtcc 1440
tcgatttaca ttgtggttaa ttcccaataa acccatcata agttaaaaat gcatataacg 1500
ttagcaacac agcagtctcc taattaatga cagcttgact taacaatttt ccaactttac 1560
catggtgtga aagaggtatg attcctaagc cctaaggagc tcctcagctt gaaatggggc 1620
tgcatcccta taaacccatc ataaagtcaa aaaatcctaa aacataagtt ggtgaccatc 1680
tgtaatcatg atgtggtggt aaatcttgga cgctacctta caataactag acaaaggaaa 1740
atcatccttt gtcctgttct gtgtaaatat ttaatgaatg atcaaaactt cagtttaaat 1800
attatgaaaa actttaaaca taaagtagta gaaataagac agtaaatact gtatcctaat 1860
atccagtcag gatacagaaa ccataccatt aacttgaaca gggataattt taatataaaa 1920
actgttaact gataatggta ttaactttta agagggatga aagagagcta tgatgtccta 1980
ggactgagag taccccagga aagaataccc ttgaaagggt ctccccttcc ccatggtgaa 2040
gtcaggccta atggagagag tggctacagc ctactcagtg attgggaaat tccctgtctt 2100
gccctgggcc agagctggtg taccgctggt ggatcaggtc ttacaagcaa agaacctcac 2160
actcccaact ggtaagccag aagcctcttg ctagggtgtg agcaaaactt ggacaggaac 2220
tctcagtaga tgtttgtgtt tgtcaagatt ctccagacaa acttccttaa aaggattggc 2280
ttgtgttgtt attattaagt ctaacaagtc caaaagctgg agtgtgaggc aggaggctgg 2340
57/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
aaacccagga aagctgatgg tgcaaggtcc agtccaaagg tatctgttgg aggattctct 2400
tgttctggga agaggacggt ctttttttct cttcagacct tcacctgact ggatcaagcc 2460
cactaacatc gaggaggaca gtctgcatta ctcagagttc actgattgat ttaaatgtca 2520
atctcatata aaacaccctc acagaaacac ctagaataat gtttgacctt ataattggaa 2580
aatcagagca aaagttaaat ctctaaaaaa aaaa 2614
<210> 84
<211> 736
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5862945CB1
<400> 84
actcggcggc ttccgtagcg ggagggcgaa agatggcggc ggcagtactg ggacagttgg 60
gtgcgttatg gatacataac ctgaggagcc gggggaagct ggccttgggt gttttacctc 120
aatcatatat ccacacaagt gcttctcttg acatttctcg aaaatgggag aagaagaata 180
aaattgttta tcctccacaa ctgcctggag aacctcggag accagcagaa atctaccact 240
gtcgaagaca aataaaatat agcaaagaca agatgtggta tttggcaaaa ttgatacgag 300
gaatgtctat tgaccaggct ttggctcagt tggaattcaa tgacaaaaaa ggggccaaaa 360
taattaaaga ggttctctta gaagcacaag atatggcagt gagagaccat aacgtggaat 420
tcaggtccaa tttatatata gctgagtcca cctcaggacg aggccagtgc ctgaaacgca 480
tccgctacca tggcagaggt cgctttggga tcatggagaa ggtttattgc cattattttg 540
tgaagttggt ggaagggccc ccacctccac ctgagccacc aaagacggca gttgcccatg 600
ccaaagagta tattcagcag cttcgcagcc ggaccatcgt tcacactcta tgatgaggag 660
attcagactc cacagtgtat atattttgcc atttattttc taaaaataaa caaaaattga 720
aggcaaaaaa aaaaaa 736
<210> 85
<211> 1046
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6319547CB1
<400> 85
ggcgtaacgc gtcacgggcg gcctggcagc tggcggcatt gaggcggacg cgtctagagg 60
tccgtctgac cgcggcgtcg ggacctggtt tccgggcatg agctgagagc accacgccga 120
ggccacgagt atttcataga cattgatgga agcagaaacc aaaactcttc ccctggagaa 180
tgcatccatc ctttcagagg gctctctgca ggaaggacac cgattatgga ttggcaacct 240
ggaccccaaa attaccgaat accacctcct caagctcctc cagaagtttg gcaaggtaaa 300
gcagtttgac ttcctcttcc acaagtcagg tgctttggag ggacagcctc gaggctactg 360
ttttgttaac tttgaaacta agcaggaagc agagcaagcc atccagtgtc tcaatggcaa 420
gttggccctg tccaagaagc tggtggtgcg atgggcacat gctcaagtaa agagatatga 480
tcataacaag aatgataaga ttcttccaat cagtctcgag ccatcctcaa gcactgagcc 540
tactcagtct aacctaagtg tcactgcaaa gataaaagcc attgaagcaa aactgaaaat 600
gatggcggaa aatcctgatg cagagtatcc agcagcgcct gtttattcct actttaagcc 660
accagataaa aaaaggacta ctccatattc tagaacagca tggaaatctc gaagatgatg 720
gttgtgaatt actgtagcag caaaagcaaa ttggtctcca cacctaaaat cgtctgcctg 780
tgtactttgt agatgtgaat ggtactattc aacggagcac aatcacatgt tagcatttgg 840
taacataatg tttttggatg ttcttatgga tgtttcttcc ctaaactatg tatggaattg 900
agcatc.atcc agaataaata gcgttgtatc ccaaattgtg atttgaaccc tgggatgctc 960
taattggctg gttggtttgg atttgtaact ccagaaacat tctatagtgt gccagagcaa 1020
aaggcaaata cacaaaatat tatttt 1046
<210> 86
<211> 2266
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 000124CB1
58/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<400> 86
cgcgttcacc agcccggaag tgcgcgtggc ggcggtggcg gctgcggcaa cagcggggcc 60
gatgtgtagt tggtgactgc ctctccagat gctgaggtgc ctgtatcatt ggcacaggcc 120
agtgctgaac cgtaggtgga gtaggctgtg ccttctgaag cagtatctat tcacaatgaa 180
gttgcagtct cccgaattcc agtcactttt cacagaagga ctgaagagtc tgacagaatt 240
atttgtcaaa gagaatcacg aattaagaat agcaggagga gcagtgaggg atttattaaa 300
tggagtaaag cctcaggata tagattttgc caccactgct acccctactc aaatgaagga 360
gatgtttcag tcggctggga ttcggatgat aaacaacaga ggagaaaagc acggaacaat 420
tactgccagg cttcatgaag aaaattttga gattactaca ctacggattg atgtcaccac 480
tgatggaaga catgctgagg tagaatttac aactgactgg cagaaagatg cggaacgcag 540
agatctcact ataaattcta tgtttttagg ttttgatggc actttatttg actactttaa 600
tggttatgaa gatttaaaaa ataagaaagt tagatttgtt ggacatgcta aacagagaat 660
acaagaggat tatcttagaa ttttaagata cttcaggttt tatgggagaa ttgtagacaa 720
acctggtgac catgatcctg agactttgga agcaattgca gaaaatgcaa aaggcttggc 780
tggaatatca ggagaaagga tttgggtgga actgaaaaaa attcttgttg gtaaccatgt 840
aaatcatttg attcacctta tctatgatct tgatgtggct ccttatatag gtttacctgc 900
taatgcaagt ttagaagaat ttgacaaagt cagtaaaaat gttgatggtt tttcaccaaa 960
gccagtgact cttttggcct cattattcaa agtacaagat gatgtcacaa aattggattt 1020
gaggttgaag atcgcgaaag aggagaaaaa ccttggctta tttatagtta aaaataggaa 1080
agatttaatt aaagcaacag atagttcaga cccattgaaa ccctatcaag acttcattat 1140
agattctagg gaacetgatg caactactcg tgtatgtgaa ctactgaagt accaaggaga 2200
gcactgtctc ctaaaggaaa tgcagcagtg gtccattcct ccatttcctg taagtggcca 1260
tgacatcaga aaagtgggca tttcttcagg aaaagaaatt ggggctctat tacaacagtt 1320
gcgagaacag tggaaaaaaa gtggttacca aatggaaaaa gatgaacttc tgagttacat 1380
aaagaagacc taaaactgat ggctactaaa aagcagagca tttctggtaa gactaaattt 1440
tctcccctcc ctcttaatga ggttttagag actacaccag aataaaagac agtttagggg 1500
acctctgtag aacaacaagg gtcttatttt gtgaattata tatttcaaga actaaacaga 1560
gatccacctt tctggatctg atttatatca ctgaaatgta cagttctttt ggaatagttt 1620
cacctgagaa aacatagttg gctattatct atcttaacct gttcaggctt ttaaaaaaaa 1680
ctgtttttgc atagggtagt actaagatct taaaaagtgg taactgtctt gaagaaaaaa 1740
cgtttattgt ttgtttgcaa ttgaaataac agggttacct taacaatgac tgtctatgat 1800
gtgtcagttc ttatctgaat tccaaaataa acctgtgctt aaaaaagaaa taattgacca 1860
agtaagtttg cataaaatgt gaatactaaa tgtgtcccca gttgctggca ttcatatgta 1920
caggatttgt tctagcaagc tatgcttcag tatgtggttg atatttttct gtcacaatga 1980
tttctttatg catgcagagc ctgggaaagt catgggatta acttgagggt cactattgag 2040
cctattaatt aattaattat tgttttaata aaacaaacat tggtattgga agataaatat 2100
gtttatgtgg tatctgacaa tgtgtattag gtgtcatata caatggtaat atgcctgtct 2160
ttaaagtgtt attttattaa ttaaaaggat atggctatta ttatatattc tctaaagatt 2220
tattctctaa agatttgagt cctaaatgct ttcatcacgg cacgag 2266
<210> 87
<211> 1041
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1659474CB1
<400> 87
caagcagcat ggctgcaggt tgctcagagg cgccgcggcc aacggcggct tctgatgggt 60
ctctggtagg gcaggctggc gtcctgcctt gcctagagtt gccgacttat gccgctgctt 120
gtgcgctggt gaacagtcgc tactcatgcc tggtggccgg gccgcaccaa aggcacatcg 180
cgctgtcgcc ccgctacctt aacaggaaac gcaccggcat tcgagaacag cttgatgcgg 240
agctccttcg ctattctgag agccttttag gtgtccctat tgcatatgat aacatcaaag 300
ttgtgggaga gcttggagat atttatgatg atcaaggaca cattcatctt aacattgaag 360
ccgattttgt tattttctgc cctgaaccgg ggcagaagct tatgggtata gttaataaag 420
tgtcttctag ccacattggc tgtttagtac atgggtgttt caatgcctcc attcctaaac 480
ctgagcagtt gtcagctgag cagtggcaaa ccatggagat aaacatgggt gatgaactag 540
aatttgaagt atttcgttta gactcagatg ctgctggagt attctgcatt cggggaaaac 600
taaatatcac aagtttacaa ttcaagcgct ctgaagtttc tgaagaagtt acagaaaatg 660
gcactgagga agctgctaaa aaacctaaaa agaagaaaaa gaagaaagac ccagagacat 720
atgaagtgga cagtggtacc acaaagctag cagatgatgc agatgacact ccaatggaag 780
agtcagccct gcagaatact aataatgcga atggcatctg ggaggaggag ccaaagaaaa 840
agaagaagaa gaaaaagcac caggaagttc aggaccagga ccctgttttc caaggcagtg 900
actccagtgg ttaccaaagt gaccataaaa agaaaaaaaa agaaaagaaa accaacagtg 960
aagaggccga atttacccca cctttgaaat gctcaccaaa aagaaaaggg aaaagtaatt 1020
59/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
ttctttagtg tattttaaac a 1041
<210> 88
<211> 2722
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2267892CB1
<400> 88
cgctttctgg gtaaagatgg acgtccacga tctctttcgc cggctcggcg cgggggccaa 60
attcgacacg agacgcttct cggcagacgc agctcgattc cagataggaa aaaggaaata 120
tgactttgat tcttcggagg tgcttcaggg actggacttt tttggaaaca agaagtctgt 180
cccaggtgtg tgtggagcat cacaaacaca tcagaagccc caaaatggag agaaaaaaga 240
agagagccta actgaaagga agagggagca gagcaagaaa aaaaggaaga cgatgacttc 300
agaaattgct tcccaagaag aaggtgctac tatacagtgg atgtcatctg tagaagcaaa 360
gattgaagac aaaaaagttc agagagaaag taaactaact tccggaaagt tggagaatct 420
cagaaaagaa aagataaact tcttgcggaa taaacacaaa attcacgtcc aaggaaccga 480
tcttcctgac ccaattgcta catttcagca acttgaccag gaatataaaa tcaattctcg 540
actacttcag aacattctag atgcaggttt ccaaatgcct acgccaatcc aaatgcaagc 600
catcccagtt atgctgcatg gtcgggaact tctggcttct gctccaactg gatctggaaa 660
aacattagct tttagcattc ctattttaat gcagctgaaa caacccgcaa ataaaggctt 720
cagagccctg attatatcac caacacgaga acttgccagc cagattcaca gagagttaat 780
aaaaatttct gagggaacag gattcagaat acacatgatc cacaaagcag cagtggcagc 840
caagaaattt ggacctaaat catctaaaaa gtttgatatt cttgtgacta ctccaaatcg 900
actaatctat ttattaaagc aagatccccc cggaatcgac ctagcaagtg ttgagtggct 960
tgtagtagac gaatcagata aactgtttga agatggcaaa actgggttca gagaccagct 1020
ggcttccatt ttcctggcct gcacatccca caaggtccga agagctatgt tcagtgcaac 1080
ttttgcatat gatgttgaac agtggtgcaa actcaacctg gacaatgtca tcagtgtgtc 1140
cattggagca aggaattctg cagtagaaac tgtagaacaa gagcttctct ttgttggatc 1200
tgagaccgga aaacttctgg ccatgagaga acttgttaaa aagggtttca atccacctgt 1260
tcttgttttt gttcagtcca ttgaaagggc taaagaactt tttcatgagc tcatatatga 1320
aggtattaat gtggatgtta ttcatgcaga gagaacacaa caacagagag ataacacagt 1380
ccacagtttc agagcaggaa aaatctgggt tctgatttgt acagccttgc tagcaagagg 1440
gattgatttt aaaggtgtga acttggtgat caactatgac tttccaacta gctcagtgga 1500
atatatccac aggataggtc gaactggaag agcagggaat aagggaaaag caattacatt 1560
tttcactgag gatgataagc cattattaag aagcgttgct aatgttatac agcaggctgg 1620
gtgtcctgta ccagaataca taaaaggttt tcagaaacta ctaagcaaac aaaagaaaaa 1680
gatgattaag aaaccattgg aaagggagag cattagtaca actccaaaat gtttcttaga 1740.
aaaagctaag gataaacaga aaaaggtcac tggtcagaac agcaagaaga aagtagctct 180f
tgaagacaaa agttaaaaac agactttaaa aatactgtcc cagaaatgta attttatgat 1860
cccagcatga atgttatttt catggaatac ttgaagtctt acagtcacct gtaccaaaca 1920
tttgaaatca actacaagta catgggactg gtgataaatg atcctaaact atcaagtcag 1980
tttcaatttg taggtgcctt ttttttttcc tgtagagatg agggtcttgc catgttgtcc 2040
aggctggtct tgaactcctg acctcacaca atcctcctgc cttagcctcc tgagtaactg 2100
agattacagg cacaagctgc tgcacccagc tctgtaggtg acttttaaat gattatacaa 2160
tggaaataac attcattgac atttctgtgg tttgaatcca gagagatact tcttatagaa 2220
aaacaaatgt ttatgctaaa aataacacca aaatgtggtg aactcttaag gacttttccc 2280
ttcaagtgtg aaggaaggtg tgatgaatgc tgtggagagg catctggaac agaaattcaa 2340
aataaagcct tgacattaaa taccccttcc actgctcact ttgtggatgg tagcatgagc 2400
tgtctaccaa gaagaaacct gctgctctct taattttaat atttcctaat ttgttgatgg 2460
ccttttgtgt tgtgaaccac aacaaagaga ggcctctttt gtggctggtt attccagttc 2520
cctgggattt taaattcttt ggtctattaa gtatccttgt attggatacg taatacctta 2580
gtgctgtcat aatgttgtac aagatcatga tcagcttctc cctttcttca ttttctgtga 2640
tttaaccatg ttctttcctg tctctttcca tttaagatat tttatttgaa tactgataaa 2700
cattttatcc cccccctttg gg 2722
<210> 89
<211> 1287
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2670307CB1
60/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<400> 89
ccaagagtct aggtaagagt ttgttcccgt ggtgcggagg gtcaaggccc acacccggaa 60
acctagcgag gtaaagttgc gtcttggttg tagagacgac aacttctccg cttcctcggc 120
gatggcggcg tccgggagcg gtatggccca gaaaacctgg gaactggcca acaacatgca 180
ggaagctcag agtatcgatg aaatctacaa atacgacaag aaacagcagc aagaaatcct 240
ggcggcgaag cctggactaa ggattcacca ttactttaag tactgcaaaa tctcagcatt 300
ggctctgctg aagatggtga tgcatgccag atcgggaggc aacttggaag tgatgggtct 360
gatgctagga aaggtggatg gtgaaaccat gatcattatg gacagttttg ctttgcctgt 420
ggagggcact gaaacccgag taaatgctca ggctgctgca tatgaataca tggctgcata 480
catagaaaat gcaaaacagg ttggccgcct tgaaaatgca atcgggtggt atcatagcca 540
ccctggctat ggctgctggc tttctgggat tgatgttagt actcagatgc tcaatcagca 600
gttccaggaa ccatttgtag cagtggtgat tgatccaaca agaacaatat ccgcagggaa 660
agtgaatctt ggcgccttta ggacataccc aaagggctac aaacctcctg atgaaggacc 720
ttctgagtac cagactattc cacttaataa aatagaagat tttggtgtac actgcaaaca 780
atattatgcc ttagaagtct catatttcaa atcctctttg gatcgcaaat tgcttgagct 840
gttgtggaat aaatactggg tgaatacgtt gagttcttct agcttgctta ctaatgcaga 900
ctataccact ggtcaggtct ttgatttgtc tgaaaagtta gagcagtcag aagcccagct 960
gggacgaggg agtttcatgt tgggtttaga aacgcatgac cgaaaatcag aagacaaact 1020
tgccaaagct acaagagaca gctgtaaaac taccatagaa gctatccatg gattgatgtc 1080
tcaggttatt aaggataaac tgtttaatca aattaacatc tcttaaacag tctctgagaa 1140
gtactttacc tgaaagacag tatgagaaaa atattcaagt aacactttaa aaccagttac 1200
ccaaaatctg attagaagta taaggtgctc tgaagtgtcc taaatattaa tatcctgtaa 1260
taaagctctt taaaatgaaa aaaaaaa 1287
<210> 90
<211> 2226
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte TD No: 4524210CB1
<400> 90
cggctcgagc cggaagcgac tttccgccga gaaatagggg gcgcgtgttt ggaaattgat 60
agaaaagata aagggaccga gctgctgtca gcctggctta ctgatctgcg tccgtttcac 120
cacggattca gttactaagc atttttttct ttttttggtt ctttgcaacg tgagtggcat 180
tggctcagtg atttccatga gcatctctac cagaaaacat tgcctcgatg aggtgtggtg 240
gaagcccggc agccccttct aatcggctag gcttgagaaa gcgtgtacct ctgcatttcc 300
gaaattaact cagcgtgatc ggcaagattt tcctcagcat ctggtgtcaa gacactcgtc 360
actattaatt cggaaagaaa aaaaaaaaca aaacaccgtt ttccagcatt tctctttgtg 420
gagaactaaa caacaggaaa aatgtctatt ttccctaaga tatctttgag acctgaggtt 480
gaaaactatc ttaaggaagg ctttatgaat aaggagattg tgactgcttt aggtaaacaa 540
gaagcagaaa ggaagtttga aactttgtta aagcacctgt cacatcctcc atcatttaca 600
actgtcagag tgaatacaca tttagcctca gtacaacatg tgaaaaatct gttacttgat 660
gaacttcaga agcagtttaa tggattaagt gttcctattc ttcaacatcc agaccttcaa 720
gatgtgttac ttattcctgt tattggaccc agaaagaata ttaaaaaaca acagtgtgaa 780
gccattgttg gagcccagtg tggcaatgca gttttaagag gagcccatgt ctatgcccca 840
ggaattgtgt cagcatcaca atttatgaaa gctggagatg ttatttctgt atactctgat 900
attaaaggaa aatgtaagaa aggagccaaa gaatttgatg gaacaaaagt atttcttgga 960
aatgggattt ctgaactaag ccgcaaagaa atcttcagtg gattacctga actgaaaggc 1020
atgggcataa gaatgacaga accagtatat ctcagccctt catttgacag tgtactgccc 1080
cgttacttat ttttacaaaa tttgccatct gccttagtaa gtcatgtact aaatcctcaa 1140
cctggagaga agattctaga cttgtgtgca gcacctggag ggaaaacaac acacattgca 1200
gcactaatgc atgatcaggg agaagttata gcactggata aaatcttcaa caaagtagaa 1260
aaaatcaaac agaatgcctt attgttaggg ctgaattcca tcagggcatt ttgttttgat 1320
ggaacaaagg cggttaaact tgatatggtg gaggacacag aaggagaacc tccatttcta 1380
ccagaatcct ttgaccgaat tcttctggat gcaccctgta gtggaatggg acagagacca 1440
aacatggcct gtacttggtc tgtgaaggaa gtggcatcat atcagccatt acagcgaaaa 1500
ctcttcactg cagcggttca gctgctgaag ccagagggtg tgctggttta tagcacgtgc 1560
actataacac tggccgaaaa tgaagaacag gttgcctggg ccctgacaaa atttccttgc 1620
cttcagcttc agccccagga accgcagatt ggaggagaag gaatgagggg agctgggctc 1680
tcatgtgaac agttgaaaca gctgcagcga tttgatccat cggctgtgcc attaccggac 1740
actgacatgg actctcttag agaggccaga agagaagaca tgttgcgtct ggctaataag 1800
gactctatag gtttttttat tgcaaaattt gtaaaatgca aaagcacata ggagagggat 1860
ggatgctcag aaatgaaaat tccaaacatt tgctgtctgt ggtttttttt tttttttttt 1920
taaccaaagt gttgtcaggc caactgaatg atgatgtggt tgctatggaa acagaaaagg 1980
61/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
ctgccagctg ttttaccagg gatccagaga catagaggaa gtagggggtg gtatgagatt 2040
atattttctg tttttaaaag attttttttt tttatgtatt tagtagagta taaagaaaag 2100
cagatgccta tagatgtctg gagcatattt tcatttgtga tctaatgttt taatttgtaa 2160
agtgtacaag tcatttttaa tgttaaaaat tagtgaatct aacaaaagga ataaattagc 2220
aatatt 2226
<210> 91
<211> 2362
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5584860CB1
<400> 91
cccgggtcga cccacgcgtc cgaaataaga cgccgaccgg cgcggcgcta gcctcggggc 60
ttgacgggat tgtggcggtc ctctctccca attcggaagc tacagctacc tccggacgct 120
etcaagatgg cgacctctct gggttccaac acctacaaca ggcagaactg ggaggatgcg 180
gacttcccca ttctgtgcca gacatgtctt ggagaaaacc catatatccg aatgaccaaa 240
gaaaagtatg ggaaggaatg caaaatctgt gccaggccat tcacagtgtt tcgctggtgc 300
cctggagtcc gcatgcgttt caagaagact gaagtgtgcc aaacctgcag taaattgaag 360
aatgtctgtc agacctgcct cttagaccta gagtatggcc tgcecatcca ggttcgtgac 420
gcaggattgt cttttaaaga tgacatgcca aagtcagatg tcaacaaaga gtactataca 480
cagaatatgg agagagagat ttctaactct gatggaacac ggccagttgg catgctgggg 540
aaagccacat ctaccagtga catgctgctc aaactggccc ggaccacacc ctactacaaa 600
aggaatcgac cccacatttg ctccttctgg gtgaaaggag agtgtaagag aggagaggaa 660
tgtccataca gacatgagaa gcctacagat ccagatgacc cccttgctga tcagaatatt 720
aaagaccgtt attacggaat caatgatcct gtagctgaca agcttctaaa gcgggcttca 780
acaatgcctc ggctggaccc accagaggat aaaactatca ccacactata tgttggtggt 840
ctaggtgata ccattactga gacagattta agaaatcatt tctaccagtt cggagagatc 900
cggacgatca ctgttgtgca gagacagcag tgtgctttca tccagtttgc cacacggcag 960
gctgcagaag tggctgctga gaagtccttt aataagttga ttgtaaatgg ccgcagactg 1020
aatgtgaaat ggggaagatc ccaggcagcc agaggaaaag aaaaagagaa agatggaact 1080
acagactctg ggatcaaact agaacctgtt ccaggattgc caggagctct tcctcctcct 1140
cctgcagcag aagaagaagc ctctgccaac tacttcaact tgcccccaag tggtcctcca 1200
gctgtggtga acattgctct gccaccgccc cctggcattg ctccaccccc acccccaggt 1260
tttgggccac acatgttcca cccaatggga ccaccccctc ctttcatgcg ggctccagga 1320
ccaatccact atccttctca ggaccctcag aggatgggag ctcatgctgg aaaacacagc 1380
agcccctagc accttgtcac cactctgggg ctctgtggaa gaaagggcac ttaaaactcc 1440
cagtaaatct tggaataaat atatttttcc ttcccttgta gtttccatgg tagctgaatg 1500
tgctcagatg tgagcagtca gagactgaca gccatgcttt cctatacttg ttcaaaggat 1560
cgatggaccg taaataagct gccattaaca catctggtta ctgctgtaac atgactaata 1620
aaaccgaacg cctgttcccc ttacccgtgt gggggacacg cagatgagtg aattggaatg 1680
tccagcagag ttaccctccc aattatatgt tcattttgta tattttttgg tcgggggaaa 1740
aattgacctg cagtaaaaaa acctttgacc atttttatgt ccattggata ctttcctttt 1800
tatcatctta aaaaaagata actagtacta atcattgtag tggcctaagt gtgatttaac 1860
tcttgaagtc acaccctccg aaagatgagt agaaaccagc accagcacag cccagatctt 1920
ctctttcctc tccttttcct catttattcc taaaggaatc tgaccatttt acgtctctac 1980
ggcccaaaaa aagacaaaaa taaaaattcc tttttattcc tgtcaactgg atggaaacac 2040
aaatttcatg gagctgtgta ccatcgaaga aacctggtgt ctggcatgaa attactgtaa 2100
agaacttcct gtaaaacacg ttctttaaca aactgaaatg aaaagcattg gagcgtctga 2160
atgaaagacg tgacctcctg ctgggactct gatggtcttc agcattcacc ttcgtgtgtc 2220
ttcagtgtct cattgtcatc cctgcttctg tttggtctta gagtgtttgg atataactga 2280
attgtagatg gtaaaggaaa tttgatgtgt tttttgtttt taaataatta aaacgggtca 2340
atttttcaaa aaaaaaaaaa as 2362
<210> 92
<211> 731
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5807892CB1
<400> 92
62/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
tagggcggca agcggaggag gcgtggcgag cggatcatcc gcttccggag tcgaggtttt 60
cgggcttgta ccgcttggcg gtgcggcctg gtgtcggctt gcaggttctt tctgtgtttg 120
ttctctgccc tgccaaggcc gtagagctgg tgcgtgcggg tagcggggct ctccgaggag 180
ccgcacgccg gcggcaccat ggtccacctc actactctcc tctgcaaggc ctaccgtggg 240
ggccacttaa ccatccgcct tgccctgggt ggctgcacca atcggccgtt ctaccgcatt 300
gtggctgctc acaacaagtg tcccagggat ggccgtttcg tagagcagct gggctcctat 360
gatccattgc ccaacagtca tggagaaaaa ctcgttgccc tcaacctaga caggatccgt 420
cattggattg gctgcggggc ccacctctct aagcctatgg aaaagcttct gggtcttgct 480
ggctttttcc ctctgcatcc tatgatgatc acaaatgctg agagactgcg aaggaaacgg 540
gcacgtgaag tcctgttagc ttctcagaaa acagatgcag aagctacaga tacagaggct 600
acagaaacat aaatgagctg actttagtga gcatagcagt gggaacaagg tcaaggtcct 660
tttgaaacac tgcagcgatc ttaattttgt tagatttgga gttcaataaa tggagtatcc 720
tgaaaaaaaa a 731
<210> 93
<211> 2088
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3210044CB1
<400> 93
ctttccagaa aatcaaatga aagattcaga gtatattcat gaattaattt tttttcaaaa 60
ccctaaattt aatcagctgg aattacttta aaagtgtcat tctatttaac ttttgggaat 120
gatgaatttg ccttttaata gggatgctgt attttatcat gaagatgaaa caaactgtct 180
tttgttaatt atggcacctt catttaccgc ccgcattcag ttgttcctct tgcgggcgct 240
aggctttctc ataggcttag taggccgagc agctttagtc ttagggggcc caaagtttgc 300
ctcaaagacc~cctcggccgg tgactgaacc attgcttctg ctttcgggga tgcagctggc 360
caagctgatc cgacagagaa aggtgaaatg tatagatgtt gttcaggctt atatcaacag 420
aatcaaggac gtgaacccaa tgatcaatgg aattgtcaag tacaggtttg aggaagcgat 480
gaaggaggct catgctgtag atcaaaagct tgcagagaag caggaagatg aagccaccct 540
ggaaaataaa tggcccttcc ttggggttcc tttgacagtc aaggaagctt tccagctaca 600
aggaatgccc aattcttctg gactcatgaa ccgtcgtgat gccattgcca aaacagatgc 660
cactgtggtg gcattactga agggagctgg tgccattcct cttggcataa ccaactgtag 720
tgagttgtgt atgtggtatg aatccagtaa caagatctat ggccgatcaa acaacccata 780
tgatttacag catattgtag gtggaagttc tggtggtgag ggctgcacac tggcagctgc 840
ctgctcagtt attggtgtgg gctctgatat tggtggtagc attcgaatgc ctgctttctt 900
caatggtata tttggacaca agccttctcc aggtgtggtt cccaacaaag gtcagtttcc 960
cttggctgtg ggagcccagg agttgtttct gtgcactggt cctatgtgcc gctatgctga 1020
agacctggcc cccatgttga aggtcatggc aggacctggg atcaaaaggt taaaactaga 1080
cacaaaggta catttaaaag acttaaaatt ttactggatg gaacatgatg gaggctcatt 1140
tttaatgtcc aaagtggacc aagatctcat tatgactcag aaaaaggttg tggttcacct 1200
tgaaactatt ctaggagcct cagttcaaca tgttaaactg aagaaaatga agtactcttt 1260
tcagttgtgg atcgcaatga tgtcagcaaa gggacatgat gggaaggaac ctgtgaaatt 1320
tgtagatttg cttggtgacc atgggaaaca tgtcagtcct ctgtgggagt tgatcaaatg 1380
gtgcctgggt ctgtcagtgt acaccatccc ttccattgga ctggctttgt tggaagaaaa 1440
gctcagatat agcaatgaga aataccaaaa gtttaaggca gtggaagaaa gcctgcgtaa 1500
agagctggtg gatatgctag gtgatgatgg tgtgttctta tatccctcac atcccacagt 1560
ggcacctaag catcatgtcc ctctaacacg gcctttcaac tttgcttaca caggtgtctt 1620
cagtgccctg ggtttgcctg tgacccaatg cccactggga ctgaatgcca aaggactccc 1680
tttaggcatc caggttgtgg ctggaccctt taatgatcat ctgaccctgg ctgtggccca 1740
gtacttggag aaaacttttg ggggctgggt ctgtccagga aagttttagg aggaccttct 1800
gcaaggttaa tgtgtgtgtg tgtttgtgtt cgtgtggtgg tgtttctatt aattgggtga 1860
aatcaagcac cagcagacaa gcagagaaac aactggggaa tttattgact catttagtta 1920
ttctttctac ttttatttcc ttctctaact gttggtctta ctaaaatggt aatatttgct 1980
tcttgctttt atgttactgg aaaattagga catgtaaatg gataagtgca ataaagtttc 2040
ctaaatgctg aaaaaaaaaa acacaaaaaa aacaaaaaaa aaaaaaaa 2088
<210> 94
<211> 660
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
63/64
CA 02407435 2002-10-24
WO 01/83524 PCT/USO1/13862
<223> Incyte ID No: 4942454CB1
<400> 94
ccgtcaatag cctccgcctc tccttccagt gtccgccgtc gtgcgctcgc tacccctctc 60
cctcgaggcc tttgccggcg aagagcgccc agtcgcccac caggatgaag tttgttgctg 120
cctacctgct tgctgtcctc gctgggaact ccagcccctc tgccgaggac ttgacagcca 180
ttctggagtc agttggctgt gaagttgaca atgaaaagat ggaactcctt ctgtcccaac 240
tgagcggtaa ggacattacc gagctcattg ctgctggcag ggagaagttt gcttcagtcc 300
catgtggcgg tggcggtgtg gctgttgcgg cagctgcccc tgctgctggc ggcgctcctg 360
cagctgaggc gaagaaagaa gagaaggtgg aggagaagga agaaagtgat gacgacatgg 420
gcttcagcct cttcgactaa gcctgtgcaa tagtcaagag tattgttttt gagtcgcgga 480
agcagaggga agaaaaatcg tagtcatgtt tggactttaa ctttgtttta tgttggaaag 540
tacttgaaag acttttcctg tggtaattct aggcgtaggt tgctgtgctg gttggggttt 600
actggtgaac cagagttttt ctatctccca ctatgaattt gttacetcaa gttacctgtg 660
64/64
