Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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HUMAN RNA-ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human RNA-
associated
proteins and to the use of these sequences in the diagnosis, treatment, and
prevention of cell
proliferative, autoimmune/inflammatory, and infectious disorders.
BACKGROUND OF THE INVENTION
Ribonucleic acid (RNA) is a linear single-stranded polymer of four
nucleotides, ATP,
GTP, 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 codon and the amino
acid that
matches that codon.
The unspliced precursors of mature mRNA transcripts are called heterogeneous
rruelear
RNA (hnRNA) transcripts. hnRNA is generally larger and more unstable than
mRNA.
Immediately upon its synthesis, hnRNA is assembled into protein-containing
complexes called
heterogeneous nuclear ribonucleoprotein particles (hnRNPs). (See, for example,
Honore, B. et al.
( 1995) J. Biol. Chem. 270:28780-28789.) hnRNPs associate with small nuclear
ribonucleoprotein
particles (snRNPs) which are stable RNA-protein complexes that function
primarily in splicing
introns from hnRNA. Each snRNP contains a single species of RNA and about l0
proteins. The
RNA components of snRNPs recognize and base pair with specific sequences of
the hnRNA
intron. Five different snRNPs associate at the intron of hnRNA to form the
spliceosome, a
multicomponent RNP complex which catalyzes the removal of introns and the
rejoining of exons.
Also associated with the snRNPs are various accessory factors that stabilize
intron-snRNP
interactions. In humans, these factors include spliceosome associated protein
49 (SAP 49) and
SAP 145. (Champion-Arnaud, P, and Reed, R. (1994) Genes Dev. 8:1974-1983.)
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
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structural, catalytic, and regulatory purposes. RNA polymerases are proteins
that transcribe RNA
from a DNA copy. The HIV Tat protein binds specific sites in the viral RNA to
prevent premature
transcriptional termination. 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.
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).
The process of splicing may involve more than the removal of an intron from an
RNA
transcript. For example, an RNA transcript may be subject to alternative
patterns of splicing,
resulting in the generation of different species of mRNA from a single primary
transcript. In
addition, certain transcripts may be subject to traps-splicing, in which an
exon from one transcript
is joined to an exon of another. Often, specific protein cofactors are
required to mediate splicing
under these special circumstances. For example, a new splicing factor, PR264,
has been
implicated in the traps-splicing of a thymus-specific c-myb transcript in
humans. (Vellard, M. et
al. (1992) Proc. Natl. Acad. Sci. USA 89:2511-2515.)
Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified that
have roles
in splicing, exporting ofthe 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 A 1,
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).
Nascent tRNA transcripts are spliced by unconventional mechanisms that are
distinct from
those employed by the spliceosome. 1n this case, splicing is carried out by
specific endonucleases
and ligases that recognize secondary structural features of the tRNA. This
process contrasts with
the spliceosomal reaction, in which specific nucleotide sequences of the
intron are recognized. In
addition, tRNAs are further processed by removal of 5' sequences and by
chemical modification of
some of the nucleotide bases. tRNA processing has been extensively studied in
yeast, in which
tRNA-specific splicing factors have been identified. (See, for example, Shen,
W. C. et al. (1993)
2
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J. Biol. Chem. 268:19436-19444.)
Proteins are also a part of the translation machinery of the cell. 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, the ribosome also
contains more than
fifty proteins. The ribosomal proteins have a prefix which denotes the subunit
to which they
belong, either L (large) or S (small). Initiation factors, many of which
contain multiple subunits,
are proteins which are involved in bringing together an initiator tRNA, the
mRNA, and the
ribosomal 40S subunit. Eukaryotic initiation factor 2 (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. Other
initiation factors include eIFIA, elF3, eIF4F (a complex including eIF4E,
elF4A, and eIF4G), and
eIFS. The elongation factors EFIa, EF1 (3 y, and EF2 are involved in
elongating the polypeptide
chain following initiation, and the release factor eRF carries out termination
of translation. (See
V. M. Pain ( 1996) Eur. J. Biochem. 236:747-771.)
Other important RNA-associated enzymes with roles in translation are the
aminoacyl-
transfer RNA (tRNA) synthetases. Protein biosynthesis depends on each amino
acid forming a
linkage with the appropriate tRNA. The aminoacyl-tRNA synthetases are
responsible for the
activation and correct attachment of an amino acid with its cognate tRNA. The
20 aminoacyl-
tRNA synthetase enzymes can be divided into two structural classes, and 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'. Class II
enzymes contain a
central catalytic domain, which consists of a seven-stranded antiparallel 13-
sheet motif, as well as -
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 - and C-terminal regulatory domains (Hartlein, M. and
Cusack, S. ( 1995) J.
Mol. Evol. 40:519-530). Autoantibodies against aminoacyl-tRNAs are generated
by patients with
dermatomyositis and polymyositis, and correlate strongly with complicating
interstitial lung
disease (ILD). These antibodies appear to be generated in response to viral
infection, and
coxsackie virus has been used to induce experimental viral myositis in
animals.
In many cases, mRNA translation, localization, and stability are controlled by
regulatory
proteins that bind to the 5' and 3' untranslated (UTR) regions of mRNA. An
example of such a
protein is Spnr, a mouse spermatid perinuclear RNA-binding protein, which may
be involved in
RNA transport, translational activation, or localization of RNA to cytoplasmic
microtubules
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(Schumacher, J. M. et al. (1995) J. Cell Biol. 129:1023-1032). RNA-associated
proteins may alter
and regulate RNA conformation and secondary structure. These processes are
mediated by RNA
helicases which utilize 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 embryogenesis. All DEAD-box helicases contain several
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
serine/arginine/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
IS 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 (DDX 1 ) may play a role in the
progression of
neuroblastoma (Nb) and retinoblastoma (Rb) tumors (Godbout, R. et al. (1998)
J. Biol. Chem.
273:21161-21168). These observations suggest that DDX 1 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
tumorigenesis. (Discussed in Godbout, supra.) For example, murine p68 is
mutated in ultraviolet
light-induced tumors, and human DDX6 is located at a chromosomal breakpoint
associated with
B-cell lymphoma. Similarly, a chimeric protein comprised of DDX10 and NUP98, a
nucleoporin
protein, may be involved in the pathogenesis of certain myeloid malignancies.
Ribonucleases (RNases) are RNA-associated enzymes which catalyze the
hydrolysis of
phosphodiester bonds in RNA chains, thus cleaving the RNA. For example, RNase
P is a
ribonucleoprotein enzyme which cleaves the S' 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 N 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
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investigated as a means to control tumor angiogenesis, allergic reactions,
viral infection and
replication, and fungal infections.
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.) T'he
RRM is about 80
amino acids in length and forms four ~i-strands and two a-helices arranged in
an a/~i sandwich.
The RRM contains a core RNP-I 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 which
have been identified in lower eukaryotes such as Drosophila melano a~ ster and
Caenorhabditis
ele 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.) The RRM includes the ribonucleoprotein-1 (RNP-1) RNA binding
motifwhich
is found in snRNP proteins, hnRNP proteins, splicing factors, mRNA binding
proteins, and
transcriptional regulatory proteins. Other hallmarks of RNA binding proteins
include regions of
repeated arginine and serine residues (RS repeats).
The discovery of new human RNA-associated 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 cell proliferative, autoimmune/inflammatory, and
infectious
disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, human RNA-
associated
proteins, referred to collectively as "RNAAP" and individually as "RNAAP-1,"
"RNAAP-2,"
"RNAAP-3," "RNAAP-4," "RNAAP-5,""RNAAP-6," "RNAAP-7," "RNAAP-8," "RNAAP-9,"
"RNAAP-10," "RNAAP-11," "RNAAP-12," "RNAAP-13," "RNAAP-14," "RNAAP-15,"
"RNAAP-16," "RNAAP-17," "RNAAP-18," "RNAAP-19," "RNAAP-20," "RNAAP-
21,""RNAAP-22" "RNAAP-23" "RNAAP-24"and RNAAP-25" In one aspect, the invention
provides a substantially purified polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-25 and fragments thereof.
The invention further provides a substantially purified variant having at
least 90% amino
acid identity to at least one of the amino acid sequences selected from the
group consisting of SEQ
ID NO:1-25 and fragments thereof. The invention also provides an isolated and
purified
polynucleotide encoding the polypeptide comprising an amino acid sequence
selected from the
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group consisting of SEQ ID NO:1-25 and fragments thereof. The invention also
includes an
isolated and purified polynucleotide variant having at least 70%
polynucleotide sequence identity
to the polynucleotide encoding the polypeptide comprising an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-25 and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide
which
hybridizes under stringent conditions to the polynucleotide encoding the
polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID NO:1-25
and fragments
thereof. The invention also provides an isolated and purified polynucleotide
having a sequence
which is complementary to the polynucleotide encoding the polypeptide
comprising the amino
acid sequence selected from the group consisting of SEQ ID NO:I-25 and
fragments thereof.
The invention also provides a method for detecting a polynucleotide in a
sample
containing nucleic acids, the method comprising the steps of (a} hybridizing
the complement of the
polynucleotide sequence to at least one of the polynucleotides of the sample,
thereby forming a
hybridization complex; and (b) detecting the hybridization complex, wherein
the presence of the
hybridization complex correlates with the presence of a polynucleotide in the
sample. In one
aspect, the method further comprises amplifying the polynucleotide prior to
hybridization.
The invention also provides an isolated and purified polynucleotide comprising
a
polynucleotide sequence selected from the group consisting of SEQ ID N0:26-50
and fragments
thereof. The invention further provides an isolated and purified
polynucleotide variant having at
least 70% polynucleotide sequence identity to the polynucleotide sequence
selected from the
group consisting of SEQ ID N0:26-50 and fragments thereof. The invention also
provides an
isolated and purified polynucleotide having a sequence which is complementary
to the
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ
ID N0:26-50 and fragments thereof.
The invention further provides an expression vector containing at least a
fragment of the
polynucleotide encoding the polypeptide comprising an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-25 and fragments thereof. 1n another aspect,
the expression
vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method
comprising
the steps of: (a) culturing the host cell containing an expression vector
containing at least a
fragment of a polynucleotide under conditions suitable for the expression of
the polypeptide; and
(b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a
substantially
purified polypeptide having the amino acid sequence selected from the group
consisting of SEQ
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ID NO:1-25 and fragments thereof, in conjunction with a suitable
pharmaceutical carrier.
The invention further includes a purified antibody which binds to a
polypeptide selected
from the group consisting of SEQ ID NO:1-25 and fragments thereof. The
invention also provides
a purified agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing a disorder
associated with
decreased expression or activity of RNAAP, the method comprising administering
to a subject in
need of such treatment an effective amount of a pharmaceutical composition
comprising a
substantially purifed polypeptide having the amino acid sequence selected from
the group
consisting of SEQ ID NO:I-25 and fragments thereof, in conjunction with a
suitable
pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder
associated with
increased expression or activity of RNAAP, the method comprising administering
to a subject in
need of such treatment an effective amount of an antagonist of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-25 and fragments
thereof.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID
NOs), clone identification numbers (clone IDs), cDNA libraries, and cDNA
fragments used to
assemble full-length sequences encoding RNAAP.
Table 2 shows features of each polypeptide sequence, including potential
motifs,
homologous sequences, and methods and algorithms used for identification of
RNAAP.
Table 3 shows the tissue-specific expression patterns of each nucleic acid
sequence as
determined by northern analysis, diseases, disorders, or conditions associated
with these tissues,
and the vector into which each cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA
clones encoding RNAAP were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze RNAAP, along
with
applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is
understood that this invention is not limited to the particular machines,
materials and methods
described, as these may vary. It is also to be understood that the terminology
used herein is for the
purpose of describing particular embodiments only, and is not intended to
limit the scope of the
present invention which will be limited only by the appended claims.
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It must be noted that as used herein and in the appended claims, the singular
forms "a,"
"an," and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such host cells,
and a reference to "an
antibody" is a reference to one or more antibodies and equivalents thereof
known to those skilled
in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although any machines, materials, and methods similar or equivalent
to those described
herein can be used to practice or test the present invention, the preferred
machines, materials and
methods are now described. All publications mentioned herein are cited for the
purpose of
describing and disclosing the cell lines, protocols, reagents and vectors
which are reported in the
publications and which might be used in connection with the invention. Nothing
herein is to be
construed as an admission that the invention is not entitled to antedate such
disclosure by virtue of
prior invention.
DEFINITIONS
"RNAAP" refers to the amino acid sequences of substantially purified RNAAP
obtained
from any species, particularly a mammalian species, including bovine, ovine,
porcine, murine,
equine, and preferably the human species, from any source, whether natural,
synthetic,
semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which, when bound to RNAAP, increases
or
prolongs the duration of the effect of RNAAP. Agonists may include proteins,
nucleic acids,
carbohydrates, or any other molecules which bind to and modulate the effect of
RNAAP.
An "allelic variant" is an alternative form of the gene encoding RNAAP.
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. Any given
natural or recombinant gene may have none, one, or many allelic forms. 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 atone,
or in combination
with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding RNAAP include those sequences with
deletions, insertions, or substitutions of different nucleotides, resulting in
a polynucleotide the
same as RNAAP or a polypeptide with at least one functional characteristic of
RNAAP. Included
within this definition are polymorphisms which may or may not be readily
detectable using a
particular oligonucleotide probe of the polynucleotide encoding RNAAP, and
improper or
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unexpected hybridization to allelic variants, with a locus other than the
normal chromosomal locus
for the polynucleotide sequence encoding RNAAP. 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 RNAAP. 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 RNAAP is retained. For example, negatively charged
amino acids may
include aspartic acid and glutamic acid, positively charged amino acids may
include lysine and
arginine, and amino acids with uncharged polar head groups having similar
hydrophilicity values
may include leucine, isoleucine, and valine; glycine and alanine; asparagine
and glutamine; serine
and threonine; 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. In this context, "fragments," "immunogenic fragments," or
"antigenic
fragments" refer to fragments of RNAAP which are preferably at least 5 to
about 15 amino acids
in length, most preferably at least 14 amino acids, and which retain some
biological activity or
immunological activity of RNAAP. Where "amino acid sequence" is recited to
refer to an amino
acid sequence of a naturally occurring protein molecule, "amino acid sequence"
and like terms are
not meant to limit the amino acid sequence to the complete native amino acid
sequence associated
with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which, when bound to RNAAP,
decreases the
amount or the duration of the effect of the biological or immunological
activity of RNAAP.
Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or
any other molecules
which decrease the effect of RNAAP.
The term "antibody" refers to intact molecules as well as to fragments
thereof, such as
Fab, F(ab'),, and Fv fragments, which are capable of binding the epitopic
determinant. Antibodies
that bind RNAAP 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,
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and keyhole limpet hemocyanin (ICLH). The coupled peptide is then used to
immunize the animal.
The term "antigenic determinant" refers to that fragment 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 (given 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 containing a nucleic acid
sequence which
is complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules
may be produced by any method including synthesis or transcription. Once
introduced into a cell,
the complementary nucleotides combine with natural sequences produced by the
cell to form
duplexes and to block either transcription or translation. The designation
"negative" can refer to
the antisense strand, and the designation "positive" can refer to the sense
strand.
The term "biologically active" refers to a protein having structural,
regulatory, or
biochemical functions of a naturally occurring molecule. Likewise,
"immunologically active"
refers to the capability of the natural, recombinant, or synthetic RNAAP, or
of any oligopeptide
thereof, to induce a specific immune response in appropriate animals or cells
and to bind with
specific antibodies.
The terms "complementary" and "complementarity" refer to the natural binding
of
polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds
to the
complementary sequence "3' T-C-A 5'." Complementarity between two single-
stranded molecules
may be "partial," such that only some of the nucleic acids bind, or it may be
"complete," such that
total complementarity exists between the single stranded molecules. The degree
of
complementarity between nucleic acid strands has significant effects on the
efficiency and strength
of the hybridization between the nucleic acid strands. This is of particular
importance in
amplification reactions, which depend upon binding between nucleic acids
strands, and in the
design and use of peptide nucleic acid (PNA) molecules.
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
RNAAP or
fragments of RNAAP may be employed as hybridization probes. The probes may be
stored in
freeze-dried form and may be associated with a stabilizing agent such as a
carbohydrate. In
hybridizations, the probe may be deployed in an aqueous solution containing
salts (e.g., NaCI),
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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
resequenced to
resolve uncalled bases, extended using the XL-PCR kit (Perkin-Elmer, Norwalk
CT) in the 5'
and/or the 3' direction, and resequenced, or which has been assembled from the
overlapping
sequences of more than one Incyte Clone using a computer program for fragment
assembly, such
as the GELVIEW fragment assembly system (GCG, Madison WI). Some sequences have
been
both extended and assembled to produce the consensus sequence.
The term "correlates with expression of a polynucleotide" indicates that the
detection of
the presence of nucleic acids, the same or related to a nucleic acid sequence
encoding RNAAP, by
northern analysis is indicative of the presence of nucleic acids encoding
RNAAP in a sample, and
thereby correlates with expression of the transcript from the polynucleotide
encoding RNAAP.
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.
IS The term "derivative" refers to the chemical modification of a polypeptide
sequence, or a
polynucleotide sequence. Chemical modifications of a polynucleotide sequence
can include, for
example, replacement of hydrogen by an alkyl, acyl, 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.
The term "similarity" refers to a degree of complementarity. There may be
partial
similarity or complete similarity. The word "identity" may substitute for the
word "similarity." A
partially complementary sequence that at least partially inhibits an identical
sequence from
hybridizing to a target nucleic acid is referred to as "substantially
similar." The inhibition of
hybridization of the completely complementary sequence to the target sequence
may be examined
using a hybridization assay (Southern or northern blot, solution
hybridization, and the like) under
conditions of reduced stringency. A substantially similar sequence or
hybridization probe will
compete for and inhibit the binding of a completely similar (identical)
sequence to the target
sequence under conditions of reduced stringency. This is not to say that
conditions of reduced
stringency are such that non-specific binding is permitted, as reduced
stringency conditions
require that the binding of two sequences to one another be a specific (i.e.,
a selective) interaction.
The absence of non-specific binding may be tested by the use of a second
target sequence which
lacks even a partial degree of complementarity (e.g., less than about 30%
similarity or identity).
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In the absence of non-specific binding, the substantially similar sequence or
probe will not
hybridize to the second non-complementary target sequence.
The phrases "percent identity" and "% identity" refer to the percentage of
sequence
similarity found in a comparison of two or more amino acid or nucleic acid
sequences. Percent
identity can be determined electronically, e.g., by using the MEGAL1GN program
(DNASTAR,
Madison Wl) which creates alignments between two or more sequences according
to methods
selected by the user, e.g., the clustal method. (See, e.g., Higgins, D.G. and
P.M. Sharp ( 1988)
Gene 73:237-244.) Parameters for each method may be the default parameters
provided by
MEGALIGN or may be specified by the user. The clustal algorithm groups
sequences into
clusters by examining the distances between al) pairs. The clusters are
aligned pairwise and then
in groups. The percentage similarity between two amino acid sequences, e.g.,
sequence A and
sequence B, is calculated by dividing the length of sequence A, minus the
number of gap residues
in sequence A, minus the number of gap residues in sequence B, into the sum of
the residue
matches between sequence A and sequence B, times one hundred. Gaps of low or
of no similarity
between the two amino acid sequences are not included in determining
percentage similarity.
Percent identity between nucleic acid sequences can also be counted or
calculated by other
methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (
1990) Methods
Enzymol. 183:626-645.) Identity between sequences can also be determined by
other methods
known in the art, e.g., by varying hybridization conditions.
"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 stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules 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 any process by which a strand of nucleic acid binds
with a
complementary strand through base pairing.
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., C°t or Rat
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
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sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively,
to the sequence found in the naturally occurring molecule.
"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.
The term "microarray" refers to an arrangement of distinct polynucleotides on
a substrate.
The terms "element" and "array element" in a microarray context, refer to
hybridizable
polynucleotides arranged on the surface of a substrate.
i0 The term "modulate" refers to a change in the activity of RNAAP. For
example,
modulation may cause an increase or a decrease in protein activity, binding
characteristics, or any
other biological, functional, or immunological properties of RNAAP.
The phrases "nucleic acid" or "nucleic acid sequence," as used herein, refer
to a
nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These
phrases also refer to
I S 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. In this context, "fragments" refers to those
nucleic acid
sequences which comprise a region of unique polynucleotide sequence that
specifically identifies
SEQ ID N0:26-50, for example, as distinct from any other sequence in the same
genome. For
20 example, a fragment of SEQ ID N0:26-50 is useful in hybridization and
amplification
technologies and in analogous methods that distinguish SEQ ID N0:26-50 from
related
polynucleotide sequences. A fragment of SEQ ID N0:26-50 is at least about 15-
20 nucleotides in
length. The precise length of the fragment of SEQ ID N0:26-50 and the region
of SEQ ID
N0:26-SO to which the fragment corresponds are routinely determinable by one
of ordinary skill
25 in the art based on the intended purpose for the fragment. In some cases, a
fragment, when
translated, would produce polypeptides retaining some functional
characteristic, e.g., antigenicity,
or structural domain characteristic, e.g., ATP-binding site, of the full-
length polypeptide.
The terms "operably associated" and "operably linked" refer to functionally
related
nucleic acid sequences. A promoter is operably associated or operably linked
with a coding
30 sequence if the promoter controls the translation of the encoded
polypeptide. While operably
associated or operably linked nucleic acid sequences can be contiguous and in
the same reading
frame, certain genetic elements, e.g., repressor genes, are not contiguously
finked to the sequence
encoding the polypeptide but still bind to operator sequences that control
expression of the
polypeptide.
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The term "oligonucleotide" refers to a nucleic acid sequence of at least about
6
nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most
preferably about 20
to 25 nucleotides, which can be used in PCR amplification or in a
hybridization assay or
microarray. "Oligonucleotide" is substantially equivalent to the terms
"amplimer," "primer,"
"oligomer," and "probe," as these terms are commonly defined in the art.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in iength 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.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding RNAAP, or fragments thereof, or RNAAP itself, 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, or an antagonist. 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 containing 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 "stringent conditions" refers to conditions which permit
hybridization between
polynucleotides and the claimed polynucleotides. Stringent conditions can be
defined by salt
concentration, the concentration of organic solvent, e.g., formamide,
temperature, and other
conditions well known in the art. In particular, stringency can be increased
by reducing the
concentration of salt, increasing the concentration of formamide, or raising
the hybridization
temperature.
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 about 60%
free, preferably about 75% free, and most preferably about 90% free from other
components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or
nucleotides by
different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
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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.
"Transformation" describes a process by which exogenous DNA enters and changes
a
recipient cell. Transformation may occur under natural or artificial
conditions according to
various methods well known in the art, and may rely on any known method for
the insertion of
foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The
method for
transformation is selected based on the type of host cell being transformed
and may include, but is
not limited to, viral infection, electroporation, heat shock, lipofection, and
particle bombardment.
The term "transformed" cells includes stably transformed cells in which the
inserted DNA is
capable of replication either as an autonomously replicating plasmid or as
part of the host
chromosome, as well as transiently transformed cells which express the
inserted DNA or RNA for
limited periods of time.
A "variant" of RNAAP polypeptides refers to an amino acid sequence that is
altered by
IS one or more amino acid residues. The variant may have "conservative"
changes, wherein a
substituted amino acid has similar structural or chemical properties (e.g.,
replacement of leucine
with isoleucine). More rarely, a variant may have "nonconservative" changes
(e.g., replacement
of glycine with tryptophan). Analogous minor variations may also include amino
acid deletions or
insertions, or both. Guidance in determining which amino acid residues may be
substituted,
inserted, or deleted without abolishing biological or immunological activity
may be found using
computer programs well known in the art, for example, LASERGENE software
(DNASTAR).
The term "variant," when used in the context of a polynucleotide sequence, may
encompass a polynucleotide sequence related to RNAAP. This definition may also
include, for
example, "allelic" (as defined above), "splice," "species," or "polymorphic"
variants. A splice
variant may have significant identity to a reference molecule, but will
generally have a greater or
lesser number of polynucleotides due to alternate splicing of exons during
mRNA processing. The
corresponding poiypeptide may possess additional functional domains or an
absence of domains.
Species variants are polynucleotide sequences that vary from one species to
another. The resulting
polypeptides generally will have significant amino acid identity relative to
each other. A
polymorphic variant is a variation in the polynucleotide sequence of a
particular gene between
individuals of a given species. Polymorphic variants also may encompass
"single nucleotide
poiymorphisms" (SNPs) in which the polynucleotide sequence varies by one base.
The presence
of SNPs may be indicative of, for example, a certain population, a disease
state, or a propensity for
a disease state.
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THE INVENTION
The invention is based on the discovery of new human RNA-associated proteins
(RNAAP), the polynucleotides encoding RNAAP, and the use of these compositions
for the
diagnosis, treatment, or prevention of cell proliferative,
autoimmune/inflammatory, and infectious
disorders.
Table I lists the Incyte clones used to assemble full length nucleotide
sequences encoding
RNAAP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs)
of the
polypeptide and nucleotide sequences, respectively. Column 3 shows the clone
IDs of the Incyte
clones in which nucleic acids encoding each RNAAP were identified, and column
4 shows the
cDNA libraries from which these clones were isolated. Column 5 shows Incyte
clones and their
corresponding cDNA libraries. Clones for which cDNA libraries are not
indicated were derived
from pooled cDNA libraries. The clones in column S were used to assemble the
consensus
nucleotide sequence of each RNAAP and are useful as fragments in hybridization
technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the
invention: column I references the SEQ ID NO; column 2 shows the number of
amino acid
residues in each polypeptide; column 3 shows potential phosphorylation sites;
column 4 shows
potential glycosylation sites; column 5 shows the amino acid residues
comprising signature
sequences and motifs; column 6 shows the identity of each protein; and column
7 shows analytical
methods used to identify each protein through sequence homology and protein
motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding RNAAP. The first column of Table
3 lists the
nucleotide SEQ ID NOs. Column 2 lists tissue categories which express RNAAP as
a fraction of
total tissue categories expressing RNAAP. Column 3 lists diseases, disorders,
or conditions
associated with those tissues expressing RNAAP. Column 4 lists the vectors
used to subclone the
cDNA library.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA
libraries from which cDNA clones encoding RNAAP were isolated. Column I
references the
nucleotide SEQ ID NOs, column 2 shows the cDNA libraries from which these
clones were
isolated, and column 3 shows the tissue origins and other descriptive
information relevant to the
cDNA libraries in column 2.
The following fragments of the nucleotide sequences encoding RNAAP are useful,
for
example, in hybridization or amplification technologies to identify SEQ ID
N0:26-50 and to
distinguish between SEQ ID N0:26-50 and related polynucleotide sequences. The
useful
fragments include the fragment of SEQ ID N0:26 from about nucleotide 586 to
about nucleotide
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615; the fragment of SEQ ID N0:27 from about nucleotide 399 to about
nucleotide 428; the
fragment of SEQ ID N0:28 from about nucleotide 234 to about nucleotide 263;
the fragment of
SEQ ID N0:29 from about nucleotide 40 to about nucleotide 69; and the Fragment
of SEQ ID
N0:30 from about nucleotide 20 to about nucleotide 49, the fragment of SEQ ID
N0:31 from
about nucleotide 40 to about nucleotide 80, the fragment of SEQ ID N0:32 from
about nucleotide
672 to about nucleotide 713, the fragment of SEQ ID N0:33 from about
nucleotide 226 to about
nucleotide 276, the fragment of SEQ ID N0:34 from about nucleotide 719 to
about nucleotide
761, the fragments of SEQ ID N0:35 from about nucleotide 77 to about
nucleotide 167 and from
about nucleotide 168 to about nucleotide 259, the fragment of SEQ ID N0:36
from about
nucleotide 465 to about nucleotide 506, the fragment of SEQ ID N0:37 from
about nucleotide 76
to about nucleotide 117, the fragment of SEQ ID N0:38 from about nucleotide
136 to about
nucleotide 180, the fragments of SEQ ID N0:39 from about nucleotide 215 to
about nucleotide
262, from about nucleotide 683 to about nucleotide 727, and from about
nucleotide 1805 to about
nucleotide 1852, the fragment of SEQ ID N0:40 from about nucleotide 162 to
about nucleotide
206, the fragment of SEQ ID N0:41 from about nucleotide 379 to about
nucleotide 423, the
fragment of SEQ ID N0:42 from about nucleotide 164 to about nucleotide 208,
the fragment of
SEQ ID N0:43 from about nucleotide 1 to about nucleotide 42, the fragments of
SEQ ID N0:44
from about nucleotide 249 to about 296 and from about nucleotide 816 to about
nucleotide 862,
the fragment of SEQ 1D N0:45 from about nucleotide 196 to about nucleotide
240, the fragment
of SEQ ID N0:46 from about nucleotide 1 to about nucleotide 54, the fragment
of SEQ ID N0:47
from about nucleotide 463 to about nucleotide 507, the fragments of SEQ ID
N0:48 from about
nucleotide 551 to about nucleotide 595, from about nucleotide 866 to about
nucleotide 910, and
from about nucleotide 1406 to about nucleotide 1450, the fragments of SEQ ID
N0:49 from about
nucleotide 218 to about nucleotide 263, from about nucleotide 758 to about
nucleotide 802, and
from about nucleotide I 190 to about nucleotide 1234, and the fragment of SEQ
ID NO:50 from
about nucleotide 11 to about nucleotide 70.
The invention alsa encompasses RNAAP variants. A preferred RNAAP variant is
one
which has at least about 80%, more preferably at least about 90%, and most
preferably at least
about 95% amino acid sequence identity to the RNAAP amino acid sequence, and
which contains
at least one functional or structural characteristic of RNAAP.
The invention also encompasses polynucleotides which encode RNAAP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence
selected from the group consisting of SEQ ID N0:26-S0, which encodes RNAAP.
The invention also encompasses a variant of a polynucieotide sequence encoding
RNAAP.
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In particular, such a variant polynucleotide sequence will have at least about
70%, more preferably
at least about 85%, and most preferably at least about 95% polynucleotide
sequence identity to the
polynucleotide sequence encoding RNAAP. A particular aspect of the invention
encompasses a
variant of a polynucleotide sequence comprising a sequence selected from the
group consisting of
SEQ ID N0:26-50 which has at least about 70%, more preferably at least about
85%, and most
preferably at least about 95% polynucleotide sequence identity to a nucleic
acid sequence selected
from the group consisting of SEQ ID N0:26-50. Any one of the polynucleotide
variants described
above can encode an amino acid sequence which contains at least one functional
or structural
characteristic of RNAAP.
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 RNAAP, 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 RNAAP, and all such variations
are to be
considered as being specifically disclosed.
Although nucleotide sequences which encode RNAAP and its variants are
preferably
capable of hybridizing to the nucleotide sequence of the naturally occurring
RNAAP under
appropriately selected conditions of stringency, it may be advantageous to
produce nucleotide
sequences encoding RNAAP or its derivatives possessing a substantially
different codon usage,
e.g., inclusion of non-naturally occurring codons. Codons may be selected to
increase the rate at
which expression of the peptide occurs in a particular prokaryotic or
eukaryotic host in accordance
with the frequency with which particular codons are utilized by the host.
Other reasons for
substantially altering the nucleotide sequence encoding RNAAP 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 RNAAP
and RNAAP 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 RNAAP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
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hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:26-50 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.) For example, stringent salt concentration will ordinarily be
less than about 750 mM
NaCi and 75 mM trisodium citrate, preferably less than about 500 mM NaCI and
50 mM trisodium
citrate, and most preferably less than about 250 mM NaCI and 25 mM trisodium
citrate. Low
stringency hybridization can be obtained in the absence of organic solvent,
e.g., formamide, while
high stringency hybridization can be obtained in the presence of at least
about 35% formamide,
and most preferably at least about 50% formamide. Stringent temperature
conditions will
ordinarily include temperatures of at least about 30°C, more preferably
of at least about 37°C, and
most preferably of at least about 42°C. Varying additional parameters,
such as hybridization time,
the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the
inclusion or exclusion
of carrier DNA, are well known to those skilled in the art. Various levels of
stringency are
accomplished by combining these various conditions as needed. In a preferred
embodiment,
hybridization will occur at 30°C in 750 mM NaCI, 75 mM trisodium
citrate, and 1% SDS. In a
more preferred embodiment, hybridization will occur at 37°C in 500 mM
NaCI, SO mM trisodium
citrate, 1% SDS, 35% formamide, and 100 /cg/ml denatured salmon sperm DNA
(ssDNA). In a
most preferred embodiment, hybridization will occur at 42°C in 250 mM
NaCI, 25 mM trisodium
citrate, 1% SDS, 50 % formamide, and 200 ~g/ml ssDNA. Useful variations on
these conditions
will be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash
stringency conditions can be defined by salt concentration and by temperature.
As above, wash
stringency can be increased by decreasing salt concentration or by increasing
temperature. For
example, stringent salt concentration for the wash steps will preferably be
less than about 30 mM
NaCI and 3 mM trisodium citrate, and most preferably less than about 15 mM
NaCI and 1.5 mM
trisodium citrate. Stringent temperature conditions for the wash steps will
ordinarily include
temperature of at least about 25°C, more preferably of at least about
42°C, and most preferably of
at least about 68°C. In a preferred embodiment, wash steps will occur
at 25°C in 30 mM NaCI, 3
mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps
will occur at
42°C in 15 mM NaCI, I.5 mM trisodium citrate, and 0.1% SDS. In a most
preferred embodiment,
wash steps will occur at 68°C in IS mM NaCI, 1.5 mM trisodium citrate,
and 0.1% SDS.
Additional variations on these conditions will be readily apparent to those
skilled in the art.
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
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fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq
polymerase
(Perkin-Elmer), 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 Hamilton MICROLAB 2200
(Hamilton, Reno
NV), Peltier Thermal Cycler 200 (PTC200; MJ Research, Watertown MA) and the
ABI
CATALYST 800 (Perkin-Elmer). Sequencing is then carried out using either ABI
373 or 377
DNA sequencing systems (Perkin-Elmer), 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 BioloQV, John Wiley & Sons,
New York NY,
unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH,
New York NY,
pp. 856-853.)
The nucleic acid sequences encoding RNAAP may be extended utilizing a partial
nucleotide sequence and employing various PCR-based methods known in the art
to detect
upstream sequences, such as promoters and regulatory elements. For example,
one method which
may be employed, restriction-site PCR, uses universal and nested primers to
amplify unknown
sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G.
(1993) PCR Methods
Applic. 2:318-322.) Another method, inverse PCR, uses primers that extend in
divergent
directions to amplify unknown sequence from a circularized template. The
template is derived
from restriction fragments comprising a known genomic locus and surrounding
sequences. (See,
e.g., Triglia, T. et al. ( 1988) Nucleic Acids Res. 16:8186.) A third method,
capture PCR, involves
PCR amplification of DNA fragments adjacent to known sequences in human and
yeast artificial
chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic.
1:1 I 1-119.) In
this method, multiple restriction enzyme digestions and legations may be used
to insert an
engineered double-stranded sequence into a region of unknown sequence before
performing PCR.
Other methods which may be used to retrieve unknown sequences are known in the
art. (See, e.g.,
Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-306). Additionally, one
may use PCR,
nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk
genomic
DNA. This procedure avoids the need to screen libraries and is useful in
finding intron/exon
junctions. For all PCR-based methods, primers may be designed using
commercially available
software, such as OLIGO 4.06 Primer Analysis software (National Biosciences,
Plymouth MN) or
another appropriate program, to be about 22 to 30 nucleotides in length, to
have a GC content of
about 50% or more, and to anneal to the template at temperatures of about
68°C to 72°C.
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When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of
sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to
analyze the size or confirm the nucleotide sequence of sequencing or PCR
products. In particular,
capillary sequencing may employ flowable polymers for electrophoretic
separation, four different
nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled
device camera for
detection ofthe emitted wavelengths. Output/light intensity may be converted
to electrical signal
using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-
Elmer),
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 RNAAP may be cloned in recombinant DNA molecules that direct
expression of
RNAAP, 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
RNAAP.
The nucleotide sequences of the present invention can be engineered using
methods
generally known in the art in order to alter RNAAP-encoding sequences for a
variety of purposes
including, but not limited to, modification of the cloning, processing, and/or
expression ofthe
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.
In another embodiment, sequences encoding RNAAP 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) Nucl.
Acids Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids Res.
Symp. Ser. 225-232.)
Alternatively, RNAAP itself or a fragment thereof may be synthesized using
chemical methods.
For example, peptide synthesis can be performed using various solid-phase
techniques. (See, e.g.,
Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be
achieved using
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the ABI 431A Peptide Synthesizer (Perkin-Elmer). Additionally, the amino acid
sequence of
RNAAP, 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
poiypeptide.
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, T. (1984) Proteins. Structures and
Molecular Properties, WH
Freeman, New York NY.)
In order to express a biologically active RNAAP, the nucleotide sequences
encoding
RNAAP 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 RNAAP. Such elements may vary in
their strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding RNAAP. Such signals include the ATG initiation codon and
adjacent
sequences, e.g. the Kozak sequence. In cases where sequences encoding RNAAP
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 RNAAP and appropriate
transcriptional and
translational control elements. These methods include in vitro recombinant DNA
techniques,
synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook,
J. et al. (1989)
Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, Plainview
NY, ch. 4, 8, and
16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolo~y,
John Wiley & Sons,
New York NY, ch. 9, 13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express
sequences encoding RNAAP. These include, but are not limited to,
microorganisms such as
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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. The
invention is not
limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected
depending upon the use intended for polynucleotide sequences encoding RNAAP.
For example,
routine cloning, subcloning, and propagation of polynucleotide sequences
encoding RNAAP can
be achieved using a multifunctional E. coli vector such as PBLUESCRIPT
(Stratagene, La Jolla
CA) or pSPORTI plasmid (Life Technologies). Ligation of sequences encoding
RNAAP 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 RNAAP are
needed, e.g. for the production of antibodies, vectors which direct high level
expression of
RNAAP may be used. For example, vectors containing the strong, inducible TS or
T7
bacteriophage promoter may be used.
Yeast expression systems may be used for production of RNAAP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or
Pichia~astoris. 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; Grant et al. ( 1987) Methods Enzymol. 153:5 I 6-54; and Scorer,
C. A. et al. ( 1994)
Bio/Technoiogy 12:181-184.)
Plant systems may also be used for expression of RNAAP. Transcription of
sequences
encoding RNAAP may be driven 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 aI. (1984) EMBO
J. 3:1671-1680;
Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991)
Results Probl. Cell
Differ. 17:85-105.) These constructs can be introduced into plant cells by
direct DNA
transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill
Yearbook of
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Science and Technoloey (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 RNAAP
may be ligated
into an adenovirus transcription/translation complex consisting of the late
promoter and tripartite
leader sequence. Insertion in a non-essential E1 or E3 region of the viral
genome may be used to
obtain infective virus which expresses RNAAP in host cells. (See, e.g., Logan,
J. and T. Shenk
(1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcription
enhancers, such as the
Rous sarcoma virus (RSV) enhancer, may be used to increase expression in
mammalian host cells.
SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments
of DNA than can be contained in and expressed from a plasmid. HACs of about 6
kb to 10 Mb
are constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat Genet.
15:345-355.)
I S For long term production of recombinant proteins in mammalian systems,
stable
expression of RNAAP in cell lines is preferred. For example, sequences
encoding RNAAP 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. Fol lowing the introduction of the vector, cells may be al lowed to
grow for about 1 to 2
days in enriched media before being switched to selective media. The purpose
ofthe 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~ or apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I, et al. (1980) Cell 22:817-823.) Also,
antimetabolite,
antibiotic, or herbicide resistance can be used as the basis for selection.
For example, dhfr confers
resistance to methotrexate; neo confers resistance to the aminoglycosides
neomycin and G-418;
and als or pat confer resistance to ehlorsulfuron and phosphinotricin
acetyltransferase,
respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
77:3567-3570;
Colbere-Garapin, F. et al. (1981) J. MoI. 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. 85:8047-8051.)
Visible markers,
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e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), (3
glucuronidase and its substrate
(3-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.
Bio1.55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of
interest is also present, the presence and expression of the gene may need to
be confirmed. For
example, if the sequence encoding RNAAP is inserted within a marker gene
sequence,
transformed cells containing sequences encoding RNAAP can be identified by the
absence of
marker gene function. Alternatively, a marker gene can be placed in tandem
with a sequence
encoding RNAAP under the control of a single promoter. Expression of the
marker gene in
response to induction or selection usually indicates expression of the tandem
gene as well.
In general, host cells that contain the nucleic acid sequence encoding RNAAP
and that
express RNAAP 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 RNAAP
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
RNAAP 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
Immunoloey, 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 RNAAP
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled
nucleotide. Alternatively, the sequences encoding RNAAP, 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
CA 02340277 2001-02-20
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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 RNAAP 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 RNAAP may be designed to
contain signal
sequences which direct secretion of RNAAP through a prokaryotic or eukaryotic
cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications
of the polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro"
form of the protein may also be used to specify protein targeting, folding,
and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are
available from
the American Type Culture Collection (ATCC, Manassas, VA) and may be chosen to
ensure the
correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding RNAAP 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 RNAAP
protein containing a heterologous moiety that can be recognized by a
commercially available
antibody may facilitate the screening of peptide libraries for inhibitors of
RNAAP 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 oftheir cognate fusion proteins on immobilized
glutathione, maltose,
phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG,
c-myc, and
hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using
commercially
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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
RNAAP encoding sequence and the heterologous protein sequence, so that RNAAP
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 RNAAP may
be
achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ
extract systems
(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, preferably 'SS-methionine.
Fragments of RNAAP may be produced not only by recombinant production, but
also by
direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton,
su ra pp. ~S-60.)
Protein synthesis may be performed by manual techniques or by automation.
Automated synthesis
may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin-
Elmer). Various
fragments of RNAAP may be synthesized separately and then combined to produce
the full length
molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of RNAAP and RNA-associated proteins. In addition, the
expression of RNAAP
is closely associated with cancer, fetal development, cell proliferation,
inflammation, and immune
response. Therefore, RNAAP appears to play a role in cell proliferative,
autoimmune/inflammatory, and infectious disorders. In the treatment of
disorders associated with
increased RNAAP expression or activity, it is desirable to decrease the
expression or activity of
RNAAP. In the treatment of the above conditions associated with decreased
RNAAP expression
or activity, it is desirable to increase the expression or activity of RNAAP.
Therefore, in one embodiment, RNAAP 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 RNAAP. Examples of such disorders include, but are not limited to,
a cell proliferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis,
mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria,
polycythemia vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers
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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;
an
autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankyiosing
spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune
thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
(APECED),
bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis,
diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis
fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,
Goodpasture's syndrome, gout,
Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, multiple
sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid
arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and
helminthic infections, and trauma; and an infectious disorder such as
infections by viral agents
classified as adenovirus, arenavirus, bunyavirus, caiicivirus, coronavirus,
filovirus, hepadnavirus,
herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus,
paramyxovirus, picornavirus,
poxvirus, reovirus, retrovirus, rhabdovirus, and togavirus; infections by
bacterial agents classified
as pneumococcus, staphylococcus, streptococcus, bacillus, corynebacterium,
clostridium,
meningococcus, gonococcus, listeria, moraxella, kingella, haemophilus,
legionella, bordetella,
gram-negative enterobacterium including shigella, salmonella, and
campylobacter, pseudomonas,
vibrio, brucella, francisella, yersinia, bartonella, norcardium, actinomyces,
mycobacterium,
spirochaetale, rickettsia, chlamydia, and mycoplasma; infections by fungal
agents classified as
aspergillus, blastomyces, dermatophytes, cryptococcus, coccidioides,
malasezzia, histoplasma, and
other fungal agents causing various mycoses; and infections by parasites
classified as plasmodium
or malaria-causing, parasitic entamoeba, leishmania, trypanosoma, toxoplasma,
pneumocystis
carinii, intestinal protozoa such as giardia, trichomonas, tissue nematodes
such as trichinella,
intestinal nematodes such as ascaris, lymphatic filaria) nematodes, trematodes
such as
schistosoma, and cestodes (tapeworm).
In another embodiment, a vector capable of expressing RNAAP 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 RNAAP including, but not limited to, those
described above.
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In a further embodiment, a pharmaceutical composition comprising a
substantially
purified RNAAP 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 RNAAP
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of RNAAP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of RNAAP including, but not limited to, those listed above.
In a further embodiment, an antagonist of RNAAP may be administered to a
subject to
treat or prevent a disorder associated with increased expression or activity
of RNAAP. Examples
of such disorders include, but are not limited to, those cell proliferative,
autoimmune/inflammatory, and infectious disorders described above. In one
aspect, an antibody
which specifically binds RNAAP may be used directly as an antagonist or
indirectly as a targeting
or delivery mechanism for bringing a pharmaceutical agent to cells or tissue
which express
RNAAP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding RNAAP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of RNAAP including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary sequences, or vectors of the invention may be administered in
combination with
other appropriate therapeutic agents. Selection of the appropriate agents for
use in combination
therapy may be made by one of ordinary skill in the art, according to
conventional pharmaceutical
principles. The combination of therapeutic agents may act synergistically to
effect the treatment
or prevention of the various disorders described above. Using this approach,
one may be able to
achieve therapeutic efficacy with lower dosages of each agent, thus reducing
the potential for
adverse side effects.
An antagonist of RNAAP may be produced using methods which are generally known
in
the art. In particular, purified RNAAP may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind RNAAP.
Antibodies to RNAAP
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 especially 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 RNAAP or with any
fragment or
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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
RNAAP have an amino acid sequence consisting of at least about 5 amino acids,
and, more
preferably, 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 and
contain the entire amino acid sequence of a small, naturally occurring
molecule. Short stretches of
RNAAP 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 RNAAP 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.
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. 81:6851-6855; Neuberger, M.S. et al. ( 1984) Nature
312:604-608; and
Takeda, S. et al. ( 1985) Nature 314:452-454.) Alternatively, techniques
described for the
production of single chain antibodies may be adapted, using methods known in
the art, to produce
RNAAP-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. 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. 86:
3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for RNAAP may also be
generated. For example, such fragments include, but are not limited to,
F(ab')2 fragments
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produced by pepsin digestion of the antibody molecule and Fab fragments
generated by reducing
the disulfide bridges ofthe 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 polyclona) or monoclonal antibodies with established
specificities are well known in
the art. Such immunoassays typically involve the measurement of complex
formation between
RNAAP and its specific antibody. A two-site, monoclonal-based immunoassay
utilizing
monoclonal antibodies reactive to two non-interfering RNAAP epitopes is
preferred, 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 RNAAP.
Affinity is expressed as
an association constant, Ka, which is defined as the molar concentration of
RNAAP-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 RNAAP epitopes, represents the
average affinity, or
avidity, of the antibodies for RNAAP. The Ka determined for a preparation of
monoclonal
antibodies, which are monospecific for a particular RNAAP epitope, represents
a true measure of
affinity. High-affinity antibody preparations with K, ranging from about 109
to 10'' L/mole are
preferred for use in immunoassays in which the RNAAP-antibody complex must
withstand
rigorous manipulations. Low-affinity antibody preparations with Ka ranging
from about I OG to 107
L/mole are preferred for use in immunopurification and similar procedures
which ultimately
require dissociation of RNAAP, preferably in active form, from the antibody
(Catty, D. ( 1988)
Antibodies, Volume I: A Practical Approach, IRL Press, Washington, DC;
Liddell, J. E. and
Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies, John Wiley &
Sons, New York
NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least I-2 mg specific
antibody/ml,
preferably S-10 mg specific antibody/ml, is preferred for use in procedures
requiring precipitation
of RNAAP-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.)
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In another embodiment of the invention, the polynucleotides encoding RNAAP, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, the
complement of the polynucleotide encoding RNAAP may be used in situations in
which it would
be desirable to block the transcription of the mRNA. In particular, cells may
be transformed with
sequences complementary to polynucleotides encoding RNAAP. Thus, complementary
molecules
or fragments may be used to modulate RNAAP activity, or to achieve regulation
of gene function.
Such technology is now well known in the art, and sense or antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding RNAAP.
Expression vectors derived from retroviruses, adenoviruses, or herpes or
vaccinia viruses,
or from various bacteria! plasmids, may be used for delivery of nucleotide
sequences to the
targeted organ, tissue, or cell population. Methods which are well known to
those skilled in the art
can be used to construct vectors to express nucleic acid sequences
complementary to the
polynucleotides encoding RNAAP. (See, e.g., Sambrook, supra; Ausubel, 1995,
s_upra.)
Genes encoding RNAAP can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding RNAAP.
Such constructs may be used to introduce untranslatable sense or antisense
sequences into a cell.
Even in the absence of integration into the DNA, such vectors may continue to
transcribe RNA
molecules until they are disabled by endogenous nucleases. Transient
expression may last for a
month or more with a non-replicating vector, and may last even longer if
appropriate replication
elements are part of the vector system.
As mentioned above, modifications of gene expression can be obtained by
designing
complementary sequences or antisense molecules (DNA, RNA, or PNA) to the
control, S', or
regulatory regions of the gene encoding RNAAP. Oligonucleotides derived from
the transcription
initiation site, e.g., between about positions -10 and +10 from the start
site, are preferred.
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 Immunoloaic 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
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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 RNAAP.
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 RNAAP. Such DNA sequences
may be
incorporated into a wide variety of vectors with suitable RNA polymerase
promoters such as T7 or
SP6. Alternatively, these cDNA constructs that synthesize complementary RNA,
constitutively or
inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as
inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and
similarly modified forms
of adenine, cytidine, guanine, thymine, and uridine which are not as easily
recognized by
endogenous endonucleases.
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) Nature Biotechnology 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 dogs, cats, cows,
horses, rabbits,
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monkeys, and most preferably, humans.
An additional embodiment of the invention relates to the administration of a
pharmaceutical or sterile composition, in conjunction with a pharmaceutically
acceptable carrier,
for any of the therapeutic effects discussed above. Such pharmaceutical
compositions may consist
of RNAAP, antibodies to RNAAP, and mimetics, agonists, antagonists, or
inhibitors of RNAAP.
The compositions may be administered alone or in combination with at least one
other agent, such
as a stabilizing compound, which may be administered in any sterile,
biocompatible
pharmaceutical carrier including, but not limited to, saline, buffered saline,
dextrose, and water.
The compositions may be administered to a patient alone, or in combination
with other agents,
drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered
by any
number of routes including, but not limited to, oral, intravenous,
intramuscular, intra-arterial,
intrameduliary, intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or recta! means.
In addition to the active ingredients, these pharmaceutical compositions may
contain
suitable pharmaceutically-acceptable carriers comprising excipients and
auxiliaries which
facilitate processing of the active compounds into preparations which can be
used
pharmaceutically. Further details on techniques for formulation and
administration may be found
in the latest edition of Reminaton's Pharmaceutical Sciences (Maack
Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using
pharmaceutically acceptable carriers well known in the art in dosages suitable
for oral
administration. Such carriers enable the pharmaceutical compositions to be
formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and
the like, for ingestion by
the patient.
Pharmaceutical preparations for oral use can be obtained through combining
active
compounds with solid excipient and processing the resultant mixture of
granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be
added, if desired. Suitable
excipients include carbohydrate or protein fillers, such as sugars, including
lactose, sucrose,
mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other
plants; cellulose, such as
methyl cellulose, hydroxypropylmethyl-cellulose, or sodium
carboxymethylcellulose; gums,
including arabic and tragacanth; and proteins, such as gelatin and collagen.
If desired,
disintegrating or solubilizing agents may be added, such as the cross-linked
polyvinyl pyrrolidone,
agar, and alginic acid or a salt thereof, such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as
concentrated
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sugar solutions, which may also contain gum arabic, talc,
polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable
organic solvents or
solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee
coatings for
product identification or to characterize the quantity of active compound,
i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of
gelatin, as well as soft, sealed capsules made of gelatin and a coating, such
as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or
binders, such as lactose or
starches, lubricants, such as talc or magnesium stearate, and, optionally,
stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in suitable
liquids, such as fatty
oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be
formulated in
aqueous solutions, preferably in physiologically compatible buffers such as
Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous injection
suspensions may contain
substances which increase the viscosity of the suspension, such as sodium
carboxymethyl
I S cellulose, sorbitol, or dextran. Additionally, suspensions of the active
compounds may be
prepared as appropriate oily injection suspensions. Suitable lipophilic
solvents or vehicles include
fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl
oleate, triglycerides, or
liposomes. Non-lipid polycationic amino polymers may also be used for
delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to increase the
solubility of the
compounds and allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art.
The pharmaceutical compositions of the present invention may be manufactured
in a
manner that is known in the art, e.g., by means of conventional mixing,
dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping, or
lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed
with many
acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic,
tartaric, malic, and
succinic acid. Salts tend to be more soluble in aqueous or other protonic
solvents than are the
corresponding free base forms. In other cases, the preferred preparation may
be a lyophilized
powder which may contain any or all of the following: I mM to 50 mM histidine,
0.1 % to 2%
sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined
with buffer prior to
use.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate container and labeled for treatment of an indicated condition. For
administration of
CA 02340277 2001-02-20
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RNAAP, such labeling would include amount, frequency, and method of
administration.
Pharmaceutical 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.
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, 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
RNAAP or fragments thereof, antibodies of RNAAP, and agonists, antagonists or
inhibitors of
RNAAP, 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 SO% of the population) statistics. The dose ratio of
toxic to therapeutic
effects is the therapeutic index, which can be expressed as the
LDS°/EDSO ratio. Pharmaceutical
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 EDs° 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 pharmaceutical
compositions may be
administered every 3 to 4 days, every week, or biweekly depending on the half
life and clearance
rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 ~cg, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
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inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular
cells, conditions, locations, etc.
DIAGNOSTICS
1n another embodiment, antibodies which specifically bind RNAAP may be used
for the
diagnosis of disorders characterized by expression of RNAAP, or in assays to
monitor patients
being treated with RNAAP or agonists, antagonists, or inhibitors of RNAAP.
Antibodies useful
for diagnostic purposes may be prepared in the same manner as described above
for therapeutics.
Diagnostic assays for RNAAP include methods which utilize the antibody and a
label to detect
RNAAP 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 RNAAP, including ELISAs, RIAs, and FACS,
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of RNAAP
IS expression. Normal or standard values for RNAAP expression are established
by combining body
fluids or cell extracts taken from normal mammalian subjects, preferably
human, with antibody to
RNAAP under conditions suitable for complex formation. The amount of standard
complex
formation may be quantitated by various methods, preferably by photometric
means. Quantities of
RNAAP 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 RNAAP 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 quantitate gene expression in biopsied tissues in which
expression of RNAAP
may be correlated with disease. The diagnostic assay may be used to determine
absence,
presence, and excess expression of RNAAP, and to monitor regulation of RNAAP
levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide sequences, including genomic sequences, encoding RNAAP or
closely related
molecules may be used to identify nucleic acid sequences which encode RNAAP.
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 (maximal, high, intermediate, or low), will determine whether
the probe identifies
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only naturally occurring sequences encoding RNAAP, allelic variants, or
related sequences.
Probes may also be used for the detection of related sequences, and should
preferably
have at least 50% sequence identity to any of the RNAAP encoding sequences.
The hybridization
probes of the subject invention may be DNA or RNA and may be derived from the
sequence of
SEQ ID N0:26-50 or from genomic sequences including promoters, enhancers, and
introns of the
RNAAP gene.
Means for producing specific hybridization probes for DNAs encoding RNAAP
include
the cloning of polynucleotide sequences encoding RNAAP or RNAAP 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'zP
or'SS, or by enzymatic
labels, such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and
the like.
I S Polynucleotide sequences encoding RNAAP may be used for the diagnosis of
disorders
associated with expression of RNAAP. Examples of such disorders include, but
are not limited to,
a cell proliferative disorder, such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed connective tissue disease (MC'TD), 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; an
autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondyiitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune
thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
(APECED),
bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis,
diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis
fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,
Goodpasture's syndrome, gout,
Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, multiple
sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid
arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis,
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thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and
helminthic infections, and trauma; and an infectious disorder such as
infections by viral agents
classified as adenovirus, arenavirus, bunyavirus, calicivirus, coronavirus,
filovirus, hepadnavirus,
herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus,
paramyxovirus, picornavirus,
poxvirus, reovirus, retrovirus, rhabdovirus, and togavirus; infections by
bacterial agents classified
as pneumococcus, staphylococcus, streptococcus, bacillus, corynebacterium,
clostridium,
meningococcus, gonococcus, listeria, moraxella, kingella, haemophilus,
legionella, bordetella,
gram-negative enterobacterium including shigella, salmonella, and
campylobacter, pseudomonas,
vibrio, brucella, francisella, yersinia, bartonella, norcardium, actinomyces,
mycobacterium,
spirochaetale, rickettsia, chlamydia, and mycoplasma; infections by fungal
agents classified as
aspergillus, btastomyces, dermatophytes, cryptococcus, coccidioides,
malasezzia, histoplasma, and
other fungal agents causing various mycoses; and infections by parasites
classified as plasmodium
or malaria-causing, parasitic entamoeba, leishmania, trypanosoma, toxoplasma,
pneumocystis
carinii, intestinal protozoa such as giardia, trichomonas, tissue nematodes
such as trichinella,
intestinal nematodes such as ascaris, lymphatic filarial nematodes, trematodes
such as
schistosoma, and cestodes (tapeworm). The polynucleotide sequences encoding
RNAAP 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 RNAAP expression. Such
qualitative or
quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding RNAAP may be useful
in assays
that detect the presence of associated disorders, particularly those mentioned
above. The
nucleotide sequences encoding RNAAP 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 quantitated
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 RNAAP in the sample indicates the presence of the
associated disorder. Such
assays may also be used to evaluate the efficacy of a particular therapeutic
treatment regimen in
animal studies, in clinical trials, or to monitor the treatment of an
individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
RNAAP, 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
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sequence, or a fragment thereof, encoding RNAAP, 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
RNAAP 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 RNAAP, or a fragment of a polynucleotide complementary
to the
polynucleotide encoding RNAAP, 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 quantitation of closely related DNA or
RNA sequences.
Methods which may also be used to quantify the expression of RNAAP include
radiolabeling or biotinylating nucleotides, coamplification of a control
nucleic acid, and
interpolating results from standard curves. (See, e.g., Melby, P.C. et al.
(1993) J. Immunol.
Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The
speed of
quantitation of multiple samples may be accelerated by running the assay in an
ELISA format
where the oligomer 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 targets in a
microarray. The
microarray can be used to monitor the expression level of Large numbers of
genes simultaneously
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and 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, and
to develop and monitor the activities of therapeutic agents.
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. 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalon,
D. et al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc.
Natl. Acad. Sci.
94:2150-2155; and Heller, M.J. et al. ( i 997) U.S. Patent No. 5,605,662.)
1n another embodiment of the invention, nucleic acid sequences encoding RNAAP
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic
sequence. The sequences may be mapped to a particular chromosome, to a
specific region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes
(HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial
P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997)
Nat Genet. 15:345-355; Price, C.M. ( 1993) Blood Rev. 7:127-134; and Trask,
B.J. ( 1991 ) Trends
Genet. 7:149-154.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich,
et al. (1995) in
Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in
various scientific
journals or at the Online Mendelian Inheritance in Man (OMIM) site.
Correlation between the
location of the gene encoding RNAAP on a physical chromosomal map and a
specific disorder, or
a predisposition to a specific disorder, may help define the region of DNA
associated with that
disorder. The nucleotide sequences of the invention may be used to detect
differences in gene
sequences among normal, carrier, and affected individuals.
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 number or arm of a particular
human
chromosome is not known. New sequences can be assigned to chromosomal arms by
physical
mapping. This provides valuable information to investigators searching for
disease genes using
positional cloning or other gene discovery techniques. Once the disease or
syndrome has been
crudely localized by genetic linkage to a particular genomic region, e.g.,
ataxia-telangiectasia to
l 1q22-23, any sequences mapping to that area may represent associated or
regulatory genes for
further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-
580.) The nucleotide
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sequence of the subject 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, RNAAP, 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 RNAAP 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 RNAAP, or
fragments thereof, and washed. Bound RNAAP is then detected by methods well
known in the
art. Purified RNAAP 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
IS peptide and immobilize it on a solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing antibodies capable of binding RNAAP specifically compete with a
test compound for
binding RNAAP. In this manner, antibodies can be used to detect the presence
of any peptide
which shares one or more antigenic determinants with RNAAP.
In additional embodiments, the nucleotide sequences which encode RNAAP 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 preferred
specific embodiments are, therefore, to be construed as merely illustrative,
and not limitative of
the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below,
in particular U.S. Ser. No. 60/115,639 and U.S. Ser No. 60/097,550, are hereby
expressly
incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some
tissues were homogenized and lysed in guanidinium isothiocyanate, while others
were
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homogenized and lysed in phenol or in a suitable mixture of denaturants, such
as TRIZOL (Life
Technologies), a monophasic solution of phenol and guanidine isothiocyanate.
The resulting
lysates were centrifuged over CsCI cushions or extracted with chloroform. RNA
was precipitated
from the lysates with either isopropanol or sodium acetate and ethanol, or by
other routine
methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was
isolated using oligo d(T}-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles
(QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively,
RNA was isolated directly from tissue lysates using other RNA isolation kits,
e.g., the
POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding
cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were
constructed with the
UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life
Technologies),
using the recommended procedures or similar methods known in the art. (See,
e.g., Ausubel,
1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oligo
d(T) or random
primers. Synthetic oligonucleotide adapters were ligated to double stranded
cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most
libraries, the cDNA
was size-selected (300-1000 bp) using SEPHACRYL S1000, 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),
PSPORTI plasmid
(Life Technologies), or pINCY (Incyte Pharmaceuticals, Paio Alto CA).
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 were recovered from host cells by in vivo excision, using the UNIZAP
vector
system (Stratagene) or 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
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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
cDNA sequencing reactions were processed using standard methods or high-
throughput
instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) 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 (Perkin-Elmer). Electrophoretic separation of cDNA sequencing
reactions and
detection of labeled polynucleotides were carried out using the MEGABACE 1000
DNA
sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing
systems
(Perkin-Eimer) in conjunction with standard ABI protocols and base calling
software; or other
sequence analysis systems known in the art. Reading frames within the cDNA
sequences were
identified using standard methods (reviewed in Ausubel, 1997, supra, unit
7.7). Some of the
cDNA sequences were selected for extension using the techniques disclosed in
Example V.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed using a combination of software programs which utilize algorithms
well known to those
skilled in the art. Table 5 summarizes the tools, programs, and algorithms
used and provides
applicable descriptions, references, and threshold parameters. The first
column of Table 5 shows
the tools, programs, and algorithms used, the second column provides brief
descriptions thereof,
the third column presents appropriate references, all of which are
incorporated by reference herein
in their entirety, and the fourth column presents, where applicable, the
scores, probability values,
and other parameters used to evaluate the strength of a match between two
sequences (the higher
the score, the greater the homology between two sequences). Sequences were
analyzed using
MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA)
and
LASERGENE software (DNASTAR).
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
were then
queried against a selection of public databases such as the GenBank primate,
rodent, mammalian,
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vertebrate, and eukaryote databases, and BLOCKS to acquire annotation using
programs based on
BLAST, FASTA, and BLIMPS. The sequences were assembled into full length
polynucleotide
sequences using programs based on Phred, Phrap, and Consed, and 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 amino acid
sequences, and these full length sequences were subsequently analyzed by
querying against
databases such as the GenBank databases (described above), SwissProt, BLOCKS,
PRINTS,
Prosite, 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, e.g., Eddy, S.R. (1996) Curr. Opin. Str. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide
and amino acid sequences were also used to identify polynucleotide sequence
fragments from
SEQ ID N0:26-50. Fragments from about 20 to about 4000 nucleotides which are
useful in
hybridization and amplification technologies were described in The Invention
section above.
IV. Northern Analysis
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which
RNAs from a particular cell type or tissue have been bound. (See, e.g.,
Sambrook, su ra, ch. 7;
Ausubel, 1995, suara, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or
related molecules in nucleotide databases such as GenBank or LIFESEQ (Incyte
Phanmaceuticals).
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 de#ined as:
% sequence identity x % maximum BLAST score
100
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. For example, with a product score of 40, the
match will be exact
within a I% to 2% error, and, with a product score of 70, the match will be
exact. Similar
molecules are usually identified by selecting those which show product scores
between 15 and 40,
although lower scores may identify related molecules.
The results of northern analyses are reported as a percentage distribution of
libraries in
which the transcript encoding RNAAP occurred. Analysis involved the
categorization of cDNA
libraries by organ/tissue and disease. The organ/tissue categories included
cardiovascular,
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dermatologic, developmental, endocrine, gastrointestinal,
hematopoietic/immune, musculoskeletal,
nervous, reproductive, and urologic. The disease/condition categories included
cancer,
inflammation/trauma, cell proliferation, neurological, and pooled. For each
category, the number
of libraries expressing the sequence of interest was counted and divided by
the total number of
libraries across all categories. Percentage values of tissue-specific and
disease- or condition-
specific expression are reported in Table 3.
V. Extension of RNAAP Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:26-50 were 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, 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 Mgz+,
(NH4)zS04, and (3-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia
Biotech),
ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with
the
following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3
min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3,
and 4 repeated 20 times; Step 6:
68 °C, 5 min; Step 7: storage at 4°C. In the alternative, the
parameters for primer pair T7 and SK+
were as follows: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step
3: 57°C, 1 min; Step 4: 68°C, 2
min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min;
Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 pl
PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes,
Eugene OR)
dissolved in 1X TE and 0.5 pl 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 ~cl to 10 ~cl aliquot of
the reaction mixture
was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which
reactions were
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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
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were relegated using T4 ligase (New England Biolabs, Beverly MA) into
pUC 18 vector
(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to
fill-in
restriction site overhangs, and transfected into competent E. coli cells.
Transformed cells were
selected on antibiotic-containing media, individual colonies were picked and
cultured overnight at
37°C in 384-well plates in LB/2x Garb 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, IS 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% dimethysulphoxide (1:2, v/v), and sequenced using DYENAM1C
energy transfer
sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or
the ABl
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequences of SEQ ID N0:26-50 are used to obtain
5'
regulatory sequences using the procedure above, oligonucleotides designed for
such extension,
and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:26-50 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-'ZP] 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 107 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
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endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xbal, 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 increasingly stringent conditions up to 0.1 x saline sodium citrate and
0.5% sodium dodecyl
sulfate. Hybridization patterns are visualized using autoradiography and
compared.
VII. Microarrays
A chemical coupling procedure and an ink jet device can be used to synthesize
array
elements on the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An
array 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 by
hand or using available methods and machines and contain any appropriate
number of elements.
After hybridization, nonhybridized probes are removed and a scanner used to
determine the levels
and patterns of fluorescence. The degree of complementarity and the relative
abundance of each
probe which hybridizes to an element on the microarray may be assessed through
analysis of the
scanned images.
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may
comprise the elements of the microarray. Fragments suitable for hybridization
can be selected
using software well known in the art such as LASERGENE software (DNASTAR).
Full-length
cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide
sequences of the
present invention, or selected at random from a cDNA library relevant to the
present invention, are
arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed
to the slide using, e.g.,
UV cross-linking followed by thermal and chemical treatments and subsequent
drying. (See, e.g.,
Schena, M. et al. ( 1995) Science 270:467-470; Shalom D. et al. ( 1996) Genome
Res. 6:639-645.)
Fluorescent probes are prepared and used for hybridization to the elements on
the substrate. The
substrate is analyzed by procedures described above.
VIII. Complementary Polynucleotides
Sequences complementary to the RNAAP-encoding sequences, or any parts thereof,
are
used to detect, decrease, or inhibit expression of naturally occurring RNAAP.
Although use of
oligonucleotides comprising from about 1 S 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
RNAAP. 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
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translation, a complementary oligonucleotide is designed to prevent ribosomal
binding to the
RNAAP-encoding transcript.
IX. Expression of RNAAP
Expression and purification of RNAAP is achieved using bacterial or virus-
based
S expression systems. For expression of RNAAP in bacteria, cDNA is subcloned
into an
appropriate vector containing an antibiotic resistance gene and an inducible
promoter that directs
high levels of cDNA transcription. Examples of such promoters include, but are
not limited to, the
trp-lac (tac) hybrid promoter and the TS or T7 bacteriophage promoter in
conjunction with the lac
operator regulatory element. Recombinant vectors are transformed into suitable
bacterial hosts,
e.g., BL21(DE3). Antibiotic resistant bacteria express RNAAP upon induction
with isopropyl
beta-D-thiogalactopyranoside (1PTG). Expression of RNAAP in eukaryotic cells
is achieved by
infecting insect or mammalian cell lines with recombinant Autog-raphica
californica nuclear
polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential
polyhedrin
gene of baculovirus is replaced with cDNA encoding RNAAP by either homologous
recombination or bacterial-mediated transposition involving transfer plasmid
intermediates. Viral
infectivity is maintained and the strong polyhedrin promoter drives high
levels of cDNA
transcription. Recombinant baculovirus is used to infect Spodoptera fru;gperda
(SP9) 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, RNAAP 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 RNAAP at specifically engineered sites. FLAG, an
8-amino acid
peptide, enables immunoaffinity purification using commercially available
monoclonal and
polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six
consecutive histidine
residues, enables purification on metal-chelate resins (QIAGEN). Methods for
protein expression
and purification are discussed in Ausubel (1995, supra, ch 10 and 16).
Purified RNAAP obtained
by these methods can be used directly in the following activity assay.
X. Demonstration of RNAAP Activity
RNAAP activity is demonstrated by the formation of an RNAAP-RNA complex as
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detected by a polyacrylamide gel mobility-shift assay. In preparation for this
assay, RNAAP is
expressed by transforming a mammalian cell line such as COS7, HeLa or CHO with
a eukaryotic
expression vector containing RNAAP cDNA. The cells are incubated for 48-72
hours after
transformation under conditions which allow expression and accumulation of
RNAAP. Extracts
containing solubilized proteins can be prepared from cells expressing RNAAP by
methods well
known in the art. Portions of the extract containing RNAAP are added to ['2P)-
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 S-10
minutes. After incubation, the samples are analyzed by polyacrylamide gel
electrophoresis
followed by autoradiography. The presence of a high molecular weight band on
the
autoradiogram indicates the formation of a complex between RNAAP and the
radioactive
transcript. A band of significantly lower molecular weight will be present in
samples prepared
using control extracts prepared from untransformed cells. The amount of RNAAP-
RNA complex
can be quantified using phospho-image analysis and is proportional to the
activity of RNAAP.
Alternatively, RNAAP, or biologically active fragments thereof, are labeled
with 'ZSI
Bolton-Hunter reagent. (See, e.g., Bolton et al. ( 1973) Biochem. J. 133:529.)
Candidate
molecules previously arrayed in the wells of a multi-well plate are incubated
with the labeled
RNAAP, washed, and any wells with labeled RNAAP complex are assayed. Data
obtained using
different concentrations of RNAAP are used to calculate values for the number,
affinity, and
association of RNAAP with the candidate molecules.
XI. Functional Assays
RNAAP function is assessed by expressing the sequences encoding RNAAP 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.
S-10 ug of
recombinant vector are transiently transfected into a human cell line,
preferably of endothelial or
hematopoietic origin, using either iiposome formulations or electroporation. I-
2 ~g of an
additional plasmid containing sequences encoding a marker protein are co-
transfected. Expression
of a marker protein provides a means to distinguish transfected cells from
nontransfected cells and
is a reliable predictor of cDNA expression from the recombinant vector. Marker
proteins of
choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a
CD64-GFP fusion
protein. Flow cytometry (FCM), an automated, laser optics-based technique, is
used to identify
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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 RNAAP on gene expression can be assessed using highly
purified
populations of cells transfected with sequences encoding RNAAP and either CD64
or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind
to conserved
regions of human immunoglobulin G (IgG). Transfected cells are efficiently
separated from
nontransfected cells using magnetic beads coated with either human IgG or
antibody against CD64
(DYNAL, Lake Success NY). mRNA can be purified from the cells using methods
well known
by those of skill in the art. Expression of mRNA encoding RNAAP and other
genes of interest
can be analyzed by northern analysis or microarray techniques.
XII. Production of ItNAAP Specific Antibodies
RNAAP 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 RNAAP amino acid sequence is analyzed using I,ASERGENE
software (DNASTAR) to determine regions of high immunogenicity, and a
corresponding
oligopeptide is synthesized and used to raise antibodies 6y 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 15 residues in length are synthesized using an ABI
431A peptide
synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH (Sigma-
Aldrich, St. Louis
MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to
increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with
the oligopeptide-
KLH complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide activity
by, for example, binding the peptide to plastic, blocking with 1% BSA,
reacting with rabbit
antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
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XIII. Purification of Naturally Occurring ItNAAP Using Specific Antibodies
Naturally occurring or recombinant RNAAP is substantially purified by
immunoaffinity
chromatography using antibodies specific for RNAAP. An immunoaffinity column
is constructed
by covalently coupling anti-RNAAP 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 RNAAP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of RNAAP (e.g.,
high ionic
strength buffers in the presence of detergent). The column is eluted under
conditions that disrupt
antibody/RNAAP 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 RNAAP is collected.
XIV. Identification of Molecules Which Interact with ItNAAP
RNAAP, or biologically active fragments thereof, are labeled with ''-SI Bolton-
Hunter
reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate
molecules previously
arrayed in the wells of a multi-well plate are incubated with the labeled
RNAAP, washed, and any
wells with labeled RNAAP complex are assayed. Data obtained using different
concentrations of
RNAAP are used to calculate values for the number, affinity, and association
of RNAAP with the
candidate molecules.
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
specific preferred
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.
52
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
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SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
HILLMAN, Jennifer L.
YUE, Henry
TANG, Y. Tom
CORLEY, Neil C.
GUEGLER, Karl J.
GORGONE, Gina A.
PATTERSON, Chandra
BAUGHN, Mariah R.
LAL, Preeti
BANDMAN, Olga
REDDY, Roopa
AZIMZAI, Yalda
SHIH, Leo L.
YANG, Junming
LU, Dyung Aina M.
<120> HUMAN RNA-ASSOCIATED PROTEINS
<130> PF-0579 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/097,550; 60/115,639
<151> 1998-08-21; 1999-O1-12
<160> 50
<170> PERL Program
<210> 1
<211> 216
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 399781CD1
<400> 1
Met Ser Arg Tyr Leu Arg Pro Pro Asn Thr Ser Leu Phe Val Arg
1 5 10 15
Asn Val Ala Asp Asp Thr Arg Ser Glu Asp Leu Arg Arg Glu Phe
20 25 30
Gly Arg Tyr Gly Pro Ile Val Asp Val Tyr Val Pro Leu Asp Phe
35 40 45
Tyr Thr Arg Arg Pro Arg Gly Phe Ala Tyr Val Gln Phe Glu Asp
50 55 60
Val Arg Asp Ala Glu Asp Ala Leu His Asn Leu Asp Arg Lys Trp
65 70 75
Ile Cys Gly Arg Gln Ile Glu Ile Gln Phe Ala Gln Gly Asp Arg
80 85 90
1 /47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
Lys Thr Pro Asn Gln Met Lys Ala Lys Glu Gly Arg Asn Val Tyr
95 100 105
Ser Ser Ser Arg Tyr Asp Asp Tyr Asp Arg Tyr Arg Arg Ser Arg
110 115 120
Ser Arg Ser Tyr Glu Arg Arg Arg Ser Arg Ser Arg Ser Phe Asp
125 130 135
Tyr Asn Tyr Arg Arg Ser Tyr Ser Pro Arg Asn Ser Arg Pro Thr
140 145 150
Gly Arg Pro Arg Arg Arg Glu Ala Ile Pro Thr Met Ile Asp Gln
155 160 165
Thr Ala Ala Gly Ile Pro Ser Thr Val Leu Leu Thr Thr Leu Gln
170 175 180
Glu Arg Ser Glu Ser Gly Lys Arg Thr Lys Glu Gly Gln Phe Lys
185 190 195
Arg Pro Lys Gly Gly Trp Lys Val Leu Gln Tyr Glu Tyr Cys Thr
200 205 210
Asn Ile Leu Thr Leu Val
215
<210> 2
<211> 962
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 1806542CD1
<400> 2
Met Asp Glu Gln Ala Leu Leu Gly Leu Asn Pro Asn Ala Asp Ser
1 5 10 15
Asp Phe Arg Gln Arg Ala Leu Ala Tyr Phe Glu Gln Leu Lys Ile
20 25 30
Ser Pro Asp Ala Trp Gln Val Cys Ala Glu A1a Leu Ala Gln Arg
35 40 45
Thr Tyr Ser Asp Asp His Val Lys Phe Phe Cys Phe Gln Val Leu
50 55 60
Glu His Gln Val Lys Tyr Lys Tyr Ser Glu Leu Thr Thr Val Gln
65 70 75
Gln Gln Leu Ile Arg Glu Thr Leu Ile Ser Trp Leu Gln Ala Gln
80 85 90
Met Leu Asn Pro Gln Pro Glu Lys Thr Phe Ile Arg Asn Lys Ala
95 100 105
Ala Gln Val Phe Ala Leu Leu Phe Val Thr Glu Tyr Leu Thr Lys
110 115 120
Trp Pro Lys Phe Phe Phe Asp Ile Leu Ser Val Val Asp Leu Asn
125 130 135
Pro Arg Gly Val Asp Leu Tyr Leu Arg Ile Leu Met Ala Ile Asp
140 145 150
Ser Glu Leu Val Asp Arg Asp Val Val His Thr Ser Glu Glu Ala
155 160 165
Arg Arg Asn Thr Leu Ile Lys Asp Thr Met Arg Glu Gln Cys Ile
170 175 180
Pro Asn Leu Val Glu Ser Trp Tyr Gln Ile Leu Gln Asn Tyr Gln
185 190 195
Phe Thr Asn Ser Glu Val Thr Cys Gln Cys Leu Glu Val Val Gly
2/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
200 205 210
Ala Tyr Val Ser Trp Ile Asp Leu Ser Leu Ile Ala Asn Asp Arg
215 220 225
Phe Ile Asn Met Leu Leu Gly His Met Ser Ile Glu Val Leu Arg
230 235 240
Glu Glu Ala Cys Asp Cys Leu Phe Glu Val Val Asn Lys Gly Met
245 250 255
Asp Pro Val Asp Lys Met Lys Leu Val Glu Ser Leu Cys Gln Val
260 265 270
Leu Gln Ser Ala Gly Phe Phe Ser Ile Asp Gln Glu Glu Asp Val
275 280 2$5
Asp Phe Leu Ala Arg Phe Ser Lys Leu Val Asn Gly Met Gly Gln
290 295 300
Ser Leu Ile Val Ser Trp Ser Lys Leu Ile Lys Asn Gly Asp Ile
305 310 315
Lys Asn Ala Gln Glu Ala Leu Gln Ala Ile Glu Thr Lys Val Ala
320 325 330
Leu Met Leu Gln Leu Leu Ile His Glu Asp Asp Asp Ile Ser Ser
335 340 345
Asn Ile Ile Gly Phe Cys Tyr Asp Tyr Leu His Ile Leu Lys Gln
350 355 360
Leu Thr Val Leu Ser Asp Gln Gln Lys Ala Asn Val Glu Ala Ile
365 370 375
Met Leu Ala Val Met Lys Lys Leu Thr Tyr Asp Glu Glu Tyr Asn
380 385 390
Phe Glu Asn Glu Gly Glu Asp Glu Ala Met Phe Val Glu Tyr Arg
395 400 405
Lys Gln Leu Lys Leu Leu Leu Asp Arg Leu Ala Gln Val Ser Pro
410 415 420
Glu Leu Leu Leu Ala Ser Val Arg Arg Val Phe Ser Ser Thr Leu
425 430 435
Gln Asn Trp Gln Thr Thr Arg Phe Met Glu Val Glu Val Ala Ile
440 445 450
Arg Leu Leu Tyr Met Leu Ala Glu Ala Leu Pro Val Ser His Gly
455 460 465
Ala His Phe Ser Gly Asp Val Ser Lys Ala Ser Ala Leu Gln Asp
470 475 480
Met Met Arg Thr Leu Val Thr Ser Gly Val Ser Ser Tyr Gln His
485 490 495
Thr Ser Val Thr Leu Glu Phe Phe Glu Thr Val Val Arg Tyr Glu
500 505 510
Lys Phe Phe Thr Val Glu Pro Gln His Ile Pro Cys Val Leu Met
515 520 525
Ala Phe Leu Asp His Arg Gly Leu Arg His Ser Ser Ala Lys Val
530 535 54C
Arg Ser Arg Thr Ala Tyr Leu Phe Ser Arg Phe Val Lys Ser Leu
545 550 555
Asn Lys Gln Met Asn Pro Phe Ile Glu Asp Ile Leu Asn Arg Ile
560 565 570
Gln Asp Leu Leu Glu Leu Ser Pro Pro Glu Asn Gly His Gln Ser
575 580 585
Leu Leu Ser Ser Asp Asp Gln Leu Phe Ile Tyr Glu Thr Ala Gly
590 595 600
Val Leu Ile Val Asn Ser Glu Tyr Pro Ala Glu Arg Lys Gln Ala
605 610 615
Leu Met Arg Asn Leu Leu Thr Pro Leu Met Glu Lys Phe Lys Ile
620 625 630
3/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
Leu Leu Glu Lys Leu Met Leu Ala Gln Asp Glu Glu Arg Gln Ala
635 640 645
Ser Leu Ala Asp Cys Leu Asn His Ala Val Gly Phe Ala Ser Arg
650 655 660
Thr Ser Lys Ala Phe Ser Asn Lys Gln Thr Val Lys Gln Cys Gly
665 670 675
Cys Ser Glu Val Tyr Leu Asp Cys Leu Gln Thr Phe Leu Pro Ala
680 685 690
Leu Ser Cys Pro Leu Gln Lys Asp Ile Leu Arg Ser Gly Val Arg
695 700 705
Thr Phe Leu His Arg Met Ile Ile Cys Leu Glu Glu Glu Val Leu
710 715 720
Pro Phe Ile Pro Ser Ala Ser Glu His Met Leu Lys Asp Cys Glu
725 730 735
Ala Lys Asp Leu Gln Glu Phe Ile Pro Leu Ile Asn Gln Ile Thr
740 745 750
Ala Lys Phe Lys Ile Gln Val Ser Pro Phe Leu Gln Gln Met Phe
755 760 765
Met Pro Leu Leu His Ala Ile Phe Glu Val Leu Leu Arg Pro Ala
770 775 780
Glu Glu Asn Asp Gln Ser Ala Ala Leu Glu Lys Gln Met Leu Arg
785 790 795
Arg Ser Tyr Phe Ala Phe Leu Gln Thr Val Thr Gly Ser Gly Met
800 805 810
Ser Glu Val Ile Ala Asn Gln Gly Ala Glu Asn Val Glu Arg Val
815 820 825
Leu Val Thr Val Ile Gln Gly Ala Val Glu Tyr Pro Asp Pro Ile
830 835 840
Ala Gln Lys Thr Cys Phe Ile Ile Leu Ser Lys Leu Val Glu Leu
845 850 855
Trp Gly Gly Lys Asp Gly Pro Val Gly Phe Ala Asp Phe Val Tyr
860 865 870
Lys His Ile Val Pro Ala Cys Phe Leu Ala Pro Leu Lys Gln Thr
875 880 885
Phe Asp Leu Ala Asp Ala Gln Thr Val Leu Ala Leu Ser Glu Cys
890 895 900
Ala Val Thr Leu Lys Thr Ile His Leu Lys Arg Gly Pro Glu Cys
905 910 915
Val Gln Tyr Leu Gln Gln Glu Tyr Leu Pro Ser Leu Gln Val Ala
920 925 930
Pro Glu Ile Ile Gln Glu Phe Cys Gln Ala Leu Gln Gln Pro Asp
935 940 945
Ala Lys Val Phe Lys Asn Tyr Leu Lys Val Phe Phe Gln Arg Ala
950 955 960
Lys Pro
<210> 3
<211> 285
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2263514CD1
4/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
<400> 3
Met Asp Trp Val Met Lys His Asn Gly Pro Asn Asp Ala Ser Asp
1 5 10 15
Gly Thr Val Arg Leu Arg Gly Leu Pro Phe Gly Cys Ser Lys Glu
20 25 30
Glu Ile Val Arg Val Leu Ser Arg Tyr Ile Glu Ile Phe Arg Ser
35 40 45
Ser Arg Ser Glu Ile Lys Gly Phe Tyr Asp Pro Pro Arg Arg Leu
50 55 60
Leu Gly Gln Arg Pro Gly Pro Tyr Asp Arg Pro Ile Gly Gly Arg
65 70 75
Gly Gly Tyr Tyr Gly Ala Gly Arg Gly Ser Tyr Gly Gly Phe Asp
80 85 90
Asp Tyr Gly Gly Tyr Asn Asn Tyr Gly Tyr Gly Asn Asp Gly Phe
95 100 105
Asp Asp Arg Met Arg Asp Gly Arg Gly Met Gly Gly His Gly Tyr
110 115 120
Gly Gly Ala Gly Asp Ala Ser Ser Gly Phe His Gly Gly His Phe
125 130 135
Val His Met Arg Gly Leu Pro Phe Arg Ala Thr Glu Asn Ala Ile
140 245 150
Ala Asn Phe Phe Ser Pro Leu Asn Pro Ile Arg Val His Ile Asp
155 160 165
Ile Gly Ala Asp Gly Arg Ala Thr Gly Glu Ala Asp Val Glu Phe
170 175 180
Val Thr His Glu Asp Ala Val Ala Ala Met Ser Lys Asp Lys Asn
185 190 195
Asn Met Gln His Arg Tyr Ile Glu Leu Phe Leu Asn Ser Thr Pro
200 205 210
Gly Gly Gly Ser Gly Met Gly Gly Ser Gly Met Gly Gly Tyr Gly
215 220 225
Arg Asp Gly Met Asp Asn Gln Gly Gly Tyr Gly Ser Val Gly Arg
230 235 240
Met Gly Met Gly Asn Asn Tyr Ser Gly Gly Tyr Gly Thr Pro Asp
245 250 255
Gly Leu Gly Gly Tyr Gly Arg Gly Gly Gly Gly Ser Gly Gly Tyr
260 265 270
Tyr Gly Gln Gly Gly Met Ser Gly Gly Gly Trp Arg Gly Met Tyr
275 280 285
<210> 4
<211> 267
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2738270CD1
<400> 4
Met Gly Ala Ala Ala Ala Glu Ala Asp Arg Thr Leu Phe Val Gly
1 5 10 15
Asn Leu Glu Thr Lys Val Thr Glu Glu Leu Leu Phe Glu Leu Phe
20 25 30
His Gln Ala Gly Pro Val Ile Lys Val Lys Ile Pro Lys Asp Lys
35 40 45
S/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
Asp Gly Lys Pro Lys Gln Phe Ala Phe Val Asn Phe Lys His Glu
50 55 60
Val Ser Val Pro Tyr Ala Met Asn Leu Leu Asn Gly Ile Lys Leu
65 70 75
Tyr Gly Arg Pro Ile Lys Ile Gln Phe Arg Ser Gly Ser Ser His
80 85 90
Ala Pro Gln Asp Val Ser Leu Ser Tyr Pro Gln His His Val Gly
95 100 105
Asn Ser Ser Pro Thr Ser Thr Ser Pro Ser Ser Arg Tyr Glu Arg
110 115 120
Thr Met Asp Asn Met Thr Ser Ser Ala Gln Ile Ile Gln Arg Ser
125 130 135
Phe Ser Ser Pro Glu Asn Phe Gln Arg Gln Ala Val Met Asn Ser
140 145 150
Ala Leu Arg Gln Met Ser Tyr Gly Gly Lys Phe Gly Ser Ser Pro
155 160 165
Leu Asp Gln Ser Gly Phe Ser Pro Ser Val Gln Ser His Ser His
170 175 180
Ser Phe Asn Gln Ser Ser Ser Sex Gln Trp Arg Gln Gly Thr Pro
185 190 195
Ser Ser Gln Arg Lys Val Arg Met Asn Ser Tyr Pro Tyr Leu Ala
200 205 210
Asp Arg His Tyr Ser Arg Glu Gln Arg Tyr Thr Asp His Gly Ser
215 220 225
Asp His His Tyr Arg Gly Lys Arg Asp Asp Phe Phe Tyr Glu Asp
230 235 240
Arg Asn His Asp Asp Trp Ser His Asp Tyr Asp Asn Arg Arg Asp
245 250 255
Ser Ser Arg Asp Gly Lys Trp Arg Ser Ser Arg His
260 265
<210> 5
<211> 369
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2824412CD1
<400> 5
Met Pro Pro Gln Pro Gln Gly Pro Ala Pro Leu Arg Arg Pro Asp
1 5 10 15
Ser Ser Asp Asp Arg Tyr Val Met Thr Lys His Ala Thr Ile Tyr
20 25 30
Pro Thr Glu Glu Glu Leu Gln Ala Val Gln Lys Ile Val Ser Ile
35 40 45
Thr Glu Arg Ala Leu Lys Leu Val Ser Asp Ser Leu Ser Glu His
50 55 60
Glu Lys Asn Lys Asn Lys Glu Gly Asp Asp Lys Lys Glu Gly Gly
65 70 75
Lys Asp Arg Ala Leu Lys Gly Val Leu Arg Val Gly Val Leu Ala
80 85 90
Lys Gly Leu Leu Leu Arg Gly Asp Arg Asn Val Asn Leu Val Leu
6/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
95 100 105
Leu Cys Ser Glu Lys Pro Ser Lys Thr Leu Leu Ser Arg Ile Ala
110 115 120
Glu Asn Leu Pro Lys Gln Leu Ala Val Ile Ser Pro Glu Lys Tyr
125 130 135
Asp Ile Lys Cys Ala Val Ser Glu Ala Ala Ile Ile Leu Asn Ser
140 145 150
Cys Val Glu Pro Lys Met Gln Val Thr Ile Thr Leu Thr Ser Pro
155 160 165
Ile Ile Arg Glu Glu Asn Met Arg Glu Gly Asp Val Thr Ser Gly
170 175 180
Met Val Lys Asp Pro Pro Asp Val Leu Asp Arg Gln Lys Cys Leu
185 190 195
Asp Ala Leu Ala Ala Leu Arg His Ala Lys Trp Phe Gln Ala Arg
200 205 210
Ala Asn Gly Leu Gln Ser Cys Val Ile Ile Ile Arg Ile Leu Arg
215 220 225
Asp Leu Cys Gln Arg Val Pro Thr Trp Ser Asp Phe Pro Ser Trp
230 235 240
Ala Met Glu Leu Leu Val Glu Lys Ala Ile Ser Ser Ala Ser Ser
245 250 255
Pro Gln Ser Pro Gly Asp Ala Leu Arg Arg Val Phe Glu Cys Ile
260 265 270
Ser Ser Gly Ile Ile Leu Lys Gly Ser Pro Gly Leu Leu Asp Pro
275 280 285
Cys Glu Lys Asp Pro Phe Asp Thr Leu Ala Thr Met Thr Asp Gln
290 295 300
Gln Arg Glu Asp Ile Thr Ser Ser Ala Gln Phe Ala Leu Arg Leu
305 310 315
Leu Ala Phe Arg Gln Ile His Lys Val Leu Gly Met Asp Pro Leu
320 325 330
Pro Gln Met Ser Gln Arg Phe Asn Ile His Asn Asn Arg Lys Arg
335 340 345
Arg Arg Asp Ser Asp Gly Val Asp Gly Phe Glu Ala Glu Gly Lys
350 355 360
Lys Asp Lys Lys Asp Tyr Asp Asn Phe
365
<210> 6
<211> 175
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 002690CD1
<400> 6
Met Arg Leu Ser Val Ala Ala Ala Ile Ser His Gly Arg Val Phe
1 5 10 15
Arg Arg Met Gly Leu Gly Pro Glu Ser Arg Ile His Leu Leu Arg
20 25 30
Asn Leu Leu Thr Gly Leu Val Arg His Glu Arg Ile Glu Ala Pro
35 40 45
7/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
Trp Ala Arg Val Asp Glu Met Arg Gly Tyr Ala Glu Lys Leu Ile
50 55 60
Asp Tyr Gly Lys Leu Gly Asp Thr Asn Glu Arg Ala Met Arg Met
65 70 75
Ala Asp Phe Trp Leu Thr Glu Lys Asp Leu Ile Pro Lys Leu Phe
80 85 90
Gln Val Leu Ala Pro Arg Tyr Lys Asp Gln Thr Gly Gly Tyr Thr
95 100 105
Arg Met Leu Gln Ile Pro Asn Arg Ser Leu Asp Arg Ala Lys Met
110 115 120
Ala Val Ile Glu Tyr Lys Gly Asn Cys Leu Pro Pro Leu Pro Leu
125 130 135
Pro Arg Arg Asp Ser His Leu Thr Leu Leu Asn Gln Leu Leu Gln
140 145 150
Gly Leu Arg Gln Asp Leu Arg Gln Ser Gln Glu Ala Ser Asn His
155 160 165
Ser Ser His Thr Ala Gln Thr Pro Gly Ile
I70 175
<210> 7
<211> 311
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 041108CD1
<400> 7
Met Leu Gln Phe Val Arg Ala Gly Ala Arg Ala Trp Leu Arg Pro
1 5 10 15
Thr Gly Ser Gln Gly Leu Ser Ser Leu Ala Glu Glu Ala Ala Arg
20 25 30
Ala Thr Glu Asn Pro Glu Gln Val Ala Ser Glu Gly Leu Pro Glu
35 40 45
Pro Val Leu Arg Lys Val Glu Leu Pro Val Pro Thr His Arg Arg
50 55 60
Pro Val Gln Ala Trp Val Glu Ser Leu Arg Gly Phe Glu Gln Glu
65 70 75
Arg Val Gly Leu Ala Asp Leu His Pro Asp Val Phe Ala Thr Ala
80 85 90
Pro Arg Leu Asp Ile Leu His Gln Val Ala Met Trp Gln Lys Asn
95 100 105
Phe Lys Arg Ile Ser Tyr Ala Lys Thr Lys Thr Arg Ala Glu Val
110 115 120
Arg Gly Gly Gly Arg Lys Pro Trp Pro Gln Lys Gly Thr Gly Arg
125 130 135
Ala Arg His Gly Ser Ile Arg Ser Pro Leu Trp Arg Gly Gly Gly
140 145 150
Val Ala His Gly Pro Arg Gly Pro Thr Ser Tyr Tyr Tyr Met Leu
155 160 165
Pro Met Lys Val Arg Ala Leu Gly Leu Lys Val Ala Leu Thr Val
170 175 180
Lys Leu Ala Gln Asp Asp Leu His Ile Met Asp 5er Leu Glu Leu
8/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
185 190 195
Pro Thr Gly Asp Pro Gln Tyr Leu Thr Glu Leu Ala His Tyr Arg
200 205 210
Arg Trp Gly Asp Ser Val Leu Leu Val Asp Leu Thr His Glu Glu
2I5 220 225
Met Pro Gln Ser Ile Val Glu Ala Thr Ser Arg Leu Lys Thr Phe
230 235 240
Asn Leu Ile Pro Ala Val Gly Leu Asn Val His Ser Met Leu Lys
245 250 255
His Gln Thr Leu Val Leu Thr Leu Pro Thr Val Ala Phe Leu Glu
260 265 270
Asp Lys Leu Leu Trp Gln Asp Ser Arg Tyr Arg Pro Leu Tyr Pro
275 280 285
Phe Ser Leu Pro Tyr Ser Asp Phe Pro Arg Pro Leu Pro His Ala
290 295 300
Thr Gln Gly Pro Ala Ala Thr Pro Tyr His Cys
305 310
<210> 8
<211> 330
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 869138CD1
<400> 8
Met Ser Thr Lys Asn Phe Arg Val Ser Asp Gly Asp Trp Ile Cys
1 5 10 15
Pro Asp Lys Lys Cys Gly Asn Val Asn Phe Ala Arg Arg Thr Ser
20 25 30
Cys Asn Arg Cys Gly Arg Glu Lys Thr Thr Glu Ala Lys Met Met
35 40 45
Lys Ala Gly Gly Thr Glu Ile Gly Lys Thr Leu Ala Glu Lys Ser
50 55 60
Arg Gly Leu Phe Ser Ala Asn Asp Trp Gln Cys Lys Thr Cys Ser
65 70 75
Asn Val Asn Trp Ala Arg Arg Ser Glu Cys Asn Met Cys Asn Thr
80 85 90
Pro Lys Tyr Ala Lys Leu Glu Glu Arg Thr Gly Tyr Gly Gly Gly
95 100 105
Phe Asn Glu Arg Glu Asn Val Glu Tyr Ile Glu Arg Glu Glu Ser
110 115 120
Asp Gly Glu Tyr Asp Glu Phe Gly Arg Lys Lys Lys Lys Tyr Arg
125 130 135
Gly Lys Ala Val Gly Pro Ala Ser Ile Leu Lys Glu Val Glu Asp
140 145 150
Lys Glu Ser Glu Gly Glu Glu Glu Asp Glu Asp Glu Asp Leu Ser
155 160 165
Lys Tyr Lys Leu Asp Glu Asp Glu Asp Glu Asp Asp Ala Asp Leu
170 175 180
Ser Lys Tyr Asn Leu Asp Ala Ser Glu Glu Glu Asp Ser Asn Lys
185 190 195
9/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
Lys Lys Ser Asn Arg Arg Ser Arg Ser Lys Ser Arg Ser Ser His
200 205 210
Ser Arg Ser Ser Ser Arg Ser Ser Ser Pro Ser Ser Ser Arg Ser
215 220 225
Arg Ser Arg Ser Arg Ser Arg Ser Ser Ser Ser Ser Gln Ser Arg
230 235 240
Ser Arg Ser Ser Ser Arg Glu Arg Ser Arg Ser Arg Gly Ser Lys
245 250 255
Ser Arg Ser Ser Ser Arg Ser His Arg Gly Ser Ser Ser Pro Arg
260 265 270
Lys Arg Ser Tyr Ser Ser Ser Ser Ser Ser Pro Glu Arg Asn Arg
275 280 285
Lys Arg Ser Arg Ser Arg Ser Ser Ser Ser Gly Asp Arg Lys Lys
290 295 300
Arg Arg Thr Arg Ser Arg Ser Pro Glu Arg Arg His Arg Ser Ser
305 310 315
Ser Gly Ser Ser His Ser Gly Ser Arg Ser Ser Ser Lys Lys Lys
320 325 330
<210> 9
<211> 183
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 934406CD1
<400> 9
Met Ser Arg Tyr Leu Arg Pro Pro Asn Thr Ser Leu Phe Val Arg
1 5 10 15
Asn Val Ala Asp Asp Thr Arg Ser Glu Asp Leu Arg Arg Glu Phe
20 25 30
Gly Arg Tyr Gly Pro Ile Val Asp Val Tyr Val Pro Leu Asp Phe
35 40 45
Tyr Thr Arg Arg Pro Arg Gly Phe Ala Tyr Val Gln Phe Glu Asp
50 55 60
Val Arg Asp Ala Glu Asp Ala Leu His Asn Leu Asp Arg Lys Trp
65 70 75
Ile Cys Gly Arg Gln Ile Glu Ile Gln Phe Ala Gln Gly Asp Arg
80 85 90
Lys Thr Pro Asn Gln Met Lys Ala Lys Glu Gly Arg Asn Val Tyr
95 100 105
Ser Ser Ser Arg Tyr Asp Asp Tyr Asp Arg Tyr Arg Arg Ser Arg
110 115 120
Ser Arg Ser Tyr Glu Arg Arg Arg Ser Arg Ser Arg Ser Phe Asp
125 130 135
Tyr Asn Tyr Arg Arg Ser Tyr Ser Pro Arg Asn Ser Arg Pro Thr
140 145 150
Gly Arg Pro Arg Arg Ser Arg Ser His Ser Asp Asn Asp Arg Pro
155 160 165
Asn Cys Ser Trp Asn Thr Gln Tyr Ser Ser Ala Tyr Tyr Thr Ser
170 175 180
Arg Lys Ile
10/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
<210> IO
<211> 670
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 1315083CD1
<400> 10
Met Ser His Leu Pro Met Lys Leu Leu Arg Lys Lys Ile Glu Lys
1 5 10 15
Arg Asn Leu Lys Leu Arg Gln Arg Asn Leu Lys Phe Gln Gly Ala
20 25 30
Ser Asn Leu Thr Leu Ser Glu Thr Gln Asn Gly Asp Val Ser Glu
35 40 45
Glu Thr Met Gly Ser Arg Lys Val Lys Lys Ser Lys Gln Lys Pro
50 55 60
Met Asn Val Gly Leu Ser Glu Thr Gln Asn Gly Gly Met Ser Gln
65 70 75
Glu Ala Val Gly Asn Ile Lys Val Thr Lys Ser Pro Gln Lys Ser
80 85 90
Thr Val Leu Thr Asn Gly Glu Ala Ala Met Gln Ser Ser Asn Ser
95 100 105
Glu Ser Lys Lys Lys Lys Lys Lys Lys Arg Lys Met Val Asn Asp
110 115 120
Ala Glu Pro Asp Thr Lys Lys Ala Lys Thr Glu Asn Lys Gly Lys
125 130 135
Ser Glu Glu Glu Ser Ala Glu Thr Thr Lys Glu Thr Glu Asn Asn
140 145 150
Val Glu Lys Pro Asp Asn Asp Glu Asp Glu Ser Glu Val Pro Ser
155 160 165
Leu Pro Leu Gly Leu Thr Gly Ala Phe Glu Asp Thr Ser Phe Ala
170 175 180
Ser Leu Cys Asn Leu Val Asn Glu Asn Thr Leu Lys Ala Ile Lys
185 190 195
Glu Met Gly Phe Thr Asn Met Thr Glu Ile Gln His Lys Ser Ile
200 205 210
Arg Pro Leu Leu Glu Gly Arg Asp Leu Leu Ala Ala Ala Lys Thr
215 220 225
Gly Ser Gly Lys Thr Leu Ala Phe Leu Ile Pro Ala Val Glu Leu
230 235 240
Ile VaI Lys Leu Arg Phe Met Pro Arg Asn Gly Thr Gly Val Leu
245 250 255
Ile Leu Ser Pro Thr Arg Glu Leu Ala Met Gln Thr Phe Gly Val
260 265 270
Leu Lys Glu Leu Met Thr His His Val His Thr Tyr Gly Leu Ile
275 280 285
Met Gly Gly Ser Asn Arg Ser Ala Glu Ala Gln Lys Leu Gly Asn
290 295 300
Gly Ile Asn Ile Ile Val Ala Thr Pro Gly Arg Leu Leu Asp His
305 310 315
Met Gln Asn Thr Pro Gly Phe Met Tyr Lys Asn Leu Gln Cys Leu
320 325 330
11 /47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
Val Ile Asp Glu Ala Asp Arg Ile Leu Asp Val Gly Phe Glu Glu
335 340 345
Glu Leu Lys Gln Ile Ile Lys Leu Leu Pro Thr Arg Arg Gln Thr
350 355 360
Met Leu Phe Ser Ala Thr Gln Thr Arg Lys Val Glu Asp Leu Ala
365 370 375
Arg Ile Ser Leu Lys Lys Glu Pro Leu Tyr Val Gly Val Asp Asp
380 385 390
Asp Lys Ala Asn Ala Thr Val Asp Gly Leu Glu Gln Gly Tyr Val
395 400 405
Val Cys Pro Ser Glu Lys Arg Phe Leu Leu Leu Phe Thr Phe Leu
410 415 420
Lys Lys Asn Arg Lys Lys Lys Leu Met Val Phe Phe Ser Ser Cys
425 430 435
Met Ser Val Lys Tyr His Tyr Glu Leu Leu Asn Tyr Ile Asp Leu
440 445 450
Pro Val Leu Ala Ile His Gly Lys Gln Lys Gln Asn Lys Arg Thr
455 460 465
Thr Thr Phe Phe Gln Phe Cys Asn Ala Asp Ser Gly Thr Leu Leu
470 475 480
Cys Thr Asp Val Ala Ala Arg Gly Leu Asp Ile Pro Glu Val Asp
485 490 495
Trp Ile Val Gln Tyr Asp Pro Pro Asp Asp Pro Lys Glu Tyr Ile
500 505 510
His Arg Val Gly Arg Thr Ala Arg Gly Leu Asn Gly Arg Gly His
515 520 525
Ala Leu Leu Ile Leu Arg Pro Glu Glu Leu Gly Phe Leu Arg Tyr
530 535 540
Leu Lys Gln Ser Lys Val Pro Leu Ser Glu Phe Asp Phe Ser Trp
545 550 555
Ser Lys Ile Ser Asp Ile Gln Ser Gln Leu Glu Lys Leu Ile Glu
560 565 570
Lys Asn Tyr Phe Leu His Lys Ser Ala Gln Glu Ala Tyr Lys Ser
575 580 585
Tyr Ile Arg Ala Tyr Asp Ser His Ser Leu Lys Gln Ile Phe Asn
590 595 600
Val Asn Asn Leu Asn Leu Pro Gln Val Ala Leu Ser Phe Gly Phe
605 610 615
Lys Val Pro Pro Phe Val Asp Leu Asn Val Asn Ser Asn Glu Gly
620 625 630
Lys Gln Lys Lys Arg Gly Gly Gly Gly Gly Phe Gly Tyr Gln Lys
635 640 645
Thr Lys Lys Val Glu Lys Ser Lys Ile Phe Lys His Ile Ser Lys
650 655 660
Lys Ser Ser Asp Ser Arg Gln Phe Ser His
665 670
<210> 11
<211> 452
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
12/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
<223> Incyte Identification No.: 1444908CD1
<400> 11
Met Glu Phe Gln Ala Val Val Met Ala Val Gly Gly Gly Ser Arg
1 5 10 15
Met Thr Asp Leu Thr Ser Ser Ile Pro Lys Pro Leu Leu Pro Val
20 25 30
Gly Asn Lys Pro Leu Ile Trp Tyr Pro Leu Asn Leu Leu Glu Arg
35 40 45
Val Gly Phe Glu Glu Val Ile Val Val Thr Thr Arg Asp Val Gln
50 55 60
Lys Ala Leu Cys Ala Glu Phe Lys Met Lys Met Lys Pro Asp Ile
65 70 75
Val Cys Ile Pro Asp Asp Ala Asp Met Gly Thr Ala Asp Ser Leu
80 85 90
Arg Tyr Ile Tyr Pro Lys Leu Lys Thr Asp Val Leu Val Leu Ser
95 100 105
Cys Asp Leu Ile Thr Asp Val Ala Leu His Glu Val Val Asp Leu
110 115 120
Phe Arg Ala Tyr Asp Ala Ser Leu Ala Met Leu Met Arg Lys Gly
125 130 135
Gln Asp Ser Ile Glu Pro Val Pro Gly Gln Lys Gly Lys Lys Lys
140 145 150
Ala Val Glu Gln Arg Asp Phe Ile Gly Val Asp Ser Thr Gly Lys
155 160 165
Arg Leu Leu Phe Met Ala Asn Glu Ala Asp Leu Asp Glu Glu Leu
170 175 180
Val Ile Lys Gly Ser Ile Leu Gln Lys His Pro Arg Ile Arg Phe
185 190 195
His Thr Gly Leu Val Asp Ala His Leu Tyr Cys Leu Lys Lys Tyr
200 205 210
Ile Val Asp Phe Leu Met Glu Asn Gly Ser Ile Thr Ser Ile Arg
215 220 225
Ser Glu Leu Ile Pro Tyr Leu Val Arg Lys Gln Phe Ser Ser Ala
230 235 240
Ser Ser Gln Gln Gly Gln Glu Glu Lys Glu Glu Asp Leu Lys Lys
245 250 255
Lys Glu Leu Lys Ser Leu Asp Ile Tyr Ser Phe Ile Lys Glu Ala
260 265 270
Asn Thr Leu Asn Leu Ala Pro Tyr Asp Ala Cys Trp Asn Ala Cys
275 280 285
Arg Gly Asp Arg Trp Glu Asp Leu Ser Arg Ser Gln Val Arg Cys
290 295 300
Tyr Val His Ile Met Lys Glu Gly Leu Cys Ser Arg Val Ser Thr
305 310 315
Leu Gly Leu Tyr Met Glu Ala Asn Arg Gln Val Pro Lys Leu Leu
320 325 330
Ser Ala Leu Cys Pro Glu Glu Pro Pro Val His Ser Ser Ala Gln
335 340 345
Ile Val Ser Lys His Leu Val Gly Val Asp Ser Leu Ile Gly Pro
350 355 360
Glu Thr Gln Ile Gly Glu Lys Ser Ser Ile Lys Arg Ser Val Ile
365 370 375
Gly Ser Ser Cys Leu Ile Lys Asp Arg Val Thr Ile Thr Asn Cys
380 385 390
Leu Leu Met Asn Ser Val Thr Val Glu Glu Gly Ser Asn Ile Gln
395 400 405
13/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
Gly Ser Val Ile Cys Asn Asn Ala Val Ile Glu Lys Gly Ala Asp
410 415 420
Ile Lys Asp Cys Leu Ile Gly Ser Gly Gln Arg Ile Glu Ala Lys
425 430 435
Ala Lys Arg Val Asn Glu Val Ile Val Gly Asn Asp Gln Leu Met
440 445 450
Glu IIe
<210> 12
<211> ?48
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 15574BICD1
<400> 12
Met Ala Asp Ser Ser Gly Gln Gln Gly Lys Gly Arg Arg Val Gln
1 5 10 15
Pro Gln Trp Ser Pro Pro Ala Gly Thr Gln Pro Cys Arg Leu His
20 25 30
Leu Tyr Asn Ser Leu Thr Arg Asn Lys Glu Val Phe Ile Pro Gln
35 40 45
Asp Gly Lys Lys Val Thr Trp Tyr Cys Cys Gly Pro Thr Val Tyr
50 55 60
Asp Ala Ser His Met Gly His Ala Arg Ser Tyr Ile Ser Phe Asp
65 70 75
Ile Leu Arg Arg Val Leu Lys Asp Tyr Phe Lys Phe Asp Val Phe
80 B5 90
Tyr Cys Met Asn Ile Thr Asp Ile Asp Asp Lys Ile Ile Lys Arg
95 100 105
Ala Arg Gln Asn His Leu Phe Glu Gln Tyr Arg Glu Lys Arg Pro
110 115 120
Glu Ala Ala Gln Leu Leu Glu Asp Val Gln Ala Ala Leu Lys Pro
125 130 135
Phe Ser Val Lys Leu Asn Glu Thr Thr Asp Pro Asp Lys Lys Gln
140 145 150
Met Leu Glu Arg Ile Gln His Ala Val Gln Leu Ala Thr Glu Pro
155 160 165
Leu Glu Lys Ala Val Gln Ser Arg Leu Thr Gly Glu Glu Val Asn
170 175 180
Ser Cys Val Glu Val Leu Leu Glu Glu Ala Lys Asp Leu Leu Ser
185 190 195
Asp Trp Leu Asp Ser Thr Leu Gly Cys Asp Val Thr Asp Asn Ser
200 205 210
Ile Phe Ser Lys Leu Pro Lys Phe Trp Glu Gly Asp Phe His Arg
215 220 225
Asp Met Glu Ala Leu Asn Val Leu Pro Pro Asp Val Leu Thr Arg
230 235 240
Val Ser Glu Tyr Val Pro Glu Ile Val Asn Phe Val Gln Lys Ile
245 250 255
Val Asp Asn Gly Tyr Gly Tyr Val Ser Asn Gly Ser Val Tyr Phe
260 265 270
14/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
Asp Thr Ala Lys Phe Ala Ser Ser Glu Lys His Ser Tyr Gly Lys
275 280 285
Leu Val Pro Glu Ala Val Gly Asp Gln Lys Ala Leu Gln Glu Gly
290 295 300
Glu Gly Asp Leu Ser Ile Ser Ala Asp Arg Leu Ser Glu Lys Arg
305 310 315
Ser Pro Asn Asp Phe Ala Leu Trp Lys Ala Ser Lys Pro Gly Glu
320 325 330
Pro Ser Trp Pro Cys Pro Trp GIy Lys Gly Arg Pro Gly Trp His
335 340 345
Ile Glu Cys Ser Ala Met Ala Gly Thr Leu Leu Gly Ala Ser Met
350 355 360
Asp Ile His Gly Gly Gly Phe Asp Leu Arg Phe Pro His His Asp
365 370 375
Asn Glu Leu Ala Gln Ser Glu Ala Tyr Phe Glu Asn Asp Cys Trp
380 385 390
Val Arg Tyr Phe Leu His Thr Gly His Leu Thr Ile Ala Gly Cys
395 400 405
Lys Met Ser Lys Ser Leu Lys Asn Phe Ile Thr Ile Lys Asp Ala
410 415 420
Leu Lys Lys His Ser Ala Arg Gln Leu Arg Leu Ala Phe Leu Met
425 430 435
His Ser Trp Lys Asp Thr Leu Asp Tyr Ser Ser Asn Thr Met Glu
440 445 450
Ser Ala Leu Gln Tyr Glu Lys Phe Leu Asn Glu Phe Phe Leu Asn
455 460 465
Val Lys Asp Ile Leu Arg Ala Pro Val Asp Ile Thr Gly Gln Phe
470 475 480
Glu Lys Trp Gly Glu Glu Glu Ala Glu Leu Asn Lys Asn Phe Tyr
485 490 495
Asp Lys Lys Thr Ala Ile His Lys Ala Leu Cys Asp Asn Val Asp
500 505 510
Thr Arg Thr Val Met Glu Glu Met Arg Ala Leu Val Ser Gln Cys
515 520 525
Asn Leu Tyr Met Ala Ala Arg Lys Ala Val Arg Lys Arg Pro Asn
530 535 540
Gln Ala Leu Leu Glu Asn Ile Ala Leu Tyr Leu Thr His Met Leu
545 550 555
Lys IIe Phe Gly Ala Val Glu Glu Asp Ser Ser Leu Gly Phe Pro
560 565 570
Val Gly Gly Pro Gly Thr Ser Leu Ser Leu Glu Ala Thr Val Met
575 580 585
Pro Tyr Leu Gln Val Leu Ser Glu Phe Arg Glu Gly Val Arg Lys
590 595 600
Ile Ala Arg Glu Gln Lys Val Pro Glu Ile Leu Gln Leu Ser Asp
605 610 615
Ala Leu Arg Asp Asn Ile Leu Pro Glu Leu Gly Val Arg Phe Glu
620 625 630
Asp His Glu Gly Leu Pro Thr Val Val Lys Leu Val Asp Arg Asn
635 640 645
Thr Leu Leu Lys Glu Arg Glu Glu Lys Arg Arg Val Glu Glu Glu
650 655 660
Lys Arg Lys Lys Lys Glu Glu Ala Ala Arg Arg Lys Gln Glu Gln
665 670 675
Glu Ala Ala Lys Leu Ala Lys Met Lys Ile Pro Pro Ser Glu Met
680 685 690
Phe Leu Ser Glu Thr Asp Lys Tyr Ser Lys Phe Asp Glu Asn Gly
15/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
695 700 705
Leu Pro Thr His Asp Met Glu Gly Lys Glu Leu Ser Lys Gly Gln
710 715 720
Ala Lys Lys Leu Lys Lys Leu Phe Glu Ala Gln Glu Lys Leu Tyr
725 730 735
Lys Glu Tyr Leu Gln Met Ala Gln Asn Gly Ser Phe Gln
740 745
<210> 13
<211> 328
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 1747456CD1
<400> 13
Met Glu Ala Asn Gly Ser Gln Gly Thr Ser Gly Ser Ala Asn Asp
1 5 10 15
Ser Gln His Asp Pro Gly Lys Met Phe Ile Gly Gly Leu Ser Trp
20 25 30
Gln Thr Ser Pro Asp Ser Leu Arg Asp Tyr Phe Ser Lys Phe Gly
35 40 45
Glu Ile Arg Glu Cys Met Val Met Arg Asp Pro Thr Thr Lys Arg
50 55 60
Ser Arg Giy Phe Gly Phe Val Thr Phe Ala Asp Pro Ala Ser Val
65 70 75
Asp Lys Val Leu Gly Gln Pro His His Glu Leu Asp Ser Lys Thr
80 85 90
Ile Asp Pro Lys Val Ala Phe Pro Arg Arg Ala Gln Pro Lys Met
95 100 105
Val Thr Arg Thr Lys Lys Ile Phe Val Gly Gly Leu Ser Ala Asn
110 115 120
Thr Val Val Glu Asp Val Lys Gln Tyr Phe Glu Gln Phe Gly Lys
125 130 135
Val Glu Asp Ala Met Leu Met Phe Asp Lys Thr Thr Asn Arg His
140 145 150
Arg Gly Phe Gly Phe Val Thr Phe Glu Asn Glu Asp Val Val Glu
155 160 165
Lys Val Cys Glu Ile His Phe His Glu Ile Asn Asn Lys Met Val
170 175 180
Glu Cys Lys Lys Ala Gln Pro Lys Glu Val Met Phe Pro Pro Gly
185 190 195
Thr Arg Gly Arg Ala Arg Gly Leu Pro Tyr Thr Met Asp Ala Phe
200 205 210
Met Leu Gly Met Gly Met Leu Gly Tyr Pro Asn Phe Val Ala Thr
215 220 225
Tyr Gly Arg Gly Tyr Pro Gly Phe Ala Pro Ser Tyr Gly Tyr Gln
230 235 240
Phe Pro Gly Phe Pro Ala Ala Ala Tyr Gly Pro Val Ala Ala Ala
245 250 255
Ala Val Ala Ala Ala Arg Gly Ser Gly Ser Asn Pro Ala Arg Pro
260 265 270
16/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
Gly Gly Phe Pro Gly Ala Asn Ser Pro Gly Pro Val Ala Asp Leu
275 280 285
Tyr Gly Pro Ala Ser Gln Asp Ser Gly Val Gly Asn Tyr Ile Ser
290 295 300
Ala Ala Ser Pro Gln Pro Gly Ser Gly Phe Gly His Gly Ile Ala
305 310 3I5
Gly Pro Leu Ile Ala Thr Ala Phe Thr Asn Gly Tyr His
320 325
<210> 14
<211> 563
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 1748626CD1
<400> 14
Met Pro Glu Asp Asp Gln Arg Ala Thr Arg Asn Leu Phe Ile Gly
1 5 10 15
Asn Leu Asp His Ser Val Ser Glu Val Glu Leu Arg Arg Ala Phe
20 25 30
Glu Lys Tyr Gly Ile Ile Glu Glu Val Val Ile Lys Arg Pro Ala
35 40 45
Arg Gly Gln Gly Gly Ala Tyr Ala Phe Leu Lys Phe Gln Asn Leu
50 55 60
Asp Met Ala His Arg Ala Lys Val Ala Met Ser Gly Arg Val Ile
65 70 75
Gly Arg Asn Pro Ile Lys Ile Gly Tyr Gly Lys Ala Asn Pro Thr
80 85 90
Thr Arg Leu Trp Val Gly Gly Leu Gly Pro Asn Thr Ser Leu Ala
95 100 105
Ala Leu Ala Arg Glu Phe Asp Arg Phe Gly Ser Ile Arg Thr Ile
110 115 120
Asp His Val Lys Gly Asp Ser Phe Ala Tyr Ile Gln Tyr Glu Ser
125 130 135
Leu Asp Ala Ala Gln Ala Ala Cys Ala Lys Met Arg Gly Phe Pro
140 145 150
Leu Gly Gly Pro Asp Arg Arg Leu Arg Val Asp Phe Ala Lys Ala
155 160 165
Glu Glu Thr Arg Tyr Pro Gln Gln Tyr Gln Pro Ser Pro Leu Pro
170 175 180
Val His Tyr Glu Leu Leu Thr Asp Gly Tyr Thr Arg His Arg Asn
185 190 195
Leu Asp Ala Asp Leu Val Arg Asp Arg Thr Pro Pro His Leu Leu
200 205 210
Tyr Ser Asp Arg Asp Arg Thr Phe Leu Glu Gly Asp Trp Thr Ser
215 220 225
Pro Ser Lys Ser Ser Asp Arg Arg Asn Ser Leu Glu Gly Tyr Ser
230 235 240
Arg Ser Val Arg Ser Arg Ser Gly Glu Arg Trp Gly Ala Asp Gly
245 250 255
Asp Arg Gly Leu Pro Lys Pro Trp Glu Glu Arg Arg Lys Arg Arg
17/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
260 265 270
Ser Leu Ser Ser Asp Arg Gly Arg Thr Thr His Ser Pro Tyr Glu
275 280 285
Glu Arg Ser Arg Thr Lys Gly Ser Gly Gln Gln Ser Glu Arg Gly
290 295 300
Ser Asp Arg Thr Pro Glu Arg Ser Arg Lys Glu Asn His Ser Ser
305 310 315
Glu Gly Thr Lys Glu Ser Ser Ser Asn Ser Leu Ser Asn Ser Arg
320 325 330
His Gly Ala Glu Glu Arg Gly His His His His His His Glu Ala
335 340 345
Ala Asp Ser Ser His Gly Lys Lys Ala Arg Asp Ser Glu Arg Asn
350 355 360
His Arg Thr Thr Glu Ala Glu Pro Lys Pro Leu Glu Glu Pro Lys
365 370 375
His Glu Thr Lys Lys Leu Lys Asn Leu Ser Glu Tyr Ala Gln Thr
380 385 390
Leu Gln Leu Gly Trp Asn Gly Leu Leu Val Leu Lys Asn Ser Cys
395 400 405
Phe Pro Thr Ser Met His Ile Leu Glu Gly Asp Gln Gly Val Ile
410 415 420
Ser Ser Leu Leu Lys Asp His Thr Ser Gly Ser Lys Leu Thr Gln
425 430 435
Leu Lys Ile Ala Gln Arg Leu Arg Leu Asp Gln Pro Lys Leu Asp
440 445 450
Glu Val Thr Arg Arg Ile Lys Gln Gly Ser Pro Asn Gly Tyr Ala
455 460 465
Val Leu Leu Ala Thr Gln Ala Thr Pro Ser Gly Leu Gly Thr Glu
470 475 480
Gly Met Pro Thr Val Glu Pro Gly Leu Gln Arg Arg Leu Leu Arg
485 490 495
Asn Leu Val Ser Tyr Leu Lys Gln Lys Gln Ala Ala Gly Val Ile
500 505 510
Ser Leu Pro Val Gly Gly Ser Lys Gly Arg Asp Gly Thr Gly Met
515 520 525
Leu Tyr Ala Phe Pro Pro Cys Asp Phe Ser Gln Gln Tyr Leu GIn
530 535 540
Ser Ala Leu Arg Thr Leu Gly Lys Leu Glu Glu Glu His Met Val
545 550 555
Ile Val Ile Val Arg Asp Thr Ala
560
<210> 15
<211> 153
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 1879135CD1
<400> 15
Met Met Ser Gln Ser Gly His Glu Tyr Asp Pro Ile Asn Tyr Met
1 5 10 15
18/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
Lys Lys Pro Leu Gly Pro Pro Pro Pro Ser Tyr Thr Cys Phe Arg
20 25 30
Cys Gly Lys Pro Gly His Tyr Ile Lys Asn Cys Pro Thr Asn Gly
35 40 45
Asp Lys Asn Phe Glu Ser Gly Pro Arg Ile Lys Lys Ser Thr Gly
50 55 60
Ile Pro Arg Ser Phe Met Met Glu Val Lys Asp Pro Asn Met Lys
65 70 75
Gly Ala Met Leu Thr Asn Thr Gly Lys Tyr Ala Ile Pro Thr Ile
BO 85 90
Asp Ala Glu Ala Tyr Ala Ile Gly Lys Lys Glu Lys Pro Pro Phe
95 100 105
Leu Pro Glu Glu Pro Ser Ser Ser Ser Glu Glu Asp Asp Pro Ile
110 115 120
Pro Asp Glu Leu Leu Cys Leu Ile Cys Lys Asp Ile Met Thr Asp
125 130 135
Ala Val Val Ile Pro Cys Cys Gly Asn Ser Tyr Cys Asp Glu Cys
140 145 150
Lys Lys Cys
<210> 16
<211> 286
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2073417CD1
<400> 16
Met Ser Trp Leu Leu Phe Leu Ala His Arg Val Ala Leu Ala Ala
1 5 10 15
Leu Pro Cys Arg Arg Gly Ser Arg Gly Phe Gly Met Phe Tyr Ala
20 25 30
Val Arg Arg Gly Arg Lys Thr Gly Val Phe Leu Thr Trp Asn Glu
35 40 45
Cys Arg Ala Gln Val Asp Arg Phe Pro Ala Ala Arg Phe Lys Lys
50 55 60
Phe Ala Thr Glu Asp Glu Ala Trp Ala Phe Val Arg Lys Ser Ala
65 70 75
Ser Pro Glu Val Ser Glu Gly His Glu Asn Gln His Gly Gln Glu
80 85 90
Ser Glu Ala Lys Ala Ser Lys Arg Leu Arg Glu Pro Leu Asp Gly
95 100 105
Asp Gly His Glu Ser Ala Glu Pro Tyr Ala Lys His Met Lys Pro
110 115 120
Ser Met Glu Pro Ala Pro Pro Val Ser Arg Asp Thr Phe Ser Tyr
125 130 135
Met Gly Asp Phe Val Val Val Tyr Thr Asp Gly Cys Cys Ser Ser
140 145 150
Asn Gly Arg Arg Arg Pro Arg Ala Gly Ile Gly Val Tyr Trp Gly
155 160 165
Pro Gly His Pro Leu Asn Val Gly Ile Arg Leu Pro Gly Arg Gln
170 175 180
19/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
Thr Asn Gln Arg Ala Glu Ile His Ala Ala Cys Lys Ala Ile Glu
185 190 195
Gln Ala Lys Thr Gln Asn Ile Asn Lys Leu Val Leu Tyr Thr Asp
200 205 210
Ser Met Phe Thr Ile Asn Gly Ile Thr Asn Trp Val Gln Gly Trp
215 220 225
Lys Lys Asn Gly Trp Lys Thr Ser Ala Gly Lys Glu Val Ile Asn
230 235 240
Lys Glu Asp Phe Val Ala Leu Glu Arg Leu Thr Gln Gly Met Asp
245 250 255
Ile Gln Trp Met His Val Pro GIy His Ser Gly Phe Ile Gly Asn
260 265 270
Glu Glu Ala Asp Arg Leu Ala Arg Glu Gly Ala .Lys Gln Ser Glu
275 280 285
Asp
<210> 17
<211> 537
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2129080CD1
<400> 17
Met Leu Ala Arg Glu Thr Tyr Glu Glu Asp Arg Glu Tyr Glu Ser
1 5 10 15
Gln Ala Lys Arg Leu Lys Thr Glu Glu Gly Glu Ile Asp Tyr Ser
20 25 30
Ala Glu Glu Gly Glu Asn Arg Arg Glu Ala Thr Pro Arg Gly Gly
35 40 45
Gly Asp Gly Gly Gly Gly Gly Arg Ser Phe Ser Gln Pro Glu Ala
50 55 60
Gly Gly Ser His His Lys Val Ser Val Ser Pro Val Val His Val
65 70 75
Arg Gly Leu Cys Glu Ser Val Val Glu Ala Asp Leu Val Glu Ala
80 85 90
Leu Glu Lys Phe Gly Thr Ile Cys Tyr Val Met Met Met Pro Phe
95 100 105
Lys Arg Gln Ala Leu Val Glu Phe Glu Asn Ile Asp Ser Ala Lys
110 115 120
Glu Cys Val Thr Phe Ala Ala Asp Glu Pro Val Tyr Ile Ala Gly
125 130 135
Gln Gln Ala Phe Phe Asn Tyr Ser Thr Ser Lys Arg Ile Thr Arg
140 145 150
Pro Gly Asn Thr Asp Asp Pro Ser Gly Gly Asn Lys Val Leu Leu
155 160 165
Leu Ser Ile Gln Asn Pro Leu Tyr Pro Ile Thr Val Asp Val Leu
170 175 180
Tyr Thr Val Cys Asn Pro Val Gly Lys Val Gln Arg Ile Val Ile
185 190 195
Phe Lys Arg Asn Gly Ile Gln Ala Met Val Glu Phe Glu Ser Val
200 205 210
20/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
Leu Cys Ala Gln Lys Ala Lys Ala Ala Leu Asn Gly Ala Asp Ile
215 220 225
Tyr Ala Gly Cys Cys Thr Leu Lys Ile Glu Tyr Ala Arg Pro Thr
230 235 240
Arg Leu Asn Val Ile Arg Asn Asp Asn Asp Ser Trp Asp Tyr Thr
245 250 255
Lys Pro Tyr Leu Gly Arg Arg Asp Arg Gly Lys Gly Arg Gln Arg
260 265 270
Gln Ala Ile Leu Gly Glu His Pro Ser Ser Phe Arg His Asp Gly
275 280 285
Tyr Gly Ser His Gly Pro Leu Leu Pro Leu Pro Ser Arg Tyr Arg
290 295 300
Met Gly Ser Arg Asp Thr Pro Glu Leu Val Ala Tyr Pro Leu Pro
305 310 315
Gln Ala Ser Ser Ser Tyr Met His Gly Gly Asn Pro Ser Gly Ser
320 325 330
Val Val Met Val Ser Gly Leu His Gln Leu Lys Met Asn Cys Ser
335 340 345
Arg Val Phe Asn Leu Phe Cys Leu Tyr Gly Asn Ile Glu Lys Val
350 355 360
Lys Phe Met Lys Thr Ile Pro Gly Thr Ala Leu Val Glu Met Gly
365 370 375
Asp Glu Tyr Ala Val Glu Arg Ala Val Thr His Leu Asn Asn Val
380 385 390
Lys Leu Phe Gly Lys Arg Leu Asn Val Cys Val Ser Lys Gln His
395 400 405
Ser Val Val Pro Ser Gln Ile Phe Glu Leu Glu Asp Gly Thr Ser
4I0 415 420
Ser Tyr Lys Asp Phe Ala Met Ser Lys Asn Asn Arg Phe Thr Ser
425 430 435
Ala Gly Gln Ala Ser Lys Asn Ile Ile Gln Pro Pro Ser Cys Val
440 445 450
Leu His Tyr Tyr Asn Val Pro Leu Cys Val Thr Glu Glu Thr Phe
455 460 465
Thr Lys Leu Cys Asn Asp His Glu Val Leu Thr Phe Ile Lys Tyr
470 475 480
Lys Val Phe Asp Ala Lys Pro Ser Ala Lys Thr Leu Ser Gly Leu
485 490 495
Leu Glu Trp Glu Cys Lys Thr Asp Ala Val Glu Ala Leu Thr Ala
500 505 510
Leu Asn His Tyr Gln Ile Arg Val Pro Asn Gly Ser Asn Pro Tyr
515 520 525
Thr Leu Lys Leu Cys Phe Ser Thr Ser Ser His Leu
530 535
<210> 18
<211> 163
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2472867CD1
21 /47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
<400> 18
Met Arg Ile Glu Lys Cys Tyr Phe Cys Ser Gly Pro Ile Tyr Pro
1 5 10 15
Gly His Gly Met Met Phe Val Arg Asn Asp Cys Lys Val Phe Arg
20 25 30
Phe Cys Lys Ser Lys Cys His Lys Asn Phe Lys Lys Lys Arg Asn
35 40 45
Pro Arg Lys Val Arg Trp Thr Lys Ala Phe Arg Lys Ala Ala Gly
50 55 60
Lys Glu Leu Thr Val Asp Asn Ser Phe Glu Phe Glu Lys Arg Arg
65 70 75
Asn Glu Pro Ile Lys Tyr Gln Arg Glu Leu Trp Asn Lys Thr Ile
80 85 90
Asp Ala Met Lys Arg Val Glu Glu Ile Lys Gln Lys Arg Gln Ala
95 100 105
Lys Phe Ile Met Asn Arg Leu Lys Lys Asn Lys Glu Leu Gln Lys
110 115 120
Val Gln Asp Ile Lys Glu Val Lys Gln Asn Ile His Leu Ile Arg
125 130 135
Ala Pro Leu Ala Gly Lys Gly Lys Gln Leu Glu Glu Lys Met Val
140 145 150
Gln Gln Leu Gln Glu Asp Val Asp Met Glu Asp Ala Pro
155 160
<210> 19
<211> 178
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2764755CD1
<400> 19
Met Ser Ser Phe Ser Arg Ala Pro Gln Gln Trp Ala Thr Phe Ala
1 5 10 15
Arg Ile Trp Tyr Leu Leu Asp Gly Lys Met Gln Pro Pro Gly Lys
20 25 30
Leu Ala Ala Met Ala Ser Ile Arg Leu Gln Gly Leu His Lys Pro
35 40 45
Val Tyr His Ala Leu Ser Asp Cys Gly Asp His Val Val Ile Met
50 55 60
Asn Thr Arg His Ile Ala Phe Ser Gly Asn Lys Trp Glu Gln Lys
65 70 75
Val Tyr Ser Ser His Thr Gly Tyr Pro Gly Gly Phe Arg Gln Val
80 85 90
Thr Ala Ala Gln Leu His Leu Arg Asp Pro Val Ala Ile Val Lys
95 100 105
Leu Ala Ile Tyr Gly Met Leu Pro Lys Asn Leu His Arg Arg Thr
110 115 120
Met Met GIu Arg Leu His Leu Phe Pro Asp Glu Tyr Ile Pro Glu
125 130 135
Asp Ile Leu Lys Asn Leu Val Glu Glu Leu Pro Gln Pro Arg Lys
140 145 150
22/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
Ile Pro Lys Arg Leu Asp Glu Tyr Thr Gln Glu Glu Ile Asp Ala
155 160 165
Phe Pro Arg Leu Trp Thr Pro Pro Glu Asp Tyr Arg Leu
170 175
<210> 20
<211> 140
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2875939CD1
<400> 20
Met Ala Glu Asn Arg Glu Pro Arg Gly Ala Val Glu Ala Glu Leu
1 5 10 15
Asp Pro Val Glu Tyr Thr Leu Arg Lys Arg Leu Pro Ser Arg Leu
20 25 30
Pro Arg Arg Pro Asn Asp Ile Tyr Val Asn Met Lys Thr Asp Phe
35 40 45
Lys Ala Gln Leu Ala Arg Cys Gln Lys Leu Leu Asp Gly Gly Ala
50 55 60
Arg Gly Gln Asn Ala Cys Ser Glu Ile Tyr Ile His Gly Leu Gly
65 70 75
Leu Ala Ile Asn Arg Ala Ile Asn Ile Ala Leu Gln Leu Gln Ala
80 85 90
Gly Ser Phe Gly Ser Leu Gln Val Ala Ala Asn Thr Ser Thr Val
95 100 105
Glu Leu Val Asp Glu Leu Glu Pro Glu Thr Asp Thr Arg Glu Pro
110 115 120
Leu Thr Arg Ile Arg Asn Asn Ser Ala Ile His Ile Arg Val Phe
125 130 135
Arg Val Thr Pro Lys
140
<210> 21
<211> 209
<212> PRT
w213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 3591363CD1
<400> 21
Met Ala Ala Ala Met Ala Ala Ser Ser Leu Thr Val Thr Leu Gly
1 5 10 15
Arg Leu Ala Ser Ala Cys Ser His Ser Ile Leu Arg Pro Ser Gly
20 25 30
Pro Gly Ala Ala Ser Leu Trp Ser Ala Ser Arg Arg Phe Asn Ser
23/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
35 40 45
Gln Ser Thr Ser Tyr Leu Pro Gly Tyr Val Pro Lys Thr Ser Leu
50 55 60
Ser Ser Pro Pro Trp Pro Glu Val Val Leu Pro Asp Pro Val Glu
65 70 75
Glu Thr Arg His His Ala Glu Val Val Lys Lys Val Asn Glu Met
80 85 90
Ile Val Thr Gly Gln Tyr Gly Arg Leu Phe Ala Val Val His Phe
95 100 105
Ala Ser Arg Gln Trp Lys Val Thr Ser Glu Asp Leu Ile Leu Ile
110 115 120
Gly Asn Glu Leu Asp Leu Ala Cys Gly Glu Arg Ile Arg Leu Glu
125 130 135
Lys Val Leu Leu Val Gly Ala Asp Asn Phe Thr Leu Leu Gly Lys
140 145 150
Pro Leu Leu Gly Lys Asp Leu Val Arg Val Glu Ala Thr Val Ile
155 160 165
Glu Lys Thr Glu Ser Trp Pro Arg Ile Ile Met Arg Phe Arg Lys
170 175 180
Arg Lys Asn Phe Lys Lys Lys Arg Ile Val Thr Thr Pro Gln Thr
185 190 195
Val Leu Arg Ile Asn Ser Ile Glu Ile Ala Pro Cys Leu Leu
200 205
<210> 22
<211> 162
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 3702292CD1
<400> 22
Met Ala Pro Lys Ala Lys Glu Ala Pro Ala His Pro Lys Ala Glu
1 5 10 15
Ala Lys Ala Lys Ala Leu Lys Ala Lys Lys Ala Val Leu Lys Gly
20 25 30
Val Arg Ser His Thr Gln Lys Gln Lys Ile Arg Met Ser Leu Thr
35 40 45
Phe Arg Arg Pro Lys Thr Leu Arg Leu Arg Arg Gln Pro Arg Tyr
50 55 60
Pro Arg Lys Ser Thr Pro Arg Arg Asn Lys Leu Gly His Tyr Ala
65 70 75
Ile Ile Lys Phe Pro Leu Ala Thr Glu Ser Ala Val Lys Lys Ile
80 85 90
Glu Glu Asn Asn Thr Leu Val Phe Thr Val Asp Val Lys Ala Asn
95 100 105
Lys His Gln Ile Arg Gln Ala Val Lys Lys Leu Tyr Asp Ser Asp
110 115 120
Val Ala Lys Val Thr Thr Leu Ile Cys Pro Asp Lys Glu Asn Lys
125 130 135
Ala Tyr Val Arg Leu Ala Pro Asp Tyr Asp Ala Phe Asp Val Val
140 145 150
24/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99II9361
Thr Lys Leu Gly Ser Pro Lys Leu Ser Pro Ala Gly
155 160
<210> 23
<211> 623
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 3778908CD1
<400> 23
Met Ala Thr Glu His Val Asn Gly Asn Gly Thr Glu Glu Pro Met
1 5 10 15
Asp Thr Thr Ser Ala Val Ile His Ser Glu Asn Phe Gln Thr Leu
20 25 30
Leu Asp Ala Gly Leu Pro Gln Lys Val Ala Glu Lys Leu Asp Glu
35 40 45
Ile Tyr Val Ala Gly Leu Val Ala His Ser Asp Leu Asp Glu Arg
50 55 60
Ala Ile Glu Ala Leu Lys Glu Phe Asn Glu Asp Gly Ala Leu Ala
65 70 75
Val Leu Gln Gln Phe Lys Asp Ser Asp Leu Ser His Val Gln Asn
80 85 90
Lys Ser Ala Phe Leu Cys Gly Val Met Lys Thr Tyr Arg Gln Arg
95 100 105
Glu Lys Gln Gly Thr Lys Val Ala Asp Ser Ser Lys Gly Pro Asp
110 115 120
Glu Ala Lys Ile Lys Ala Leu Leu Glu Arg Thr Gly Tyr Thr Leu
125 130 135
Asp Val Thr Thr Gly Gln Arg Lys Tyr Gly Gly Pro Pro Pro Asp
140 145 150
Ser Val Tyr Ser Gly Gln Gln Pro Ser Val Gly Thr Glu Ile Phe
155 160 165
Val Gly Lys Ile Pro Arg Asp Leu Phe Glu Asp Glu Leu Val Pro
170 175 180
Leu Phe Glu Lys Ala Gly Pro Ile Trp Asp Leu Arg Leu Met Met
185 190 195
Asp Pro Leu Thr Gly Leu Asn Arg Gly Tyr Ala Phe Val Thr Phe
200 205 210
Cys Thr Lys Giu Ala Ala Gln Glu Ala Val Lys Leu Tyr Asn Asn
215 220 225
His Glu Ile Arg Ser Gly Lys His Ile Gly Val Cys Ile Ser Val
230 235 240
Ala Asn Asn Arg Leu Phe Val Gly Ser Ile Pro Lys Ser Lys Thr
245 250 255
Lys Glu Gln Ile Leu Glu Glu Phe Ser Lys Val Thr Glu Gly Leu
260 265 270
Thr Asp Val Ile Leu Tyr His Gln Pro Asp Asp Lys Lys Lys Asn
275 280 285
Arg Gly Phe Cys Phe Leu Glu Tyr Glu Asp His Lys Thr Ala Ala
290 295 300
Gln Ala Arg Arg Arg Leu Met Ser Gly Lys Val Lys Val Trp Gly
25/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
305 310 315
Asn Val Gly Thr Val Glu Trp Ala Asp Pro Ile Glu Asp Pro Asp
320 325 330
Pro Glu Val Met Ala Lys Val Lys Val Leu Phe Val Arg Asn Leu
335 340 345
Ala Asn Thr Val Thr Glu Glu Ile Leu Glu Lys Ala Phe Ser Gln
350 355 360
Phe Gly Lys Leu Glu Arg Val Lys Lys Leu Lys Asp Tyr Ala Phe
365 370 375
Ile His Phe Asp Glu Arg Asp Gly Ala Val Lys Ala Met Glu Glu
380 385 390
Met Asn GIy Lys Asp Leu Glu Gly Glu Asn Ile Glu Ile Val Phe
395 400 405
Ala Lys Pro Pro Asp Gln Lys Arg Lys Glu Arg Lys Ala Gln Arg
410 415 420
Gln Ala Ala Lys Asn Gln Met Tyr Asp Asp Tyr Tyr Tyr Tyr Gly
425 430 435
Pro Pro His Met Pro Pro Pro Thr Arg Gly Arg Gly Arg Gly Gly
440 445 450
Arg Gly Gly Tyr Gly Tyr Pro Pro Asp Tyr Tyr Gly Tyr Glu Asp
455 460 465
Tyr Tyr Asp Tyr Tyr Gly Tyr Asp Tyr His Asn Tyr Arg Gly Gly
470 475 480
Tyr Glu Asp Pro Tyr Tyr Gly Tyr Glu Asp Phe Gln Val Gly Ala
485 490 495
Arg Gly Arg Gly Gly Arg Gly Ala Arg Gly Ala Ala Pro Ser Arg
500 505 510
Gly Arg Gly Ala Ala Pro Pro Arg Gly Arg Ala Gly Tyr Ser Gln
515 520 525
Arg Gly Gly Pro Gly Ser Ala Arg Gly Val Arg Gly Ala Arg Gly
530 535 540
Gly Ala Gln Gln Gln Arg Gly Arg Gly Val Arg Gly Ala Arg Gly
545 550 555
Gly Arg Gly Gly Asn Val Gly Gly Lys Arg Lys Ala Asp Gly Tyr
560 565 570
Asn Gln Pro Asp Ser Lys Arg Arg Gln Thr Asn Asn Gln Asn Trp
575 580 585
Gly Ser Gln Pro Ile Ala Gln Gln Pro Leu Gln Gly Gly Asp His
590 595 600
Ser Gly Asn Tyr Gly Tyr Lys Ser Glu Asn Gln Glu Phe Tyr Gln
605 610 615
Asp Thr Phe Gly Gln Gln Trp Lys
620
<220> 24
<211> 786
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 4163642CD1
<400> 24
26/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
Met Ser Phe Ser Arg Ala Leu Leu Trp Ala Arg Leu Pro Ala Gly
1 5 10 15
Arg Gln Ala Gly His Arg Ala Ala Ile Cys Ser Ala Leu Arg Pro
20 25 30
His Phe Gly Pro Phe Pro Gly Val Leu Gly Gln Val Ser Val Leu
35 40 45
Ala Thr Ala Ser Ser Ser Ala Ser Gly Gly Ser Lys Ile Pro Asn
50 55 60
Thr Ser Leu Phe Val Pro Leu Thr Val Lys Pro Gln Gly Pro Ser
65 70 75
Ala Asp Gly Asp Val Gly Ala Glu Leu Thr Arg Pro Leu Asp Lys
80 85 90
Asn Glu Val Lys Lys Val Leu Asp Lys Phe Tyr Lys Arg Lys Glu
95 100 105
Ile Gln Lys Leu Gly Ala Asp Tyr Gly Leu Asp Ala Arg Leu Phe
110 115 120
His Gln Ala Phe Ile Ser Phe Arg Asn Tyr Ile Met Gln Ser His
125 130 135
Ser Leu Asp Val Asp Ile His Ile Val Leu Asn Asp Ile Cys Phe
140 145 150
Gly Ala Ala His Ala Asp Asp Leu Phe Pro Phe Phe Leu Arg His
155 160 165
Ala Lys Gln Ile Phe Pro Val Leu Asp Cys Lys Asp Asp Leu Arg
170 175 180
Lys Ile Ser Asp Leu Arg Ile Pro Pro Asn Trp Tyr Pro Asp Ala
185 190 195
Arg Ala Met Gln Arg Lys Ile Ile Phe His Ser Gly Pro Thr Asn
200 205 210
Ser Gly Lys Thr Tyr His Ala Ile Gln Lys Tyr Phe Ser Ala Lys
215 220 225
Ser Gly Val Tyr Cys Gly Pro Leu Lys Leu Leu Ala His Glu Ile
230 235 240
Phe Glu Lys Ser Asn Ala Ala Gly Val Pro Cys Asp Leu Val Thr
245 250 255
Gly Glu Glu Arg Val Thr Val Gln Pro Asn Gly Lys Gln Ala Ser
260 265 270
His Val Ser Cys Thr Val Glu Met Cys Ser Val Thr Thr Pro Tyr
275 280 285
Glu Val Ala Val Ile Asp Glu Ile Gln Met Ile Arg Asp Pro Ala
290 295 300
Arg Gly Trp Ala Trp Thr Arg Ala Leu Leu Gly Leu Cys Ala Glu
305 310 315
Glu Val His Leu Cys Gly Glu Pro Ala Ala Ile Asp Leu Val Met
320 325 330
Glu Leu Met Tyr Thr Thr Gly Glu Glu Val Glu Val Arg Asp Tyr
335 340 345
Lys Arg Leu Thr Pro Ile Ser Val Leu Asp His Ala Leu Glu Ser
350 355 360
Leu Asp Asn Leu Arg Pro Gly Asp Cys Ile Val Cys Phe Ser Lys
365 370 375
Asn Asp Ile Tyr Ser Val Ser Arg Gln Ile Glu Ile Arg Gly Leu
380 385 390
Glu Ser Ala Val Ile Tyr Gly Ser Leu Pro Pro Gly Thr Lys Leu
395 400 405
Ala Gln Ala Lys Lys Phe Asn Asp Pro Asn Asp Pro Cys Lys Ile
410 415 420
Leu Val Ala Thr Asp Ala Ile Gly Met Gly Leu Asn Leu Ser Ile
27/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
425 430 435
Arg Arg Ile Ile Phe Tyr Ser Leu Ile Lys Pro Ser Ile Asn Glu
440 445 450
Lys Gly Glu Arg Glu Leu Glu Pro Ile Thr Thr Ser Gln Ala Leu
455 460 465
Gln Ile Ala Gly Arg Ala Gly Arg Phe Ser Ser Arg Phe Lys Glu
470 475 480
Gly Glu Val Thr Thr Met Asn His Glu Asp Leu Ser Leu Leu Lys
485 490 495
Glu Ile Leu Lys Arg Pro Val Asp Pro Ile Arg Ala Ala Gly Leu
500 505 510
His Pro Thr Ala Glu Gln Ile Glu Met Phe Ala Tyr His Leu Pro
515 520 525
Asp Ala Thr Leu Ser Asn Leu Ile Asp Ile Phe Val Asp Phe Ser
530 535 540
Gln Val Asp Gly Gln Tyr Phe Val Cys Asn Met Asp Asp Phe Lys
545 550 555
Phe Ser Ala Glu Leu Ile Gln His Ile Pro Leu Ser Leu Arg Val
560 565 570
Arg Tyr Val Phe Cys Thr Ala Pro Ile Asn Lys Lys Gln Pro Phe
575 580 585
Val Cys Ser Ser Leu Leu Gln Phe Ala Arg Gln Tyr Ser Arg Asn
590 595 600
Glu Pro Leu Thr Phe Ala Trp Leu Arg Arg Tyr Ile Lys Trp Pro
605 610 615
Leu Leu Pro Pro Lys Asn Ile Lys Asp Leu Met Asp Leu Glu Ala
620 625 630
Val His Asp Val Leu Asp Leu Tyr Leu Trp Leu Ser Tyr Arg Phe
635 640 645
Met Asp Met Phe Pro Asp Ala Ser Leu Ile Arg Asp Leu Gln Lys
650 655 660
Glu Leu Asp Gly Ile Ile Gln Asp Gly Val His Asn Ile Thr Lys
665 670 675
Leu Ile Lys Met Ser Glu Thr His Lys Leu Leu Asn Leu Glu Gly
680 685 690
Phe Pro Ser Gly Ser Gln Ser Arg Leu Ser Gly Thr Leu Lys Ser
695 700 705
Gln Ala Arg Arg Thr Arg Gly Thr Lys Ala Leu Gly Ser Lys Ala
710 715 720
Thr Glu Pro Pro Ser Pro Asp Ala Gly Glu Leu Ser Leu Ala Ser
725 730 735
Arg Leu Val Gln Gln Gly Leu Leu Thr Pro Asp Met Leu Lys Gln
740 745 750
Leu Glu Lys Glu Trp Met Thr Gln Gln Thr Glu His Asn Lys Glu
755 700 765
Lys Thr Glu Ser Gly Thr His Pro Lys Gly Thr Arg Arg Lys Lys
770 775 780
Lys Glu Pro Asp Ser Asp
785
<210> 25
<211> 260
<212> PRT
<213> Homo sapiens
28/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
<220>
<221> misc_feature
<223> Incyte Identification No.: 4906154CD1
<400> 25
Met Thr Pro Val Gln Arg Gly Gly Pro Gly Ala Leu Val Ala Leu
1 5 10 15
Gly Trp Gly Arg Arg Lys Ala Glu Asp Lys Glu Trp Met Pro Val
20 25 30
Thr Lys Leu Gly Arg Leu Val Lys Asp Met Lys Ile Lys Ser Leu
35 40 45
Glu Glu Ile Tyr Leu Phe Ser Leu Pro Ile Lys Glu Ser Glu Ile
50 55 60
Ile Asp Phe Phe Leu Gly Ala Ser Leu Lys Asp Glu Val Leu Lys
65 70 75
Ile Met Pro Val Gln Lys Gln Thr Arg Ala Gly Gln Arg Thr Arg
80 85 90
Phe Lys Ala Phe Val Ala Ile Gly Asp Tyr Asn Gly His Val Gly
95 100 105
Leu Gly Val Lys Cys Ser Lys Glu Val Ala Thr Ala Ile Arg Gly
110 115 120
Ala Ile Ile Leu Ala Lys Leu Ser Ile Val Pro Val Arg Arg Gly
125 130 135
Tyr Trp Gly Asn Lys Ile Gly Lys Pro His Thr Val Pro Cys Lys
140 145 150
Val Thr Gly Arg Cys Gly Ser Val Leu Val Arg Leu Ile Pro Ala
155 160 165
Pro Arg Gly Thr Gly Ile Val Ser Ala Pro Val Pro Lys Lys Leu
170 175 180
Leu Met Met Ala Gly Ile Asp Asp Cys Tyr Thr Ser Ala Arg Gly
185 190 195
Cys Thr Ala Thr Leu Gly Asn Phe Ala Lys Ala Thr Phe Asp Ala
200 205 210
Ile Ser Lys Thr Tyr Ser Tyr Leu Thr Pro Asp Leu Trp Lys Glu
215 220 225
Thr Val Phe Thr Lys Ser Pro Tyr Gln Glu Phe Thr Asp His Leu
230 235 240
Val Lys Thr His Thr Arg Val Ser Val Gln Arg Thr Gln Ala Pro
245 250 255
Ala Val Ala Thr Thr
260
<210> 26
<211> 1872
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 399781CB1
<400> 26
gccctctagc tgtgtgtgtc tgaggctcgg ccgcctgagc cgcggacggt ttgctgagcc 60
cgttagtgcg cccggccgag acacgccgcc gccatgtccc gctacctgcg tccccccaac 120
29/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
acgtctctgt tcgtcaggaa cgtggccgac gacaccaggt ctgaagactt gcggcgtgaa 180
tttggtcgtt atggtcctat agttgatgtg tatgttccac ttgatttcta cactcgccgt 240
ccaagaggat ttgcttatgt tcaatttgag gatgttcgtg atgctgaaga cgctttacat 300
aatttggaca gaaagtggat ttgtggacgg cagattgaaa tacagtttgc ccagggggat 360
cgaaagacac caaatcagat gaaagccaag gaagggagga atgtgtacag ttcttcacgc 420
tatgatgatt atgacagata cagacgttct agaagccgaa gttatgaaag gaggagatca 480
agaagtcggt cttttgatta caactataga agatcgtata gtcctagaaa cagtagaccg 540
actggaagac cacggcgtag agaagccatt ccgacaatga tagaccaaac tgcagctgga 600
atacccagta cagttctgct tactacactt caagaaagat ctgaaagcgg aaaaagaacc 660
aaagaagggc agttcaagcg accaaagggt gggtggaagg tgctgcagta tgaatactgt 720
acgaatattt tgactctggt ctgaaaagat aaaagaatgt tatcgaaaac tacatggaat 780
aattgaagtc ccttcaagtt tgaaagtaag cattttagga caaataaaag gaaattcaac 840
tttgtacttg tggaaactaa tccctaaata tgaataggtt tatattgatt catgggtaac 900
aggtccataa taaattattg gaaactagga tgtctgaata tcaaggaaga cagccatagt 960
ctcttacagt gcctctgttg gtctgtctca aactgaattg ggtgggaaaa ggtatggtcc 1020
aatataaaag ttccattttt gccattattg gcaaatcttg cctttgttta ttttggtgcc 1080
agtgttttct gcttaatcat ttgctttgtt ggcatctgtg tttatttact tgtacaccac 1140
atgcagttta catctgtctt aactactcct tcccaggtaa attccaatta tatttgacat 1200
ccagctaaga gggcccatct cttctcacct ctttcctagt cagtatattc agcaaatatt 1260
tattgagccc ttactgtggg caaatcattg tactggataa ttgagaaaaa tagataattc 1320
ccttattcag taaatgtcta ctgagcacaa tctagtgaat cattacagta tggcctcatt 1380
gttttgtttg aggtgtgtta ttcataacaa tattttacac cattcgtatc aatgtaatta 1440
tagaacacaa tatacgatca aggataagta attgtgtggt tatctgccat ttaaaagtat 1500
ccagtatttg atcacattat tataaataat gaaaaaatga tttaatctgt aataaactgg 1560
tttattgtgc agtgactgta atatactaga gttataataa attgtttact ctgcctcacc 1620
aaacacatgc taggatataa cccccaaaat aagtatttaa ctttgcatta ggtataaagg 1680
agactgggtg ctataattag attattttga ggcagacaga gagctgttat cctaactgat 1740
ttagtatgtt ctgtaattga gaaaatgttc accaaattat actttttagt gatttacatg 1800
tacattttat aggggacatg ttctgtgtat agcgaataaa taacttttat agtatcaaaa 1860
aaaaaaaaaa as 1872
<210> 27
<211> 3834
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 1806542CB1
<400> 27
ctgcggcggc ggcggcggcg ttaccggccc tcgcgctctt tcccttcctg gggcgccgac 60
cccgcccgct tgcttgcttg cttgcttgcc tgcctgcctg cctgcccggc gccacgcaag 120
agaaggtgcc aggggacgca gagcgactag aggcgcgcgg tcccggccag caccgtctct 180
ggcgttgtag ctgcggccgt ggcggaggac tacggcgaca aggacgaggg ccgctctccc 240
agctctctgc gtgccgcgcc gctccgctcc gctggctgac catctggagt gcaggctggg 300
aggcgggatg gagtgatagg gaagatgttt ataaattctt ctgtgggatc agagggcacg 360
cctattacaa ccagaaaact acaagtataa cagcgaggat ggatgaacag gctctattag 420
ggctaaatcc aaatgctgat tcagacttta gacaaagggc cctggcctat tttgagcagt 480
taaaaatttc cccagatgcc tggcaggtgt gtgcagaagc tctagcccag aggacataca 540
gtgatgatca tgtgaagttt ttctgctttc aagtactgga acatcaagtt aaatacaaat 600
actcagaact aaccactgtt caacaacagc taattaggga gacgctcata tcatggctgc 660
aagctcagat gctgaatccc caaccagaga agacctttat acgaaataaa gccgcccaag 720
tcttcgcctt gctttttgtt acagagtatc tcactaagtg gcccaagttt ttttttgaca 780
ttctctcagt agtggaccta aatccaaggg gagtagatct ctacctgcga atcctcatgg 840
ctattgattc agagttggtg gatcgtgatg tggtgcatac atcagaggag gctcgtagga 900
30/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
atactctcat aaaagatacc atgagggaac agtgcattcc aaatctggtg gaatcatggt 960
accaaatatt acaaaattat cagtttacta attctgaagt gacgtgtcag tgccttgaag 1020
tagttggggc ttatgtctct tggatagact tatcccttat agccaatgat aggtttataa 1080
atatgctgct aggtcatatg tcaatagaag ttctacggga agaagcatgt gactgtttat 1140
ttgaagttgt aaataaagga atggaccctg ttgataaaat gaaactagtg gaatctttgt 1200
gtcaagtatt acagtctgct gggtttttca gcattgacca ggaagaagat gttgacttcc 1260
tggccagatt ttctaagttg gtaaatggaa tgggacagtc attgatagtt agttggagta 1320
aattaattaa gaatggggat attaagaatg ctcaagaggc actacaagct attgaaacaa 1380
aagtggcact gatgttgcag ctactaattc atgaggatga tgatatttct tctaatatta 1440
ttggattttg ttacgattat cttcatattt tgaaacagct tacagtgctc tcggatcagc 1500
aaaaagctaa tgtagaggca atcatgttgg ccgttatgaa aaaattgact tacgatgaag 1560
aatataactt tgaaaatgag ggtgaagatg aagccatgtt tgtagaatat agaaaacaac 1620
tgaagttact gttggacagg cttgctcaag tttcaccaga gttactactg gcctctgttc 1680
gcagagtttt tagttctaca ctgcagaatt ggcagactac acggtttatg gaagttgaag 1740
tagcaataag attgctgtat atgttggcag aagctcttcc agtatctcat ggtgctcact 1800
tctcaggtga tgtttcaaaa gctagtgctt tgcaggatat gatgcgaact ctggtaacat 1860
caggagtcag ttcctatcag catacatctg tgacattgga gttcttcgaa actgttgtta 1920
gatatgaaaa gtttttcaca gttgaacctc agcacattcc atgtgtacta atggctttct 1980
tagatcacag aggtctgcgg cattccagtg caaaagttcg gagcaggacg gcttacctgt 2040
tttctagatt tgtcaaatct ctcaataagc aaatgaatcc t.ttcattgag gatattttga 2100
atagaataca agatttatta gagctttctc cacctgagaa t.ggccaccag tccttactga 2160
gcagcgatga tcaacttttt atttatgaga cagctggagt gctgattgtt aatagtgaat 2220
atccggcaga aaggaaacaa gccttaatga ggaatctgtt gactccacta atggagaagt 2280
ttaaaattct gttagaaaag ttgatgctgg cacaagatga agaaaggcaa gcctctctag 2340
cagactgtct taaccatgct gttggatttg caagtcgaac cagtaaagct ttcagcaaca 2400
aacagactgt gaaacaatgt ggctgttccg aagtttatct ggactgttta cagacattct 2460
tgccagccct cagttgtccc ttacaaaagg atattctcag aagtggagtc cgtactttcc 2520
ttcatcgaat gattatttgc ctggaggaag aagttcttcc gttcattcca tctgcttcag 2580
aacatatgct caaagattgt gaagcaaaag atctccagga gttcattcct cttatcaacc 2640
agattacggc caaattcaag atacaggtat ccccgttttt acaacagatg ttcatgcccc 2700
tgcttcatgc aatttttgaa gtgctgctcc ggccagcaga agaaaatgac cagtctgctg 2760
ctttagagaa gcagatgttg cggaggagtt actttgcttt cctgcaaaca gtcacaggca 2820
gtgggatgag cgaagttata gcaaatcaag gtgcagagaa tgtagaaaga gtgttggtta 2880
ctgttatcca aggagcagtt gaatatccag atccaattgc acagaaaaca tgttttatca 2940
tcctctcaaa gttggtagaa ctctggggag gtaaagatgg accagtggga tttgctgatt 3000
ttgtttataa gcacattgtc cccgcatgtt tcctagcacc tttaaaacaa acctttgacc 3060
tggcagatgc acaaacagta ttggctttat ctgagtgtgc agtgacactg aaaacaattc 3120
atctcaaacg gggcccagaa tgtgttcagt atcttcaaca agaatacctg ccctccttgc 3180
aagtagctcc agaaataatt caggagtttt gtcaagcgct tcagcagcct gatgctaaag 3240
tttttaaaaa ttacttaaag gtgttcttcc agagagcaaa gccctgagga ctggatttcc 3300
ctgtgcctac ttcatgatca tgaattccag ttaatttata aagaggcgat ttttgtgtgc 3360
cattcacact ggtctttttc acattgtttt gagcttattg cagtatatgt tttgggattt 3420
ttctgtaaaa tgggtgtaat tttcctaata caggtatgta acaacaaaag aagttgcctg 3480
catgccggtc caaattgttc tgtataaaga tgctcttaaa agacacaaga gttatcctag 3540
aaccttaatt cttttttatt tgaaatttta agtcaagtcc tttataaaga ccatagcagt 3600
ggaaaacagt gtacttttta aaaaattgct gaatataaaa tctttgaaaa ttttctttat 3660
gtgtgaagac acaaagtatg ggggaagaca gcaatcaaaa ctaacttttt gtagatagcc 3720
atttcatttc tttaaactgt ttcaacgcca atatgtattc tacaaaagag aatggtttta 3780
ggctccagtg ttatactttt ttttatatat atatataaaa ataaacttta cgtt 3834
<210> 28
<211> 2178
<212> DNA
<213> Homo sapiens
<220>
31/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
<221> misc_feature
<223> Incyte Identification No.: 2263514CB1
<400> 28
cagctcccta agcggttgtc accgctggag acggttggga gaaccgttgt ggcgagcgct 60
acacgaggca aacgacttct cccttctttg aactggaccc cgcgagcacc agagtcggcg 120
taactatcgc ctgacaggca tttaaatcaa acggtattga gatggattgg gttatgaaac 180
ataatggtcc aaatgacgct agtgatggga cagtacgact tcgtggacta ccatttggtt 240
gcagcaaaga ggaaatagtt cgagttcttt caaggtatat tgagatcttc agaagtagca 300
ggagtgaaat caaaggattt tatgatccac caagaagatt gctgggacag cgaccgggac 360
catatgatag accaatagga ggaagagggg gttattatgg agctgggcgt ggaagttatg 420
gaggttttga tgactatggt ggctataata attacggcta tgggaatgat ggctttgatg 480
acagaatgag agatggaaga ggtatgggag gacatggcta tggtggagct ggtgatgcaa 540
gttcaggttt tcatggtggt catttcgtac atatgagagg gttgcctttt cgtgcaactg 600
aaaatgccat tgctaatttc ttctcaccac taaatccaat acgagttcat attgatattg 660
gagctgatgg cagagccaca ggagaagcag atgtagagtt tgtgacacat gaagatgcag 720
tagctgccat gtctaaagat aaaaataaca tgcaacatcg atatattgaa ctcttcttga 780
attctactcc tggaggcggc tctggcatgg gaggttctgg aatgggaggc tacggaagag 840
atggaatgga taatcaggga ggctatggat cagttggaag aatgggaatg gggaacaatt 900
acagtggagg atatggtact cctgatggtt tgggtggtta tggccgtggt ggtggaggca 960
gtggaggtta ctatgggcaa ggcggcatga gtggaggtgg atggcgtggg atgtactgaa 1020
agcaaaaaca ccaacataca agtcttgaca acagcatctg gtctactaga ctttcttaca 1080
gatttaattt cttttgtatt ttaagaactt tataatgact gaaggaatgt gttttcaaaa 1140
tattatttgg taaagcaaca gattgtgatg ggaaaatgtt ttctgtaggt ttatttgttg 1200
catactttga cttaaaaata aatttttata ttcaaaccac tgatgttgat actttttata 1260
tactagttac tcctaaagat gtgctgcctt cataagattt gggttgatgt attttactat 1320
tagttctaca agaagtagtg tggtgtaatt ttagaggata atggttcacc tctgcgtaaa 1380
ctgcaagtct taagcagaca tctggaatag agcttgacaa ataattagtg taactttttt 1440
ctttagttcc tcctggacaa cactgtaaat ataaagccta aagatgaagt ggcttcagga 1500
gtataaattc agctaattat ttctatatta ttatttttca aatgtcattt atcaggcata 1560
gctctgaaac attgatgatc taagaggtat tgatttctga atattcataa ttgtgttacc 1620
tgggtatgag agtgttggaa gctgaattct agccctagat tttggagtaa aaccccttca 1680
gcacttgacc gaaataccaa aaatgtctcc aaaaaattga tagttgcagg ttatcgcaag 1740
atgtcttaga gtagggttaa ggttctcagt gacacaagaa ttcagtatta agtacatagg 1800
tatttactat ggagtataat tctcacaatt gtattttcag ttttctgccc aatagagttt 1860
aaataactgt ataaatgatg actttaaaaa aatgtaagca acaagtccat gtcatagtca 1920
ataaaaacaa tcctgcagtt gggttttgta tctgatccct gcttggagtt ttagtttaaa 1980
gaatctatat gtagcaagga aaaggtgctt tttaatttta atccctttga tcaatatggc 2040
ttttttccaa attggctaat ggatcaaaat gaaacctgtt gatgtgaatt cagttattga 2100
acttgttact tgtttttgcc agaaatgtta ttaataaatg tcaatgtggg agataaaaaa 2160
aaaaaaaaaa aaactggg 2178
<210> 29
<211> 1503
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2738270CB1
<400> 29
acgacctttt ggccaggtta gggagggggc gacgctgaga tgggggcggc ggcggcggaa 60
gcggatcgca ctctctttgt gggcaacctt gaaacgaaag tgaccgagga gctccttttc 120
gagcttttcc accaggctgg gccagtaata aaggtgaaaa ttccaaaaga taaggatggt 180
aaaccaaagc agtttgcgtt tgtgaatttc aaacatgaag tgtctgttcc ttatgcaatg 240
32/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
aatctactta atggaatcaa actttatgga aggcctatca aaattcaatt tagatcagga 300
agtagtcatg ccccacaaga tgtcagtttg tcatatcccc aacatcatgt tggaaattca 360
agccctacct ccacatctcc tagcagcagg tacgaaagga ctatggataa catgacttca 420
tcagcacaga taattcagag atctttctct tctccagaaa attttcagag acaagcagtg 480
atgaacagtg ctttgagaca aatgtcatat ggtggaaaat ttggttcttc acctctggat 540
caatcaggat tttcaccatc agttcaatca cacagtcata gtttcaatca gtcttcaagc 600
tcccagtggc gccaaggtac accatcatca cagcgtaaag tcagaatgaa ttcttatccc 660
tacctagcag atagacatta tagccgggaa cagcgttaca ctgatcatgg gtctgaccat 720
cattacagag gaaagagaga tgatttcttc tatgaagaca ggaatcatga tgactggagc 780
catgactatg ataacagaag agacagtagt agagatggaa aatggcgctc atctcgacac 840
taacacatgt taaaaggaca ttgtttttat agggtcattt taggcccttt gactaagttg 900
atatggaaat attttgttga aaaactgtac agagcagctt tacaagttgt cacattttct 960
ttataaattt ttttaaagct acagtttaat acaaaatgaa ttgcggtttt attacattaa 1020
taacctttca cctcagggtt ttatgaagag gaaagggttt tatgcaaaag aaagtgctac 1080
aattcctaat cattttagac actttaggag ggggtgaagt tgtatgataa agcagatatt 1140
ttaattattt gttatctttt tgtattgcaa gaaatttctt gctagtgaat caagaaaaca 1200
tccagattga cagtctaaaa tggctactgg tattttagtt aattcaaaaa tgaaactttt 1260
cagtgattca ctttactaac attctatttg agaaggctta ttggtaaagt ttggggataa 1320
aggcattgct taacttctta tataatttag gtataaattc tgtgacatgc tcttgagctt 1380
taccctagtt gaacatacat gtgtagattt acacatactg tttcattcta aaaattttag 1440
aattgttcat taaaacccca tttgaggtat aaggtcactc aggaaggtta aaatatctcc 1500
acc 1503
<210> 30
<211> 2548
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2824412CB1
<400> 30
tctgggagtc cgaccaggca tgcctcctca gcctcagggg cctgcaccct tacgtcgtcc 60
tgactcatct gatgaccgtt atgtaatgac aaaacatgcc accatttatc caactgaaga 120
ggagttacag gcagttcaga aaattgtttc tattactgaa cgtgctttaa aactcgtttc 180
agacagtttg tctgaacatg agaagaacaa gaacaaagag ggagatgata agaaagaggg 240
aggtaaagac agagctttga aaggagtttt gcgagtggga gtattggcaa aaggattact 300
tctccgagga gatagaaatg tcaaccttgt tttgctgtgc tcagagaaac cttcaaagac 360
attattaagc cgtattgcag aaaacctacc caaacagctt gctgttataa gccctgagaa 420
gtatgacata aaatgtgctg tatctgaagc ggcaataatt ttgaattcat gtgtggaacc 480
caaaatgcaa gtcactatca cactgacatc tccaattatt cgagaagaga acatgaggga 540
aggagatgta acctcgggta tggtgaaaga cccaccggac gtcttggaca ggcaaaaatg 600
ccttgacgct ctggctgctc tacgccacgc taagtggttc caggctagag ctaatggtct 660
gcagtcctgt gtgattatca tacgcattct tcgagacctc tgtcagcgag ttccaacttg 720
gtctgatttt ccaagctggg ctatggagtt actagtagag aaagcaatca gcagtgcttc 780
tagccctcag agccctgggg atgcactgag aagagttttt gaatgcattt cttcagggat 840
tattcttaaa ggtagtcctg gacttctgga tccttgtgaa aaggatccct ttgatacctt 900
ggcaacaatg actgaccagc agcgtgaaga catcacatcc agtgcacagt ttgcattgag 960
actccttgca ttccgccaga tacacaaagt tctaggcatg gatccattac cgcaaatgag 1020
ccaacgtttt aacatccaca acaacaggaa acgaagaaga gatagtgatg gagttgatgg 1080
atttgaagct gaggggaaaa aagacaaaaa agattatgat aacttttaaa aagtgtctgt 1140
aaatcttcag tgttaaaaaa acagatgccc atttgttggc tgtttttcat tcataataat 1200
gtctacattg aaaaatttat caagaattta aaggatttca tggaagaacc aagtttttct 1260
atgatattaa aaaatgtaca gtgttaggta ttatttgaat ggaaagacac ccaaaaaaaa 1320
aaatgtgctc cgactagggg gaaaacagta gttccgattt tttcccatta tttttatttt 1380
33/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
attttctggt tgccctagct tcccccccta tttttgtgtc ttttattaac tagtgcattg 1440
tcttattaaa tcttcactgt atttaatgca ggatgtgtgc ttcagttgct ctgtgtattt 1500
tgatatttta atttagaggt tttgtttgct ttttgacact agttgtaagt tactttgtta 1560
tagatggtat cctttacccc ttcttaatat tttacagcag tacgtttttt tgtaacgtga 1620
gactgcagag tttgtttttc tatatgtgaa ggattacaac acaaaaagtt atcctgccat 1680
tcgagtgctc agaactgaat gtttctgcag atcttgtggc atttgtctct agtgtgatat 1740
ataaaggtgt aattaagaca gagttctgtt aatctaatca agtttgctgt tagttgtgca 1800
ttagcagtat aaaagctaat atatactata tggtcttgca acagttttaa agcctctgca 1860
taattgataa taaaaatgca tgacattctt gtttttaata gacttttaaa atcataattt 1920
taggtttaac acgtagatct ttgtacagtt gactttttga catagcaagg ccaaaaataa 1980
ctttctgaat atttttttct tgtgtataag tggaaagggc atttttcaca tataagtggg 2040
ctaaccaata ttttcaaaag aacttcatca ttgtacaact aacaacagta actagccctt 2100
aattatggtg acagttcctt attggtgtgt gtgagattac tctagcaact attacagtat 2160
aacacagatg atcttctcca cacaccccat cacccagata atttacagtt ctgttaacag 2220
tgaggttgat aaagtattac tgataaaaaa ttatctaagg aaaaaaacag aaaattattt 2280
ggtgtggcca tcttacctgc ttatgtctcc tacacaaagc taaatattct agcagtgatg 2340
taatgaaaaa ttacatctta ctgttgatat atgtatgctc tggtacacag atgtcatttt 2400
gttgtcacag cactacagtg aaatacacaa aaaatgaaat tcatataatg acttaaatgt 2460
attatatgtt agaattgaca acataaacta cttttgcttt gaaatgatgt atgcttcagt 2520
aaaatcatat tcaaatttaa aaaaaaaa 2548
<210> 31
<211> B11
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 002690CB1
<400> 31
cgcatgcgtt ccctgaaatt gccgccaccg gctctacctt ccagtttcca gttccggcct 60
ccaaggggcg ggcagaagtt ggaaacatgc ggctgtcggt cgctgcagcg atctcccatg 120
gccgcgtatt tcgccgtatg ggcctcggtc ccgagtcccg catccatctg ttgcggaact 180
tgctcacagg gctggtgcgg cacgaacgca tcgaggcacc atgggcgcgt gtggacgaaa 240
tgaggggcta cgcggagaag ctcatcgact atgggaagct gggagacact aacgaacgag 300
ccatgcgcat ggctgacttc tggctcacag agaaggattt gatcccaaag ctgtttcaag 360
tactggcccc tcggtacaaa gatcaaactg ggggctacac aagaatgctg cagatcccaa 420
atcggagttt ggatcgggcc aagatggcag tgatcgagta taaagggaat tgcctcccac 480
ccctgcctct gcctcgcaga gacagccacc ttacactcct aaaccagctg ctgcagggtt 540
tgcggcagga cctcaggcaa agccaggaag caagcaacca cagctcccac acagctcaaa 600
caccagggat ttaactggat ctgaagagtc tgcagccctt aatcagtacc catgatcaca 660
ggcctttgga gcacttttac tctctgagaa gaactggagc tagagatgta aaatggacag 720
tcttgatggg gttgagaacc ttctggggag ccagatgacc ctctctttgc acaatagata 780
aaagtcttta tatgaatata aaaaaaaaaa a 811
<210> 32
<211> 1457
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 041108CB1
34/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
<400> 32
tacggaagct gggtcttctt gctgtgaggt cgcgttcccc agtgttacgg agggtccttg 60
aggcaggagt gaaaattggg tctgggggtt agtcctgggg tggaggtctg ggcacgccgg 120
gtcggacccc ctccatcttc ggttttgcac accccgcttt ccagcgcgga gtcgcggcgg 180
gtagggcggc gtcgcgtgcg tgacgtcatc cagcggcgcc tcgcgaggct ccagtggcct 240
tgacctcccg cggcgtggga ggctgcgcgg cgatgctgca gttcgtccgg gccggggcgc 300
gggcctggct tcggcctacc ggcagccagg gcctgagttc cctggcggaa gaggcagcgc 360
gtgcgaccga gaacccggag caggtggcga gcgagggtct cccggagccc gtgctgcgca 420
aagtcgagct cccggtaccc actcatcgac gcccagtgca ggcctgggtc gagtccttgc 480
ggggcttcga gcaggagcgc gtgggcctgg ccgacctgca ccccgatgtt ttcgccaccg 540
cgcccaggct ggacatactg caccaggttg ctatgtggca gaagaacttc aagagaatta 600
gctatgccaa gaccaagacg agagccgagg tgcggggcgg tggccggaag ccttggccgc 660
agaaaggcac tgggcgggcc cggcatggca gcatccgctc tccgctctgg cgaggaggag 720
gtgttgccca tggcccccgg ggccccacaa gttactacta catgctgccc atgaaggtgc 780
gggcgctggg tctcaaagtg gcactgaccg tcaagctggc ccaggacgac ctgcacatca 840
tggactccct agagctgccc accggagacc cacagtacct gacagagctg gcgcactacc 900
gccgctgggg ggactccgta ctcctcgtgg acttaacaca cgaggagatg ccacagagca 960
tcgtggaggc cacctctagg cttaagacct tcaacttgat cccggctgtt ggcctaaatg 1020
tgcacagcat gctcaagcac cagacgctgg tcctgacgct gcccaccgtc gccttcctgg 1080
aggacaagct gctctggcag gactcacgtt acagacccct ctaccccttc agcctgccct 1140
acagcgactt cccccgaccc ctaccccacg ctacccaggg cccagcggcc accccgtacc 1200
actgttgatg tgaagcacct cttctgagcc aggccgagcc cctggccgac ttgggagcct 1260
caggcccacg cccacccttc gaggaaggtg tcacctggac cccttcattc cacggaggaa 1320
gctgaggcca cagggagcgg ccatcgccat tgggaagggg cgactccacg gagagcccag 1380
acgggcttct gcatccattc cctctttttg tttttaaaat aaattgtatt tttgaatcaa 1440
ggaggaaaaa aaaaaaa 1457
<210> 33
<211> 1357
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 869138CB1
<400> 33
ctggtggcgt tcaagatgtc gaccaagaat ttccgagtca gtgacgggga ctggatttgc 60
cctgacaaaa aatgtggaaa tgtaaacttt gctagaagaa ccagctgtaa tcgatgtggt 120
cgggagaaaa caactgaggc caagatgatg aaagctgggg gcactgaaat aggaaagaca,180
cttgcagaaa agagccgagg cctatttagt gctaatgact ggcaatgtaa aacttgcagc 240
aatgtgaatt gggccagaag atcagagtgt aatatgtgta atactccaaa gtatgctaaa 300
ttagaagaaa gaacaggata tggtggtggt tttaatgaaa gagaaaatgt tgaatatata 360
gaaagagaag aatctgatgg tgaatatgat gagtttggac gtaaaaagaa aaaatacaga 420
gggaaagcag ttggtcctgc atctatatta aaggaagttg aagataaaga atcagaggga 480
gaagaagagg atgaggatga agatctttct aaatataagt tagatgagga tgaggatgaa 540
gatgacgctg atctctcaaa atataatctt gatgccagtg aagaagaaga tagtaataaa 600
aagaaatcta atagacgaag tcgctcaaag tctcgatctt cacattcacg atcttcatca 660
cgctcatcct ccccctcaag ttcaaggtct aggtccaggt cccgttcaag aagttcttcc 720
agttcgcagt caagatctcg ttccagttcc agagaacgtt cgagatctcg tgggtcgaaa 780
tcaagatcca gctccaggtc ccacaggggc tcttcttccc cacgaaaaag atcttattca 840
agttcatcat cttctcctga gaggaacaga aagagaagtc gttctagatc ttcttcatct 900
ggtgatcgca aaaaaagacg aacaagatca cggtcacccg aaagacgcca caggtcatca 960
tctggatcat cccattctgg ttcccgttca agttcaaaaa agaaataatg tattaaaatt 1020
tacatcttaa aaaaatccag tacagtgcat gaagcatatt tttaaagaag ttggtgtctt 1080
acttggtcag aagtgctaaa tctgctagta gaggtgcatg cctttcattg cttttcaaaa 1140
35/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
caatacagct gtgtttattt gtgaagttaa aagtaaatag cattttaagc cataatgtcc 1200
caaaatagat gttctgtcat tcattattta caaccatttg cttcatttaa aaccatttca 1260
gctataacaa agtactttgc ttcctaattt aaacccattt ttgtcatttc caaatacatc 1320
ctgtccattg gctaagacag gattacctag gcttgct 1357
<210> 34
<211> 1326
<212> DNA
<213> Homo Sapiens
<221> unsure
<222> 1313
<223> a or t or g or c, unknown, or other
<220>
<221> misc_feature
<223> Incyte Identification No.: 934406CB1
<400> 34
ttttcttcgg ggactatcct tgtctgatca ggcgggaaag acggtgccgc ccgacaatgc 60
gcggaggtag gagggggaag tggaggcggg agtgaagtct cgcgagaaga gtcggttgcc 120
gtagcagagc cctctagctg tgtgtgtctg aggctcggcc gcctgagccg cggacggttt 180
gctgagcccg ttagtgcgcc cggccgagac acgccgccgc catgtcccgc tacctgcgtc 240
cccccaacac gtctctgttc gtcaggaacg tggccgacga caccaggtct gaagacttgc 300
ggcgtgaatt tggtcgttat ggtcctatag ttgatgtgta tgttccactt gatttctaca 360
ctcgccgtcc aagaggattt gcttatgttc aatttgagga tgttcgtgat gctgaagacg 420
ctttacataa tttggacaga aagtggattt gtggacggca gattgaaata cagtttgccc 480
agggggatcg aaagacacca aatcagatga aagccaagga agggaggaat gtgtacagtt 540
cttcacgcta tgatgattat gacagataca gacgttctag aagccgaagt tatgaaagga 600
ggagatcaag aagtcggtct tttgattaca actatagaag atcgtatagt cctagaaaca 660
gtagaccgac tggaagacca cggcgtagca gaagccattc cgacaatgat agaccaaact 720
gcagctggaa tacccagtac agttctgctt actacacttc aagaaagatc tgaaagcgga 780
aaaagaacca aagaagggca gttcaagcga ccaaagggtg ggtggaaggt gctgcagtat 840
gaatactgta cgaatatttt gactctggtc tgaaaagata aaagaatgtt atcgaaaact 900
acatggaata attgaagtcc cttcaagttt gaaagtaagc attttaggac aaataaaagg 960
aaattcaact ttgtacttgt ggaaactaat ccctaaatat gaataggttt atattgattc 1020
atgggtaaca ggtccataat aaattattgg aaactaggat gtctgaatat caaggaagac 1080
agccatagtc tcttacagtg cctctgttgg tctgtctcaa actgaattgg gtgggaaaag 1140
gtatggtcca atataaaagt tccatttttg ccattattgg gcaaatcttg cctttgttta 1200
ttttggtgcc agtgttttct gcttaatcat ttgctttgtt ggcatctgtg tttatttact 1250
tgtacaccac atgcagttta catctgtctt aactactcct tcccaggtaa ttnccattat 1320
attgac 1326
<210> 35
<211> 3301
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 1315083CB1
<400> 35
gaagggaagt aacgtcagcc tgagaactga gtagctgtac tgtgtggcgc cttattctag 60
gcacttgttg ggcagaatgt cacacctgcc gatgaaactc ctgcgtaaga agatcgagaa 120
36/47
CA 02340277 2001-02-20
WO 00/1 I I71 PCT/US99/19361
gcggaacctc aaattgcggc agcggaacct aaagtttcag ggggcctcaa atctgaccct 180
atcggaaact caaaatggag atgtatctga agaaacaatg ggaagtagaa aggttaaaaa 240
atcaaaacaa aagcccatga atgtgggctt atcagaaact caaaatggag gcatgtctca 300
agaagcagtg ggaaatataa aagttacaaa gtctccccag aaatccactg tattaaccaa 360
tggagaagca gcaatgcagt cttccaattc agaatcaaaa aagaaaaaga agaaaaagag 420
aaaaatggtg aatgatgctg agcctgatac gaaaaaagca aaaactgaaa acaaagggaa 480
atctgaagaa gaaagtgccg agactactaa agaaacagaa aataatgtgg agaagccaga 540
taatgatgaa gatgagagtg aggtgcccag tctgcccctg ggactgacag gagcttttga 600
ggatacttcg tttgcttctc tatgtaatct tgtcaatgaa aacactctga aggcaataaa 660
agaaatgggt tttacaaaca tgactgaaat tcagcataaa agtatcagac cacttctgga 720
aggcagggat cttctagcag ctgcaaaaac aggcagtggt aaaaccctgg cttttctcat 780
ccctgcagtt gaactcattg ttaagttaag gttcatgccc aggaatggaa caggagtcct 840
tattctctca cctactagag aactagccat gcaaaccttt ggtgttctta aggagctgat 900
gactcaccac gtgcatacct atggcttgat aatgggtggc agtaacagat ctgctgaagc 960
acagaaactt ggtaatggga tcaacatcat tgtggccaca ccaggccgtc tgctggacca 1020
tatgcagaat accccaggat ttatgtataa aaacctgcag tgtctggtta ttgatgaagc 1080
tgatcgtatc ttggatgtgg ggtttgaaga ggaattaaag caaattatta aacttttgcc 1140
aacacgtaga cagactatgc tcttttctgc cacccaaact cgaaaagttg aagacctggc 1200
aaggatttct ctgaaaaagg agccattgta tgttggcgtt gatgatgata aagcgaatgc 1260
aacagtggat ggtcttgaac agggatatgt tgtttgtcct tctgaaaaga gattccttct 1320
gctctttaca ttccttaaga agaaccgaaa gaagaagctt atggtcttct tttcatcttg 1380
tatgtctgtg aaataccact atgagttgct gaactacatt gatttgcccg tcttggccat 1440
tcatggaaag caaaagcaaa ataagcgtac aaccacattc ttccagttct gcaatgcaga 1500
ttcgggaaca ctattgtgta cggatgtggc agcgagagga ctagacattc ctgaagtcga 1560
ctggattgtt cagtatgacc ctccggatga ccctaaggaa tatattcatc gtgtgggtag 1620
aacagccaga ggcctaaatg ggagagggca tgccttgctc attttgcgcc cagaagaatt 1680
gggttttctt cgttacttga aacaatccaa ggttccatta agtgaatttg acttttcctg 1740
gtctaaaatt tctgacattc agtctcagct tgagaaattg attgaaaaga attactttct 1800
tcataagtca gcccaggaag catataagtc atacatacga gcctatgatt cccattctct 1860
gaaacagatc tttaatgtta ataacctaaa tttgcctcag gttgctctgt catttggttt 1920
caaggtgcct cccttcgttg atctgaacgt caacagtaat gaaggcaagc agaaaaagcg 1980
aggaggtggt ggtggatttg gctaccagaa aaccaagaaa gttgagaaat ccaaaatctt 2040
taaacacatt agcaagaaat catctgacag caggcagttc tctcactgaa cacatgcctt 2100
cctttcatct tgaataactt tgtcctaaaa tgaatttttt ttccccttga tttaacagga 2160
tttttgtaga ctttagaatt tggacttacc taacaagagt ataaattgac ttgggttgca 2220
agcactgagc actgttactt ctatcacgtc tctcttttat ttctgggata taaaacaggc 2280
tttaagtttc ttggttgccc aagggcagag caaggaatat ctggtgtttc ttgtgatgat 2340
aatattttaa ttttaaatat ccctccctca tacaagtgta tgttaccatt ttaatataat 2400
tctttttgta cctttccttc ttgttttgtg aagatttttg tggcatggat tgctgtgctc 2460
actgctgtaa aaggtgacct agtgtactgg gcagctggtg gcggtgcaga aaagagtctc 2520
aggttatttt ttgtttttag ttatttcttg gaccttgaca gtatctaatg actcctcctg 2580
aaaatgctgc agtataaaag agcaaagagc tttgggaaat acctaagaag caccttaaga 2640
ttagggtggc attgctttta tagattcttg attttaaagc aacaggcctt tctcaggtgt 2700
tgcatttttt ggagcaaaaa ctatgggttg taatttgaat aaagtgtcac taagcagtta 2760
taacgtttga tggctggggg gtaggaagag gatggaattg agatgtttga gcctcattta 2820
catcaataga ggtgtaatgt actgcatttc ttcatttggt aacataacaa agactttcat 2880
acaaagaacg atgatgctcc tcattaagat ttgtttaatt caaggtggtt tggatttggt 2940
aagcctttgc actctgtaga gtacttagaa gacaagggca acttacttgg agttagagcc 3000
aagctgtcag acggtgccca gcacacatta atgttagctt ctttctgaga aaaaaatacc 3060
tcttccaggc cctgaaacaa aaaatacatt tgctgtgaag attgaaaatg aacaaagtta 3120
gaaaaaaaaa cagcaaaatc agtgatttag tcagatgagt ttttcgttgt aggagcactt 3180
gatttctagt gtgttttgta cagtatataa ctacaagata gtacattttg tagcagttca 3240
aagccaaagt tgctagcatc attttgctgt tgtgccagtt aatcatagga tcccattaag 3300
g 3301
<210> 36
37/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
<211> 1703
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 1444908CB1
<400> 36
gcttcccctc cgtcatccat cgctcggcgg tcttctcttc tccatgggtc tgctctgcgc 60
gttccatcga gtttctcggc catcgcgcgc ctgcgccatt gggctgtcag tcagaggcgg 120
cgtggagatc gctgggagcg gttgcggcgt gcggggagct gagttatagc tgtgacttct 180
gccctgccag gccgcacaca agctggctga cccggtttgt aaaaatggaa tttcaagcag 240
tagtgatggc agtaggtgga ggatctcgga tgacagacct aacttccagc attcccaaac 300
ctctgcttcc agttgggaac aaacctttaa tttggtaccc attgaacctg cttgagcgtg 360
ttggatttga agaagtcatt gtggttacaa ccagggatgt tcaaaaggct ctatgtgcag 420
aattcaagat gaaaatgaag ccagatattg tgtgtattcc tgatgatgct gacatgggaa 480
ctgcagattc tttgcgctac atatatccaa aacttaagac agatgtgctg gtgctgagct 540
gtgatctgat aacagacgtt gccttacatg aggttgtgga cctgtttaga gcttatgatg 600
catcacttgc tatgttgatg agaaaaggcc aagatagcat agaacctgtt cccggtcaaa 660
aggggaaaaa aaaagcagtg gagcagcgtg acttcattgg agtggacagc acaggaaaga 720
ggctgctctt catggctaat gaagcagact tggatgaaga gctggtcatt aagggatcca 780
tcctacagaa gcatcctaga atacgtttcc acacgggtct tgtggatgcc cacctctact 840
gtttgaaaaa atacatcgtg gatttcctaa tggaaaatgg gtcaataact tctatccgga 900
gtgaactgat tccatattta gtgagaaaac agttttcctc agcttcctca caacagggac 960
aagaagaaaa agaggaggat ctaaagaaaa aggagctgaa gtccttagat atctacagtt 1020
ttataaaaga agccaataca ctgaacctgg ctccctatga tgcctgctgg aatgcctgtc 1080
gaggagacag gtgggaagac ttgtccagat cacaggtgcg ctgctatgtc cacatcatga 1140
aagaggggct ctgctctcga gtgagcacac tgggactcta catggaagca aacagacagg 1200
tgcccaaatt gctgtctgct ctctgtccag aagaaccacc agtccattcg tcagcccaga 1260
ttgtcagcaa acacctggtt ggagttgaca gcctcattgg gccagagaca cagattggag 1320
agaagtcatc cattaagcgc tcagtcattg gctcatcctg tctcataaaa gatagagtga 1380
ctattaccaa ttgccttctc atgaactcag tcactgtgga ggaaggaagc aatatccaag 1440
gcagtgtcat ctgcaacaat gctgtgatcg agaagggtgc agacatcaag gactgcttga 1500
ttggaagtgg ccagaggatt gaagccaaag ctaaacgagt gaatgaggtg atcgtgggga 1560
atgaccagct catggagatc tgagttctga gcaagtcaga ctccttcctt ttggcctcca 1620
aagccacaga tgttggccgg cccacctgtt taactctgta tttatttccc aataaagaag 1680
ggcttccaaa ggcaaaaaaa aaa 1703
<210> 37
<211> 2536
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 1557481CB1
<400> 37
gacttccggg gcggcggttg catcagattc taggaagtgt ctgtagccgc agctgcgggt 60
ccgggattcc cagccatggc agattcctcc gggcagcagg gcaaaggccg gcgtgtgcag 120
ccccagtggt cccctcctgc tgggacccag ccatgcagac tccaccttta caacagcctc 180
accaggaaca aggaagtgtt catacctcaa gatgggaaaa aggtgacgtg gtattgctgt 240
gggccaaccg tctatgacgc atctcacatg gggcacgcca ggtcctacat ctcttttgat 300
atcttgagaa gagtgttgaa ggattacttc aaatttgatg tcttttattg catgaacatt 360
acggatattg atgacaagat catcaagagg gcccggcaga accacctgtt cgagcagtat 420
38/47
CA 02340277 2001-02-20
WO 00/11171 PGT/US99/19361
cgggagaaga ggcctgaagc ggcacagctc ttggaggatg ttcaggccgc cctgaagcca 480
ttttcagtaa aattaaatga gaccacggat cccgataaaa agcagatgct cgaacggatt 540
cagcacgcag tgcagcttgc cacagagcca cttgagaaag ctgtgcagtc cagactcacg 600
ggagaggaag tcaacagctg tgtggaggtg ttgctggaag aagccaagga tttgctctct 660
gactggctgg attctacact tggctgtgat gtcactgaca attccatctt ctccaagctg 720
cccaagttct gggaggggga cttccacaga gacatggaag ctctgaatgt tctccctcca 780
gatgtcttaa cccgggttag tgagtatgtg ccagaaattg tgaactttgt ccagaagatt 840
gtggacaacg gttacggcta tgtctccaat gggtctgtct actttgatac agcgaagttt 900
gcttctagcg agaagcactc ctatgggaag ctggtgcctg aggccgttgg agatcagaaa 960
gcccttcaag aaggggaagg tgacctgagc atctctgcag accgcctgag tgagaagcgc 1020
tctcccaacg actttgcctt atggaaggcc tctaagcccg gagaaccgtc ctggccgtgc 1080
ccttggggaa agggtcgtcc gggctggcat atcgagtgct r_ggccatggc aggcaccctc 1140
ctaggggctt cgatggacat tcacggaggt gggttcgacc tccggttccc ccaccatgac 1200
aatgagctgg cacagtcgga ggcctacttt gaaaacgact gctgggtcag gtacttcctg 1260
cacacaggcc acctgaccat tgcaggctgc aaaatgtcaa agtcactaaa aaacttcatc 1320
accattaaag atgccttgaa aaagcactca gcacggcagt tgcggctggc cttcctcatg 1380
cactcgtgga aggacaccct ggactactcc agcaacacca tggagtcagc gcttcaatat 1440
gagaagttct tgaatgagtt tttcttaaat gtgaaagata tccttcgcgc tcctgttgac 1500
atcactggtc agtttgagaa gtggggagaa gaagaagcag aactgaataa gaacttttat 1560
gacaagaaga cagcaattca caaagccctc tgtgacaatg ttgacacccg caccgtcatg 1620
gaagagatgc gggccttggt cagtcagtgc aacctctata tggcagcccg gaaagccgtg 1680
aggaagaggc ccaaccaggc tctgctggag aacatcgccc tgtacctcac ccatatgctg 1740
aagatctttg gggccgtaga agaggacagc tccctgggat tcccggtcgg agggcctgga 1800
accagcctca gtctcgaggc cacagtcatg ccctaccttc aggtgttatc agaattccga 1860
gaaggagtgc ggaagattgc ccgagagcaa aaagtccctg agattctgca gctcagcgat 1920
gccctgcggg acaacatcct gcccgagctt ggggtgcggt ttgaagacca cgaaggactg 1980
cccacagtgg tgaaactggt agacagaaac accttattaa aagagagaga agaaaagaga 2040
cgggttgaag aggagaagag gaagaagaaa gaggaggcgg cccggaggaa acaggaacaa 2100
gaagcagcaa agctggccaa gatgaagatt ccccccagtg agatgttctt gtcagaaacc 2160
gacaaatact ccaagtttga tgaaaatggt ctgcccacac atgacatgga gggcaaagag 2220
ctcagcaaag ggcaagccaa gaagctgaag aagctcttcg aggctcagga gaagctctac 2280
aaggaatatc tgcagatggc ccagaatgga agcttccagt gagggggcac aggactgact 2340
ttttaaacca ttgtggacta gtggctgctg tctgcctcag tgacaatgtc ccagcgctcc 2400
tatcatgttt acagtcaccc ttgggtccta aattaagagt tgtgttcatg taggttcgtg 2460
tcgtcgttgg ctctgagaca ttgataataa atttttctca acagtgaaaa aaaaaaaaaa 2520
gaaaaaaaaa aaaaaa 2536
<210> 38
<211> 1350
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 1747456CB1
<400> 38
cggaggagcc cgggcggggg ggaggaggag ggggaggagg gagcggagat ctcggggctc 60
ggagccggcc gccgctccgc tccgatcgct gtggggcttg gttttttggg ggtggggggg 120
cgggggggct cagatatgga ggcaaatggg agccaaggca cctcgggcag cgccaacgac 180
tcccagcacg accccggtaa aatgtttatc ggtggactga gctggcagac ctcaccagat 240
agccttagag actattttag caaatttgga gaaattagag aatgtatggt catgagagat 300
cccactacga aacgctccag aggcttcggt ttcgtcacgt tcgcagaccc agcaagtgta 360
gataaagtat taggtcagcc ccaccatgag ttagattcca agacgattga ccccaaagtt 420
gcatttcctc gtcgagcgca acccaagatg gtcacaagaa caaagaaaat atttgtaggc 480
gggttatctg cgaacacagt agtggaagat gtaaagcaat atttcgagca gtttggcaag 540
39/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
gtggaagatg caatgctgat gtttgataaa actaccaaca ggcacagagg gtttggcttt 600
gtcacttttg agaatgaaga tgttgtggag aaagtctgtg agattcattt ccatgaaatc 660
aataataaaa tggtagaatg taagaaagct cagccgaaag aagtcatgtt cccacctggg 720
acaagaggcc gggcccgggg actgccttac accatggacg cgttcatgct tggcatgggg 780
atgctgggat atcccaactt cgtggcgacc tatggccgtg gctaccccgg atttgctcca 840
agctatggct atcagttccc aggcttccca gcagcggctt atggaccagt ggcagcagcg 900
gcggtggcgg cagcaagagg atcaggctcc aacccggcgc ggcccggagg cttcccgggg 960
gccaacagcc caggacctgt cgccgatctc tacggccctg ccagccagga ctccggagtg 1020
gggaattaca taagtgcggc cagcccacag ccgggctcgg gcttcggcca cggcatagct 1080
ggacctttga ttgcaacggc ctttacaaat ggataccatt gagcaggtgc tttcgttgcc 1140
atctcactct gagagcatac ctggatgtcc aggcaagact gggcgaagtt tctgagtggc 1200
cctttgttta ggtgatgtcc tcagacctgg acccccacca gcctcactcc ccatcccaac 1260
cagagatggc tcacttcgga tcgagggttg actacatctc atcatctcac gaatctgctg 1320
taatataaga caacagcttt taaatgtgta 1350
<210> 39
<211> 2190
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 1748626CB1
<400> 39
cctgcgggag ccccgtgccc gtcacgccgc cgcagccttc gccctggatg ccgctgctgc 60
cgccgccgtg ggactgtccc gggagcgggc cctggactac tacgggctgt acgacgaccg 120
tgggcgcccc tatggctacc cagctgtgtg tgaggaggac ctgatgcccg aggatgacca 180
gcgggccacg cgcaacctct tcattggtaa cctggaccac agcgtatctg aggtggagct 240
gcgaagggcc ttcgagaaat atggcatcat cgaggaggtg gtcatcaaga ggcctgcccg 300
tggccagggc ggtgcctatg ccttcctcaa gttccagaac ctggacatgg cccatagggc 360
taaggtggcc atgtcgggcc gagtgattgg tcgcaacccc attaagatag gctatggcaa 420
ggccaacccc accactcgtc tctgggtggg tggcctggga cctaacacgt cactggcggc 480
tctggcccga gagtttgacc gctttgggag cattcggacc attgatcacg tcaaaggaga 540
tagctttgcc tatattcagt acgagagctt ggacgcagcc caggccgcct gtgctaaaat 600
gaggggtttt cccttgggtg gaccagaccg caggctccgc gtggattttg ccaaagcaga 660
ggagactcgg tacccccagc agtaccagcc ctcgccactc cctgtgcatt atgagctgct 720
cacagatgga tacacccggc accgcaacct ggacgccgac ctggtgcggg acaggacgcc 780
cccacacctt ctgtactcag accgagaccg gacttttttg gaaggggact ggaccagccc 840
cagtaaaagc tctgaccgcc gaaacagcct tgagggctac agtcgctcag tgcgcagccg 900
gagtggtgag cgttgggggg cagatggaga ccgtggtttg cccaagccct gggaagagag 960
gcggaaacgg agaagccttt ccagtgaccg tgggaggaca acccattcac catatgagga 1020
acggagtagg accaagggca gtgggcagca gtcagagcgg ggctccgacc gcacccctga 1080
gcgcagccgc aaggagaacc actccagtga agggaccaag gagtccagca gcaactccct 1140
cagcaacagc agacatgggg ctgaggaacg gggccaccac caccaccacc acgaggctgc 1200
agactcttcc cacgggaaga aggcaagaga cagcgagcgc aatcaccgga ccacagaggc 1260
cgagcccaag cctctggaag agccaaaaca cgagaccaaa aagctgaaga atctttcaga 1320
gtacgctcag acactacagc tgggttggaa tgggcttctg gtgttgaaaa acagctgctt 1380
ccccacgtct atgcatatcc tagaggggga ccagggggtg atcagcagtc tcctcaaaga 1440
ccacacttct gggagcaagc tgacccagct gaagatcgcc cagcgccttc gactggacca 1500
gcccaagctt gacgaggtca cacgacgcat caagcagggg agccccaacg gctatgcggt 1560
cctcttagcc acccaggcaa cccccagtgg gcttggcact gaggggatgc ccacagtaga 1620
gcccggtctg cagaggcggc ttctcaggaa cctggtctcc tacttgaaac agaagcaggc 1680
cgcaggggtg atcagcttgc cagtgggggg gtccaagggc agagacggca caggcatgct 1740
ctacgccttc ccaccctgcg acttttccca gcagtacctc cagtcagcac taaggacatt 1800
gggcaagcta gaagaagaac acatggtgat agtcatcgtc agagacactg cctagcccaa 1860
40/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
gcctgtcttt cccagcgtca tgtttgtgtc acaaaagcag ttattttaaa atctgatccc 1920
ctctctaccc taccactttg gtttgaatta tctcctgggt tattttggtt catttgggtg 1980
gggatcaaag tcctgtccac caccaaaact aagttcttag attttggggg attttttttt 2040
ttaaacgatg agaagggaat ccggttatgt tgatttctag tgtacaagat actgtctgct 2100
gtggttctgt atttttttat tttttgacca actgtatgga aagttgtcag taaaaccttt 2160
gacagaggat ggatttttaa aaaaaaaaaa 2190
<210> 40
<211> 680
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 1879135CB1
<400> 40
gtctttggct gtgtggtctt aaatgtgttt ctaatgtgtg tgtcaaataa ttacctgtta 60
aacagactgc caatctggct gaagccaatg cttctgaaga agataaaatt aaagcaatga 120
tgtcgcaatc tggccatgaa tacgacccaa tcaattacat gaagaaacct ctaggtccac 180
cacctccatc ttacacgtgt ttccgttgtg gtaaacctgg acattatatt aagaattgcc 240
caacaaatgg ggataaaaac tttgaatctg gtcctaggat taaaaagagc actggaattc 300
ccagaagttt catgatggaa gtgaaagatc ctaatatgaa aggtgcaatg cttaccaaca 360
ctggaaaata tgcaatacca actatagatg cagaagcata tgcaattggg aagaaagaga 420
aacctccctt cttaccagag gagccatctt cttcctcaga agaagatgat cctatcccag 480
atgaattgtt gtgtctcatc tgcaaggata ttatgactga tgctgttgtg attccctgct 540
gtggaaacag ttactgtgat gaatgtaaga agtgctgaat cttggaagat gtatatttta 600
gaatatttgt atttacttgg aatggctctt cccaacctca tatgttttaa taataaaata 660
aataatgttg aaaaaaaaaa 680
<210> 41
<211> 1150
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2073417CB1
<400> 41
gacggaagtg cggtgttgag cgccggcggc tcgcgcccac gctgggccgg gagtcgaaat 60
gcttcccggt gccgggagtg agcgatgagc tggcttctgt tcctggccca cagagtcgcc 120
ttggccgcct tgccctgccg ccgcggctct cgcgggttcg ggatgttcta tgccgtgagg 180
aggggccgca agaccggggt ctttctgacc tggaatgagt gcagagcaca ggtggaccgg 240
tttcctgctg ccagatttaa gaagtttgcc acagaggatg aggcctgggc ctttgtcagg 300
aaatctgcaa gcccggaagt ttcagaaggg catgaaaatc aacatggaca agaatcggag 360
gcgaaagcca gcaagcgact ccgtgagcca ctggatggag atggacatga aagcgcagag 420
ccgtatgcaa agcacatgaa gccgagcatg gagccggcgc ctccagttag cagagacacg 480
ttttcctaca tgggagactt cgtcgtcgtc tacactgatg gctgctgctc cagtaatggg 540
cgtagaaggc cgcgagcagg aatcggcgtt tactgggggc caggccatcc tttaaatgta 600
ggcattagac ttcctgggcg gcagacaaac caaagagcgg aaattcatgc agcctgcaaa 660
gccattgaac aagcaaagac tcaaaacatc aataaactgg ttctgtatac agacagtatg 720
tttacgataa atggtataac taactgggtt caaggttgga agaaaaatgg gtggaagaca 780
agtgcaggga aagaggtgat caacaaagag gactttgtgg cactggagag gcttacccag 840
gggatggaca ttcagtggat gcatgttcct ggtcattcgg gatttatagg caatgaagaa 900
41 /47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
gctgacagat tagccagaga aggagctaaa caatcggaag actgagccat gtgactttag 960
tccttgggag aacttgagcc agcggctgtc ttgctgcctg tacttactgg tgtggaaaat 1020
agcctgcagg taggaccatt gcagtgatgg gcagatgcgt ctttcacacg gaatcaggca 1080
cagtggcctt ctgtgacatg tgtttataaa aaatggttaa gtatataata aattgaacat 1140
ctttgagatt 1150
<210> 42
<211> 2545
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2129080CB1
<400> 42
ggcagggagc ggagacggag gaggaggagg gagaggctga atgttggctc gggagacgta 60
cgaggaggac cgggagtacg agagccaggc caagcgtctc aagaccgagg agggggagat 120
cgactactcg gccgaggaag gcgagaaccg ccgggaagcg acgccccggg gcgggggcga 180
tggcggcggc ggcggccgga gcttctctca gccggaggca ggtggaagtc atcataaagt 240
ttctgtttca cccgtcgtcc atgttcgagg actctgtgaa tctgtggtgg aagcagacct 300
cgtggaagcg ctggaaaaat ttgggacaat atgctatgtg atgatgatgc catttaaacg 360
acaggctcta gtggaatttg aaaacataga tagtgccaaa gaatgtgtga catttgctgc 420
agatgaaccc gtgtacattg ctggtcaaca ggcttttttc aactattcta caagcaaaag 480
gatcactcgg ccaggaaata ctgatgatcc atcaggaggc aacaaagttc ttctgctctc 540
aattcagaat ccgctttatc caattacagt ggatgtttta tatactgtat gcaaccctgt 600
tggcaaagtg caacgtattg ttatattcaa gagaaatggg atacaagcaa tggttgagtt 660
tgaatcagtc ctttgtgccc agaaagctaa agcagcactc aatggagctg atatatatgc 720
tggatgttgc acactaaaaa ttgaatatgc acggccaact cgtctaaatg ttattaggaa 780
tgacaatgac agttgggact acactaaacc atatttggga agacgagata gaggaaaggg 840
tcgccagaga caagccattt tgggagaaca cccttcttcg tttagacatg atggctatgg 900
atcccatggt ccattattgc ctttaccaag tcgttacaga atgggctctc gagatacacc 960
tgaacttgtt gcttatccat taccacaggc ttcttcctct tacatgcatg gaggaaatcc 1020
ctctggttca gttgtaatgg ttagtggatt acatcaacta aaaatgaatt gttcaagagt 1080
cttcaacctg ttctgcttat atggaaatat tgagaaggta aaatttatga agaccattcc 1140
tggtacagca ctggtagaaa tgggtgatga gtatgctgta gaaagagctg tcacacacct 1200
taataatgtc aaattatttg ggaaaagact taatgtttgc gtgtctaaac aacattcagt 1260
tgttccaagt caaatatttg agctggagga tggtaccagc agctacaaag attttgcaat 1320
gagcaaaaat aatcgcttta caagtgctgg ccaagcatct aagaatataa tccagccacc 1380
ctcctgtgtt ttgcattatt ataatgttcc attgtgtgtc acagaagaga ccttcacaaa 1440
gttgtgtaat gaccatgaag ttcttacatt catcaaatat aaagtgtttg atgcaaaacc 1500
ttcagccaaa acactttctg ggctattaga atgggagtgc aaaactgatg cagtagaagc 1560
ccttacggca ctgaatcact atcagataag agtgccgaat ggttccaatc cctatacatt 1620
gaagctttgc ttttctacat catcccattt ataagaagag aagagcatgt tagaatttat 1680
gttcaccttt attacaattt caaagctaca cttcattaaa aaaaaatcta aaatggttga 1740
tctcatgttg ccttgcttac tttaagatcc tgttctgtaa taaacatatt ttgcettgag 1800
taaatttgtt gtaagcttaa atattgaatt gttttcattt taagatagaa tatcataatg 1860
tagactatct acagcttcat tgtagattat acagatatat gatttctaac cttattactg 1920
gaatttttct tccacagtaa aaatatattt gcattcttaa tgctaattat ctgcaagtat 1980
tttttcattg tgtaagagat taatgcaggt gaaagtattg cattttaata tagaattcct 2040
attatatgtt tagatgttta agtatgttgc agttactcat attaaacata acttgtatat 2100
ttattatttt aatgaagttt gagaataacg ttacatatgt tgaattttaa gtactacaga 2160
tttaactgat tttatatttc tgaaaggcta acagacatgg atacacgtgt acagtatgca 2220
ttcaaactta tttaaattgg tgtatttttt tttaagtcac tgtccatttg tattgacatg 2280
cctctgtttc tagtccagtt tggagatttt ataaagttat aacaatgagt taatgtgttc 2340
attttcattt gttgcatgtg acttaaatac agctagtatt tggcattgag attttaatag 2400
42/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
aggttataat taacagttcc tcttatagat aataatctgg gatccatggg tgggcttcag 2460
agaatctgtg taccccctaa aattgtatgt agaattttgg atatttacat ttttattttt 2520
taactcttgg atccatcagt aatat 2545
<210> 43
<211> 907
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2472867CBI
<400> 43
caagcttggc gtttgtttgg tggggtcaca cgcgggttca acatgcgtat cgaaaagtgt 60
tatttctgtt cggggcccat ctatcctgga cacggcatga tgttcgtccg caacgattgc 120
aaggtgttca gattttgcaa atctaaatgt cataaaaact ttaaaaagaa gcgcaatcct 180
cgcaaagtta ggtggaccaa agcattccgg aaagcagctg gtaaagagct tacagtggat 240
aattcatttg aatttgaaaa acgtagaaat gaacctatca aataccagcg agagctatgg 300
aataaaacta ttgatgcgat gaagagagtt gaagaaatca aacagaagcg ccaagctaaa 360
tttataatga acagattgaa gaaaaataaa gagctacaga aagttcagga tatcaaagaa 420
gtcaagcaaa acatccatct tatccgagcc cctcttgcag gcaaagggaa acagttggaa 480
gagaaaatgg tacagcagtt acaagaggat gtggacatgg aagatgctcc ttaaaaatct 540
ctgtaaccat ttcttttatg tacatttgaa aatgcccttt ggatacttgg aactgctaaa 600
ttattttatt ttttacataa ggtcacttaa atgaaaagcg attaaaagac atctttcctg 660
cattgccatc tacataatat cagatattac ggatgttaga ttgcatctca gtgttaaatc 720
tttactgata gatgtactta agtaaatcat gaaaattcta cttgtaacta tagaagtgaa 780
ttgtggacgt aaaatggttg tgctatttgg ataatggcac taggcagcat ttgtatagta 840
actaatggca aaaattcatg gctagtgatg tataaaataa aatattcttt gcagtaaaaa 900
aaaaaaa 907
<210> 44
<211> 1104
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2764755CB1
<400> 44
cttcgtttag gtcggctgga aattatgtcc tccgtcggtt ttccgcagtt tttccaccaa 60
gcgagatatt tttgggagtt attccctaaa taactgcatt atatgctcct ttcatgacga 120
aattgctgcc gtggagaaga ctggaggaaa ctcgaggaag agggagaagc cgacaagtgc 180
tcgacgggct aggaactgtc ctgcttgggt gttagcgttt cccgccgggc cagtaaggct 240
gagtgacccg gcgtggctac taggagaagg acgtacggtc ctgctagtag aggaatatgt 300
cgagtttctc tagggcgccc cagcaatggg ccacttttgc tagaatatgg tatctcttag 360
atgggaaaat gcagccacct ggcaaacttg ctgctatggc atctataaga cttcagggat 420
tacataaacc tgtgtaccat gcactgagtg actgtgggga tcatgttgtt ataatgaaca 480
caagacacat tgcattttct ggaaacaaat gggaacaaaa agtatactct tcgcatactg 540
gctacccagg tggatttaga caagtaacag ctgctcagct tcacctgagg gatccagtgg 600
caattgtaaa actagctatt tatggcatgc tgccaaaaaa ccttcacaga agaacaatga 660
tggaaaggtt gcatcttttt ccagatgagt atattccaga agatattctt aagaatttag 720
tagaggagct tcctcaacca cgaaaaatac ctaaacgtct agatgagtac acacaagaag 780
aaatagacgc cttcccaaga ttgtggactc cacctgaaga ttatcggcta taagagaata 840
43/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
agaattgcag aaaataacag tgaagtgatt gaaactttct tctgatgagt ttctctaacc 900
tacaggatgg agtaaaacaa ctgctacagt tcagcacctg ttttatgtgc cgaatcactg 960
tggggaaagg tcaggaaggt gtagtccttc aataggaaat tgtaattaaa atataatttt 1020
atagaaccat ttttatgtaa tctgatttga atgttatagt tgataataat aaaatcactt 1080
acttggttga ctaaaaaaaa aaaa 1104
<210> 45
<211> 910
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 2875939CB1
<400> 45
cccacgcgtc cgcgattcat agctcgcagg gtacgggcgc gcgtgcgcac tccgcagccc 60
gttcaggacc ccggcgcggg cagggcgccc acgagctggc tggctgcttg cacccacatc 120
cttctttctc tgggacctgg ggtcgcggtt acttgggctg gccggcgaac ccttgagtgg 180
cctggcgggg agcgggcctc gcgcgcctgg agggccctgt ggaacgaaga gaggcacaca 240
gcatggcaga aaaccgagag ccccgcggtg ctgtggaggc tgaactggat ccagtggaat 300
acacccttag gaaaaggctt cccagccgcc tgccccggag acccaatgac atttatgtca 360
acatgaagac ggactttaag gcccagctgg cccgctgcca gaagctgctg gacggagggg 420
cccggggtca gaacgcgtgc tctgagatct acattcacgg cttgggcctg gccatcaacc 480
gcgccatcaa catcgcgctg cagctgcagg cgggcagctt cgggtccttg caggtggctg 540
ccaatacctc caccgtggag cttgttgatg agctggagcc agagaccgac acacgggagc 600
cactgactcg gatccgcaac aactcagcca tccacatccg agtcttcagg gtcacaccca 660
agtaattgaa aagacactcc tccacttatc ccctccgtga tatggctctt cgcatgctga 720
gtactggacc tcggaccaga gccatgtaag aaaaggcctg ttccctggaa gcccaaagga 780
ctctgcattg agggtggggg taattgtctc ttggtgggcc cagttagtgg gccttcctga 840
gtgtgtgtat gcggtctgta actattgcca tataaataaa aaatcctgtt gcactagtaa 900
aaaaaaaaaa 910
<210> 46
<211> 733
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 3591363CB1
<400> 46
gttcgcgtcg tttccgtttc cggccgaggc tgcgggaaga tggcggcggc catggcagca 60
tcttccctga cggtcacctt agggcggctg gcgtccgcgt gcagccacag catcctgaga 120
ccttcggggc ccggagcagc ctccctttgg tctgcttctc gaaggttcaa ttcacagagc 180
acttcatatc taccaggata tgttcctaaa acatccctga gttcaccacc ttggccagaa 240
gttgttctgc cagacccagt tgaggagacc agacaccatg cagaggtcgt gaagaaggtg 300
aatgagatga tcgtcacggg gcagtatggc aggctctttg ccgtggtgca ctttgccagc 360
cgccagtgga aggtgacctc tgaagacctg atcttaattg gaaatgaact agaccttgcg 420
tgtggagaga gaattcgact ggagaaggtc ctgctggttg gggcagacaa cttcacgctg 480
cttggcaagc cactcctcgg aaaggatctt gttcgagtag aagccacagt cattgaaaag 540
acagaatcat ggccaagaat cattatgaga ttcaggaaaa ggaaaaactt caagaagaaa 600
agaatcgtca cgaccccgca gactgtcctc cggataaaca gcattgagat tgctccgtgt 660
ttgttgtgat taccgagtta atacttacaa aaggataaaa ataaactcct gcttcccaag 720
44/47
CA 02340277 2001-02-20
WO 00/11171 PCTNS99/19361
gaaaaaaaaa aaa 733
<210> 47
<211> 918
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 3702292CB1
<400> 47
cgcatgcggc cgagtgcggg actggggctc ctggctgtgg gtgtggtacc gaggcttcag 60
cgggtgccgc ccgcctagag ggagtggagc ggtgagcacg tcaggggtgg ggggcgcagg 120
tcaagctttc accagttttt aattctttga tggggtaaat ttgagcaatt ttctcgactt 180
gtcgacattc gttattaact gagcaggaat caggagagga acccggtcct ctccacacag 240
cccagcagag agcctacgac tagatttgca tctttacgtc ctgcgcggag gctgctacac 300
acatgcagaa gtcatgctgg tggcctggac agtgaaggga gagaagtgga tttgggagac 360
atttaggaga tggcaccaaa agcgaaggaa gctcctgctc atcctaaagc cgaagccaaa 420
gcgaaggctt taaaggccaa gaaggcagtg ttgaaaggtg tccgcagcca cacgcaaaaa 480
cagaagatcc gcatgtcact caccttcagg cggcccaaga cactgcgact ccggaggcag 540
cccagatatc ctcggaagag cacccccagg agaaacaagc ttggccacta tgctatcatc 600
aagtttccgc tggccactga gtcggccgtg aagaagatag aagaaaacaa cacgcttgtg 660
ttcactgtgg atgttaaagc caacaagcac cagatcagac aggctgtgaa gaagctctat 720
gacagtgatg tggccaaggt caccaccctg atttgtcctg ataaggagaa caaggcatat 780
gttcgacttg ctcctgatta tgatgctttc gatgttgtaa caaaattggg atcacctaaa 840
ctgagtccag ctggctaact ctaaatatat gtgtatcttt tcagcataaa aaaataatgt 900
ttttcataaa aaaaaaaa 91g
<210> 48
<211> 2680
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 3778908CB1
<400> 48
agtggaactg gatcgggttt gctgccagcg gcgtgagctt cggccgccat tttacaacag 60
ctccactcgc gccggacaca gggagcagcg agcacgcgtt tcccgcaacc cgataccatc 120
ggacaggatt tctccgcctc agcccaacgg ggagatctct ggaaacatgg ctacagaaca 180
tgttaatgga aatggtactg aagagcccat ggatactact tctgcagtta tccattcaga 240
aaattttcag acattgcttg atgctggttt accacagaaa gttgctgaaa aactagatga 300
aatttacgtt gcagggctag ttgcacatag tgatttagat gaaagagcta ttgaagcttt 360
aaaagaattc aatgaagacg gtgcattggc agttcttcaa cagtttaaag acagtgatct 420
ctctcatgtt cagaacaaaa gtgccttttt atgtggagtc atgaagactt acaggcagag 480
agaaaaacaa gggaccaaag tagcagattc tagtaaagga ccagatgagg caaaaattaa 540
ggcactcttg gaaagaacag gctacacact tgatgtgacc actggacaga ggaagtatgg 600
aggaccacct ccagattccg tttattcagg tcagcagcct tctgttggca ctgagatatt 660
tgtgggaaag atcccaagag atctatttga ggatgaactt gttccattat ttgagaaagc 720
tggacctata tgggatcttc gtctaatgat ggatccactc actggtctca atagaggtta 780
tgcgtttgtc actttttgta caaaagaagc agctcaggag gctgttaaac tgtataataa 840
tcatgaaatt cgttctggaa aacatattgg tgtctgcatc tcagttgcca acaataggct 900
ttttgtgggc tctattccta agagtaaaac caaggaacag attcttgaag aatttagcaa 960
45/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
agtaacagag ggtcttacag acgtcatttt ataccaccaa ccggatgaca agaaaaaaaa 1020
cagaggcttt tgctttcttg aatatgaaga tcacaaaaca gctgcccagg caaggcgtag 1080
gttaatgagt ggtaaagtca aggtctgggg gaatgttgga actgttgaat gggctgatcc 1140
tatagaagat cctgatcctg aggttatggc aaaggtaaaa gtgctgtttg tacgcaacct 1200
tgccaatact gtaacagaag agattttaga aaaggcattt agtcagtttg ggaaactgga 1260
acgagtgaag aagttaaaag attatgcgtt cattcatttt gatgagcgag atggtgctgt 1320
caaggctatg gaagaaatga atggcaaaga cttggaggga gaaaatattg aaattgtttt 1380
tgccaagcca ccagatcaga aaaggaaaga aagaaaagct cagaggcaag cagcaaaaaa 1440
tcaaatgtat gacgattact actattatgg tccacctcat atgccccctc caacaagagg 1500
tcgagggcgt ggaggtagag gtggttatgg atatcctcca gattattatg gatatgaaga 1560
ttattatgat tattatggtt atgattacca taactatcgt ggtggatatg aagatccata 1620
ctatggttat gaagattttc aagttggagc tagaggaagg ggtggtagag gagcaagggg 1680
tgctgctcca tccagaggtc gtggggctgc tcctccccgc ggtagagccg gttattcaca 1740
gagaggaggt cctggatcag caagaggcgt tcgaggtgcg agaggaggtg cccaacaaca 1800
aagaggccgc ggggtacgtg gtgcgagggg tggccgcggt ggaaatgtag gaggaaagcg 1860
caaagctgat gggtacaacc agccagattc caagcggcgc c:agaccaata atcagaactg 1920
gggctcccaa cccattgctc agcaaccgct ccaaggtggt gatcattctg gtaactatgg 1980
ttacaaatct gaaaaccagg agttttatca ggatactttt gggcaacagt ggaagtagaa 2040
acagtagggc ctctgtaaaa ttggagactg ataggttgat cagaaactca ccctaaatct 2100
gaacgggtgc cgctataatt tgtgacatct ggcaagattt ccctttatgt atatatttta 2160
acaatccgct tggacacgaa caaagccaca cttctaactg cttctggcga actgatttta 2220
tttttaattt ttttcaataa agatattctt agatactgaa agaaatagtt aatgagtttg 2280
catttgtgct tgagaaaatt tggctcaagt ccatttggct gtagtgtcaa cgatgtttcc 2340
agtagtgttt agatttggtg tcttcaaagg tagttgatta aaaccaagtg tgtctttaat 2400
atcttgtatc agaataactt tgtatgttac caacttaaat tgctagaata aggtaaattg 2460
atacacaact gctattttta atttagaact ttgacctaat ttgggttttc aaaaccattt 2520
tggctacttg tattctttat gctgttgttt atttcaataa aaaattcaca cctaaatgta 2580
tacttactaa aattgtgttt acaattcgtt tttcacaaaa tttcctgcaa atttggttca 2640
aattgtatag catgtcaagg ccaattaaag ggttttgtga 2680
<210> 49
<211> 2568
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 4163642CB1
<400> 49
ccaggctgcg ccagacagtg tagaacctgc ggcctcgatg tccttctccc gtgccctatt 60
gtgggctcgg ctcccggcgg ggcgccaggc tggccaccgg gcagccatct gctctgccct 120
tcgtccccac tttgggccct ttcccggggt tctggggcaa gtttctgtcc ttgccaccgc 180
ctcctcctct gcctccggtg gctccaaaat accaaacacg tccttgttcg tgcccctgac 240
tgtgaaacct cagggcccca gcgccgacgg cgacgtcggg gccgagctaa cccggcctct 300
ggacaagaat gaagtaaaga aggtcttaga caaattttac aagaggaaag aaattcagaa 360
actgggtgct gattatggac ttgatgctcg tctcttccac caagctttca taagctttag 420
aaattatatt atgcagtctc attccctgga tgtggacatt cacattgttt tgaatgatat 480
ttgcttcggt gcagctcatg cggatgattt attcccattt ttcttgagac atgccaaaca 540
aatatttcct gtgttggact gtaaggatga tctacgtaaa atcagtgact taagaatacc 600
acctaactgg tacccagatg ctagagccat gcagcggaag ataatatttc attcaggccc 660
cacaaacagt ggaaagactt atcacgcaat ccagaaatac ttctcagcaa agtctggagt 720
gtattgtggc cctctaaaat tactggcaca tgagatcttc gaaaagagta atgctgctgg 780
tgtgccatgt gacttggtga caggtgaaga gcgtgtgaca gttcagccaa atgggaaaca 840
ggcttcacat gtttcttgta cagttgagat gtgcagtgtt acaactcctt atgaagtggc 900
tgtaattgat gaaattcaaa tgattagaga tccagccaga ggatgggcct ggaccagagc 960
46/47
CA 02340277 2001-02-20
WO 00/11171 PCT/US99/19361
acttctagga ctgtgtgctg aagaggttca tttgtgtgga gaacctgctg ctattgacct 1020
ggtgatggag cttatgtaca caacggggga ggaagtggag gttcgagact ataagaggct 1080
tacccccatt tctgtgctgg accatgcact agaatcttta gataaccttc ggcctgggga 1140
ctgcattgtc tgttttagca agaatgatat ttattctgtg agtcggcaga ttgaaattcg 1200
gggattagaa tcagctgtta tatatggcag tctcccacct gggaccaaac ttgctcaagc 1260
aaaaaagttt aatgatccca atgacccatg caaaatcttg gttgctacag atgcaattgg 1320
catgggactt aatttgagca taaggagaat tattttttac tcccttataa agcccagtat 1380
caatgaaaag ggagagagag aactagaacc aatcacaacc tctcaagccc tgcagattgc 1440
tggcagagct ggcagattca gctcacggtt taaagaagga gaggttacaa caatgaatca 1500
tgaagatctc agtttattaa aggaaatttt gaagaggcct gtggatccta taagggcagc 1560
tggtcttcat ccaactgctg agcagattga aatgtttgcc taccatctcc ctgatgcaac 1620
actgtccaat ctcattgata tttttgtaga cttttcacaa gttgatgggc agtattttgt 1680
ctgcaatatg gatgatttta aattttctgc agagttgatc cagcatattc cactaagtct 1740
gcgagtgagg tatgttttct gcacagctcc tatcaacaag aagcagcctt ttgtgtgttc 1800
ttcactgtta cagtttgcca ggcagtatag caggaatgag cccctgacct ttgcatggtt 1860
acgccgatac atcaaatggc ctttacttcc acctaagaat attaaagacc tcatggatct 1920
tgaagctgtc cacgatgtct tggatcttta cttgtggcta agctaccgat ttatggatat 1980
gtttccagat gccagcctta ttcgagatct ccagaaagaa ctagatggta ttatccaaga 2040
tggtgtgcac aatatcacta aattgattaa aatgtctgag acgcataagc tgttgaattt 2100
ggagggcttt ccatcaggga gccagtcacg attgtcagga accttaaaga gccaagctag 2160
aaggacacgc ggcaccaaag ctctagggag taaagctact gagccaccca gccccgatgc 2220
aggagagctg tcccttgctt ccagattggt gcagcaagga ctcctcactc cagacatgct 2280
gaaacagcta gaaaaagagt ggatgacaca acaaactgaa cacaacaaag aaaaaacaga 2340
gtctgggact catccaaaag ggacgagaag aaagaagaag gaacctgatt cggactagtt 2400
ttctgttcct gttttttttt tttatttaat tttgcaaata~aaaatttatt ttgaatcctt 2460
tttcctcata tgcatttact ccctcctcta gtattgtggc tatctggtac tgggggattt 2520
ttggtgtgtg tgtgtgtttg tgtgtgtgtt tttttgtttg t.tttttcc 2568
<210> 50
<211> 847
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Identification No.: 4906154CB1
<400> 50
caaatggcgg atgacgccgg tgcagcgggg gggcccgggg gccctggtgg ccctgggatg 60
gggccggagg aaggccgagg ataaggagtg gatgcccgtc accaagttgg gccgcttggt 120
caaggacatg aagatcaagt ccctggagga gatctatctc ttctccctgc ccattaagga 180
atcagagatc attgatttct tcctgggggc ctctctcaag gatgaggttt tgaagattat 240
gccagtgcag aagcagaccc gtgccggcca gcgcaccagg ttcaaggcat ttgttgctat 300
cggggactac aatggccacg tcggtctggg tgttaagtgc tccaaggagg tggccaccgc 360
catccgtggg gccatcatcc tggccaagct ctccatcgtc cccgtgcgca gaggctactg 420
ggggaacaag atcggcaagc cccacactgt cccttgcaag gtgacaggcc gctgcggctc 480
tgtgctggta cgcctcatcc ctgcacccag gggcactggc atcgtctccg cacctgtgcc 540
taagaagctg ctcatgatgg ctggtatcga tgactgctac acctcagccc ggggctgcac 600
tgccaccctg ggcaacttcg ccaaggccac ctttgatgcc atttctaaga cctacagcta 660
cctgaccccc gacctctgga aggagactgt attcaccaag tctccctatc aggagttcac 720
tgaccacctc gtcaagaccc acaccagagt ctccgtgcag cggactcagg ctccagctgt 780
ggctacaaca tagggttttt atacaagaaa aataaagtga attaagcgtg ttaaaaaaaa 840
aaaaaaa 847
47/47
