Note: Descriptions are shown in the official language in which they were submitted.
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
PROTEINS REGULATING GENE EXPRESSION
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of proteins
regulating
gene expression and to the use of these sequences in the diagnosis, treatment,
and prevention of
reproductive disorders, nervous disorders, and diseases associated with cell
proliferation and
differentiation, including cancer, immune disorders, and developmental
disorders.
BACKGROUND OF THE INVENTION
Multicellular organisms are comprised of diverse cell types that differ
dramatically both in
structure and function. The identity of a cell is determined by its
characteristic pattern of gene
expression, and different cell types express overlapping but distinctive sets
of genes throughout
development. Spatial and temporal regulation of gene expression is critical
for the control of cell
proliferation, cell differentiation, apoptosis, senescence, and other cellular
processes that
contribute to organismal development. Furthermore, gene expression is
regulated in response to
extracellular signals that mediate cell-cell communication and coordinate the
activities of different
cell types. Appropriate gene regulation also ensures that cells function
efficiently by expressing
only those genes whose functions are required at a given time.
Transcription Factors
Transcriptional regulatory proteins are essential for the control of gene
expression. Some
of these proteins function as transcription factors that initiate, activate,
repress, or terminate gene
transcription. Transcription factors generally bind to the promoter, enhancer,
and upstream
regulatory regions of a gene in a sequence-specific manner, although some
factors bind regulatory
elements within or downstream of the internal coding region of a gene.
Transcription factors may
bind to a specific region of DNA singly or as a complex with other accessory
factors. (Reviewed
in Lewin, B. (1990) Genes IV, Oxford University Press, New York, NY, pp. 554-
570.)
The double helix structure and repeated sequences of DNA create topological
and
chemical features which can be recognized by transcription factors. These
features are hydrogen
bond donor and acceptor groups, hydrophobic patches, major and minor grooves,
and regular,
repeated stretches of sequence which induce distinct bends in the helix.
Typically, transcription
factors recognize specific DNA sequence motifs about 20 nucleotides in length
or less. Multiple,
adjacent transcription factor-binding motifs may be required for gene
regulation.
Many transcription factors incorporate DNA-binding structural motifs which
comprise
either a helices or f3 sheets that bind to the major groove of DNA. Four well-
characterized
-I-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
structural motifs are helix-turn-helix, zinc finger, leucine zipper, and helix-
loop-helix. Proteins
containing these motifs may act alone as monomers, or they may form homo- or
heterodimers that
interact with DNA.
The helix-turn-helix motif consists of two a helices connected at a fixed
angle by a short
chain of amino acids. One of the helices binds to the major groove. Helix-turn-
helix motifs are
exemplified by the horneobox motif which is present in homeodomain proteins.
These proteins
are critical for specifying the anterior-posterior body axis during
development and are conserved
throughout the animal kingdom. The Antennapedia and Ultrabithorax proteins of
Drosophila
melano ag_ ster are prototypical homeodomain proteins (Patio, C.O. and R.T.
Sauer ( 1992) Ann.
Rev. Biochem. 61:1053-1095).
The zinc finger motif, which binds zinc ions, generally contains tandem
repeats of about
30 amino acids consisting of periodically spaced cysteine and histidine
residues. Examples of this
sequence pattern, designated C2H2 and C3HC4, have been described (Lewin,
supra.). Zinc finger
proteins each contain an a helix and an antiparalle) Q sheet whose proximity
and conformation are
I S maintained by the zinc ion. Contact with DNA is made by the arginine
preceding the a helix and
by the second, third, and sixth residues of the a helix. Variants of the zinc
finger motif include
poorly defined cysteine-rich motifs which bind zinc or other metal ions. These
motifs may not
contain histidine residues and are generally nonrepetitive.
The bromodomain signature is an additional conserved region of about 70 amino
acids
found in a number of transcriptional regulatory proteins (ExPASy PROSITE
document PS00633;
Haynes, S.R. et al. (1992) Nucleic Acids Res. 20:2603). Although the exact
function of this
domain is unclear, it is found in the DNA-binding region of the thyroid
hormone receptor
coactivating protein. The thyroid hormone receptor is a member of the
steroid/thyroid receptor
superfamily that regulates the expression of many target genes through binding
to thyroid
hormone response elements (Tsuyoshi, M. et al (1997) J. Biol. Chem. 272:29834-
29841). The
bromodomain signature is also found in eukaroytic transcriptional initiation
factor, TFIID, a
protein essential for progression of the G 1 phase of the cell cycle.
The leucine zipper motif comprises a stretch of amino acids rich in leucine
which can
form an amphipathic a helix. This structure provides the basis for
dimerization of two leucine
zipper proteins. The region adjacent to the leucine zipper is usually basic,
and upon protein
dimerization, is optimally positioned for binding to the major groove.
The helix-loop-helix motif consists of a short a helix connected by a loop to
a longer a
helix. The loop is flexible and allows the two helices to fold back against
each other and to bind to
DNA. The transcription factor Myc contains a prototypical HLH motif.
_2_
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
Most transcription factors contain characteristic DNA binding motifs
including, but not
limited to, those described above. Variations on the above motifs and new
motifs have been and
are currently being characterized (Faisst, S. and S. Meyer (1992) Nucl. Acids
Res. 20:3-26).
Chromatin Associated Proteins
In the nucleus, DNA is packaged into chromatin, the compact organization of
which limits
the accessibility of DNA to transcription factors and plays a key role in gene
regulation (Lewin,
su ra, pp. 409-410). The compact structure of chromatin is determined and
influenced by
chromatin-associated proteins such as the histories, the high mobility group
(HMG) proteins, and
the chromodomain proteins. There are five classes of histories, H1, H2A, H2B,
H3, and H4, all of
which are highly basic, low molecular weight proteins. The fundamental unit of
chromatin, the
nucleosome, consists of 200 base pairs of DNA associated with two copies each
of H2A, H2B,
H3, and H4. HI links adjacent nucleosomes. HMG proteins are low molecular
weight,
non-histone proteins that may play a role in unwinding DNA and stabilizing
single-stranded DNA.
Chromodomain proteins play a key role in the formation of highly compacted
heterochromatin,
which is transcriptionally silent.
RNA-Associated Proteins
Much of the regulation of gene expression in eukaroytic cells occurs at the
posttranscriptional level. Messenger RNAs (mRNA) which are produced in the
cell nucleus from
primary transcripts of protein-encoding genes are processed and transported to
the cytoplasm
where the protein synthesis machinery is located. RNA-associated proteins are
a group of proteins
that participate in the processing, splicing, editing, transport,
localization, translation, stability, and
posttranscriptional regulation of mRNAs. Such proteins include RNA helicases,
splicing factors,
nucleases, and translational regulatory proteins. In addition, the nucleolus
is a highly organized
subcompartment of the nucleus which contains protein machinery specifically
dedicated to the
transcription and processing of ribosomal RNAs. The RNA-binding activity of
these proteins is
mediated by a series of RNA-binding motifs identified within them. These
domains include the
RNP motif, the arginine-rich motif, the RGG box, and the ICH motif. (Reviewed
in Burd, C. G.
and Dreyfuss, G. (1994) Science 265:615 - 621.) The RNP motif is the most
widely found
and best characterized of these motifs. It is composed of 90-100 amino acids
which form an
RNA-binding domain, and is found in one or more copies in proteins that bind
pre-mRNA,
mRNA, pre-ribosomal RNA, and small nuclear RNAs. The RNP motif is composed of
two short
sequences (RNP-1 and RNP-2) and a number of other mostly hydrophobic,
conserved amino acids
interspersed throughout the motif (Burd, supra; ExPASy PROSITE document
PDOC0030).
Diseases and disorders related to aerie re ulation
-3-
CA 02329685 2000-12-11
WO 99/64596 PG"T/US99/13281
Many neoplastic disorders in humans can be attributed to inappropriate gene
expression.
Malignant cell growth may result from either excessive expression of tumor
promoting genes or
insufficient expression of tumor suppressor genes (Cleary, M.L. (1992) Cancer
Surv. 15:89-104).
Chromosomal translocations may also produce chimeric loci which fuse the
coding sequence of
one gene with the regulatory regions of a second unrelated gene. Such an
arrangement likely
results in inappropriate gene transcription. The Wilms tumor suppressor
gene~product, WT1, is a
protein containing a DNA-binding domain consisting of four zinc fingers and a
proline-glutamine
rich region capable of regulating transcription (ExPASy PROSITE document
PR00049).
Deletions of the WT1 gene, or point mutations which destroy the DNA-binding
activity of the
protein, are assiociated with development of the pediatric nephroblastoma,
Wilms tumor, and
Denys-Drash syndrome (Rauscher, F.J. (1993) FASEB J. 7:896-903}.
Certain proteins enriched in glutamine are associated with various
neurological disorders
including spinocerebellar ataxia, bipolar effective disorder, schizophrenia
and autism (Margolis,
R.L. et al. (1997) Human Genetics 100:114-122). These proteins contain regions
with as many as
1 S or more consecutive glutamine residues and may function as transcription
factors with a
potential role in regulation of neurodevelopment or neuroplasticity.
The immune system responds to infection or trauma by activating a cascade of
events that
coordinate the progressive selection, amplification, and mobilization of
cellular defense
mechanisms. A complex and balanced program of gene activation and repression
is involved in
this process. However, hyperactivity of the immune system as a result of
improper or insufficient
regulation of gene expression may result in considerable tissue or organ
damage. This damage is
well documented in immunological responses associated with arthritis,
allergens, heart attack,
stroke, and infections (Harrison's Princ~les of Internal Medicine, l3/e,
McGraw Hill, Inc. and
Teton Data Systems Software, 1996). In particular, a zinc finger protein
termed Staf50 (for
Stimulated trans-acting factor of 50 kDa) is a transcriptional regulator and
is induced in various
cell lines by interferon-I and -II. Staf50 appears to mediate the antiviral
activity of interferon by
down-regulating the viral transcription directed by the long terminal repeat
promoter region of
human immunodeficiency virus type-1 in transfected cells (Tissot, C. (1995) J.
Biol. Chem.
270:14891-14898).
Furthermore, the generation of multicellular organisms is based upon the
induction and
coordination of cell differentiation at the appropriate stages of development.
Central to this
process is differential gene expression, which confers the distinct identities
of cells and tissues
throughout the body. Failure to regulate gene expression during development
could result in
developmental disorders.
The discovery of new proteins regulating gene expression and the
polynucleotides
-4-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
encoding them satisfies a need in the art by providing new compositions which
are useful in the
diagnosis, prevention, and treatment of reproductive disorders, nervous
disorders, and diseases
associated with cell proliferation and differentiation, including cancer,
immune disorders, and
developmental disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, proteins
regulating gene
expression, referred to collectively as "PRGE" and individually as "PRGE-1 ",
"PRGE-2",
"PRGE-3 ", "PRGE-4", "PRGE-5", "PRGE-6", "PRGE-7", "PRGE-8", "PRGE-9", "PRGE-
10",
"PRGE-11 ", "PRGE-12", "PRGE-13", "PRGE-14", "PRGE-15", "PRGE-16", "PRGE-17",
"PRGE-18", "PRGE-19", "PRGE-20", "PRGE-21 ", "PRGE-22", "PRGE-23 ", "PRGE-24",
"PRGE-25", "PRGE-26", "PRGE-27", "PRGE-28", "PRGE-29", "PRGE-30",and "PRGE-31
". 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, SEQ ID N0:2, SEQ
ID N0:3, SEQ
ID N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ
ID
NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID NO:15,
SEQ
ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID N0:20, SEQ ID
N0:21,
SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:25, SEQ ID N0:26, SEQ ID
N0:27, SEQ ID N0:28, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31 (SEQ ID NO:1-31
), 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:1-31, and fragments thereof. The invention also provides an isolated and
purified
polynucleotide encoding the polypeptide comprising an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-31, and fragments thereof. The invention also
includes an
isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity
to the polynucleotide encoding the polypeptide comprising an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-31, 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:1-31,
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:1-31, and
fragments thereof.
The invention also provides an isolated and purified polynucleotide comprising
a
-5-
CA 02329685 2000-12-11
WO 99/64596 PGT/US99/13281 _
polynucleotide sequence selected from the group consisting of SEQ ID N0:32,
SEQ ID N0:33,
SEQ ID N0:34, SEQ ID N0:35, SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID
N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44,
SEQ
ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID
NO:50,
S SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID
N0:56, SEQ ID N0:57, SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61,
SEQ
ID N0:62 (SEQ ID N0:32-62), and fragments thereof. The invention further
provides an isolated
and purified polynucleotide variant having at least 90% polynucleotide
sequence identity to the
polynucleotide sequence selected from the group consisting of SEQ ID N0:32-62,
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 N0:32-62, 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 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:1-31, and fragments thereof. In 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
ID NO:1-31, 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:1-31, 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 PRGE, the method comprising.administering
to a subject in
need of such treatment an effective amount of a pharmaceutical composition
comprising a
-6-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
substantially purified polypeptide having the amino acid sequence selected
from the group
consisting of SEQ ID NO:I-31, 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 PRGE, 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: I-31, and fragments
thereof.
BRIEF DESCRIPTION OF THE TABLES
IO Table 1 shows nucleotide and polypeptide sequence identification numbers
(SEQ ID NO),
clone identification numbers (clone ID), cDNA libraries, and cDNA fragments
used to assemble
full-length sequences encoding PRGE.
Table 2 shows features of each polypeptide sequence including potential
motifs,
homologous sequences, and methods and algorithms used for identification of
PRGE.
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
Incyte
cDNA clones encoding PRGE were isolated.
Table 5 shows the programs, their descriptions, references, and threshold
parameters used
to analyze PRGE.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is
understood that this invention is not limited to the particular machines,
materials and methods
described, as these may vary. It is also to be understood that the terminology
used herein is for the
purpose of describing particular embodiments only, and is not intended to
limit the scope of the
present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a,"
"an," and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such host cells,
and a reference to "an
antibody" is a reference to one or more antibodies and equivalents thereof
known to those skilled
in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
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
"PRGE" refers to the amino acid sequences of substantially purified PRGE
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 PRGE, increases
or
prolongs the duration of the effect of PRGE. Agonists may include proteins,
nucleic acids,
carbohydrates, or any other molecules which bind to and modulate the effect of
PRGE.
An "allelic variant" is an alternative form of the gene encoding PRGE. 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 alone,
or in combination
with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding PRGE include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polynucleotide the same as
PRGE or a polypeptide with at least one functional characteristic of PRGE.
Included within this
definition are polymorphisms which may or may not be readily detectable using
a particular
oligonucleotide probe of the polynucleotide encoding PRGE, and improper or
unexpected
hybridization to allelic variants, with a locus other than the normal
chromosomal locus for the
polynucleotide sequence encoding PRGE. 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 PRGE. 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 PRGE 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
-8-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
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" or "amino acid sequence" refer to an oligopeptide,
peptide,
S 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 PRGE 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 PRGE. 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 PRGE,
decreases the
amount or the duration of the effect of the biological or immunological
activity of PRGE.
Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or
any other molecules
which decrease the effect of PRGE.
The term "antibody" refers to intact molecules as well as to fragments
thereof, such as
Fab, F(ab')z, and Fv fragments, which are capable of binding the epitopic
determinant. Antibodies
that bind PRGE polypeptides can be prepared using intact polypeptides or using
fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide
used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived
from the translation of
RNA, or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly
used carriers that are chemically coupled to peptides include bovine serum
albumin, thyroglobulin,
and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to
immunize the animal.
The term "antigenic determinant" refers to that 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
_g_
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
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 PRGE, 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" or "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" or 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
PRGE or
fragments of PRGE may be employed as hybridization probes. The probes may be
stored in
freeze-dried form and may be associated with a stabilizing agent such as a
carbohydrate. In
hybridizations, the probe may be deployed in an aqueous solution containing
salts (e.g., NaCI),
detergents (e.g., sodium dodecyl sulfate; SDS}, and other components (e.g.,
Denhardt's solution,
dry milk, salmon sperm DNA, etc.).
"Consensus sequence"refers to a nucleic acid sequence which has been
resequenced to
resolve uncalled bases, extended using 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 PRGE, by
-lo-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
northern analysis is indicative of the presence of nucleic acids encoding PRGE
in a sample, and
thereby correlates with expression of the transcript from the polynucleotide
encoding PRGE.
A "deletion"refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to 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).
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" or "% 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 ME(iALIGN
program (DNASTAR,
Madison WI) 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.) The clustal algorithm groups sequences into clusters by
examining the
distances between all 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
-11-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
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., Cot or Rnt 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" or "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively,
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" or "array element" in a microarray context, refer to
hybridizable
polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of PRGE. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of PRGE.
The phrases "nucleic acid" or "nucleic acid sequence," as used herein, refer
to a
-12-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 -
nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These
phrases also refer to
DNA or RNA of genomic or synthetic origin which may be single-stranded or
double-stranded
and may represent the sense or the antisense strand, to peptide nucleic acid
(PNA), or to any
DNA-like or RNA-Iike material. In this context, "fragments" refers to those
nucleic acid
sequences which, comprise a region of unique polynucleotide sequence that
specifically identifies
SEQ ID N0:32-62, for example, as distinct from any other sequence in the same
genome. For
example, a fragment of SEQ ID N0:32-62 is useful in hybridization and
amplification
technologies and in analogous methods that distinguish SEQ ID N0:32-62 from
related
polynucleotide sequences. A fragment of SEQ ID N0:32-62 is at least about 15-
20 nucleotides in
length. The precise length of the fragment of SEQ ID N0:32-62 and the region
of SEQ ID
N0:32-62 to which the fragment corresponds are routinely determinable by one
of ordinary skill
in the art based on the intended purpose for the fragment. Alternatively, 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" or "operably linked" refer to functionally
related nucleic
acid sequences. A promoter is operably associated or operably linked with a
coding 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 linked to the
sequence encoding the
polypeptide but still bind to operator sequences that control expression of
the polypeptide.
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 oligonucieotide of at least about 5 nucleotides in length linked
to a peptide backbone
of amino acid residues ending in lysine. The terminal lysine confers
solubility to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding PRGE, or fragments thereof, or PRGE 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" or "specifically binding" refer to that
interaction between a
-13-
CA 02329685 2000-12-11
WO 99164596 PCT/US99/13281
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 specif c 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 cancentration 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,
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 PRGE polypeptides refers to an amino acid sequence that is
altered by one
-19-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 -
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 PRGE. 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 polypeptide 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
polymorphisms" (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.
THE INVENTION
The invention is based on the discovery of new human proteins regulating gene
expression
{PRGE), the polynucleotides encoding PRGE, and the use of these compositions
for the diagnosis,
treatment, or prevention of reproductive disorders, nervous disorders, and
diseases associated with
cell proliferation and differentiation, including cancer, immune disorders,
and developmental
disorders.
Table 1 lists the Incyte Clones used to derive full length nucleotide
sequences encoding
PRGE. Columns 1 and 2 show the sequence identification numbers (SEQ ID NO) of
the amino
acid and nucleic acid sequences, respectively. Column 3 shows the Clone ID of
the Incyte Clone
in which nucleic acids encoding each PRGE were identified, and column 4, the
cDNA libraries
from which these clones were isolated. Column 5 shows Incyte clones, their
corresponding cDNA
libraries, and shotgun sequences. The clones and shotgun sequences are part of
the consensus
nucleotide sequence of each PRGE. The regions of the full-length nucleotide
sequence of each
PRGE to which the clones and shotgun sequences correspond are listed in Column
5. The clones
and fragments are useful as fragments in hybridization technologies.
-15-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
The columns of Table 2 show various properties of the polypeptides of the
invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3, potential phosphorylation sites; column 4, potential
glycosylation sites;
column 5, the amino acid residues comprising signature sequences and motifs;
column 6, the
identity of each protein; and column 7, 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 PRGE. The first column of Table
3 lists the
nucleotide sequence identifiers. The second column lists tissue categories
which express PRGE as
a fraction of total tissue categories expressing PRGE. The third column lists
the diseases,
disorders, or conditions associated with those tissues expressing PRGE. The
fourth column lists
the vectors used to subclone the cDNA library.
The following fragments of the nucleotide sequences encoding PRGE are useful
in
hybridization or amplification technologies to identify SEQ ID N0:56-62 and to
distinguish
between SEQ ID N0:56-62 and related polynucleotide sequences. The useful
fragments are the
fragment of SEQ ID N0:56 from about nucleotide 1675 to about nucleotide 1719;
the fragment of
SEQ ID N0:57 from about nucleotide 379 to about nucleotide 423; the fragment
of SEQ ID
N0:58 from about nucleotide 596 to about nucleotide 640; the fragment of SEQ
ID N0:59 from
about nucleotide 219 to about nucleotide 263; the fragment of SEQ ID N0:60
from about
nucleotide 732 to about nucleotide 776; the fragment of SEQ ID N0:61 from
about nucleotide 197
to about nucleotide 244; and the fragment of SEQ ID N0:62 from about
nucleotide 217 to about
nucleotide 261.
The invention also encompasses PRGE variants. A preferred PRGE 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 PRGE amino acid sequence, and which
contains at least one
functional or structural characteristic of PRGE.
The invention also encompasses polynucleotides which encode PRGE. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence
selected from the group consisting of SEQ ID N0:32-62, which encodes PRGE.
3U The invention also encompasses a variant of a polynucleotide sequence
encoding PRGE.
In particular, such a variant polynucleotide sequence will have at least about
80%, more preferably
at least about 90%, and most preferably at least about 95% polynucleotide
sequence identity to the
polynucleotide sequence encoding PRGE. A particular aspect of the invention
encompasses a
variant of a polynucleotide sequence comprising a sequence selected from the
group consisting of
SEQ ID N0:32-62 which has at least about 80%, more preferably at least about
90%, and most
-16-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 -
preferably at least about 95% polynucleotide sequence identity to a nucleic
acid sequence selected
from the group consisting of SEQ ID N0:32-62. Any one of the polynucleotide
variants described
above can encode an amino acid sequence which contains at least one functional
or structural
characteristic of PRGE.
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 PRGE, 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 PRGE, and all such variations
are to be considered
as being specifically disclosed.
Although nucleotide sequences which encode PRGE and its variants are
preferably
capable of hybridizing to the nucleotide sequence of the naturally occurring
PRGE under
appropriately selected conditions of stringency, it may be advantageous to
produce nucleotide
sequences encoding PRGE 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 PRGE 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 PRGE
and
PRGE 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 PRGE or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:32-62 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
-17-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
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, 50 mM trisodium
citrate, 1% SDS, 35% formamide, and 100 ~g/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, 1.5 mM trisodium citrate, and 0.1% SDS. In a most
preferred embodiment,
wash steps will occur at 68°C in 15 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
fragment of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq
polymerise
(Perkin-Elmer), thermostable T7 polymerise (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerises 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, Witertown MA) and the
ABI
CATALYST 800 (Perkin-Elmer). Sequencing is then carried out using either ABI
373 or 377
-18-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
DNA sequencing systems (Perkin-Elmer) or the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA). The resulting sequences are analyzed using
a variety of
algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997)
Short Protocols in
Molecular Biolo~y, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
(1995) Molecular
Biolog~and Biotechnoloev, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding PRGE 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.
1:111-119.) In
this method, multiple restriction enzyme digestions and ligations may be used
to insert an
engineered double-stranded sequence into a region of unknown sequence before
performing PCR.
Other methods which may be used to retrieve unknown sequences are known in the
art. (See, e.g.,
Parker, J.D. et al. ( 1991 ) Nucleic Acids Res. 19:3055-306). 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.
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
-19-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
detection of the emitted wavelengths. Outputllight 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 PRGE may be cloned in recombinant DNA molecules that direct
expression of
PRGE, 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
PRGE.
The nucleotide sequences of the present invention can be engineered using
methods
generally known in the art in order to alter PRGE-encoding sequences for a
variety of purposes
including, but not limited to, modification of the cloning, processing, and/or
expression of the
gene product. DNA shuffling by random fragmentation and PCR reassembly of gene
fragments
and synthetic oligonucleotides may be used to engineer the nucleotide
sequences. For example,
oligonucleotide-mediated site-directed mutagenesis may be used to introduce
mutations that create
new restriction sites, alter glycosylation patterns, change codon preference,
produce splice
variants, and so forth.
In another embodiment, sequences encoding PRGE 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. 21 S-223, and Horn, T. et al. ( 1980) Nucl. Acids Res.
Symp. Ser. 225-232.)
Alternatively, PRGE 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
the ABI 431A Peptide Synthesizer (Perkin-Elmer). Additionally, the amino acid
sequence of
PRGE, or any part thereof, may be altered during direct synthesis and/or
combined with sequences
from other proteins, or any part thereof, to produce a variant polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
3U 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 PRGE, the nucleotide sequences
encoding PRGE
or derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which
-20-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
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 PRGE. Such elements may vary in their
strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding PRGE. Such signals include the ATG initiation codon and
adjacent
sequences, e.g. the Kozak sequence. In cases where sequences encoding PRGE 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 PRGE and appropriate
transcriptional and
translational control elements. These methods include in vitro recombinant DNA
techniques,
synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook,
J. et al. (1989)
Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, Plainview
NY, ch. 4, 8, and
16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolo~v,
John Wiley & Sons,
New York NY, ch. 9, 13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express
sequences encoding PRGE. These include, but are not limited to, microorganisms
such as bacteria
transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast
transformed with yeast expression vectors; insect cell systems infected with
viral expression
vectors (e.g., baculovirus); plant cell systems transformed with viral
expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or tobacco mosaic virus,TMV) or with bacterial
expression
vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. 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 PRGE.
For example,
routine cloning, subcioning, and propagation of polynucleotide sequences
encoding PRGE can be
achieved using a multifunctional E. coli vector such as PBLUESCRIPT
(Stratagene, La Jolla CA)
or pSPORTl plasmid (Life Technologies). Ligation of sequences encoding PRGE
into the
-21-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
vector's multiple cloning site disrupts the IacZ 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 PRGE are
needed, e.g. for the production of antibodies, vectors which direct high level
expression of PRGE
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 PRGE. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH,
may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In
addition, such vectors
direct either the secretion or intracellular retention of expressed proteins
and enable integration of
foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Grant et al. ( 1987) Methods Enzymol. I 53:516-54; and Scorer, C. A. et al. (
1994) Bio/Technology
12:181-184.)
Plant systems may also be used for expression of PRGE. Transcription of
sequences
encoding PRGE may be driven viral promoters, e.g., the 355 and 19S promoters
of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
( 1987)
EMBO J. 6:307-311). Alternatively, plant promoters such as the small subunit
of RUBISCO or
heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO
J. 3:1671-1680;
Brogue, R. et al. (1984} Science 224:838-843; and Winter, J. et al. (1991}
Results Probl. Cell
Differ. 17:85-105.) These constructs can be introduced into plant cells by
direct DNA
transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill
Yearbook of
Science and Technoloszv (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 PRGE
may be ligated
into an adenovirus transcription/translation complex consisting of the late
promoter and tripartite
leader sequence. Insertion in a non-essential E 1 or E3 region of the viral
genome may be used to
obtain infective virus which expresses PRGE 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
-22-
CA 02329685 2000-12-11
WO 99/64596 PCTlUS99/13281 -
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of PRGE in cell lines is preferred. For example, sequences encoding
PRGE can be
transformed into cell lines using expression vectors which may contain viral
origins of replication
and/or endogenous expression elements and a selectable marker gene on the same
or on a separate
vector. Following the introduction of the vector, cells may be allowed to grow
for about 1 to 2
days in enriched media before being switched to selective media. The purpose
of the selectable
marker is to confer resistance to a selective agent, and its presence allows
growth and recovery of
cells which successfully express the introduced sequences. Resistant clones of
stably transformed
cells may be propagated using tissue culture techniques appropriate to the
cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk 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 chlorsulfuron 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. Mol. Biol. 150:1-14.) Additional
selectable genes have been
described, e.g., trpB and hisD, which alter cellular requirements for
metabolites. (See, e.g.,
Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.)
Visible markers,
e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), Q
glucuronidase and its substrate
13-glucuronide, or luciferase and its substrate luciferin may be used. These
markers can be used
not only to identify transformants, but also to quantify the amount of
transient or stable protein
expression attributable to a specific vector system. (See, e.g., Rhodes, C.A.
(1995) Methods Mol.
Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of
interest is also present, the presence and expression of the gene may need to
be confirmed. For
example, if the sequence encoding PRGE is inserted within a marker gene
sequence, transformed
cells containing sequences encoding PRGE can be identified by the absence of
marker gene
function. Alternatively, a marker gene can be placed in tandem with a sequence
encoding PRGE
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 PRGE
and that
-23-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
express PRGE 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 PRGE 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 PRGE 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
ImmunoloQV, Greene Pub.
Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998)
Immunochemical
IS 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 PRGE
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled
nucleotide. Alternatively, the sequences encoding PRGE, or any fragments
thereof, may be
cloned into a vector for the production of an mRNA probe. Such vectors are
known in the art, are
commercially available, and may be used to synthesize RNA probes in vitro by
addition of an
appropriate RNA polymerise such as T7, T3, or SP6 and labeled nucleotides.
These procedures
may be conducted using a variety of commercially available kits, such as those
provided by
Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable
reporter
molecules or labels which may be used for ease of detection include
radionuclides, enzymes,
fluorescent, chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors,
magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding PRGE 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 PRGE may be designed to
contain signal
sequences which direct secretion of PRGE through a prokaryotic or eukaryotic
cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
_29_
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications
ofthe polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro"
fonm 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, Bethesda MD) 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 PRGE 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 PRGE protein
containing a heterologous moiety that can be recognized by a commercially
available antibody
may facilitate the screening of peptide libraries for inhibitors of PRGE
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, (:BP, 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 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 PRGE
encoding sequence
and the heterologous protein sequence, so that PRGE 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 PRGE 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 PRGE may be produced not only by recombinant production, but also
by
direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton,
s-upra, pp. 55-60.)
Protein synthesis may be performed by manual techniques or by automation.
Automated synthesis
may be achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin-
Elmer). Various
-25-
CA 02329685 2000-12-11
WO 99/64596 PCTNS99/13281
fragments of PRGE 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 PRGE and proteins regulating gene expression. In addition,
the expression of
PRGE is closely associated with cancer and other cell pro(iferative
conditions, differentiated cells,
inflammation and the immune response, and is found in reproductive and nervous
system tissues.
Therefore, PRGE appears to play a role in reproductive disorders, nervous
disorders, and diseases
associated with cell proliferation and differentiation, including cancer,
immune disorders, and
developmental disorders. In the treatment of the above conditions associated
with increased
PRGE expression or activity, it is desirable to decrease the expression or
activity of PRGE. In the
treatment of the above conditions associated with decreased PRGE expression or
activity, it is
desirable to increase the expression or activity of PRGE.
Therefore, in one embodiment, PRGE 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 PRGE. Examples of such a disorder include, but are not limited to,
a reproductive
disorder such as disorders of prolactin production; infertility, including
tubal disease, ovulatory
defects, and endometriosis; disruptions of the estrous cycle, disruptions of
the menstrual cycle,
polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and
ovarian tumors,
uterine fibroids, autoimmune disorders, ectopic pregnancies, and
teratogenesis; cancer of the
breast, fibrocystic breast disease, and galactorrhea; disruptions of
spermatogenesis, abnormal
sperm physiology, cancer of the testis, cancer of the prostate, benign
prostatic hyperplasia,
prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and
gynecomastia; and a
nervous disorder such as epilepsy, ischemic cerebrovascular disease, stroke,
cerebral neoplasms,
Alzheimer's disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and
other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor
neuron disorders,
progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias,
multiple sclerosis
and other demyelinating diseases, bacterial and viral meningitis, brain
abscess, subdural empyema,
epidural abscess, suppurative intracranial thrombophlebitis, myelitis and
radiculitis, viral central
nervous system disease; prion diseases including kuru, Creutzfeldt-Jakob
disease, and Gerstmann-
Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the
central nervous system, cerebral palsy, neuroskeletal disorders, autonomic
nervous system
disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy
and other
-26-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 -
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis;
inherited, metabolic, endocrine, and toxic myopathies; myasthenia gravis,
periodic paralysis;
mental disorders including mood, anxiety, and schizophrenic disorders;
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, and Tourette's disorder; 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; 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 immune disorder such as acquired
immunodeficiency
syndrome (AIDS), Addison's disease, adult respiratory distress syndrome,
allergies, ankylosing
spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune
hemolytic anemia,
autoimmune thyroiditis, 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, S,jogren'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, trauma, X-
linked
agammaglobinemia of Bruton, common variable immunodeficiency (CVI), DiGeorge's
syndrome
(thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe
combined
immunodeficiency disease (SCID), immunodeficiency with thrombocytopenia and
eczema
(Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous
diseases,
hereditary angioneurotic edema, and immunodeficiency associated with Cushing's
disease; and a
developmental disorder such as renal tubular acidosis, anemia, Cushing's
syndrome,
achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy,
gonadal
dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary
abnormalities, and mental
retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary
mucoepithelial
dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-
Marie-Tooth disease
and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such
as Syndenham's
-27-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis,
congenital glaucoma,
cataract, sensorineural hearing loss, and any disorder associated with cell
growth and
differentiation, embryogenesis, and morphogenesis involving any tissue, organ,
or system of a
subject, e.g., the brain, adrenal gland, kidney, skeletal or reproductive
system.
In another embodiment, a vector capable of expressing PRGE 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 PRGE including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially
purified PRGE 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 PRGE
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of PRGE
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of PRGE including, but not limited to, those listed above.
In a further embodiment, an antagonist of PRGE may be administered to a
subject to treat
or prevent a disorder associated with increased expression or activity of
PRGE. Examples of such
disorders include, but are not limited to, those described above. In one
aspect, an antibody which
specifically binds PRGE 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 PRGE.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding PRGE may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of PRGE 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 PRGE may be produced using methods which are generally known
in the
art. In particular, purified PRGE may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind PRGE.
Antibodies to PRGE 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,
-28-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
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 PRGE or with any
fragment or
oligopeptide thereof which has immunogenic properties. Depending on the host
species, various
adjuvants may be used to increase immunological response. Such adjuvants
include, but are not
limited to, Freund's, mineral gels such as aluminum hydroxide, and surface
active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH,
and dinitrophenol.
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum
are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
PRGE 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
I S contain the entire amino acid sequence of a small, naturally occurring
molecule. Short stretches of
PRGE 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 PRGE 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
PRGE-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:
-29-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for PRGE may also be
generated.
For example, such fragments include, but are not limited to, F(ab')2 fragments
produced by
pepsin digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide
S bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries
may be constructed to
allow rapid and easy identification of monoclonal Fab fragments with the
desired specificity.
(See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the
desired specificity. Numerous protocols for competitive binding or
immunoradiometric assays
using either polyclonal or monoclonal antibodies with established
specificities are well known in
the art. Such immunoassays typically involve the measurement of complex
formation between
PRGE and its specific antibody. A two-site, monoclonal-based immunoassay
utilizing monoclonal
antibodies reactive to two non-interfering PRGE 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 PRGE. Affinity
is expressed as an
association constant, K" which is defined as the molar concentration of PRGE-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium
conditions. The K, determined for a preparation of polyclonal antibodies,
.which are
heterogeneous in their affinities for multiple PRGE epitopes, represents the
average affinity, or
avidity, of the antibodies for PRGE. The K, determined for a preparation of
monoclonal
antibodies, which are monospecific for a particular PRGE epitope, represents a
true measure of
affinity. High-affinity antibody preparations with K, ranging from about 109
to 10'2 L/mole are
preferred for use in immunoassays in which the PRGE-antibody complex must
withstand rigorous
manipulations. Low-affinity antibody preparations with K, ranging from about
106 to 10' L/mole
are preferred for use in immunopurification and similar procedures which
ultimately require
dissociation of PRGE, 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 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is preferred for use in procedures
requiring precipitation
of PRGE-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity,
and guidelines for antibody quality and usage in various applications, are
generally available.
-30-
CA 02329685 2000-12-11
WO 99/64596 PCTIUS99/13281 -
(See, e.g., Catty, supra, and Coligan et al. su ra.)
In another embodiment of the invention, the polynucleotides encoding PRGE, or
any
fragment or complement.thereof, may be used for therapeutic purposes. In one
aspect, the
complement of the polynucleotide encoding PRGE 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 PRGE. Thus, complementary
molecules
or fragments may be used to modulate PRGE 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 PRGE.
Expression vectors derived from retroviruses, adenoviruses, or herpes or
vaccinia viruses,
or from various bacterial plasmids, may be used for delivery of nucleotide
sequences to the
targeted organ, tissue, or cell population. 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 PRGE. (See, e.g., Sambrook, supra; Ausubel, 1995, su
ra.)
Genes encoding PRGE can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding PRGE. 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, 5', or
regulatory regions of the gene encoding PRGE. 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 polymerises, transcription factors, or regulatory
molecules. Recent therapeutic
advances using Mplex DNA have been described in the literature. (See, e.g.,
Gee, J.E. et al.
(1994) in Huber, B.E. and B.I. Can, Molecular and Immunologic Awroaches,
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
-31-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 -
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 PRGE..
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
IS and in vivo transcription of DNA sequences encoding PRGE. Such DNA
sequences may be
incorporated into a wide variety of vectors with suitable RNA polymerise
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,
monkeys, and most preferably, humans.
-32-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
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 PRGE, antibodies to PRGE, and mimetics, agonists, antagonists, or
inhibitors of PRGE. 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, infra-arterial,
intramedullary, intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain
suitable pharmaceutically-acceptable carriers comprising excipients and
auxiliaries which
IS 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 Remin on'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
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
-33-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
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 parenterai 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
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 foamed
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: 1 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
PRGE, 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
-39-
CA 02329685 2000-12-11
WO 99/64596 PCTIUS99/13281 _
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
PRGE or fragments thereof, antibodies of PRGE, and agonists, antagonists or
inhibitors of PRGE,
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 EDS° (the dose therapeutically effective in
50% of the population) or
LDs° (the dose lethal to 50% of the population) statistics. The dose
ratio of toxic to therapeutic
effects is the therapeutic index, and it can be expressed as the
LD5°/ED5° ratio. Pharmaceutical
compositions which exhibit large therapeutic indices are preferred. The data
obtained from cell
IS 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 /cg to 100,000 /sg, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular
cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind PRGE may be used for
the
diagnosis of disorders characterized by expression of PRGE, or in assays to
monitor patients being
-35-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
treated with PRGE or agonists, antagonists, or inhibitors of PRGE. Antibodies
useful for
diagnostic purposes may be prepared in the same manner as described above for
therapeutics.
Diagnostic assays for PRGE include methods which utilize the antibody and a
label to detect
PRGE 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 PRGE, including ELISAs, RIAs, and FACS,
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of PRGE
expression. Normal or standard values for PRGE expression are established by
combining body
fluids or cell extracts taken from normal mammalian subjects, preferably
human, with antibody to
PRGE under conditions suitable for complex formation. The amount of standard
complex
formation may be quantitated by various methods, preferably by photometric
means. Quantities of
PRGE 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 PRGE 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 PRGE
may be correlated with disease. The diagnostic assay may be used to determine
absence,
presence, and excess expression of PRGE, and to monitor regulation of PRGE
levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide sequences, including genomic sequences, encoding PRGE or
closely related
molecules may be used to identify nucieic acid sequences which encode PRGE.
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
only naturally occurring sequences encoding PRGE, 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 PRGE encoding sequences. The
hybridization
probes of the subject invention may be DNA or RNA and may be derived from the
sequence of
SEQ ID N0:32-62 or from genomic sequences including promoters, enhancers, and
introns of the
PRGE gene.
-36-
CA 02329685 2000-12-11
WO 99/64596 PCT/ITS99/13281 _
Means for producing specific hybridization probes for DNAs encoding PRGE
include the
cloning of polynucleotide sequences encoding PRGE or PRGE 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.
Polynucleotide sequences encoding PRGE may be used for the diagnosis of
disorders
associated with expression of PRGE. Examples of such a disorder include, but
are not limited to, a
reproductive disorder such as disorders of prolactin production; infertility,
including tubal disease,
ovulatory defects, and endometriosis; disruptions of the estrous cycle,
disruptions of the menstrual
cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome,
endometrial and ovarian
tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, and
teratogenesis; cancer of
the breast, fibrocystic breast disease, and galactorrhea; disruptions of
spermatogenesis, abnormal
sperm physiology, cancer of the testis, cancer of the prostate, benign
prostatic hyperplasia,
prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and
gynecomastia; and a
nervous disorder such as epilepsy, ischemic cerebrovascular disease, stroke,
cerebral neoplasms,
Alzheimer's disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and
other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor
neuron disorders,
progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias,
multiple sclerosis
and other demyelinating diseases, bacterial and viral meningitis, brain
abscess, subdural empyema,
epidural abscess, suppurative intracranial thrombophlebitis, myelitis and
radiculitis, viral central
nervous system disease; prion diseases including kuru, Creutzfeldt-Jakob
disease, and Gerstmann-
Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the
central nervous system, cerebral palsy, neuroskeletal disorders, autonomic
nervous system
disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy
and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis;
inherited, metabolic, endocrine, and toxic myopathies; myasthenia gravis,
periodic paralysis;
mental disorders including mood, anxiety, and schizophrenic disorders;
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, and Tourette's disorder; 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,
-37-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13Z81 _
primary thrombocythemia; 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 immune disorder such as acquired
immunodeficiency
syndrome (AIDS), Addison's disease, adult respiratory distress syndrome,
allergies, ankylosing
spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune
hemolytic anemia,
autoimmune thyroiditis, 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, trauma, X-
linked
agammaglobinemia of Bruton, common variable immunodeficiency (CVI), DiGeorge's
syndrome
(thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe
combined
immunodeficiency disease (SCID), immunodeficiency with thrombocytopenia and
eczema
(Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous
diseases,
hereditary angioneurotic edema, and immunodeficiency associated with Cushing's
disease; and a
developmental disorder such as renal tubular acidosis, anemia, Cushing's
syndrome,
achondroplastic dwa~sm, Duchenne and Becker muscular dystrophy, epilepsy,
gonadal
dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary
abnormalities, and mental
retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary
mucoepithelial
dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-
Marie-Tooth disease
and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such
as Syndenham's
chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis,
congenital glaucoma,
cataract, sensorineural hearing lass, and any disorder associated with cell
growth and
differentiation, embryogenesis, and morphogenesis involving any tissue, organ,
or system of a
subject, e.g., the brain, adrenal gland, kidney, skeletal or reproductive
system.
The polynucleotide sequences encoding PRGE 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
-38-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
detect altered PRGE expression. Such qualitative or quantitative methods are
well known in the
art.
In a particular aspect, the nucleotide sequences encoding PRGE may be useful
in assays
that detect the presence of associated disorders, particularly those mentioned
above. The
nucleotide sequences encoding PRGE 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 PRGE 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
PRGE, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding PRGE, 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
PRGE 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
-39-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
polynucleotide encoding PRGE, or a fragment of a polynucleotide complementary
to the
polynucleotide encoding PRGE, 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 quantitate the expression of PRGE 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. 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
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; Shalom
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. ( 1997) U.S. Patent No. 5,605,662.)
In another embodiment of the invention, nucleic acid sequences encoding PRGE
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, su ra, pp. 965-968.} Examples of genetic map data can be found in
various scientific
journals or at the Online Mendelian Inheritance in Man (OMIM) site.
Correlation between the
location of the gene encoding PRGE on a physical chromosomal map and a
specific disorder, or a
-40-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/I3281 _
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
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, PRGE, 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 PRGE 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 PRGE, or
fragments thereof, and washed. Bound PRGE is then detected by methods well
known in the art.
Purified PRGE can also be coated directly onto plates for use in the
aforementioned drug
screening techniques. Alternatively, non-neutralizing antibodies can be used
to capture the
peptide and immobilize it on a solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing antibodies capable of binding PRGE specifically compete with a
test compound for
binding PRGE. In this manner, antibodies can be used to detect the presence of
any peptide which
shares one or more antigenic determinants with PRGE.
In additional embodiments, the nucleotide sequences which encode PRGE 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
-41-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 -
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. 60/089,029, U.S. Ser. No. 60/094,575, and U.S.
Ser. No. 60/104,624,
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
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, Valencia 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, su ra, units 5.1-6.6). Reverse transcription was initiated using oligo
d(T) or random
primers. Synthetic oligonucleotide adapters were ligated to double stranded
cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most
libraries, the cDNA
was size-selected (300-1000 bp) using SEPHACRYL 51000, 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, Palo Alto CA).
Recombinant plasmids
-42-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
were transformed into competent E. coli cells including XL1-Blue, XL1-BIueMRF,
or SOLR from
Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies. II.
Isolation of cDNA Clones
Plasmids 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 REAL Prep 96
plasmid 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, piasmid DNA was amplified from host cell lysates using direct
link PCR in
a high-throughput format (Rao, V.B. (1994} Anal. Biochem. 216:1-14). Host cell
lysis and
thermal cycling steps were carried out in a single reaction mixture. Samples
were processed and
stored in 384-well plates, and the concentration of amplified plasmid DNA was
quantified
fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a
Fluoroskan II
fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
The cDNAs were prepared for sequencing using the ABI CATALYST 800 (Perkin-
Elmer)
or the HYDRA microdispenser (Robbins Scientific) or MICROLAB 2200 (Hamilton)
systems in
combination with the PTC-200 thermal cyclers (MJ Research). The cDNAs were
sequenced using
the ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer) and standard ABI
protocols, base
calling software, and kits. In one alternative, cDNAs were sequenced using the
MEGABACE
1000 DNA sequencing system (Molecular Dynamics). In another alternative, the
cDNAs were
amplified and sequenced using the ABI PRISM BIGDYE Terminator cycle sequencing
ready
reaction kit (Perkin-Elmer). In yet another alternative, cDNAs were sequenced
using solutions
and dyes from Amersham Pharmacia Biotech. Reading frames for the ESTs were
determined
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, extension, and shotgun
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 software
programs, descriptions,
references, and threshold parameters used. The first column of Table 5 shows
the tools, programs,
and algorithms used, the second column provides a brief description thereof,
the third column
presents the references which are incorporated by reference herein, and the
fourth column
presents, where applicable, the scores, probability values, and other
parameters used to evaluate
-43-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
the strength of a match between two sequences (the higher the probability the
greater the
homology). 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 GenBank primate,
rodent, mammalian,
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,
IS Prosite, and Hidden Markov Model (HMM)-based protein family databases such
as PFAM.
HMM is a probalistic approach which analyzes consensus primary structures of
gene families.
(See, e.g., Eddy, S.R. (1996) Cur. 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 N0:32-62. 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, supra, ch. 7;
Ausubel, 1995, supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or
related molecules in nucleotide databases such as GenBank or LIFESEQ database
(Incyte
Pharmaceuticals). This analysis is much faster than multiple membrane-based
hybridizations. In
addition, the sensitivity of the computer search can be modified to determine
whether any
particular match is categorized as exact or similar. The basis of the search
is the product score,
which is defined as:
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
-44-
CA 02329685 2000-12-11
WO 99/64596 PGT/US99/13281 _
within a 1% 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 PRGE occurred. Analysis involved the
categorization of cDNA
libraries by organ/tissue and disease. The organ/tissue categories included
cardiovascular,
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 PRGE Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:56-62 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, lnc.). The
reaction mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+,
(NH4)zS04, and ~i-mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia
Biotech),
ELONGASE enzyme (Life Technologies), and Pfu DNA polymerise (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
-4 5-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
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 ~I to 10 ,ul aliquot of
the reaction mixture
was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which
reactions were
successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were religated using T4 ligase (New England Biolabs, Beverly MA) into
pUC 18 vector
(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to
fill-in
restriction site overhangs, and transfected into competent E. coli cells.
Transformed cells were
selected on antibiotic-containing media, individual colonies were picked and
cultured overnight at
37°C in 384-well plates in LB/2x curb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA poiymerase (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step S: 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 DYENAMIC
energy transfer
sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or
the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequences of SEQ ID NO:56-62 are used to obtain
S'
regulatory sequences using the procedure above, oligonucleotides designed for
such extension,
and an appropriate genomic library.
The nucleic acid sequences of SEQ ID N0:32-55 were used to design
oligonucleotide
primers for extending a partial nucleotide sequence to full length. For each
nucleic acid sequence,
one primer was synthesized to initiate extension of an antisense
polynucleotide, and the other was
synthesized to initiate extension of a sense polynucleotide. Primers were used
to facilitate the
extension of the known sequence "outward" generating amplicons containing new
unknown
nucleotide sequence for the region of interest. The initial primers were
designed from the cDNA
using OLIGOTM 4.06 (National Biosciences, Plymouth, MN), or another
appropriate program, to
be about 22 to 30 nucleotides in length, to have a GC content of about SU% or
more, and to anneal
to the target sequence at temperatures of about 68 °C to about 72
°C. Any stretch of nucleotides
-46-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/I3281 _
which would result in hairpin structures and primer-primer dimerizations was
avoided.
Selected human cDNA libraries (GIBCO BRL) were used to extend the sequence. If
more
than one extension is necessary or desired, additional sets of primers are
designed to further
extend the known region.
High fidelity amplification was obtained by following the instructions for the
XL-PCRTM
kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix. PCR was
performed
using the Peltier Thermal Cycler (PTC200; M.J. Research, Watertown, MA),
beginning with 40
pmol of each primer and the recommended concentrations of al l other
components of the kit, with
the following parameters:
Step 1 94 C for 1 min (initial denaturation)
Step 2 65 C for 1 min
Step 3 68 C for 6 min
Step 4 94 C for 15 sec
Step S 65 C for 1 min
Step 6 68 C for 7 min
Step 7 Repeat steps 4 through 6 for an additional
1 S cycles
Step 8 94 C for 15 sec
Step 9 65 C for 1 min
Step 10 68 C for 7:15 min
Step 11 Repeat steps 8 through I 0 for an
additional 12 cycles
Step 12 72 C for 8 min
Step 13 4 C (and holding)
A 5 ~l to 10 ~I aliquot of the reaction mixture was analyzed by
electrophoresis on a low
concentration (about 0.6% to 0.8%) agarose mini-gel to determine which
reactions were successful
in extending the sequence. Bands thought to contain the largest products were
excised from the
gel, purified using QIAQUICKT"' (QIAGEN Inc.), and trimmed of overhangs using
Klenow
enzyme to facilitate religation and cloning.
After ethanol precipitation, the products were redissolved in 13 ul of
ligation buffer, l~cl
T4-DNA ligase (I S units) and 1/cl T4 polynucleotide kinase were added, and
the mixture was
incubated at room temperature for 2 to 3 hours, or overnight at 16° C.
Competent E, coli cells (in
/cl of appropriate media) were transformed with 3 ~1 of ligation mixture and
cultured in 80 ~I
of SOC medium. (See, e.g., Sambrook, supra, Appendix A, p. 2.) After
incubation for one hour at
37°C, the E. coli mixture was plated on Luria Bertani (LB) agar (See,
e.g., Sambrook, supr-a,
35 Appendix A, p. 1) containing carbenicillin (2x carb). The following day,
several colonies were
randomly picked from each plate and cultured in I50 ~1 of liquid LB/2x carb
medium placed in an
individual well of an appropriate commercially-available sterile 96-well
microtiter plate. The
following day, 5 ~l of each overnight culture was transferred into a non-
sterile 96-well plate and,
after dilution 1:10 with water, S /c) from each sample was transferred into a
PCR array.
40 For PCR amplification, 18 ~1 of concentrated PCR reaction mix (3.3x)
containing 4 units
-47-
CA 02329685 2000-12-11
WO 99/64596 PCT/tTS99/13281 _
of rTth DNA polymerise, a vector primer, and one or both of the gene specific
primers used for
the extension reaction were added to each well. Amplification was performed
using the following
conditions:
Step 1 94 C for 60 sec
Step 2 94 C for 20 sec
Step 3 55 C for 30 sec
Step 4 72 C for 90 sec
Step 5 Repeat steps 2 through 4 for an additional
29 cycles
Step 6 72 C for 180 sec
Step 7 4 C (and holding)
Aliquots of the PCR reactions were run on agarose gels together with molecular
weight
markers. The sizes of the PCR products were compared to the original partial
cDNAs, and
appropriate clones were selected, ligated into plasmid, and sequenced.
In like manner, the nucleotide sequences of SEQ ID NO:32-55 are used to obtain
S'
regulatory sequences using the procedure above, oligonucleotides designed for
5' extension, and
an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:32-62 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
r.Y-32P1 adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 supe~ne size exclusion dextrin bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-
based hybridization analysis of human genomic DNA digested with one of the
following
endonucleases: Ase I, Bgl II, Eco RI, Pst I, 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. After XOMAT-AR film (Eastman Kodak, Rochester NY) is exposed to the
blots to film
for several hours, hybridization patterns are compared visually.
VII. Microarrays
A chemical coupling procedure and an ink jet device can be used to synthesize
array
-48-
CA 02329685 2000-12-11
WO 99/64596 PGT/US99/13281
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; Shalon, D. et aI. ( 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 PRGE-encoding sequences, or any parts thereof,
are
used to detect, decrease, or inhibit expression of naturally occurring PRGE.
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
PRGE. To inhibit transcription, a complementary oligonucleotide is designed
from the most
unique 5' sequence and used to prevent promoter binding to the coding
sequence. To inhibit
translation, a complementary oligonucleotide is designed to prevent ribosomal
binding to the
PRGE-encoding transcript.
IX. Expression of PRGE
Expression and purification of PRGE is achieved using bacterial or virus-based
expression systems. For expression of PRGE 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 PRGE upon induction
with isopropyl beta-
-49-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
D-thiogalactopyranoside (IPTG). Expression of PRGE in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autogrraphica californica
nuclear polyhedrosis
virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin
gene of
baculovirus is replaced with cDNA encoding PRGE 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 fruyiperda (S~) 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, PRGE 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 Lponicum, 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 PRGE 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, s- u~ra, ch 10 and 16).
Purified PRGE obtained by
these methods can be used directly in the following activity assay.
X. Demonstration of PRGE Activity
PRGE activity is measured by its ability to stimulate transcription of a
reporter gene (Liu,
H.Y. et al. (1997) EMBO J. 16(17):5289-5298.) The assay entails the use of a
well characterized
reporter gene construct, LexAop LacZ, that consists of LexA DNA
transcriptional control
elements (LexAoP) fused to sequences encoding the E. coli LacZ enzyme. The
methods for
constructing and expressing fusion genes, introducing them into cells, and
measuring LacZ
enzyme activity, are well known to those skilled in the art. Sequences
encoding PRGE are cloned
into a plasmid that directs the synthesis of a fusion protein, LexA-PRGE,
consisting of PRGE and
a DNA binding domain derived from the LexA transcription factor. The resulting
plasmid,
encoding a LexA-PRGE fusion protein, is introduced into yeast cells along with
a plasmid
containing the LexAoP LacZ reporter gene. The amount of LacZ enzyme activity
associated with
LexA-PRGE transfected cells, relative to control cells, is proportional to the
amount of
transcription stimulated by the PRGE.
XI. Functional Assays
-50-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
PRGE function is assessed by expressing the sequences encoding PRGE at
physiologically elevated levels in mammalian cell culture systems. cDNA is
subcloned into a
mammalian expression vector containing a strong promoter that drives high
levels of cDNA
expression. Vectors of choice include pCMV SPORT (Life Technologies) and
pCR3.1
(Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter.
5-10 /cg of
recombinant vector are transiently transfected into a human cell line,
preferably of endothelial or
hematopoietic origin, using either liposome formulations or electroporation. 1-
2 ~g of an
additional plasmid containing sequences encoding a marker protein are co-
transfected. Expression
of a marker protein provides a means to distinguish transfected cells from
nontransfected cells and
is a reliable predictor of cDNA expression from the recombinant vector. Marker
proteins of
choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a
CD64-GFP fusion
protein. Flow cytometry (FCM), an automated, laser optics-based technique, is
used to identify
transfected cells expressing GFP or CD64-GFP, and to evaluate properties, for
example, their
apoptotic state. 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 C~rtometry, Oxford, New York NY.
The influence of PRGE on gene expression can be assessed using highly purified
populations of cells transfected with sequences encoding PRGE 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 PRGE and other genes
of interest can
be analyzed by northern analysis or microarray techniques.
XII. Production of PRGE Specific Antibodies
PRGE 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 PRGE amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
-51-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are
well described in the art. (See, e.g., Ausubel, 1995, supra, ch. I 1.)
Typically, oligopeptides 15 residues in length are synthesized using an ABI
431A
Peptide Synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to ICLH
(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-KL,H 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,
l0 reacting with rabbit antisera, washing, and xeacting with radio-iodinated
goat anti-rabbit IgG.
XIII. Purification of Naturally Occurring PRGE Using Specific Antibodies
Naturally occurring or recombinant PRGE is substantially purified by
immunoaffinity
chromatography using antibodies specific for PRGE. An immunoaffinity column is
constructed
by covalently coupling anti-PRGE 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 PRGE are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of PRGE (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/PRGE 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 PRGE is collected.
XIV. Identification of Molecules Which Interact with PRGE
PRGE, 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
PRGE, washed, and any
wells with labeled PRGE complex are assayed. Data obtained using different
concentrations of
PRGE are used to calculate values for the number, affinity, and association of
PRGE 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 02329685 2000-12-11
WO 99/64596 PC'TNS99/13281
O H H H W -. ~ _ W O N
tf~ .-1 O a fy Lay Gu H W rl H -1~ ~ M I
11~ (Y.,, . H W 00 N tn a O a O -.1 ~. OD OI H
rl t0 N 1 N M N N .-I ~ l0 '-I W lfl
I M M OI W l7 r-1 Q~ OD lO W O -O W l0 O rl a I ~ O
W cr ~ ~~ W N Gu O H M ~ t0 ~ri ~ I N H W ~ N
M H O (n W 01 ~ O O O O N .~.~ V~ .--I Q ~ 01 W
W ~ r 2 U .-i o r.~ r.~ r.~ ~ 1 O ~ ~ oo U Z y~ M O a~
H m w a~ o ~n a D ~ o, U .a a ~ cn N TS
O to Ll O '1 'C1 H O rl: r.>: F~ M .-i O ~ (L O . V~ -r-1
~ .. o H °' ~~I O o m ~n cn ° U Z '-' a~ H a~ ~ m ~
~ 'LJ N M O O J.~ Z FC ~ N ~ O ~ 'O ~ D ~ 'L3 cr l0 O
N ~.i O .-I d ~ H O a 'J ~ O ~ G Z ~ ~.-i N ~ ~ ~ri ~-I I N
M +~ E-~ W N I N U' a' M M M M I -rl W 1 +~ O N +J H 01 .-I
1 O O ~ ~ i ~ r-i G.' CO M M M M ~ -1.~ ~ pG r O H H 1 O O I~ U
'r N Z N ~-I 'n U C7 .. .. .. .. ~ O ~~ D c~ °~ N W 5C M N 2 v~ ~
~ rl U M W ~° ~ v - O O -O O 'n N o f.~ M '~ H Gu ao H C7 G
Ur.>r ~~ O U G M2zzz ~Z'''.. U(~~OU~ ~ UZ N
~ a O W .~ to M ~ U O O ~~ ~ r.J; .-~i r~ ~ ~ D 'U --
~wz~~.~ _ ~w ..or~r~a ~z~oz Gwo .. aa~~I~
-- v--I 1~ ~ rl I~ Q H H H H -'~ ~ W H ~ x O Q W v JJ O
D O~ O ~ O I~ 2 ~ OG O Ll ~ '-' C Z O O H
O N N H I U O H M a a a a ° ~ Ll 00 H O N M O M .~ N
O o ,-1 ,-1 .-I O D W f~ W W W W U ,-a ~ H O o ~ D '~ O H rl H
H x OI O U H H v~ H cn (n U7 v7 H O v N OI H H Cu . H U H U U Cl1
U U ~ W m ~ U l>~ oo U H w W U ~ vwn ~ ~ u7 O O
H l'~ U~ ~ ~ ~C N d W W W W ~ O ~ H U7 C H OD M OI ~ H H C f3:
U'oo N ~ WO000 HO4M HM~~W Ht~ W
z ~o W Ts ~ ~ p . u~ z o W u~ r~ o u~ ~ z o, .-. ,--
~ ~ O O ~rl N v M O f~ 01 ~ ~ D 00 . O ~ W N Z N W O v~ l0
a M J..1 O M W 00 00 O O H r-1 V~ H O W
o--HN OE-. o.-n ~~ O~IOOa~M o?~NMO~ o--MC1 O H~~-IH04M
f~ N O ~ aC O .-1 .--i H N H v ~n .. M H I-..~ ~ O M p
O lfl . t~ H Z O ~ 2 H I I I I O ~ O N O .~ . m H ~ . Z N Z
Z I~ N I U Cl) Z u7 N c~ f~ M ~ Z .-i M Z I Z x ~ d M O H ~ U' H
l0 M C~ ~ I>a' H 00 D lD OD 00 ~f7 O H x lO U 00 M W N Z C~ M 2 lD D
~H p'u1H 1 MOV'~-1 ~N .DM Mf~ ~~Cn I 001 ~~D~t'H
rt H M O ~ ~ x .-i r-i ~ N ~ N ~ u~ ~r H oo a I1 O ~ '~ I~ O a ~r1
h Z ~ N ao OI ~ M M ~ M Z W t~ Z v H pl
N VM NNV WNNONU lo~~OIO '~~ 00 ~ W
U N l.~ b O H ~ fn 'b T3 'd 'O TJ ~ O W 'd N Ll -O '1 N Gl ~-i ~ Cn
'-t H -r1 H x ~ M ~rl ~.-I ~rl -r1 ~rl '~ 01 Z ~ -.-i H rl H ~ ~rl x e-i H ~
l~
x l~ p H ~' M W J-~ 1~ ~ J-~ .J-J x 1~~ 1'.' l0 .4.J ~ ~ M W
O ~ .a o z O M ~~ O O O O O O N .O W O ~ .pIM O ~ .OI.-~I ~~ O
N W N U N N O N N N N N c~ H O N i.n W ~-I N lO W I~ O
CT N M U1 r~i ø~ 01 M ~ Z O .-i .-i r-I .-1 .-1 ~ M r-i H M fn I H O M V7 01 z
N
ro H U a t0 ~~ N ~o U U U U U ~ ~~ pl M U °° ~~ ,n U M ~~ O a~
W ~ O W ~ 0.1 N O ~ D H ~ ~ ~ ~ ~ ~ O W c~ ~ ~ O W H ~ ~ O W u7 D M
Z O ~ v N Z H .-a G ~ ~ C G Z tn ~r G z O r C Z O .-I H N
.-I v-I '-I N C
O O O O .-1
~, H H H H E-~
O ~ a ~ O
ro z H H H z
N cn ~ Z H U'
.p IY FC CJ M 2
E-m'~ H W O
a ~ o x H a
A
H OD Ln
O l0 N I~ .-1
rU o~ u~ ~ r
L', N OD M M 01
O ,mn to r o
.-i 01 rl ~ N
U uo ao rn .-I ~-I
O ..
'a O
-.i Z
O L1
N H
H
Ua
W N M d' tn l0
2 U1 M M M M M
O
z
-.-I o
N H
oa
N W
LL U1 .-I N M c~ u~
-53-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/I3281
'O W N -O W 'O H
H l0 .-. . C/~ I a C ri H OJ rl ~ ri
-ri i J Ll
1 N N 00 l0 N
l0
J. r .y O tn J-~ N x OI
a 00 ~ T1 N H M W ~ ~ 'O H
O O1 x ~ I O N -1
r a
'
O O r . W
I N -r1 E x O v~ f-i N -r1 1 N E-a N rl
-1 N OD O Ly In OI i'
~ r u~ M -t H 1 ~
~
~
. + . +- r .-1 O oo ~r
O -.~ W
O
U N O Z r1 z N ~ N U ~ O .N W 00 U z 0
N ~ ~ O W Cn I
N z ~ M W n o~ TS ~
p z r N O O a~ ~ D r p
C ~p - -t O
-1 a d D vo t C U
1 o N Z
r r ~ . C O er z W
~ r ~ N
O N I U Q M H .-I ~ J-~ I U .-i ~ U ~ N M O
O ~ H ~ C/~ ~
I OWND O
~V 5
~p r ~ M ~b-.D Dr I
-- ,.i -, U "
H N N ~ C '
l 1n wl OI OD N Cs l ~
C 'r
r .i r -rl M
~ . I O . H N d
N I
~
+~ O W 00 ri C7 ~ .-i O H C .t~ '-1 r1 I
-- .-i
O E-~ ~ -- ~ . U) o ~ U E-~ ~ --. N O E-~ to ~
U z m d o~ U 01 to to
00 NGuOD r~ C5M CO.-IM NN--. N~Lv W.-i I
W N
U.-Iz ~O01MW Ca fn O CE-~x OON-1 .-iEWDOCn 01
.d UD ~H~-1 O N ~O .d ENO .bHMO ~ UH~Dz Nay
-,~ W '"i O T7 -~ ~ Cn -,1 o .O ~ v W Tf
~ a ~ Z 4-1 M z M H N
t o~ ~
~ a
vo O
t .~ v ,- H ao G
Z w ~1 M W U r Ca O -.~
a7 ~ z M oo +.r ~ o E..~ o rWo ~ a7 o H
o nG o 0 0 ~
a r 0 c O E~ H '-' H N H -- r N O 'L1
v' H M .-I H
'
tn ~ p rl H I N ,1 O ~ ,~ FC ~ 00 .-a OI
-i O M d I z M N ri
O lO OI z ~O M
i
U
. r .- O a . H O lfl W N .-i
U E z U O G U -- J-~
x N W '~
U -- f
d W U H N W t
. u O cn Er
(n ~I v s, . c/) I U
N O
C z C v-i M Cn C H 61 C t0 C H M a W H
l0 C W M 01 ~. C
r O
~-I
O C tL C
W
MM u (U E-nN NMW ErQr-IV~HO~ O N U
FC~o U7 ~--M .. M.--Izx NO T3--~
~OOWT7--v O-CS p ~
O a o N Z O -ri tn o ~ O O ap ~
r O . -,~ W-1 M N O , N z u7 ri
(n l0 ~ O ~ M C
z -I 00 r
r J. z V M L1 f.Il l0 N 1~
E' J.. E' N Z N
v E' O
E'
-- .-i u1 D ~1 O E-~ ~C N N o~ H -- ~1 L1 0~
/'1 O Ll ~ M ~ O ~ M O H ~1
n G Q E'
i N ~
O
M
O M
.- M v H .-1 Q7 O Cs.i
~ l0 v 1 0 M D --I
v H H U rl v E '
N H M OI
H C r ri E-~ i OD H
V' O r-i rl
t~ r ~-1 Ot I U u~ r ~ y
ov z i U 00 0, ,- ~ 1 r-i z V
I .-i w Cra ,-1 a o,
w U c9 ~
~ ~ wr aoN ,~ a ~ a N
rMa Mar cnNa ~~wr ~zN
O ~~O .U7N CR:--if~M aa' WN 0 . W ~.--i .~7M COO
.. C
W
~" M O v 01 Q ~ O W M
Q Cl) ~ OD N W O H (n r . C/) H
l0
t0 z M W ~ V N ~ .-1 z v~ O .-
~.. x c~ z W
U7 1D r H O N N OI O) ~ W N ~ .-1 ao O U M Cl7
W N W
~o c0 ~
o
-1 v t,-, x ~ ~ ~ EN., ~'-' a; c~, z M
x o ~ o
-1 H o
o cn . ~
-
x ~
"'1 ~ m M M u -,
o ~ -1
r M o
~ ,-
~ E
~ r ~
N 61 ' + J ~ U O OD J-~ Q N
~Olr J , N .OIM Ozm
O piv~ O Ll,--IOO~
O
L1N Ozv~ .. W
Oz
r W ~ ~ o m
M H .-i N z ~ O ~ H I H ~ W ~ N .-1
r M O N FC O W ~o ~ u~ ~I ,-I
Cl1 ~ I N M ~
I 7 ~
a
.-i r Z f/~ T z 1 v U7 I .-i W
"fl~ ~ r z .--I 1 r-i M d to v~ c~ ~
~ ~ r U H to ~
N OI ao U O ~
M U W
m U x m
O W W -i C U r C> D W O W r tn W M C E-WO
~ M C C~ C O~
Cx.~,~ '..i H H N O
Z O fn OD C: ~" -1 H O M CO r1 O O
n-I H r L,' '~ ('.. ~ V~ z O 01 Li "
N .f. .~.
N ri .--I M
O N O O N
>, E-~ H H E~ H
ro z ~
z z z
a H
:a ~ z z
a p H H o
a m t~ w cn a
0
H r O~ M N N
lD rl e1 N In
N M .--I OD M In
C ~ v~ ~r M v
O r M rr oo pp
rl ~o r o~ ~o ~o
U H rl rl N N
O
..
'
O
.-I
z
t~
O
D
N
H
ri
U
of
~ r ao o~ o rl
w
z M M M .
~n
0
z
-~1
0
N
H
l~
oa
s~ o
w
w vo r au ~ ,--I
-54-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
w~ob p~ov ob~ ~a
r-1 ~ LO ri H to -.-/ C rl O l0 C' W
xN ' l I rl cJ
~ ' r
~ '
W d ~r aNt . .
J- c NH t/1V
a1 x
I o a~ ~ oNx~ I oHr-lo ~ o00
~ ' 0
N
't
,~ a a r Noc ~t0 NOh ~W h W2 t
.. N '
~
M O r-1 -.i ~ aD r-I h .i z ~ O ~ ~ O O
O z N U E-~ M a ~
~ O
o~ U W
z
~ ~,,~ N W ao U v7 ao f o ~ H H
r~ + o~ ~ C.a N --. tn D ~I
'L1 ~ O c~ -I ~ O t~ H u~
'U t~
a
~ N W r ~ o
ao4 -~I a a~ czo~o ,- O z z
H H
cxh M.,~r-,~
~, ,~ N f1r N a H +~ z ~ U cn
N H -1~ r-I !v G] 01 H d' a O
H
1 N '" H
v
w
'
.oi ~ a ;-W =,x .c ~H zap
W ~ ~ a ~~o
.~
n
i a
~z
~ W ~-I O H 1~ O '-' y N t0 uf M ri ~ ~ LL
!~' . W H ~ IL
M c/) ~ ~ ~ ~
U H U O H c' O E-~ H ~r W ao
tn U tn c W
~ ~ I h ~
~Dto ~D
O ~ ~ NOo .. pao ~O4 00 W
~ H N ~ N W ' O ~- yn ~
w C H ~r rl f~, ~ .-i z t'~ p G
~r Gu W al o
-o omo ~ Uz~o .o Um~,ztn a~ --zhr, ,-,x
o H o
~ c~ ~ ,~ a ~ G N -a G ~ ~ ~ aNO r' ~ ~ o
~ p z N .~ h ~
-W ~
a~ o m o~ x c ~ ~ , x ~ 0 0
h z w co H ,-I ~ o o
x H ~
a 00 '~ H -- ~'~' -- -- ,-I I o H o, ' r-' to
N x o, H 00 I N ,-a
~ ~' ~
H I v~ o I a, 1 ~ aN a~oMa.-, o
~ W ~ ~o io W -
U U '-i "~
Or M
. o o o o m
U h '~ H Cv N . w o .-~ E-~ r~ ~ h t~
ch v1 '~ Z G.. w ~ N .--a
M W m U H Is, u~ cn ~
U f.~ h tn h
.-1 ~ z t!7 ~ O h . ~ ~ O cr N ~ ~ ~ y-i
l0 V~ v v~ f/~ tl7 fh rl N
H ~ H o .. ~ z m ~r H m . w s~ 9 o ,-a
.. W
' r
'
~o~ ~ z o;'.". ' ~ , .~~,~
N o z~ o~ 'y~z 'o-~' .~
~ ~ ~
rl 00 l0 f3i tf1 .-i 00 -.~ tI~ '~ ~ N O
-rl ~ .-i ~D O -.-i . ri -ri ~ .-1
O ch Gl ~N H I~ 01 H CO OD Gl ('r1 l0 N O H
1J G7 Z 00 J-t J-~ O .-i ~ l0 H O
H O O .JJ r-1
E' N
O O Lv ~ H -- N O~ ~ ~-I H r'7 O H O O H O
~" ~ M H O x ~ c'7 O O H
E-' E' p H H
E''
1 M N h M DO N N NO~ ,-i U HO OZO
-~ z ~" ~o -
~ a ~ a H
-,
-
.,..a, . . _ z , Z x z U
w , , . a I .-, z o0 ~n z
~ o r ~-I z
I
.ic o~~aN ~ o~~~M a~ oz ~ no ~ao
a~~<
n
. ~ N oo W ao v h C x ov L1 o ~
~ 0 ~ 2 a w D
x
Q p H U ~-, p
N C Q W . c'~ O CO 10 O W N H 01 H O LL ~
W ~ ~i Pa Ll
a ~ U x ~ ~ W ..
~-I 2 O N 2 ~ .r N a
~.. O N Z O ~
M ~ ~ ~ v 01
~ p v h W N N ...
O N M OI N h
o . D o~ N O O Tf W n D ~-I 27 O vo .-i mo
r"~~ ~ z O h o ~ . W -t~ o
H ~ M H O ~
H
-1
~ x N H -rl ~-I H ao -r1 H x w x X
O C h O v H x u1 U1 ri H .-t C....
O 1 'y H n
1~ W OD G
Ov C~ M
1
J 'J
. . P In t
DZ ~ OINzN H .d~''1 - pooh OD
Of~ . NOOtO
J
~ .Qlr-I OHtc'7
W OH
~.1 H ~ O W I PG s7 W 01 u7 w I N f.Y, h N l0 l~
I N z O O N Cn ~ N
1 ~ ~ N ~
CJ O
'
~ h O O ~ ~ i .-i (O N r-1 ry ~O M M O
"'~~ ~Ota Z ~ ~o UH V tO
~ MUN h z ri PG
~ h U~o to U
H ~sroo~ooao
W W H Ll W N O W N ~ O W N ~ O ~ L7 h M f~'1
Cn ~-i ~G l0 ~ 'J rl tn
~ ~
N U1 h H O N ,Z O M ' h N .-i
"~ '-' N C"., v z O r-I C'., '-' OD H rl
N H 01 [; ~- N
l'~ V' N h .--1 N
O O O O O O
l-a O O W p E-' H
O O
~ O a ~ ~ U
.fa
a H ~ o
x ~ w
0
H O l0 C' OD In
~-I c~ M u7 tn O
N f'7 M N 01 ~ cp
C O M C ~ N M
O f'7 l~ O1 ri 61 O
.-i ao o~ o~ .-i h m
U N N N ~1' h .-I
..
'~
O
-.a
z
O
C7
N
H
H
Ua
W N fh V u7 l0 h
Z ~r c v c a
Cn
O
z
0
-~I
o
N
H
1~
oa
~1 r-I N M C
W
GL ri ri ri r-1 H ri
U1
-55-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
'-' lD ~-1 r ~ N '-1 v~ M N l~ ~ ~ N M I~ N ~ f~ I~ N ~
O O O ~ O O O O ~ O E H O ~ O N O O r-i O p N
H H E. H H H H H 2 ~ H p p H H H H H H H H H
H H z H Z z H z z H H ~ H 2 ~ H H Z H 2 ~' Cwv 2
zzHOc zxz~c~ ~~ ~ . ~h ~,HH~,~'fliE-~ a['J
aaar~ a~z~~z wz ~,~ ~z x a~~Cv, z
oocn~ owoxa H~ ox ~H H~~x>x ~a x
~_Uvo ~_aUHa ._a ,..N ~~n .~mwHO_m ~.a
,..
d.
l0 l0 ~-i ~ l0 ri ri t0 ~ ~--I ~ N ~ rl i ~ l0 .-~ '-I rl l4 '1 .-~
~,., w H x w w x ~c H p x ~ o ~ x ~ H x x x H °~ x
I~ f~ d' p ~-I N t~ h ~ r-1 ~.,~ ~(,' N t~ M N OD r-i O~ f'~ '~ O1
lfl N N C h ~ f~ f~ W M N 00 1f7 O 00 N OD
.-1 OD a ~ r v~ ri c 'W f7 ~ OD U~ OD f~
OO l0 ,~ ~ ~ M O O c~ 01 l0 N t9 O
~-i M ~C7 .--I l~ M O M fn t!) M 1~ I~ 0~ M r O
rl l~ 01 ~ I~ O t17 N ~ O N ~ O~ ~ O ~ O 01 .-t p Qt M
M l0 O 01 M ~ 01 l0 ~ N lD r ~ 00 W 17 ~ Qv 01 .-i M ap
.--i N N ~ ri ri N M ~ N N '-I H N N W --I N N N M ~ N M
. . . . . . . ~ . ~ tn ~ ~ . _~ . .
_ _ _ _ . ~..~ M l0 ~
M l0 M M ~ O .-a ra ~ N ~ .-~ ~ O M ~ ~ '-i N N f~ N (~ t~ ~ M O ~ M
p O O O O p .~ O O O N H O H O N H e--1 O ~ O O O O r-i ~ p ,--1 N ,--~
H H H H z H H H H H H a H ~ O H H E-, H H H H H H H H H
o ~ ~ ~ o o a D o ~ o H ~ z FC ~C w o x w o 0 0 ~ o o w o
,~ zHHHZ zHHZHZ zr~ ~ww HHH zzzzHZ zwz zH
~zzHU ~v~zUC~H a~ ~,~r~ zHw aUxhxH ~~z
~,-, a a ~ U z o z o ~ ~ z v~ m cn W r~ r~ z ~ cn z a o
°'c°~~m_~ ~w~wo_ao ~_a H . . ~a~o~_ ~woc~o_~ ~ac_°'n xw
V ~ ~ ,. .... w .... ~ .r ~ " r., ,_., -- .... ~ .... ,.. .r
N l0 ~ .WD H to l0 l0 l~ ri ~ r1 H O~D 00 ~ e-I H ~ lD .-i r-t l0 l0 ~ l0 r-I
~ .-1
.-, ~ x H x x w H r=.. w w w x ,n x ", ~ a7 ~ x x ~ w x x w w w x w x
N ~7 O l'- Q1 M ~ .-i N O d' OD p OD N M N ~, dO 01 r N r-I I~ 01 l~ x M l0 p
.-i
tc~ l0 c~ N V~ IW -1 M C 117 r-1 v-i N ~C7 CD M OD O m ~ r-I 01 ~ l0
~ OD ~-i O a0 N 01 V~ N M OD M c~ ~ O O ~ 00 cT ~ 1~ l0 t0 N t~ N 01 N ~ I
(I! ,-~ ~ .1 M OO ~, I~ t~ I1 l0 l0 N If7 H FC ~C H M I~ p 01 ~ 1~ CO M N .-1
O ~ c~
M .-i H N OD O M N I~ O~ W W ~r M ~ O OJ Ov .-I ~ u~7 a M ~p
Cu 0 M to rl l~ ~ t0 O1 ~-i IW -i ~ 01 N ~ ~ N f~ l0 ~ C~ 01 OD 01 ~-I ~ QO M
~ 1
ri r-t N M ~i ~-i N N C~ H Cn Cfj N v~ r~l H N N M .-i M M M
t0 W--W ~ ~r l~ O 1~
O O O O ~i O ,1 N
?i H H E-~ H E-~ H H H
N O O C~ ~ ~ p
FC H H H
a z z ~ cHn ~ z z
a p ~ a o ado ~ ~ H
o v7 x
0
H ~ N OO ~-i o0 01 l0 O
f ~ M N O O1
N .--I v~ v~ ~ m N N
C'., .-i f1 Ln l0 M OD O l0
O e--i O o~ N sr 01 ~r M
r-i t0 01 CT f~ I~ 01 M Lf1
U '"'~ ~ ~ N N N M M
-.~ z
o r~
N H
~a
W ~ ~ O H N M aW fJ
z v~ ~r ~r In In In In In u7
0
z
c
-.~ o
N H
oa
~1 w h 00 01 O .-1 N M
~ Cn '"'~ .-1 .-1 N N N N N
-56-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
ro ~ w
~ H ' x M to ~ ' . . ~ ,.r> '., ~ '
~ M ~ W x w ~ .... ~ ~ ~ a x h
~ N V' ° O h l0 l9 rl mi ri ~ M 00 M OD M
O O h E V~ h H l0 O O O Q O O M ~
O ~ H M u7 v~ to ~a~ H H H H
H H O ~ z M W m c ~ z z p E~ ~ ~ M In H
2 x N ~ M o l0 lNp ~ U ra H Z '-I N 01 ~
~ t~ D ~ h ,~ a p c~ '-' a n. o M ~ O ,-,
azw~s ~,bvN ozx ~oxH ~ ~x
aaa a~ ~UWp UHW .
y-l ~- ro ~ ro ro ' ~. a1 .~ ~ ~, ~ t"~ M
H '-' e-i ~ (yJ O
l0 H O '1 ~-I ~ .-1 O l0 l0 ~-1 ~ l0 .--1 p n-i H ~ p
H x H x x ~o o H C~. C~ x '~ C..~ x H H O H O
w x h M W ~ a~ rx H O ~ ~ ~ ~ a~ O a4 O
O M ~a c~ G.~ N M h Q z 01 O N ~ ~r N v Z ~ m C7
y-I cr M H ~ OO I'~ 2 W t0 M f~ H .-1 O Z Q,'
N h v~ 00 ~ ~ M v~ H O4 l~ M a ~ .--1 N l0 ,~
~ OO l~ (YJ ~ V' 01 W O lfl .-i 00 m 01 h N tn
N M .-1 N v M O H ~ H N N H r-i N O~ v .-I '-'~ H
H v ~ ~ w W
. ri _' . . ,-I . . . . _ . . N r-I ~ 01 _' N
~ M M 'x-II N M x (Ya h M l0 M M H ~ v-i 01 01 01 ~ 01 ~ pp
p .~ O O l0 ~ .-i h tn h .-i O O O ~ '~ O O N M ° r-I '-'~ N
O H H N O H h N c H H H En H O H H h N O E O
/"1 H H p O c H D v~ ao o~ D O O O O H cn O T1 0~ °~ ~ O r.~
~°nz4xo ~z~cw~HC~r~~~~.,~ 2°H°H ro°'
M ~ ~ O .~ '"'~ ~, W H ~ ~ ~ ~ ~ '~ Ul V1
V ~ ~ '.~ ~ ~ .... ~ .., ~ -r
~/ ~ M ~ tn ri M H v r-~ 01 ~i H Lc~ H Cr.t
l0 t0 N l0 N O O t0 rl tD l~ h r-f M O O ~r N
r..~ ~ x l0 f~ f~ H ~ G4 H H H H x fzi Gr M ~ Gr x x H E~ ~ M ~ M
O ~ L1; M N O h O O O O ~ tn a~ tn O m h O M O H ~ v' ~ sr
~~n~z ~nzzz~M~~ ~~a,zm o M
~ o~ h o C7 o M z D H ~r ~n o ~ °~ °' a~ o N z z ° o ~ o
ro N N ~ o z ,n o~ p H v) ~ h ~r o~ ° N ao M o a H ~
N l0 00 "J h H H W N .-i h 01 N In 01 LL ,.~C~, h
N l~ 00 a ~ U7 x LL H l0 Q1 c~ O M ~ ~ e-I O1 Cl) U) ~ ~l ~ FC
u7 ,-1 .--1 ~- N ~- ~~ ~ r-.1 r-1 N M rl N N ~ Cn CO
ao M ~r
O ~ ~' H
O O
H
'ri 'J H
a x w w H co
n
H p1 O1 pp
lf~ h l0 lfl l0
N t17 h e--i O l0
G rl v~ 01 N l0
O N m O~
ri 00 ~ M c' u7
U o O
N
~ O
w z
op
H
U
h o0 0, o
z yn ~n imn
0
az
.'~ p
H
i~
O
h OD 01
~ N N N N N
-57-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
N ~" tD
. ~ H
-~ H
M N O M ~ O
01
O
-1
N .-i
N r
O
Hz~zH~
a z z vn M
Wo~ox
~Uwaw . ,=,
.
fa.~ O N
l0 e--1 E O
.--I ,-I ~ H
00
W fs-~ x
x x N
~ I~ Z
-a ~ t~
O
. z
M N ~ N O
O
tn t~ l~ ~ F
O F~
o~ t~ W ~ W
.n W
N l0 01
M OJ ~
H H e-~I H 'w'
N N Cn
x~
.
N W
M f~ M .--1O O
M N
N O O O
O O
OJ
~ H H H H u7
H H
ozz2~w
N
Yn ~ ~ z N
~
''aHOO~H
V H,..~~'.~ ro
~
,
w
,D mo ~o w o
,~ ,-mn 0
~ N
~''
O lD '~ O
.-1 In V~ O
~
M N f H ?..,
M 00 M N pG
O N .--I D
M u~ ~ H
O
~
v~ M OJ z
ro l0 t~ FI,'
.
N
u,
~, O
O N W
O .-i W (I~
~ cr 01
O I~ 01
F(,'
H H N N
N tn
N ~-i
O O
~r H H
O
ro H z
N U H
f.~ H
a w cn
n
H V~ N
H W
N N N
G ~ H
O r ao
a~
U ~ N
U
~
O
w
z
0
0
H
H
U
--i N
z o to
y
O
~z
~
A
H
O
Q'
s~ o
CL M M
~
-58-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13Z81
H
~ H
z U z
H
O H
ro ~ ~ P0 W
U
rl w
N
r1 H U7 ,~'", U7 ,~,' ,~F.,'
,f''., '~',
ro W U7
H H O
N O ~ ~ H H
P0 00 PO PD W ~
GL W
P0
r-1
G
G ~ v
U
~ -
~
G U ~ 1J ro S-I 1~
1-i
o ~-~ ro.~ v a ro
a
-~ v r, .cZ s~ r, . p
G v ~
N s~
ro O bl N ~"' ri
s~ -~ ro
v
~ ~ ~ ro v ro ~ v ~ v
rn
o v o v
o -~
~ .a . a~ ~ o ~
a '~ v~ a
3
'
,~ ~ vU oa
, a
~
v v ~ o U >
ro
z~ ~~ ~ '
z x x ~
N
M
H
v ap c~
?i
v
W N d0 ri
1J l0 -I
.(", W a4
ro E,
N
w x
G ~ ~ .-i o. i ~ o
vo
o~o~,
wwa
xHx
0
.,.,
.-I N
ro
ro~
yf N
z
o u~
N U D I~ N .1
W
J~ N N
~
.Lf
~ n z
a z z z
Sue, O M
O v-I N V~ M 01 '~ N 01 M Ov
ri N d~ V~ t0 ri Ea O . v-i M tD 00 '-i
lD
iJ M f-i M I~ ~O I~ ' U] N M M M 00
M M
ro cn V E~ ~ ~ r ~--i o E ua H
H v~ .~
E u~ o E d~ ,-t v7
.-m o oo ~ N r ~ ~ E ~ t~ ,-mn
U1 Lf1 if1 OJ
~ M e-1
O N '-I N ri N M ri e-i M
N M M N N
W tn tn cn ~ ~ vI w oo u~ E E-r
tn E .1 E-~
H V1 u)
N N ao N o o av E o .wo o t~
N oo a
1! Lh O 10 In r-1 N N O M er tl1
O d~ N CO l~ o-I 00
11
O .~' N v-I N M 01 ~ ,-I
-rl N M I11 rl ~' M
h
w am cn cn tn to Ea rn c E ~ v c
cn cn H Y~ cn n n t
a H
N
v
0 2s
a 2s
-.~
~ u~ o, ~ o ,~ ,-,
U O ~ M M M n-1
M .-1 N v-i
v
-.~
o
.~
z
a
va
(1,
H
r~iy
b'
O N
N M er
-59-
CA 02329685 2000-12-11
WO 99/b4S96 PCT/US99/13281
x
x
v a
0 0
a ~ a
v o as
v
~ 2s
~
r Cl) U7 ',F'., ,''. U1 ,$". U1
i .C H Vl
P4 GO W ~ 00 00 C4 W f~7 G1
00
N
U N
A C ~ w
1J ~ 1~ ro N
w q u~ ~ O
~
O N L1 ~ - ?~ .r - N v ~
'
rl f-I .~ O v ri a v S-I UJ O
~ N
~ ~ N ~
~
~C --i . t ~i O s
-I 0 ~ N ~ 1-
U N 3 ~ a ~ w
- - di p, b~ ~ N .~
'
'
w v O H r-~ .~ ~" v r~ O N
~ O U
rl ~ .rl 'O U v ~ -~-i N U1 O S-I
i-I U ~
o v v o ~n .~ m v v o .~ ~ ~n
a G
a ~ ~ a v ~ ~ ~, v o ~, ~
v o
v u~ o ~ ro ~ x u~ ~ ~ a
a ~ ro w b
xaN H ~ ~ ,
x o ~ ~ aro~v~
.n
v
I~ N
j 1-~ 0~ QY 01 M L~ Ll1 N
N Y.1
N
U ch U7 N cp M 01 e~ N M
~ r-i
iJ N rl M (~ 00 Ln 4L'
j," 1J !.~ ~ .Y.
~o c? x r
C71 bW -i ~ a V
~ ~ F OD V~ 0 ~
0
. 1 I7
~ v N O rl 01 M N O 0
rl V~ M 1
e-I N N
~ v u~ x ~n a a H x v
N H
M
,~ O
N
z
b y o
~ o z
~n
N U oo O
N r
Ll N O N ~ LfW l7
~
JJ
~~ z
w z z zz z
o ~ o m t~ o co t~
00
.ri rl d~ H t1 .-I N !~ 00 M
t!7 01 Lf7 M eh O
.1J U7 N 01 N l0 d~ M d~ N d~ M l~ 01
N N N V~ e-I
ro cn ~ o N w E ~ u~ ~ a1 u~ n .-~
u~ E cmn y
o E .-I U1 .~ E ~n u1 cn Ea
'
rl 00 N U7 r-I rl Vl 1p VI t0
, CO N I~ N
T~
ro E-W-1 O O1 01 l~. O 1!1 M 10 d~
~1 N O I~ s~ L~ ~O
ri N N N N a0 N M N 01 wi N V~ c-I d~
O t~ N v-1 M N II1
J.1 Ln U1 N ri U1 Ov E~ ~N V7 U1 e-i
.L; v-i v-I U1 U1 E-r H M H
t0 E~
F." d~ io U7 E U1 En u1 u1
61 ~ cI~
U1
v N Ea ch N 10 O t0 01 O ~ O If1 If1
N v0
.1.) O lt1 01 O M L(1 00 N '-I 10 M ri N 00 00
O N l0 !f) lf1 O M V~
JJ
O J"'..,01 N N tf1 N ch N M N wi ~ N ~O '-i
-ri d~ M d~ ri tft ri M l0 L!1
a~ E~ H m1 vi En E-~ E En L~ u7 cn
w E~ Y Ea U1 ~. vi U1 E E~
cn E cn
m
N
O ''O
G Tl
~
-ri 1D M I~ 01
N
U N N oo .-i
Ri N ri M d~ Lf1 M
v
~j
.ri
~z
a
N A
H
ri
O v
U7 ~D (~ 0~ 01
-60-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
E
z
H
r~ [li
ro w
U
.-I
N
1J Vl U7 U1 UI
~
A H E
r U n
U c
a ~ ~ ~ W
i
0 04 P
G p GO
I
b1 S-1 1.1 G1 d~
N
f~ O ~ G N U -~I
-~I ~ ~ -~ x x N
''~ U 1 'L~ N M O
O N w U U
~
C: N a
-
~
-ri i-a O N r~ S-1 O
-rl
~ ~
~
O
rt ~ .~ ~ N l
-. U
.i x J.
~
U N a -~ U N b~ b~
O 1a
a -,~ ~ ~ a ~ a ~.
. ~I o
w o ~t ro v o
N a
.~I U G ~-I w .~I w .~I
N .~
~I v N -~I v ~I v v v ,o
x
v ~ >~ ~ .~ v ~ U t~ U 1.1
a -~I .rl
v ~ ro s~ a ~ ~ o a o
a ~I x
o s~ s~ v -~ o r~ .,~ s~ -~I
-~ v s~
H zw E ~ ~ N.~ zw N a N a.~l
I ~ N
01 01
r-1
M ao
U
U x
v o t . o
s~ d~ . r ~ d~
v
~ G H M
-.t U ~I U
~
~1 N ~-
~ I-1 01
i
in a v x a v
c x
i
N
0
-rl M
H
r1 ~ M '~., O lfl
ro
N
1J z z 01 z M z
o N z z
v U to 00 N 00
N O
J~ 01 M rl l~ O I~
.~1 l0
1J
O r-I N M ~O H N ~ N
-.~
w~~n z z zz z zz
a
O 00 ri O tf1 N Lf7
rl N 01 d~ '-i O~ OD VW t~
f1 M N
1f M O~ N ri th ~-1 lf1 d~ M Lf1
M N M
ro ~r u~ .o E-mn 0o v~ ~n oo .-~
~ m ,1 ~I v~ cn cn
r-I ov N ow-I oo E .-~ E
-I 1 a~ v~ ~r ~ E 01 N 01 ri N
?W v1 N l11
~-I
ro o o m m wo vo ov E o d~
~I ov
-.1 .-i tlW -I lf1 r1 N H N
O M M M l~ '-I 'wl
M N
.u d~ c4 ~ a) U1 ~ E u~ .-t d~ ua
.~ ~n M cn tn V7
cn
>~ v~ E~ E U1 E~ 00
a
u1
N Ul M 01 N l~ lD ~O 01 (~ OD
N O 10 \D O
J-1 Lf1 v-i dt V~ M tf1 M 00
O L~ I~ 10 OD ~O I~ ~O
J-1 00 M M
00
.' rl wl V~ .~ N '-I v--I tfW -~i
-rl M N v-I Lf7 N 10 .--I .-1
N M I~
w w cn E-~ cn E-~ to E v1 cn E u~
u~ ~ E Ea tn v1 H cn cn
~ cn
N
v
O
~ 'ti
-~
.ri N In r-1 01 d~
-ri
Ul
R', tf1 M M N N
R'a
v
.rl
~z
a
vA
, H
r~i
CT
O v N M d~ tn l0
L4 '-I e--I v-i ri e-1
U1
-61-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
ro
U
rl
U1
~ x ~ v
a z
~ a z
~ w ~ w
an
w
N
x
~ ro
o
b N
Sa N ri N iI
N
O
U ~
b N .
G ,
b~ ~ ~
~
rl W rl
ri (".., ro U .,
.~.
r
l .~"
v
U 1J i1 v
~'
r U7 J-
ro f l ro
x
N F: O ~ ~'
N
,''., .
i ~I u) U
N ~ U ~ ~ to U .L1
N ~O V~ N O t0 V~ N O ~ . t0
OD ~O d~ N O
S-1 M ~D 01 N l~ O M 10 v-I N If1
N OD n-i d~ I~ O M
lf1
N N N M M d~ ~ ~ d~ ,--I N
Lf1 t!7 f1 ~p
.~ x x x x x x x x x x a~
a x x x
p ~s 1 I I I I I I I I I I ,.a w I
v I I
~D VI N O ~O d~ N O cr I I ('
b1 00 ~O ~ N O .d,
ri d~ I~ O ~f1 OD
i dl 1D O~ N 1f1 00
r O rl ~f1
ri N N N M M M d~ V~ dl '-I N
v V~ tf1 tI1 t!'1 rl
U U U U U U U U U U U x x H
U V U ,~
N
N
,_, o
fC M
r1
N OD
z .1
~ O
N
v a uwo
v
iJ N O 1f1 N
~
l~
n z z
z z
a o, M
O M a0 00 01 N d~ l0 a0 1D O O
L~ M M
ri tfW 1 N d~ O ri H M M If1 01 ,-1
O 00 cp
1-1 M lW -1 N M l11 N M eh 10 ci M H
d~ e~
c0 U1 .-1 M v) t! v) H 01 U1 U1 H H
~ V1 U~ ',al
r-I lf1 U7 H O lf1 OD d0 H N d~ dl V~ 01
',~I M I~ O CO O
IIf M d~ 01 O 10 l0 M l~ I~ N tl1 U7
~! d0 !f1 Ln I~
ri M d~ O n-i .-1 M M lf1 10 M M tl1 l~ M
O N d~ l0 I~
a.~ vi N o~ H m v1 U~ U1 d~ H cn V7 H
~ H v~ H
i H ~ H H
N
N
t ~I ~O h d~ M In H
n N 01 t~ N OD t-WO ov
iJ VI tf1 cf' .--1 10 OW 00 t11 ~O 01 V~
O O tf1 rl O N M 01
1~
ri v-1 rl M ~-i r-1 N M N wi M d~ l~ I~
tl1 N V~ Lf1 ri l0
GL tl~ H u1 u7 v1 v~ H Cn E-~ V7 H v1 u1
W H H ~ cl~ ?~ H
u~
N
N
O ro
aro~
U o
N
N D l~ ri
~ I1W
N
ro.
w O
~z
c~
N A
G1,
H
~
.,
r
i
b'
O N I~ Op Oy
W tn r-I H
-62-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
ro
U
r1
W
H U ~ ~ U 2
O H
~.' '~i LL G0 P~ tI1 04 ~ W
ri f-1
tC N
b~
v
x s
U
G,"
1~
O .~
U
~ Ol C~
S.~ i~
J-1 f-I -ri ri 1J
N
~
H ~ m x
~ ~''
'~
~ ~a,
- a
~
'~ -~ -~ G --
it o 0
N N ri N '-i
(', .L"'
-
Ul G' N
.~., r-i
rl
'Cj -ri 11 . U
H N a U ,~
(I1 ~ .!-1
O M rl O1 L~ Lf1 M ri 01 M Lf1
t~ Lf1 M wl d'1 I~ tf7
1-I t!1 aD O M 1D 41 N d~ (~ O O
O O M lD 00 rl V~
U v-I wl N N N N M M M d~ M M
V~ ~ dW n cI7
x x x x x x x x x x x x a a
x x x
f.," M e-I 01 (~ Ll1 M ~i U1 M I~
~ r lt1 M r1 01 (~ tf1
dl M 10 OD '-1 d~ t~ O N tf1 CO l~
OO r1 d~ l0 01 N
rl v-1 ~-i r-I N N N M M M N N
N M d~ d~ cT t~ tf1
~ U U U U U U U U U U U U W U
U U U
a G
O
N
fa M
ri
~ N
cat ~ o z
~
N U N
N
J~ N 01
'fit
J~
n '
w z z
~
a
O N '-i M I~ OD N
ri lD dW -I O~ 10 00 10 00 CO
1~ V~ LW -1 M M t11 N M OD r-I
N E t~ u1 u~ U~ U~ ~ H .-i ~n
ri d~
~
7, d~ t~ .~ r o7 r ~r ~ W n
mo
ftl M I~ U1 lD V~ lf1 ~D OD l~ M
f-1 lf1
'i N v-1 lt1 N M 117 N V~ f-1 M N
O N
a w E ~ ~ E v~ E ~ ~ v
~n
N N r w o7 .~ ~ t~ 07 vo ~ d~
N
1J rl d~ lD N V~ M O M (W -i O
O 00
J~
rl rl ri If1 N M off ~-1 d~ ri !~ N
N
w w ~ E W E V v~ y. ?~ E-~ f~ cn
W E
Z7
U U1
N
U
O '0
~',
rl
N IW p
N o0
ai U1 M
N
rtj
w O
~z
~a
H
~i
O N O ,--
p~ N N
U1
-63-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
x
~ ~
H ro
U W W O u1
W ~'. N
tn
O a ~
H >, l H f~ x E-
-I t~ C
x -~
~ .>~ f~ H fs.~ H v1 w
ro U U z ~
~ O ~
-~ ~
E O E-~ ~
v O O H O
x o w x o w a w sI
a a x
z w ~ w a, z w w w w
cn oo w
a
.
o ui a~
N
a
ri O iJ
~
~ o
-. ro x w~ ...~ v.,
o
1~ fa f-m1 U
v O
w x n
' 0 ~ ~ L
~
~
1. ~ o s~ ~n -~ w
1 o
S-l
U
l
i
~ o m v
a
'~ b1 rl ~ I N 1~ i-a U U7
,
~ O
v O
. T3 ~, S.a ~
~ i.1
W ~ W rl H ~,' ~"', 1~ N
ro 1.1 ~
N 10 t~
N
S-I n-i N 00 l0
N O
~
a ~ ~ n ~ a ~
~ v H x n
l
N o z
o a
o
b1 l0 O N O t0 I O
.ri 01 OD '-i r-i 01 M
N r1
cn cn d c~ w a H v
~n
U
o
N .r.,
o ~ .-i o0
ro
.1 ,~, ro o w n
~
z fl
..
c~ a o z
~
E ~
~
~ . I
;,
z "'
~
a z z
. o,
~
~r o
o G ~ M
OD N a-i O H VW -I lf1
O O rl N
U1 .-i OD -ri M rl 00 E N
(~ V~ l0 ~O
N M d~ cr JJ M 10 l0 cP M d~ M
N M
ov E-~ E-~ ro E-~ ,-, o~ m uo d~ o
a1 W y~ E-~ u1
~o
~
E 01 O OD ri I~ U1 01 M U) H
L11 10 ~ u-i N
oW vo o~ ro N d~ oo ao t~ u1
C~ N
O v--I N rl U] N 01 v-i M I~ d~
d~ M e-1 Q N M
H c!~ v7 ~ p ~ ~ E U1 v1 v
v1 S~ v1
~ Ui t~ f
17
tO N l0 O v U! tn O ~ ch N N Ln M
.-I v O
~ tD ~ O J~ I~ N 00 M O V~ O N
N ip O 10 M
11
r-i e-i N O .r.,lf1 M v-1 e-I N Ll1 V~
d~ M V~ -ri e-1 ri N M
U7 H u~ clW) ~ W u7 vI U) U~ E-~ u1
U1 U1 H E-~ U1 Ew
H
U ul
rC
v
oro
~ N o. d~ 00
~n o,
R: '-i N V~
v
.~
o
~z
a
v A
G1,
H
N O N M d~ In
N ~ U~ N N N
-64-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
x ~n
U G4 ~
O H ro
ro a E U
U PO O U7
-rl ,~,'' v
(n
~ro
H ~
E w
c z
n
w ~ w
x
O U
1~ 1a G
ro
U v ro
rl
~
.
ro
ro v o v
a
v ~ 0 0 0
ro
H bl C~ .C7 R~
N O
~i v o
U o0
a
-~ro v
(31 O
rl v OD
UI LL
(," ri d~
o a,
o~ o m N z
z~,M~
ro o N N
'
- M z z z
~
~
~ o z z
cd~ ~~o~o
U N M O~ V~ 01 O
.IJ ri O O N 00 10
~ JJ
O ri 01 H rl r-I N 00
r1
w ~ z z z z z z
~n
('., r-I N
O I~ O N II7 N O II1 Q1 ~-1 d1 l~ 01 L~ M OD N 01
01 10 01 00 l~
rl O I~ cH rl O O O N lf1 l0 I~ O M rl 00 M l0 M N
O M O lp
11 10 N M V~ Ll1 IW -I N M N t0 N d~ 1f1 ~O l~ OD
V~ '-I .-1 tf1 V~ 00 OW i
ro ~ tn cn E ~n E u~ ~o mo v1 M E ~n cn cn cn v~
E E E o ~ E r m E m
-t u~ ~ ov ~ ~ r
r1 lfl tl7 N O O Lf1 r1 N N M tl7 tD h 00 tf1 01
r-1 W1 OD N M 00 v-1 O E d~ M N lf1
ro O~ N M 01 00 01 V~ rl rl 1D O O 00 O N cp M OO
N tl1 O 01 e-i V~ ~O OD O .-i
-rl M N M M d~ ~D 01 U) E H rl rl N ~ d~ ~O I~ 00
O M V~ 01 U1 Y~ rl 01 O N 1D Ov O1
~ .s~ ~ to v1 cn E U1 cn U1 E E o~ E v~ E E cn tn u1
E E ~ w cn a1 u1 ui
G C~ t~ t~ M oo u~ E t4
m
N fl1 01 Ch N V~ 'd~ OD l0 In v-1 h Op O M ~ t11 tl1
UI lf1 00 f~ tl1 O 00 CO I~ N Q1 01
J~ O tI1 01 OD GO N 00 O O N O O 00 01 CO 01 rl ~l7
i~ O O I~ ~O '-I N ~D -1 O d~ t11 01 O
O .Li OD rl N M V~ ~D f~ rl rl v-i N a-i M cN tf1 l~
rl ri M M 10 rl rt N [~ 01 N ri tl1 00 01
w w E u1 u1 E vi v~ v1 Cn v1 E cn cn cn E u~ v7 E
~n E V1 VI E a~ t~ ~ Vi tn E E v7 E
U UI
N
O
p -a of
.ri of
~n
N N L(1
p; v--I O~
N
w O
~z
a
v A
H
~
r-
1
o v
W V1 N N
-65-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
E
U U GL
~" ~
,
-~-I
m
U H E U ~ H
U
U
1
z
~ o a w ~ o ~
~ ~ ~ w
a w ~ ao ~n m ~ a
z 0o r~ oo w
w cn
o '
a
a
- i o 4o w
-
~ ~
R'
- p
-
-
U ~ ~
W-
-~I d~ A - ,..,
m
-
1J t-I UI ,~ N I
rl
H
~
~ G ~ ~
E
H ~C I -- ~ W
E
~ N
D1 M N ~ M M
hl l0
N
M c-i l11
M N i 10 u-~I
i N
r V O e-I M
a-W -i
ro (Y., E z N ~ N ri x x
N 00 .~ c~
C, 1 W I M f/I OD U1 (I
~
i
Ol M I di I O N N
O I d
~
I M M V
a ~ 00 r-I
u~ ~ z E-i ~ C ~ r. ~ M
Q r > ~. o~ ,~
n ~ U U U
W ~7
U
N o
~
N rl z
ro
'_"'
t,
~ o z ~r
~n z
11 M
?I
JJ
M
z
a ~,
O ~ C~ N Ll) I~ M l!1 l~ M 00
s-1 l~ ~O di ~O
Vi 01 M 01 O t~ Lf1 N 01 ill 00 '-1
O I~ (~ M
JJ tf1 01 Vi r1 e-i M M di l0 M N M M
V~ tf1 tI1 00 (~
ro o M vo o cn uwn E v~ cn u1 0~ E cn ~ E
.-i E w cn cn ~
~
E U7 E Cf1 N 00 O~ di O ~ tf1 N O E 01
U7 M N O M N
ro 01 M O~ rl tIt 00 lf7 00 O N 00 l~
S-1 00 01 10 O O
-r-1 O l0 M t0 10 tip '-1 e-1 N M M N N N wi
O O lf7 1f1 N di tl1 00
l~
Q ~ H ~ ~ u7 cn E rn ~ cn ua ~n
H ~ ~ E (n V1 vwn cn
~ E
, m
N N r1 LI1 OD M lf1 '-i M t0 M O1 O
N e-1 di l0 L~ L~ 'di
J-1 O !~ N OD N 01 O Vi M M 00 Lf1 OWE 01 O
O N OD a0 N ~ eh N '-i
J-1
O ,~'aN Vi I~ M M OD r-I N M M Ci N v-I N M
-ri Vi '-I N Vi tl1 10 01 r-1
r1
c~ v1 E ~n cn cn u1 u~ to to u~ t/~ cn E
w E cn E E E E u1 v~ E ra
v~ rn
ro
.r.,
U W
~C
N
O 't'S
~ -~-i
UJ N ~O t~ N
N dD CO e-I p
a. N e-1 01 M
N
-'i
O
~z
a
N A
H
ri
O N OO 01 O
W U1 N N M M
-66-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
H
p H
U
U
f~' LL Ri .'~ '.~ '~ ,'~'~W ,'a~'~ 'i~
O O O U U U U O U U U
z z z z a z z z
CO V1 U7 H H H H fI1 H H H
a a a a a a, a, a a, a a,
O I~ 1D e-1 M t0 M O
W
-I N 0~ ~D cp N M Lf1
M M N N v-1 M N N
E
O O O O O O O O
w --
O _ _
G ~ M ~ 01 01
O O M O .i N O O O O O
O -rl r1 -ri N ct~ ri -r-I -r1 -rl ri
J-1 iJ O .U J..)il 1.1 1J 1J
ro o o ro
ro ~
ro ro ~ ~ ~, ro ro ro m
-~ ~.
rl rl rl ro v n--Ir-i ri ri
01 M
w w ro w ~ U w w w w w
,.-~ ~r
~ H
~
H H E~ H G4 H H H H
o U
U o
_ _ _ _ _ _
~ O ~ ~ O N V~ t~ lJ1 O
~ ~ ~ .-. .-. .-.
N 01 l0 00 01 ~ N v-1 t11 M t!1
V ~O N V~ O .('.lD 10 M O
d~ d~ tD N V~ I~ t11 I~ ~ Lf1 M
CO N d O O -1 01 l0 Lf1
O N v--1 . . ~ ,~
N -,..~-~..~
O O O O O 1J O O O O O
J..7
ro ~ v O O ~ ~- O v
O O ro O O O
v _ '''
v N v v v r-a N v v v v
ri ri r-1 r~ r1 r~ r~
U U U U U ro U U U U U
A ro to ro ri r-I ro ro ro ro
~ G ~ ~ ~ ~ ~ ~ ~ G ~
~ ~ ~ w w a ~ ~ ~r
ro ro ro rd ro v ro ro ro ro ro
v v v a ~ v v v v
U U U U U fs, U U U U U
Gu fsa fsa H H Ga fs, G~ Gu
0
'
c~-I o
e N
0 0
" ~ ~o
~ .-. ~ ro
E ~ ~r N w ~ b M ~ ~ ~ v
o~
U N ro U .~ ri O ro ~-f 'J
.-1
w rl N r~ -rl J~ ~ N r~ iJ O -rl
.--1
O +~ ~ ~ m U ~ N ..
~
N O U v v tn O U N U
O M
ri U! rl J-1 ro ~-~ N 1J
.~ V~
O O td O ~ ~ ro p ro ZS
t~ ~ .-i O
RWo N > i1 ~ O m D .~ .-a O
~ M v -~ M ..
.a.) O ~ O O O -ri "~ O O ~ i-I
n-I N V~ C,' O M .-I O
U J.~ O ri ~ f-1 'O O rl ~.1 U
N ~-1 ~ ~- d~ "'
ro ro 5 ro ro a s~ > 2s ~ u~ ,
o .-~ . ~ ~ o ~
,-
s~ s~ ~ u~ it ~ s~ ~n ro 04
. o ~ ~ . ,- ~.
w v vo ro v-- roH U v~ roo ro >o o
ro
-- x z U x c~ ~ z U c~ o _
~ -- ~ ~ -- s~ ~ ~
~ ro ~ o ~' ;~
~ ,~
-, ~ ~ ~ ~ , ~ ~ ~ o
v - v v
'
O r-i 1D O l~ Lf1 N V~ 10 01 H N
~ G JJ J~ U1 O (.," ~ O
,
-rl ao ao o u1 D V~ O co M ro
U ~ U1 y N v ~- ~ U U O
M N M N 1J M N N
ro v -r1 UI
' ~ ~ ~
~
N O O O O O -r O O O ... H
, I~ ~ (1~ 1 ro H ',~ ro
~
p ~ ~ ~ ' O ~' '-' .- l0 H
~p O O ~ w O G: w
rl U .--1ri 1.1 r-1 -rl U -ri Lft f0
11 -r1 U
v v v v v ro v v v N .-a
~a -~ v ro .u +.~ ~, 2s ~ -~
w > D ~ ~ D ~ > > > ~n ~
~ a~ o > ~ u~ u~ ~ s~ +~
ro -~ ~ -,~ -~ ~ .~ ~ .a o U
v -- v v ro v v ro v v
v ~ ~ .u ~ ~ v ~ ~ ~ --
U .~ A x c~ ~ -~, U
~ ~ ~
N '~ .'1 '~1 .h ~ ~ ~ ~ O (Il '7
~ i1~ rl ~ -rl fly ~ r
i
tA 'd '~ Zf 't'~'W O 't3 'LJ 23 ~ r
O O tT d~ D M O O ~ O O
O
w O O O O O .-I O O O O ri
av w O .-i N ep N ~ t~ H 1~
H N ft S1 N ~.I N h ~1 ~-I ,5 'Cj
~-i ro ri N wi e-I JJ ro '-) J~
l~ Lb Cb i1 C1~ ~ CL i1~ G1 i-i
~ O U1 ~
v v v v v v v v v v ro
o v s~ o o o ro v o ro
x x x x x A x x rx z U
x x -- -- c~ x -- c~ x
v
.
~
o
.~
z
A
m
i
a
a
z N M .~ ~ ~ ~ ~ ~, o .~ N
~
M M M M M M M M cr d~
-67-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
o r, r,
Ew E
U ~ ~ ~ P.' ~ ~ ~ lx .'
U U U O U U U O U U U
z z z w z z z w z z z
H H H V1 H H H tn H H H
a a a a a a a a a a a
ro
M N V~ M ~D l0 M
O Lf1 d~ M I~ M t~ 00
E N N N N N N ro N
~i
0 0 0 0 0 o E. o
O _ _ _ _
C', ~.,'('., rr ~,' N I~ O O
O O O O O O rn a~ u1 ~n O
-rl -rl -rl -ri -ri -rl w-1 r-I N N -rl
1J .~.J.1.J 1J 11
U O O O O
ro ~
ro ro ro ~ ~ ~ ~ ro
-. .. ~
w ~ r-, ,-, .-m -.~ ~ ro ro ro v
N N ~
w w w w W W J-1 11 J-I U w
Ov 01 \O
C G N N v p G
~ ~ .-~
H H H H H H G'' ~ ~
~
O O U
O d~ M lfl M I~ rl 01 M I~
U ~ N 01 .~ ~ ~. ~.
d~ N t11 tf1 O r-I ri N OD l~ C~
OD 00 V~ Vi N l~ C ~ (,' 01
d~ d~ n ~r d~ u~ .a~ vn w .1 M
ov .m .~ ,~ N O O O O co
,.i . .
N N
ro o 0 00 0 00 00 o~ o~ oro o o
0 0 0
_ _ _
t-' N s s s s s s s s s V
" ro ~ ~ ~ o
~ ~ ~ ~ ~ .~ ~ ~ ~
A v v v v v v v v v :mn v
~ ~ ~ ~ ~ ~ ro ro ro
U U U U U U U U U ro U
ro N ro ro r-I r1 r-I N ro
~ ~ ~ a a ~ ~ G a a
ro ro ro ~ y .u w w w . ro
v ro ro ro ro ro ro ro ro v v
~ s~ ~ v v ~ a ~ o
U U U U U U U U U G4 U
G4 H E O fs. fsi H H H -- Cz.
M
.. ~
r- v r ~o
y o > ~o
.-,
C~ ro . ~-' . o
[-~ ~' . ~ -
0
E-'~~ . . ., ~ . ., S-I l0
tf1 . . I'~ . M
N f-1 00
1D e--I 01 N r-I O l~ l~ ri ro N
w rl N rl N ro N ri .-I ro H N
o . . . . ~, a . . " ~
0 0 0 0 ~ v o o ~ o
~ ~
UJ UI VJ UJ a U7 tl) a ~J N
~
~ O r-i ~ ~ O O O
u1
0 0 0 0 .-, ~ o o .~ -,~ o
0
5 D D ~ v N 5 ~ v 2f
N
S-I f-1 f-I N .~ ~I S-I 'J f-1 f-1
~ ~ ~ ~ ~
w v N N N N O N N N ro Ul
01 O ~ l~ 01 t~ N
'~ z z z z C~ "' ~f-, z Ca U z
~ V~ "' ~ I~ M M 10 M
M .-. rl M O v-I ~- W ri
-1
N
O O N I~ M N ~,' N 1D M M d~
v-i N O L', .-i O O O I~ O
N V~ l~ f~ N ~ 01 M M M ~D
'-I '-' a O '-' ~ ~ ~D N
N N N N N ~ rl N M M
O ~ O ~ rl
v O ~ ~ ~ ~
v O O O O O H O O O O O
~ ro H N ro ro ro O ro
ya ,.. .-. .... .... ~ ...
~ ~ ~ ~ C
a ro H - U ro U r - --.
O 1 -i
v v v v Ul -.~ OJ N N v N
r-1 ro tJ -rl .-~ 'J 1.J 1~ t~ U 1~
'J ,~ ~ ~ ~J 1.1 5 ' 'J 5 'J
~ 1~ Ul l~ ~ !-1 N U! Ul -.-IUl
rl -1 rl ri -ri N ri -r-I rl -ri -'i
U f..,"N v U v N N N b1 v
v .~ ~ ~ ~ ~ -~ +~ ~ ~ ~
~n v ~ .~ u~ z .u ~ ~r o
U U U U U O U U U U U
ro ~ G O ro G ~ G r1
a ~ ~ ~ ~
~ -~I -.i -rl O
't1 '~ '~ '0 21 O Tl 2J 'd '~ '~
O O O O O v-i O O O ~ O
O O O O O .~-1 O O O O O
r-I rl S-~ 1.~ -.~ d~ N ~.1 f-I f-~
E S1 1.1 1-I ~i ~i ro S-1 f-I 1-1 ~-I S-I
~ v 1J 'O N J-7 .!-1 1J .l~
~ ~ ~
~
v ~ ~ ~ o ~ ~ ~ v ~
~ rd ro ro rt ~ ~ ro
x x x x x x x x x x x
U A c~ x U -- ~ a c~ A c~
~s
o
~
z
O
A
N
H
r~
U
O~
z M ~ ~, ~ ~ ~ ~, ~, N M
~
-68-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/I3281
E- E
H H
P.' fx
U U
N U1 U1
O ~ ?~ W W
H H H H H
Ql H pa (~ H
a a a a a a a a a
a a a G
0 0 0 0 0
.~.,
ro ro b ~ ro ro
~
a ~
~ o
o w w w w w
v
H H H H (~ H H O
O G', 10
w O O N N
O M ri N cr Lf1 O M
1~ O 01 O O 10 M O
O ro t0 l0 ~O IW D
0 0 0 o 0
~ O
~
U ro r-1
u~
.u w v N N ro N N ~
t~ o
s~ a~ ~ ~ > ~ s~ 5 ~ ro
~ ,~
C4 G4 H rl rl rl E ri ri ~
O 13 1.1 1J 1J 1~ O
ro ro ro ~ it ro --
U! N ~ N N N vo N N M
G C
ro d~ ~ w w w ~r w W M
O O
. . .,1 .,i .,~ . .,~ .,~
.,i
U O O rl rt ri O r-i r1 O
JJ JJ
-- -- o o o o o
ro
_ _ _ _ _ _
U ~ S 1
l -1 .1 a a a 11 a a ro
ro ~ N M ~ ~O M O O1
ro ~ ~
M l I UJ N ~-I
U U r1 r-I ri M ri r-1 ~
'-i M ~-I N U v-i N r-i
~-1
m ~ ~ ~ ~ .-~ q .-~ ~-, ro
w ro . w
ro N N v ro v N s
~ o o o o o o
~
La U U U U U U U U E~
H v v ~ ~- ~- ~- H
M
~
N l
r J-1
JJ
H ~ ~ U1
ro M b l0 '~ O O 00 N
11 rl M O f~ O O d~ OD
O ro M f.1 rl S-I N r-1 rl O
E
~ o v o N 0 0 0
ao
w N -- cx -- 0; -- .r ._.
m
o E ,~ it
o >
vo ~ .-i ~
o
~ ~ ~ O O U
N 1~ N ~ 5 N
_
U N ~ O O o ~ N N 'J
.~ cr ro .-I
ro A b --M z~ --~ b~ z-. z~ o
I1 O 00 O rl O O v-1 rl
M ei
a O M
~
ov G p, o d~ ~
~ o ~ ,. ~ o o
i
t~ Ql O N '~3'4) 10 M ro
" Q1 '-' ''
N (x ~ M ~ (1,'N N U
'-' H ~ O O
v v
H H
rl O ~ H O H ~ O O ~
O M Ul ro ri ro ro
UI ~ M \ ~', \ M ~I G,' f~
LI1 M ~ O li
u! .1 M U ~ U M ro -a ~
M O S~ ro
N N M i-1 QI r1 M Q) N V~
'J 11 J.1 e-i rI 1J J.~
'J O J-1 ' 11 ~ ,5 'J UI
O ~I N N ~ Ul
-- o N -~ N o ~ .a o
-- N N ro U U N N
" ,- ~,z ~~ ~u --
W U U O U O ro U U G
U G ro ~
-rl U1 a ~ a N ~ ~ UI
-ri ~ ri ~ 'J J ri rl
N 'O "~ O rd O ~ '~ 'd ~
b1 b1 d~ O r-I O O O O
O O 1.~ O 1~ O O O O
O O O 1a t~ ri -.i f~ Sa
UJ ~.1 'J ro ~1 ro 'J fi ~1 'J
rl rl N 1J rl 'O 'L~ iJ ~1
v v ~ v ~ i ~ ~'
ro ~
s s o o a m v ~
a a r ro r
a
H x z x x x z x x z
~ ~ ~- a -- U U c~ c~
~s
o
.~
z
O
A
N
H
ri
Ua
W V~ tf7 10 I~ OO 01 O r-1 N
z ~,
~
-69-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
N
~u '~' ~ ~ ~ v
N -
ro O N N ~ ~, m
~
ro a ~1 v 1J
L
~ ro
'
N -~, vN ~ ro ~ .a r-I,~,-.I
~ m v ~
v zs ~ ~ro ro o
o .~ N
~-..IN ro ro> v
~
1.~ ro w-I.-1O S-IN N U N .1N 'L3
ri N -~-i
U U 1.1ritJ U N v 23 iJ1.1ro -'i
~ ,~ w
N O ro ~N f~U i-I~ a~rom .i
1.1 m ro 1-~
.a ~.,> N ro~ -~Iv ,--rv ,-,o, v
ro -~I a ~
+~ ~ o rom a ro x ~ U ~ -~,
w ~ ~I a~
' v
G -rl ~ U v O ~ ri v
1~ p
.s~
N ~ b~O ~ N ~I ~ ~ ~ w
G ~I ~
~ O ~~ ~ O .i
O
S.I .t S m m H O ~, S-~O~O
.-~I tn -I ro
l
w ro w ~.1U roro w f-I~1 W ~",w -ri
ro S-1 N O m
t-I
.-1 N ~~ v v >~ O ~ U ro
ro ~ ~
-ri
~ m ~ ~ci~ m N ~ a ~ 'dN ro o
25 ~ p 8
~
v -~Iv -,~.civ v m v ~ ~ w
N 2s -~I
ro ~s ~ vN ~u ~ a -,~ ,~~ o N
a rn
ro
ro ro ro ~I~ . ro .>~ ro G v
U t~ o
r-I ~, ,~ rov ~ v rl tn r,v -~I
o -~I ~a N
ro
o N o E~ N m o >~ .u o r,U ro
~, 5 ~
m v ~n vro v ro m -~,G cnroN x
~ ~ ~ o .u
N
-~I ~ -~Iws., ~Iv ~I s.lv ,~~ ro
o o .u
v
ro N tn N m ~ ~
~I 5 m
zs
m ~ ~a ro~I ~ ro .u ~ ~ v a
N ro
O N -~_ > ~ ~ rt U
5 N ro
m
, m~I , N N W - ~ -~I
N rt7 N ~.1 m
.u .~ rl
O
rnzs ro ~ roro roro tT ro ~ roo ~
o a N rt
ro
~i C.'C.'UU C,''r~ ~",,~,ri .~""U ,'~
v ~ - ro .L; m ri
- o
I
I
~ ~I ~'.I o ~ r-Iv v -.~~ N v
~ v a m
m N m to~ N U m w m m ro
ro ,~ >., v
>
~ O ~ U~ O m ~ ~ m
N
rl
O U rl U ro 27 O O O
~ ro ~ a
ro
zs ro ro zs~ > ro ~I 5 roroN o
~ ~
v -rlv rlO U O v O N r-I23 -~-I
3 ~ N N
orl -~IN ~ ~ ~ .uo v ~
o ~
U >~ U IO 1J,Q U ~",O U 1 .J O
N N ~ ~
ro
~ ~ ~ N-I O v ~ ~ O ~ N ro v
.--I ~-I w ~-I
N
1-I ro 1.1roO i-I1.11.1C.,"r-1 S.IroU .-1
ro O N 1.i
1~
~ a ~ v,~ v v ~ o .o .uv -~I
N r-I v v
~I
U1 O m ~1~ riU m ~ m 'y'C3
N N ~ - ~ U
Ci ~.,.~',1ro U (.,-'I f-I [,'I ,
w J-1 N m ('.,
J-1
O ~ O Mw m O co ro O '-Iw
m ,>~ ~
~
U U V~~ O ~ U r~ r-I U M b~ la
'O N
O ~
N
I
-I
~
-
~ N O ~ ~ G N
m ~ m row N ~ m w
O 'd ~ ro m
O 3 >
, m ro~I b~
ro N ro ~ C ~ G
I ~ O
l
, r . ro a ro O ~
a -rl
3 ro 3 ~O 1~U g ~ ro 3 ~ -~ rl
N U U 2s ro N rn
~u ~ ~' wv ~a~ ~ 4N1x ~ w ~ ~
~, o m a
'u -~
v
a ro ro N v . ro U ro ro ro -~
I N sa ~ ~a
, v ~
~
N
a
~
s~
~ , , zv ~ m N ro ~ N ~dw o
-~I N ~I ~
N JW ~ ~7 UJ>~ ~ m >a N G ~ O m ~-I
n U v 23 ~ N
O
U rl rl ,~a ~-iN rf ,5 ro ri,5m ro
r-i N U ~ ~,' O
. 1.l
ro ~ ~ ~ '-iO ro ''~ U
rl
v ~ ~O 1J N ~ ~ ~
~ U L
-rl
A .-i ~I ~I NI w m '-I ~ N N O ~
w O O U U
- .C
O ri O S.IO O la bt O N 1~ U
O ~' rl
ri
?i E-W r H b~ 'r- E O .>~ E-~ U -ri
.u N N ro
t~
N O ~I p N~ ?.IN D v U D N N ~
v U 'L3 O r-IO 'L3
G ro
ro ?, O E- ~ O N E-~~ >~ E-~~ rl O
N O 1-I r1 1~-ri O U O
U1 ~ ~ U
m ro N i
m
a
1~
-
l
r s z m v E m .>~
~ ~ .-I la
3
.4 rx m m m ~ A m N n m U a
m w .--i v ~ w O N
m
a.~
-~ H ~I ~ ro w ro H ro w N v
w a ~ ~ y -r.l G -.~ ~ a
ro ro
a ~ O ~m U x 1-Ia H J.~,
~ U ~ LL ro O G
.O a~ m tn
rl ,r~
H H H N
O
H
a 5 0
w
H
A
H
O t0 N L~-
v o m n
~' N OD M M
O .-~ lf1 ~D L~
ri 01 ,--I ~ N
U m o0
O
.
't7
O
-~
z
O
A
v
H
r~
U
d
W N M
z M M M M
U7
CA 02329685 2000-12-11
WO 99/64596 PCTNS99/13281 _
o 'N v
m . m '-'
~
w v 3 o
x
~ v N ~ O ll f(fW i-1
~ N
l
N N ',?~ y '~ O
N ) N
rl
i-I 4-1JJ rl.~, (0 ~. N LI 13b rlN V~
U1 1 b
f0
U1 ,'3.N -rlU 1.rO 1-~ HN Ul".~ N .~". 4f?i
S-1 -rl C' G m
$ u1
U1 '~"~U r1 1.1 J-1 1J ri23 1dO ~.,Ul
N 11 tn ~ b
' O
r1 U1rl~, ~1U la v~-I l~O a W ~Io 10
vv~rto N O U
~
v v w m v u~ o ~
s~ >', ~ v
v ~
a s~ 2t.u a >~N rt-~ G z
~ o ~ a E
~ ~
x ~uv~ s~ o it a 0 0
>~ roa zs ~' o
~ ~
> ro~ m rlv x v ~ a
m v ro s~
rl
s~v ~ rla rl ~ro o u~ ~,v .aa
~ ,~ v ~
rl m
.-i~0 3 ~0 ca~7 ~ v~ U ~I W .-mo
-~ E ~
v
m O >~ r-I U T3O b r-I'Lf ~-i
~ 'O N
r-i
~ r-I .WO W 0 u~ ~11 ~ U m ~ ~ s~
v N r1 N
1J
O UI.41W ,17 O U 1a rlO O UI N f6 O . U
1J v -rl
W
1-1t~ ("., i-I-.~ 9 UU ~I- .Ori N U7
b G." 'Cf N
~ v
4-aU'~b O 4-Ib r-I C,' W ~ O N W 1.1
't~ -rl O .(', 1.~
r-1 .~
~v -~I rt s~ -~Ia ~ >, O G
ro it~ ~ v 2sa ~ ~ -.~ 3
m ~ v E
ro o -
v ~
I . -. ~ 0 2s~ ~ ~ o
o
v vro.aG v m a ~,~ v ~I v o v o ~
~ x s~ ~ ~
1~ -rlU N 1~10 .LJ f-1O 1~U ''>;j1-I l~''Cf
~-I W U! U
1J
rtfT3U ~ ~ G roN v Olm 0 O ~ U m
U --i N
rl rlo v ~I o rlrn x ~v ~ x rlv
s~ a ro ~ ,~
~n
0 0~n~ v -~Io t~ ~ rn,~ o ~ a ~ o ro
-.~ v ~ o sa o
-~I o
Ul ~V1t~ 1 Wn-rl la -riU VJ-ri ~ U1 u)rl
~ ,1~ ,f"., fff 1-I
~
s~roa 7, v -.~s~ o ~v~ .~I3 -~>., -~I~n
+~ ~ " w
ro o
td ~ 1-I ~ J..~ O rtf U rtf
N E N 1.~ -ri
NN N O b ~ 23 ~ ~Y-i ~ N ~,v ~ U 'O
H ~ ~ S-I
O N
G ~av '~ .~~ ~ ro o a ~ ro
w .~y , G o
-~
l~ -ri-rl r-i 2! ~, L - U -ri
-ri U ~J..1 ~ .~
~"" N
(C
C51~r~.I2TO trm -rl brtf b W U1U b~rtf
,~ S., N O O 't~
~
S~ O ~'N r-I~ ~ f-I C4.rl ~ 4-I rlv C,"N 1-1
1~ Ti -I
U
toW la.p rt-.~o a ~ -~I~ .~~ -~I'-I
7, a +~ -~I .c~
U7 .-I-.iv7~ O ~ U)~'., ~ VIfl1
ri U V! ' O
G
~ W~ _ b 2s~ m s~ rt~n ~ ro ~ .-~
o ~ o a ~I v ~
.-, o
rov s.~ -.~ a .~m -~I t~o v u~
ro o ~ ~ m ro.o -.~ ro a a
ro ,~ rl
~
u~ ~n~ b u, v ~ ro~I
v w a a ro
o
v v.-1O ~ U Olrtf N roa v m -~I~n v 3
' W to ro s~ -.~ .~I
U a
11 ~O C.ri O 7-~U -ri riO !JU 1JO ~ 'O
U O3 l U W N ri ~
" U ~
, r ~ r-I f.1 aN U ~ m N U G:
.. -rl r-I ?~ R.'
.--1
r-I1JU G W O b i-I~ O rtS W rl ~ rt3
~I b S~N N td 4-~ Olr-i N r--t S-I
-~I U -I rtf 10
tn ~ -
G I
, ~ U -a s~
.u s~w roro v ~ ~ . ~~ ,~ro O " v
o a -,~ v "
' ~ ~
,1
W v U ,~ 1!N Cj U U UlT3 U11.1 V7r-I
N O ~.' 'L3 U1 UI
ri rl
'
a 3 o s~ ~ s~rl ~n .aro a rl ~Is~ ~ ro
u~ ~ v s~ -~, m
~
0 0>~t~o a o o ~ ~ o o v v o
U -IO N ~I a ro rt v .~
S -~I ro
b '
'
r 1 21U I E., U7O U I U C,' U
N ri U L,
U! ~
.,
-rl'i~Ul f..l '~1J f-I r-110 00
U1 O J..! ~, flf
'(," (0
~~ m -~,~dm m v o~n ~nro ~ r-I u~~ s.,
rt wa ~, s~rtro u~ ~~, ~
~ ro ~
s~ v ro
>
s
~ . ~ rov ~, m
a 3 vv ~rrt ro3 ~ r, rt~ 3 w
~ 3 ?
~
I ~ v v 3 w
O .-iUf .L~ I ~~ o N ~ O
p ~ v I a
N
-11 ?~ N N G ~ ~ o ~ Ou ~ to
O ~ rt
M
, . . , p ~ ~r ~,O
1.~ N N~r'bN u1H ~ b~ H~ W r-t ~ H cn
~ td ~ n ~I
O
-.I
a m >~ ro-.~v m v ~ ,ra, rt-~I a G roro
.-, - a 4-I
u~
-rl H +.Itn1-It~ 1.1H 10 O -.i-r1 N m s~O ?.I>~
~ '~ tlS 1.1 v1
N -.i
S-I .~ G."b1~0 N ~71d S1 Wri .faU ~ .i ~ fC
a ~ L', '~.," 23
Ul
U w ~~ G~ .Q~ 1~ .~ O'~ -~~ a a.l r-I
~ N O -.~N
~ O
U1 ~-IOH ItSN t0r-IO 11 G'.,~ .-IO U r-It7~
O -rl '~.,' U7 rl -.-1
~ H
v ~I > -.~ s~ vro >~ v
~ v u~
W v
zs
A cr 4-1 '(7fa 'L'~Lf14-I L," '~ N W C;W .-I
rl -rl U la S~ N '
Tf O r1
rtv ~ o v o rocn N rov o ~
~ v ~ . ~ o
a
E 'b1-I.t~O H H b rl N H Tl i-I (~m ,L",
U t~ O N r-i U!
1.! N
o vs~ro.-IH o v u~ ros~ o v s~~ o .u
a m G u~ m o 3
ro m .
ro z 9v a ~ z ~ ~ vv > ro,-r
s~ c7 0 .~s~ v a -.~ rt -~ rn
>~ a v rt sa
~ o a
m
-
I
~ 3.n ~ o v rt
p ~ ~~ 'dro ~ ~ .u Ov .u G it E ~ Tf
u1 O l~ t0 v v N
'ty m ~ N
N v
rn ~
vv a ~I ~ v x ..a~I o v ~I,-~ H -~I
,~ s~ to .~I ro s~ ,-,
~ -.I ~ u,
.
a a ~I~n ~, m s~ v ~n~ v a -~I w a o
-~I o a a ~ a .c~ M
-~I ro m
~
N ,-I
~1 O O
z ~
Z
~
a ~
u a
A
H L!7 l~ 01 M
ri ~O
N L~ M r-1 QO
C: O W O di d,
O O L~ M Cp
ri tf1 1D ~ d1
U .-I .1 'i e-t
O
.~
z
o
A
v
H
H
as
w ~ r m o,
z M M M M
~n
~71~
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
N u7
~ ~ i~ ~
-Iv ~ - v zs ro
i
rt> ro r~ ro m a ~ ~
ro ro ~
N C:rl il N -- U G".,ro -ri 'b v Ul
fir .~'.. U
O a ~ ro
ro
UI ~ro U ~ ro .i ,~ U U! U r
Ul O U J J. N 7 't1 ! -II
l N tl1
i
-I r l N ~., f.l E-~
T w '-I o o ~
~
-
-.~rov ~ ~n~n o ro v I ,-~ i ~ ~ o
-~I v o
~v y -~I~ a ro tn ro .,~ o
~ ~ ro -- ~ a H a v
H o
O ~ U ro O .~ ~ u1 1~
r M w ~ U
-I ~
W
~
rov O b1b1 t~ U ~ 1J 5 N Sa Sa
~ rl .~ b LI
N HU .u U!G G,'O N b1 U1 H w ri roro
r-I~U v v ~ 'LSUl i~ v ~ v
- , s 1~
~ v ~
~n
, .~ v u~-~, v -~I v v a~ v
z -~I ~n ~-I
w ~G ~ ~ .-~H ~ t~-~ H 3 U o ~n
v ro -a ro 2s rl
-.~o a v ~ u~ . v~ r-I v
~ v H ~ a
2s
H ~ .~~ ~ o H ,pv ~ u~ ~ ro H
-~I v ~ o ~n x
m a
o ~.~ o roo ~ v ,~ o .1 0 H o 3
~ .~I x -~I
it
roU U -rlisN ~ U b >-I H O ,1 H
U 'O N U ri 3 ,r,
w J-1ro 'dw r~ V~ (~..,j~..- w N w UJ
Ul ri
ON U7 ro 1~ roroSi U v .-i tr> SI
S-I i 1~ >-I
O
2s ~> ro H .r~~ w a v b ~a n, ro v o
v ro ~ ro ~-I ~ ~
.~
v ~ 3 H v o .v v H v v .~ v ~
.u ~ ro ~ ~
,-.I
roJ-I 1J('.,'-' v S-I(; 1.1 1J ri Y-I 1~,
1C f-1N ~" -ri ro I
~ Ul
ro U ~ Qlroro ro O Nro ro ro ro f-I ror-i
?.I O ~ t~ !~
U N
b1ro O r-Iri M N UU r1 r-i S-I O rlrl
N tn ~ ,F"1J A ~".,ro
t11
o s~rr ro o rn v m ~ o v o v ~
ro ~ ro w r,
r~
u~ -~I~, o r~~nro v -~,av v~ m ~ v ~n~
o ~ ~ ~ -~ o ro
r1 Hr-I -riU 'CJ 1J i~ -ri rl ro C rlW
U .~., 'f~ UJ N
O U
~v H Ts ~ ro ~-Iro H ro .-~ .
ro ro ro o,
roH rl t~~ ro H .uro~ ~ o ~C -~, b
o .a ro v v a ~
~ V ~ a
vP i~ ~ rt o o No ~ o ~ 'Q o ~ 3
~ m ~ ~
~
-~v ~n ro
zs
v
. .~Ia m zsH ~-I a a H
,~ ro ~a
t51roUl -rl ~ t71r-1--1 ORi b1 b1 ro ~ b1O
ri U V1 f0 fl, .~
U
~'L7 U7~ O ~ b ~ ~ U ~ -~ ~ !~O
H O 'O v P~
w ~ ro-~,I ro v zs~ r.l -.~ a , .
~ ~n ro a .-, r-I ~
.~
u~ ~5, v v u~H .~ ro v u~ ~n o - m
> u~ H ,~ o
ro~ .~ cn~ ro ~n ~ vb a -a ~ ~ .
-~~ o -.~ ro ~ -,~~ I ~ o,
o ~ v ro ~
v -~, v
' >.I a u~
u~ ' a
~g m ~ b ~, ~ a orl ~o ~ v -~r ro~
v r, ro ~ ro ro o a
o
v itu~ w v I v ~ aa v v ~ ~ v v
o G ,~ a .~I v ~ ,-r o
.~I
1J UO O ~r1JOD ~ -r1ro~ 1J 1.1 , .ri 1Jw
~ N 1J ~ ~ b1 rl
1..1
U ~~ u7 N U u1 1~ ri U ~ U O .u U 1~
1~ v t rn .u
U
ro~ 'O N ~ S-1'O ?i x ~ O ~ .J~ U! ~ N
S.1 w N r-I W ro
v
f-IUw G 1~Saro v N N~r Sa N U ?, N r-i
N 1.1JJO N O V~ Sa r~ td v .t~
NN w (' l1N 1JW J w
rl '
., 1 ( Url l~roUl
u7 25-n U roU7N -~i 1 UO um .,~."(;
~ 1 r-i
. n O
f iN O "' " " ro .~1 ~ ~
' '
, rr. .~.O ro N t11 ~." (".,11 U ~'.,bf
. ~ N ., '>vS 'C3 Q)
u
7
O OU ,>~ ~,O O 1~ rlroN O N O ~ ri p
U IU 1 .~ " N U7 ~ ~ ,~
" m r-f
i E-~
'
r -IU ri L, ~ U-~ U 1~ U fd b1 U ro
f. O Ei -. 1J r-1 O '.1 .1.~ -
ro
Ha ro ro ~ o .~ ro H v ro -~
~ ~ rl H ~, rn
m roo ~ ~ u~~ a ~ va u~ m y .~ ~n~n
-~I o . ..~ w .~I w x
v
.-r ro va . o row u~ ~ ~~, ro ro ~ H roro
ro .~ ro .u s~ o v
c a > u~
3 ?~~n ~ H 3 v v v ro~ 3 s~ 3 a H 3 a
v ~ v ro o - v
o
o Io G o .-I~ -.~.u-~ v ~n v ro o ~
G .~ b ,~ ~a -~I ro 3
~ ~I rl
>, Nu v a ~ w o ~ m~ 7, ~, rn ~ ?~ro
o ~ s~ H > ro ,~
o ,-I
H ~a ~n H v .--Irooro H v H - v H v
rl rs ro v o ~ ~ M
ro tr~ v
c~ ro v v a ro.a o w Hw ro ro ro .~ roN
o v w ~ w o
v 0o
H roN a -~,H ~ H a- H w H ~ H ro
~u ~ s~ b v w
~
H .4 ro ~, .u.~o v . H .~ ,ct v ~
~ -~I H v
v o
a -~I~p .-~ o .~I~ 3 ~ -.~ -.a ~ ~., -~~
rl ~I H ~ a~ zs ~ a~ ro
~ s~
rl oro ~n H rlo o b1O ,-I rl v ~I ~Io
ro ~ ~ U a v rl ~
a ~, ro
N 1.rro ~ N H ri O !~rl v N ~ ro ro S.I
O U O G U Ra 1~
A ~-I >, 3 .-iM w w v ~m M H ~
w -.~ v ro w rn w
-.~
6 x v N v~ s~ H ~ o v o s~ N o rt
~ a u~ m ~ rtt
v
?~ E Tto u~ ~nE-~ro w ro v H 3 H ro ~ r-~z5
o ?~ v b~ a -~ H ~i v
~--I ~
H v v~ -~ o o v v ~n ~ o ro o s~ o w v
ro o ~H H H z H ~ v ~ .~ ~ ro H w .a
a .u > ~ o H H Z .~ o
s~ .~I .~ u~ o ~
o a ~
ro x
H H o v c70 ~ v ~v ~ a ~ ~ v z o
z v .>~ as v ~ ro ~ 25 -~I ,-~ A .~
-~ ~ v .~ o ,.d o, E
~ ~ I H ~-I -I w
~ H -
~ o ~
~
v
m
H vo o.~-.~~ ~ v x m w~ axe v ~ r H v
~ o H o v . v
H ~
aro
a ~n Hv a roa H o o E-~ ~n p, x H
E .~ .-~ .~ 3 a c~, ~
~ .c~ ao a
w rn
w
r-I
M M s>t N
o z o o w
~
z
-ri H
E., U7
a cn
A
H N N O l0
N !!1 ,-I 'di M
v M
t11 M M N
O OD Op O M
M ~p 01
r-I ~O l0 pp 01
U N N N N N
ro
o
~I
z
o
A
v
H
U
OI
W O r-1 N
z
~n
_72_
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
N
W
G ~ ~' riv ro ~ O U
f.1 >,. .,. ~ ~ '
I .I
ow ro ilroU v ~ .~ , ~
~ s ,
~
m v -~, 5 a ou~ . s~~n W o u~
o v ~ t ~ o,ro 5 o u a
ro o a ~ ,
v .~
.
~ .ro?~ roro o I ro
~ .~r-I o G ~n v .-I ,-~ > u~ ,-,m r~
ro ~ .~ ro ~, r-I .-,
o ~,
,~
U
,~ a ~ o -~v a ,~ v o ro~ o ~n ~,a ro a
v a -~ ~
v ro U ~ S-I-r1 1. y1 U .-i1.~O 1.1O ro N
ro .~ v uI -.i O
ro
G roG .-a O ~ >I O bt tnN roN 5r N
ro ~
w '~ro ~ ~, ~ aro-.~v ~ >~ s I >~
~ a ,
o rl o -~Iv ~ ~ 0 0 0
~
v ro~ rl v o ~n ~I v a s~~ U ~I~ s~ r,
~ a ro v o x
1J U O ro rorlv O W ~ W ~ro11UJ O C',W .I!
O O H ya aJ
t~ -riSa 1-I v ro O T3U >~U1 U ro ro ri
l-I ~ 1~ v
H ~
roU ~ ~ ~ v ro ~ ro -~v v t~ ro 3
a~ >~v rov ro v 3 v vro ~ U rn
-~ ~-I ,~ ro I -I
v ~I a
~
~ ~ v W
O -~-i~ rlW ro 3-1~ 1J .-i~',1J. --iri 1~ O
F," U ro ro , ro
s~ ~n O u~ ro v ro ro~Irow ..~r-,ro s
~ ~I O ~,
L
w ro .u ~n>~v ro ~-~I~, .-I
s~ ~ o -~ o ~
~ v ~ -~Iro.~ o a o v ~ s ~ o ~ b
ro o s~ I s~ rl ~ ~ a
ro 3 ~I ~ H
v ro v
. cn ,~ rn w ~ v ~ O y~
v .-Io ~ ~n~ H -~ rn - ~
a ~ tn ~
. . o m ~ ..-Iro -~I x
v o x ~-, ~ v o 3 ~' o
~ ro ~
a v ~ ~ ~o ro. ~ b ~ v
rro-I~ ~ '-i ~ ro fa ~-t~ ~ UI O v
ro ro U
m ~ ~ .
N O ~
>~U a ~ roror-It~ N G
~ - .>~ 7r
w ~ ro -~I o ro rn 3 a, Uw roro .>~o o~ o
w ro ~ ~
-~
H u~ m ror-I ~ O t~ ~ U O a~~ O w ~I
~ ~n .
~
~ ~,v O a ,-~~-I ~I N w ro v 2s ro~n ~r O
a r-I v ~ .u ro
~n ~
ro 1.1U 1.1 O ro U1 O U7 U~ U ~ O N G
~I roU N roI x ~ ~ U
v ~ v
s~ w o
ro
v o ~ ro . v ~ ro
N ' Q1 " v o
J UI rl N S-IS-I , 'i7O ro v 1~ N
a rI N 4'
i
b~ O .C ~ w roN m ro w ro .1~ ~ rnf.~.
ro N p, ~ ro >'I
U
>~ t~ ~ U N ,~ Ql v OU 0~O roO v ro
U ro Ut r-I
~' -r1
~ v rn ~ ~,a ~-I ~ v ~ Iv G ~ v ~ ro
U1 ~-iro O i'II ~ ~, U w o ~
ro 1
O
r U N~IriU a7 U ri
U tnO a N
N w w c~w N ~ ro ~ roN ~ v ~ ,O
>~ N
G ro -- m ~ro v s~ ~ s~ v~ r~s~ ro s~ ~
~n ' ~ ~n s.~ ro
o
ro -ri.~ .-I (, U lJ v ~..y,(IIO O 1J -rl
ro N U
N UJ~ N O rorl Ul W U! I',~yW N S-1.~ N rl
r-I .i~. f;"., -rl ri ri
fn ,L;.
.t~ N r1 U ro >~ b~ O u~~ >~ ro., ~ U ro
O ~ ro i-I v7
U ro(~ a ~ ~ U O U O ~ ro1~ r-Iro O G
I ~ v O a
f'-I ro -ri UlO O v U rl U n-i.~, ~ r1 U N rl
r1 e-I
11
ro W .o ~ ros~~ tn a roroo w U a .n
w O ~ ~I 3 w N u~ o ro
v a ,-~ N a ro
v ro s
~ cn G a o u~O N
N ~ ~ ~I ro a ro ~-~I-~Iro rov ro ~
~ o v G
v
, G ~ ?~ ~ ?~ro~ ~n 3 ~n 3 0~ a u~ ~ >~ 3 ~ o
I t~ v v~ s~ -.~
~
O ros~ .>~ a v ro .~ s~O >~~ O O ~-I
.~ > ~I ro +.,
U N
~
o v ro> a ro ~, x ~, wroro~n s~s~ ~, >',
ro o ro
a
o ~ sa >'Io ro s~ ~ ~I .4U ro ,~ >., w
m ~ m ~ n ~ v i -.~ ,~ >~
ro v v ro
ro
az
. ro ro ro ro roroo .H v x ro ~
s~ o ~ v
~
ro v s~ g -.~v ?, >a -~I ~I v t~a s~u~ ~I ro
~ ~ ~ v ro x
v
o
0 3 ~IU o a ~I~ ~-I >a w ,>~~,-Iv o v o v w
E ~ > ~ ro
f,,
0 o v v o U -.~o -~ -~Irorov U ~
>, ~I ro s~ -.~ >
v a >',
~, ,~~n s~ ~-Iv ~ ~ a ~ a ro~ roa ~ a o
s~ ~ ,ca ~-I o o
f~ ~ v O E ~ U -~f ~O m . ro Q N W
W N v
m
a ro -riUI .~. P4N N U ~ ~ ,'a~.1~1.1U (","tJ!O ~ N r-I
rt ,~ N rl
Ul
~I 3 ro E o ~nN ~, U ~n U o ~Io, v ~ s.l
ro v v -~I o, ro
,~ ~
z
-~ 3 .1 w ~Iv N z ~n z ro~ -.~a ~
rn a s~ -.I
ro
U ~ ~rro U11~1~ H H (UG O ~ v H N N
O ri N r-i r1
O -
~ rl
~n ~-I ~ ro -. a u~ a ~ a oro~Iv ~ ro c~, o
o v ~ m 5 w ,~
~ s~ ~
v ~ o -~I ~ ~, x ~n o ~ , ~ U
~ m ~n o ,-,ro
.>~
~
Ca l~ 1J-rl .--1ro>~' N r-I vD In~ U ro -r1>"'.,'-i ('.
v UI -rl v ro
Id U r-I
'i ro
O U ~-I O ~', O ro O ~, ro ro~ ,
~ i.l v ro ri ~1 N rl ',~ O O 1J
En UI ro ro v
'[~
~
, ~-I E-~S~.-I E-~~ E a-IO v v E a >'I
O .u 3 N ~-I
b~
N ~ H v a O ~ ro O G D w 'a~H u~ D N tn
N U ~, ~, N rI O
p
-~I
v ?.,N E S-IU ~ ro ~ ro
~ U ~-I z ON ro N U N
H ~ N ri N ~ N U
O U ro S-1 ri
~ N
~.. O
W
~I
-~
i
a D t,-'r ro W a ~ ~ ~ ~ O
~.I N ~ ro ~
' t ro U
ll ro
, . U b1-I ro U 1~
. ~ U U
I W --i N
p E
~ E
O H
.R ~
H O
O
U U
A
H 00 tf1 r N
t!1 O 1p
N O
~
lf1
N c~1 ,--I
O i
r 01 O ,-I p
H
U ~ ~ ~ ~
I
,-
v
ro
o
.~
z
O
A
v
H
Ud
w l y o
z~
-73-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
o' I
~ N ~,-ro ro
>~ .~ ,~Nv
i ~ rt o ro~~
~ .u v ~
v -.~~ a ~ v v~ N 5 A .r-Iro
~ -~,
ro H o ro .~ 3 .uro a ro -~ .~ robm p
H
~ ro~I. ~~ ~G v N a a vv ro
a.
NG .>~ri 't~ ~ .-I YI rov ro'O,1~
o rovo u N v v~
H
~
. ,raro>
o .r.l-r.l-~Io ~ ,~-..I ~ t~~ r-IN r-I. s~
ro ro
~,
H ~ ~ H+~3 v ro~3 v ~ ~ rn-~,~ N H aro v
~ ~s H v
w o G roro ~ a ro o ro o ~ N o s~>~ a
~ >., m
ro H -~I~~-Iro~ -..~~N ~ ~I~.I~ .~ro ~ -.~ro a
v ~..~ ~I ro
H w H o-~I~ b .~-.~ w Hroro~ a v ro
~-I ro
N
N ~ 'L3O ~'.,("., !J U .F., ~ 11r-IN 7,~ U
1J rl -r1 U1
1.1 23Z3b G -.a.rl -.i u1 b '~'OU N U Hro
ro ~~ ,~ r-I -ri
ro 4J GZ1~IU U -~-I .H v O ~ O N O-~I b~
'O ~ -~I T3
r-I 1JN ro~iU W U1Ori W rl 1~N1~'dX ~11J ['.,
ro ro~ roH O rn 'fir o ~ ~
o m > ~
v w .
,
-,
, rorlro ro N, o
N ,.-IroN roro a x~ ~-Iroa ~ ~ ~ -~~, ~-I
.~ . ~
H H H
~ .~I
o ~ v a ~ -.~rov o ~ o ~.~Iro~n v ~~
~s .~I v
~
N v ~>~ ~ .uva ~ v N v~ ~ v rn,
H ,~
-~I4-i~O b ~ ~ ~~ a ~Iw>~O ~ O aH H
ro ~ H
W , U - ~ ' ~ ~ , Nro
~
~ .~ UIO rl y-! .ri S- R Ci r O .1J .~'~U
. . Li 1 . i
ya
roi~-.i.iO ~ r-i>~ ro ro ~ b~ U w u~rlZf
u1 u~ ' ~,
-r-iroU S-Itn U rtfO 1~ G, ~I H O N 1JSI ro
-r1 O N N
S~ m ~IX ~ a ~N s~ O NO roG roU U
v N -~ ro
-r1 b~roaN N ro O~ N ~ blror-tU dP ?.IWb
-rl ..I .U O
H
u1 G."U O .~.,U rof-1U -m1 r~ f~"UO O o O -ri G',
'~ u1 O
1~
~ ~ ~ ~~ G o ~~ a ~ ~ w ' ro
i > ro
o I ~ v i ro ~ ~ ~;n -
o - o
>~ ~ U roo a -~ H ro :~Urororo v Hro H
H ~ w a
. ~
v w>~v ~ v v~I ro - w rov ~ oa ro
3 v v zs
J..~ 'O'O 1r W ~~ G N 't7''O U7 r11~U
U ~ N
U QI~-1N~ d~O -riX r-I tl1 N r-I ~ rl .tJ
N U -rl O
r-I y.rO ,~a N w N UI'O r1 ro ~ O~ tnH -H~ro
N U I ~ U ~ N~ w ~
ro v ~
i O ''
3
~ C 0l U I
~ H Zfp r1N1WI u1 ~ S-IO W ~ O N~ f-I
ri 1.r S-I
O
U1 IW0 W.-tro~ ~tUI~ N w 1.1rot~O O a HN v
O ~O ~ Ul
O
~1 >~ ~ N O 1-IO N XU U '~ m NU v U 'O U
t~ 1.1 i0 UY ri
r-1
O Ul?I r-Ib1-rlJ-Ivri S-I tA,7rN 'L1,~,4)-1-1f.'
ro ,f~ ,tt U
U 1~1 r-Iro 1J ~ '~ N ~, >~"I1JM . ,1~a ro
-r1 ' U .~ U1
Ul O N roG '~ro ,t,'~', S.i O NUI v b1Ori U
,C, O
v rl
Ul U v0.5O N rl S-I1J-n-i N U ~Orov A1 ~'.,f-1r1
ro '>3 1.~ f,..1
.r.
ro
ro O-~-I1 W O -.-a1.~ .u ~ '0~ O vi-a t~
U ~ ~ ro v
3 N ro~isroo .~37, >~ H Inro ro~ H ~v o
~ -~I H 3 ~
ro ro vv a v H N -~ ro ro v H ~ Ny rl
E w o
~. 3 E HH ~IH v ~a o 3 ~~ rno Inv~ o
v ' ' t~ ro ro
O C1v J-I O i.l ~, Oa 'L3 bl,~ U
t ro ro ~IH s~Lf N -~,,!~ ~ f
zs w .~I H
v N ~ v
ro
H ~ H w ~j,..~w o H, ro ~,H~ H v v ozs o
~ 3 .ca N w-~Im o H Hv N
?~ ~
a s~
C~ ,~ ro ~~ -,~a .ua G ~ ro Inv o v a~ a
o o ns ro -~ ~rs ro
rl 1~ H b OO '~ Nr-I O N Tj r-IU 3 tn O
I .O v G N U ~ N
a al ,17v a.m m N ~ro E~ ?-m1p N'L1U S-I SIro -~-i
~-I -ri r-I m
ro
O ro ro ri~ UO O U7 .~OU ro O r-I~v ~ . t!~Or-~ .1.~
U ri ro U rl
-, a o vH rl~ ~ b.~ ~I U ~.7O21~ U .--Iwp, U
v X -~I z3 ~ ~
.
E w ~ Hroo o a~ >~
~, .~, w N v~ ~'
'~
a x v ~ v va ~ > ~ vH ~ a ~ - ~ ~ va ro
I v -.~ H n H
O '>JU H mrot~H -~t N O -~t U N
t~ 'i3 ro a~
.aJ
i-I W ,'~z Ulr-IroN UJU ~7 a~ z X ~ '~ -.f~ G
.-i a -rl ri -I ~
a u~ r, H v ~, w E ~ >ro ~-I o H vv to H ,~~
r .Q ~ o
a
N a a a ~ .~~, N v H G H a ~ w -.~o .~,o a
ro -~I ro a ~
v
N .('~., Ul S-I N ' JJO ~'.. N fnr-IO U ~ U1rl r~
r-i UI W (O
A r-Irl lf1U7r-IO N N H ro-rl r-~ V~Ulro'-'f0 . OO ro
W ~ J..) ro ri ., 'i ro
-rl ~
L,'
0 0 .~roa tn5 v HH t~ v .-,~IH ~ E av
0 E-~ ~ a > ~ ~
?~ E >, E ~ t~roro..~N va v N E ~v rno ~s
~ o o ro o
W n H O .~IH ~ N L~ >~ .u o O ~ ~ . >,~ H
v . w '~ H .u ~, H
ro ~ o E H ~o .ut~ o ro .~ H E Hrov .~Im ~~ ro
a 7, -a -~I ,~ N r.l u
H c7 ~ x o o~-Iv v r,~ rn v E o~-~Izsa v x-~, .
N H o v ro .a a
v
.a m m ~ oa H ~ N oro o ~ ~n .~IroH roroa o
RX o ro roHr~ H ~ ~ ~ H
X H ~
9 ~ H v
o
~
. > x G H ro o vv ~,
a a , v v
~ .~ O ~ rov a v 3 aro a ro m ~o ~,a >~3,p ~
o .1 U ~ c.. a
y
r-I u,
H
7r N
E
5
O
.Lt ~
a o
A
H OD ,-~ pp
Ll1 M N
N ~ ~ co
O ao N
d~
r1 01 ('
U .-i N N
Tl
O
~
z
O
A
N
H
ri
U
Ot
W O u-I N
z ~,
~n
-74-
CA 02329685 2000-12-11
WO 99/64596 PCTNS99/13281
-° N a ?~ ~ N ~ ro ~, u,
rn .u s~ o ~I ro a ~n o
.~. U -r1 U !~., ro ~rl Gi U Tf ~ S~1 S-1 '~ (n r-I
11 -ri ~ 1~ N '~ 1~I N b1 UJ v (,~' 1'-1 N ro N ro N N
.C ~ S.~ ~ O -rl N N v ro v 'p ro -.i ~ ~-I U ~ I U
bl ~ 1~ N S~1 'J U r-i ~", r-I r-I ~"., 9 U ~ ~ ~ .1 S~r
-rl 'C5 UI ~ U ~.1 ~,' 1.1 (3, O C1 O, ro ro O Ul U ~r-i ro ~ c<s v
v TJ O >~ ~ U U ro ~ N v I per, ,~ -~ b x U ~ v .O
l ro v ~ .~ r, ~ v o~ ro o r~ > ~ ~o ro
o ro o ro v .u ~~ o ro a .~ ~ x n, v o o .-, U
>.I ~ >~ ,~ m ~~ u~ ~I .u rn ,~ a a ~ H s~ o
w v o ~r ~n ~ ~u ~ ro b w o U ~I b ~I ~a w I ~, ~ci o 0
w -,~ ro ro v v v ~ ~ zs a w o v ro v ~, ,~ v ~, ~~,
u~ w ~ ~-I zJ w to b b v ro o ~ w ~ s~ ~C3 l0 b~ ~ w
v >~ -~ ro ~ o ~s~ v ro ~ ~, s~ a v v v -a ~ ro
ro U ~ ro N r-1 . t~ ro ~ t,' ro 't r-I >~ U .IJ
ro -r1 >C I~~I ~ U ~ N ro tT U U O r-1 U O b ~ u7 U N O
~n N ~~I b~ v ~ v ~n r-I G -~I ~~I -~I u~ ~~I G -~I ~ N >~ +.I v~
o ro ro a s~ ~.~ ~~I u~ ro o -a ro r-I ~ v ro -.~ ,.I a o ~ 2s ~~, ro .~,
m U a~ p, ~~ ro v ~n ~ G r-1 S.I .IJ U U O W v ,-I
w ~ ~t7 v ~ ~ v N ~~I ~ -~I O o >, -~I s~ ~~I -~ ro .u ?,
~C v ~ ~ o ro o~' -~ -~ ~ ~ ro ~ w o' o v ~ w v ~ w v o
2s ~>~ ~~ '~ v c"n ro ~ ~, ~ v v ~ x ,~ ~ ~ v z~ o -,~ ~
ri O ,5 CL U N ~.~ W 'i7 U1 rtr v
O r-1 R~ ~sC ~ T! -rl .~., H S-I ro ~-I -rl O -rl G N ~" -r~ f'.1 U
tT O U7 v -rl ~4' O ro N tT .~. O 1! ~ ,~ y,! ,i," r-i -ri b1 r-I -rl ~L," ~,
O y
p I m ~ ~--I ~ p ~~ U ~n ..~ >., rt1 s~ O w m
-r~I s~ ~ ~ ~ -~I ~ >,.I ~~ s~ +~ ro -~I I U ~ -~ u~ -.~ S1 ~ b~ N
U1 ro G 1-I U G U U fIf m ro ro N W E-~ O ~ b ~ u7 p O Gq
Ol .--I ro ~.~ N S-I N ~ ~~ G1~ S~~ O G N to O ~ ~, O N ~rl
a >, b m > -~I ro ro >r u~ 1..~ v O ~r~I ro ~ N ~ -~I tn ~ b
'CS I G ~ ~ ~-1 ..i U ~ Sd 't3 ro ~ ':~ ro 5 ~ .u U ~ T1 ~ O ~
p N ~ O >'I O v ro O ro N U ~ N N -rl Id O U N 'C7 ~~ ro
a ~ u~ -~I -.~ >~I C1 w ~ N G ~ ~ ~ 3 ~ ~ ~~I 0.. >, >~I ~ -~I ~ w Z1 ~-I .1
a rn ro v ~ ,ca o U ro o o ~n ~~ ~ v U x ro v v
ro N GL m t~ ~ ro v fa ~ U ~ .~ m Zf ~ .c7 ~ w ~ .u U w
f-I N N w ro s~ O S~ O cn U O v m ~ ~ f~ ir, tn U O
/'1 ~ J.! ,~" S-I v .~, ~ v U 1.1 '=3 1~ -.~i G1 C'.~ L: v .1,J i~ N ~rl
N O ri O W U tl! r-I ro W W I ~rl -.~ ro U1 3 ~r U7 S-1 O tl1
H ~, v ~ p -rl U ~ O R~ Ql ~rl n ro ro ~ ~ ~ O ~ ro ~ rn u~
O w ~ rl O N -~I ~ 2f ~.~ O c ro b U a ~ .~ ~ S~ o ~ O ~~r rn t~ v
U O N ~ ~~ U -rl v 1J U i-I r-I O N U W U U ro U 1~ ~ ~ ~ y.l
'p ~ U -r1 E ~I ~~ 'Cf O ro 1~ f.~, >~ ~ t~ U ~~.I O 1.~
U7 N U O U ro v ~ >,1 N v ~r tl7 'L7 J~ JJ ~ri ~ri U7 w v -ri U c.7 ttJ
ro ~ N tW-1 U ri r-I N rt y, N ,~ )~ ~"., ro .~ f-I ro N S-I r-I
3 0 ~I u~ ro 7, o .a U ~ 3 I o », o ro ~ b~ ~, o, 3 rl v o rn U s~
O ?i U N G O~ >~ U o~ t> >~ ~ ~~I >a .u N ro ro
v ,~ U O ro v O -~ tn tr1 ro I ri O ~ ~, N v .O 3 U N
i-I CL t~ ~ 'O O f.l rl r~ a~ ~ N S~~ .~ N f.~ U ~
ro o - v o I~ '~ ~, s~ ro ro o ~d a v ~ ~n u~ ~ ro ~ ~ v ~, E rn
.-~ s~ v o v b o s~ v s~ ~--r v ~ U .u ~.~ .,~ ro ro r~ o G
,.o ~ I ~ ro ~ U s~ o .>~ .Q ~ ~ ~~ -~ ~, rl ~~ v E ,~ ~ s~ ro
-~I u~ O m .sr ~~I .C ~ ~ -~ O E-~ ~ ~ U ~ m O -~I O .~ b >'I
a u~ a~ ~n M ar ~ ~ u~ ro a >., v a o ~ ~, ~., ,~ a sa a ~ :a U o
~~I ~ -~I -~I ro ~.~ w ~ 'O ro ~~ O ~ 'p f.~ w v ro ~~I I ~
~ -~I ~ N ~I a - ,>~ . ~ 23 >~ v 2J P~ ~ -a ~ >~ ~-I U
U w zi ~n ro ro U ro ~ 0 3 ~ tn s~ ~ U ~ N w ~o
z s~ ~ rl ro v ro -~I ~, -.~ z v ro -~I o ~ ~ o ~ ro r-I z v aT -~ ,-I I v
I-I o ro ro ~ .~ y ~ ~-~I ~ r-I ~ ~ .C ,17 rl ~ f~ ~-I I--I ~ ~ rn o ~.,
o, m v tn .u v v ~~I v a o ~, m ~n ro I ~ ~ ~o a o ~~ o ~ w U
G ~ 'LJ >~ 'p ~ ~ N W ~ ~ U >~ ~ is N 7y E ~ N
c~ 1~ r-I O Z7 b~ ~r1 ro ~~I O N O tn N T3 ~ u7 ro In N ~ .s~ N U1 ~n
o ro ~ v a w a w a ~ c-I ~ t~ f~., N U7 N -rl O ro N 11 'O CL W 'O ~~
E-~ f.' ,4 ~ri 1~ -rl O -rl ~rl v E- U O ,~ G: 'J ~rl 'J v H N v ~
.~. ro dl N ro U r-i ~ r-I 11 O N N U ~rl ~rl ~rl ro 1l O UI O N ~ C'., ~ri N
fn
[-i -r~ 1~ N U b W i-1 ~rl 1.1 ~ ~ N .C: 1.~~ -rl ~ N 'Cf N ~ ri O ~~C ',L' U1
rt rro-II ~ 'O tb U ~ N ~ ~~.I ~~7 01 r-I 1~ v ~ U~ ~ w H u1 A t0 ~ ~ v N N ~
O O ,~ O -~ ~ w .O ~ .~ w t~r~ ~ m ~3 E U Rm-I -~ U b x ~ w ,~ ~ E tin 3
0
H
ro E O H
a W
a o ~ A
x
a
H
O
v o ~ W n
1~ N N I~ tf1
U N M L17 00
M O
v ~
ro o
~.~ z
oa
N N
ra
U pl
z tn u~ ~ w0
-75-
CA 02329685 2000-12-11
WO 99164596 PCT/US99/13281
N s~ v
~
v
ro ~ ~o o ro
z ~ v ~
o
v rl U ~ ro~ it~ ~
tn
v b1 U7 G1-1N riro f ~ ~1
O . N
I a roofw O ~ s~ ~ roO
ro ~-I-~,
~ c~ w ,~ x s~v O ,~~,w
o sa ~ ro Nro~ a ~ ro
o H a-I
~
ro ~,w w roN
o
ro ro s~ rov .u .>~o o ~Iroo
~ ~ .~ UL v ~ ~ w .L7U
U .u ~ .-I ~
-- ~
T3
~
rl -~I ~roN ro ~ ~ O
~o O -~I
U
~ ~-I w ~ roU -rl ~ -rlO ~ ror-I
b1 a r-Iro U
N
O .L! -'i O U-r11-~ O N r-I O U O
-rl -ri r-I~" O
ro
s~ zs s~ roro >'Iro o >a
~I rl .~ ro o
. ~
w a O w ~~ w w U ~ w ''~~r
N O f_I E G .u
O
1-I ~., ri-.i4) ~ tJ rlro
1-I N Q) N
ro ~ Ts o ~ ~ ro ro ~uo a.
v ,p ro ro
o
v .a ro v I N v U w v
>~ o 1 v ro ro
1 O
: J
- 1. i-I ri iJ 1Jf-IU
C L;
S-I
zs ~, ro roo N roro ~I roro~
>~ v ro -
ro L~ ~ NrlO ~ r-Iro .1v O
~ O U v 1~
.N --1 O '~O f~-I O O ~Cf O '~,.-1
O ro 1J r-i ,7,
~'.,
S-I 'L3 N I~ U N I rl N I N
ro 1J ri ro ~ U
b
O w N ri I~1~O rlN O riO N
ri U ~ U U O
tU
N JJ V~ro~.' ro 1-i V~r-I
N 0l -riri 1-1
'L~
Tl ro ~ p,O ~ N ~ r.>; N
ro r-~t ro G ''C5ri ror-I
N U r-i ~ ~ '~y,L,' ri
N rl ('.,r-i
ro
r-i
1.1 O ~ I 1.1 ~ ro
O N U ..iO
ro N wv ro oo w w v
ro ~ v
a
b~ -ri OI O~ 1-I b~N y b~O bt
~ ~ U ~ tn
U ri
~ ~ ~ ~ ~ ' -
~ N - v r-, ~ ro ~ ~ ~ v -~
n v ~, o ~ ~
N -~I ro N ,~~Iro N N rl ,-, o N Q s~
.-I .~ ro
s~
3 ~ oa~s~ ro~ w G s~~ o a~
~ ~ U O v
o
N >, r-, o r-I o v
-~ ~ ~
~
b ro ~ ~ ,-.I~ a ~o~ ~ x 2s ro
v ~-I a w ~
at o N
3
w
t rlroa~ o v ~ a ~ at~-I~
-~ ro o s~
a ~ rov ~ v ~ -~ ro ~ roro
ro a. tn
.a
U r.~ U .Nbl ~ U O ~ U U .aJtI
b
~"1 ~ G w ~ >~G r-I ~ Sr -.~ w ~ G .p
s o s . Z7 ~
ro ~,
~ ~ o-~Iro v s~>, v v s~o v
v v
.-I
1J ro 1J ilC U t~1J.4'J~ -i.U1.1S.a
N v N w N 't 1.~ 1.1
N rl '
~ N N O roN t~ >~ N w v
,~ 'C3 >~ ~ ro J-I~ ~ ro GlG ro
r-I ~ O v U
w U U
O
ro
O E~ ro O ~ N N O .N N ~ O t~.-I
ro ~ Cl~ y.l.-I
U ~ S-I U wr-I,~ O U w N .i.~U w ro
~ ri U 'C3 'C3
r.! Nro1J H N !-I N
O b N !~
N 11 N rlY-1rl a N r-1a ~iN r-Iw
00 N 2S -~ r1
ro ~ ro ~ 3 ro~I -,~ro o
~ 0 ~,
.~ N
3 0
0 3 vv ro a~3 v ro 3 v v
~ o v
~ w ~I .Cs.~ro >~ ~ -~ N ~ G
N O 3 ~ 1W .-I ro' to ~ ~
w ~ ~
J ~
1 O
- ,T~1 N N ~ 1JO
u1 i-I U O .~"',t.1?, N rof-1 N
U L' ro -ri
U N N
ro 25 ro ~ JJ blro~ ri ~ ro~ N
M ~r N JJ -rl -ri
r-I U
o ~ ow >., -~s~o a s~o -~I
J v ro ~ ~I
t .Q ro
7 1 'i .4 t-1O U r-1,~N S-I 'CS,.QS-IU
N D N
O ~
o a 25 -~Iw o ro.~w v v .~w X
~ 0 ~ sa ~,
o
v ,~ >~~I ~ rlv o ~ ~ v
s~ o
~
ro o 2io ~ ~ ro
3 oo v-~,p ro ,~ ~
a N ~
o v
r, 2s
. r,v .~ ~-~I~rv rn
o N 3 o yN ro o a .u ~ ,-I~ ~
~ ~ ~I > .u
o G ~
H I ~ H o-~Iv ~ H o N N F o ~I
o U ro o
0 0 ~r o x ~U ~, >~o ~ ~ a o ~ s~
s~ U U ~u ro
U ~ oo ~ H ~~ ro roz~v o roz v ~
w w , v v
N ~
~ ~ ~
ro b
5. H ~o o ~ v~ rl v ~. ~
>a ro o v N x
H v ~ N
~ -I
ro
a
r v ~ v v v
ro w z N oo ~a -~ roE rn -~I I m ~ ~I
> o ~ ~ o ,~
~ v >~ U v
t-1O O 1-I Nr1t-1 N N ('.,ri N O
~ v a ,Li N Nt r-f -rlO
ri ri
ro i
1J R
l U
-
r -1r N N N N r-IN N N
-ri,C,' v .L."rI'7. --1~'..t-aN N .!".r~N
U N ri -riTJ N
~ N .~", ~ N
a
a H -- ~, N row ~csH ~ ro 3 H ~ w
s~ ~ ~ -,~ ~ ro ro
U v
0
o ~ 0
~
w
00
A ov o0
~ ~o
~ ~o
N t~ ,~ N ~D
C.'d~ p~
O u~ of
ri Q1 M
'-~ ''a
U o f-I
O
z
A
H
a
w t~ o0 0, o
-76-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
~r
~ ~
ro
a ~
-
i
4-1 i~ U
U!
~ ~ U G
t; N
O ~
~
T1 Sa rt
1 t0
.~
w i U ~
~
N
m
t N
n
U N
U ~
T3 ~ .i.~
N N
' ro
'~
-~ ro
ro
~ ~ '~
~
~
~
n o ro
-
_
~ U "' ~,
~ ~ .~
- ~
o
o FC ~r
.R .~ N
~
v ~'~
v'~ ~
.c a
,
0
~ u ~
w
~
, .
ro
ri V~ UJ N S-~
U
G.'
~ ~
~
1I3 1
.r -I v 'C1
-I
N
~
~ N U~ .r
O i zJ
?i
U
N O 1J
1J
S-1
S-~ U 1J ~
U rl
O
U
~
H
C.~" -.
Ul ~1 v L~,
1.~
7-i'O 1.i ,i;
O -r1
.ri
O
~1
O '~ ~
b 0
r- ~., u
~., U .
1 I
U
U N W O l-i
O
y
fn
~ 3
3 ~ ~ b
ro
.~
~ ~
i. ..-1 rt O O
~ U W
~
W
~ 0
~ ~
~
i .r
N 1
l1-r
a ;~ ,-, '1 v ~
~
ro
_ 'd w
v '
E
~
.,
H ~
.~
~
ro
u . z ~ x
-
U ~ o 3
N
N
~
- z
~ ~ ~~~i
N U m rt3
rl G
L1~
N
~ E U ~ ~
~
.. R~ -~ n
.1 ' H ~ 4
N r-f
0 0
E
"' O
J
H
N U
A
H V~ N
ri ap
N N N
G'. f~ ri
O I~ ao
.-~ 01 '-i
U .-i N
O
z
A
H
a
W .-t N
V7 ~O t0
_77_
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
x
°° ~ I;, ~ o ~ 'o c
o .n 'R .-_ 4. ~ .? ~ ~' 000
c
I~ ao', o ~ .ate.. a ~ ~ ~ o ~ .u
3 ? yN 'y'°~~,~ ~.'~°1 q~.
> G N I~ ~ ~ ~ ~ r R ~ E
y L O
a .. ~ ~ x ~ ~ ~ C
.C 0 ~ ~ O > ~ ~ ~ ~ d o0 ~ ~ ~~ ~ ai
E-~ ~n ~ ~ ~ W ~ °' a w " 0 0
a ~ ~ 'Vo 3~ ~ o_.~c ~ ~ = o
. a
H ~ ~ C r ~ :~ ~~ II C ~ ~ ~ C id
'° aHvlu ~E~s?~n ~w~~ :v
f.. ~ ~' 3 ~ H - m > .1 O O O C O
Gr ~ ~ O ~L > ~ ~ O~ ~ fi7 ~' ~ ~ V7 ~ (/) C
~' 'Q OC
.. ~3'°~
h ~
o o = oo a vi ~ w N o°°o
00 00 ~ . 'p N ~
m v O~ O yr ti ~ . V O yr
E E ~ E o ~e~ "Q,~ ~ z~ ~-: a~
r O r ~ M ~ c~1 O ~ ~ ' N
,C N N d7
7, 7, V iv -~ O~ ~ V O C ~ ~ O M
O O _~ O ~ ~ ~ _~ ip .at 6> ~
CC m v m ~ M ~ ~ 0 '~ C x ~ '~ N ..., ~ N
v ~ h ~' E IT y ~ id V' ~~ w
N ~ a C~~7 yr ~ N ~ N C ~ ~ ~ ~ ~ y ~ N
G. d ~ ~ V1 (~ ,~ N . ~ Q vi
a n. ~d ~ oo eo -~ Y ~ E a~
_ ~~ b 'p ~, M _. OG
as ~a as ~~°~ . 'o ~
v V y V cc: o ~ ~ '~ ~ ~ $; ~ a
aa, E ~~ ,y L_: E ~~ 'i'. g a ~ ~ ~ ~'' ~ 'n ~ a
r wV WV wV M ~~t-.,g
r 5 y c y c ~ o~ 'u o _a ~ _. ~ ;~ ~ ~ s .. 'a>
t~ v io uo uO ~v'~ ~~~E~ vo~,.d~v °">>
GG a c~. a ~a. a. cc. ~ it Z a Z ... v~ d z .-. Z ~ U ~ .- Z
> s
eD
c ~ o, y., v c o ~ '~d
~o ~ ~ .~ ~ ',8 a ~
-p C .E~y r~~~= v1
C 7 ~ ~~ '~ v A
C N 00 _~ V a ,~ 'C ~ ~ 9 ~t~
ti '~ y
o'a .~~' '~ g~ '~ ~ ca~a< ~ EZ m .a
- o
a o
a
._e ~ a o .o .o ~ if s ~ a
v 'E ~ '° ~ ~' ~ e~ ~ a
o a ~ = E E ~ a~ cya °' ~ .e ° r
a w3: ~~~~-,~ ae ~ ~ °'~ g~ ~ Y~.
H
a ~ ~,v° ~ ~ d v.a ~~ o ~ ~ ~' n~ ~.a,
_O fn
s_~ .,_~ ~_
~ o ~ ~ c "w ~w
'' o.=Q ~ a ° ~~ ~v~$ ~'y' y~° ~ H ~ e' ~~u
as rs' ~ m~E~ ~ °. ~ Q.e ~'R'°°° z o
a ~ a ~ a <'Hm~ a'~ ~~ ~ A g~ a a
0
a
E o
a a' < ~ ~ a
o a a a m ~ m a
_78_
CA 02329685 2000-12-11
WO 99/64596 PCT/US99113281
s
a
o ~ a
ca
w ~ v a
a
0 '
0 0 a
~ D
w N O
h
$ II II
I
V V V
e4
o. '~ v,
a
.ai > on
N
'v c
. 3 ~ ..
a~a '
' o =
z ,
E~ ~
9~s
C
~ T 0C
h ~
(d
_ v
fiyr ~p ~ LLl C
A
m d O C ~ 3
O ~ o ya
_ -3
~ ~y .
' ~ R ~ ~ ~ o
H ~E '
m U
~
c~~ 3~ V
o. ~ ' ~ ~ ~ =
~ ~.=
o'
yr ~ ~ ~ 3
~ C fA Os ~
N
0 . > ~ ~ G
0 ~ E ~ ..-~q ~
'= ~ '~ '3
~ c ~
~
c ~ .. V
y,d d m h V _ id td '~ Ov
N ~ nj M O
~
00
~
O ~, pip ~ i7 a> > '
N ~ ~ op
~ N N ~
O. H
t O
v '
v
~ ~C C
fl " m a ~a 9
' ; :
'
~
u3
U y o ~ E.. a
o~o a
OC ~
a'~ ~ ~ c $ ~ $ ~ ~ '~
~ ~ ~ ~ ~
' euo
a d
V
~
vv~u . 'E~~'~d ,5u
3'~'y,
oc V~ a c~cc~ v~a3 roc zv maa
~v,
,, _
E ~ ~ ._=
r
._ a ~ ,~ E
. ~
a v ~ ~ N E :_ ~ .a ~' '
~ d
a A ~
C
~
.p ~ ~ C ~ .~ CC7
'
z m
' t '~ Q ~ H
y
~
~ V ~ e
~ r 'S Ci
.
~ ~
E .~ ' ~ 0 a '- a
~ .5 c
V
~~e o E E ~ ~~ ~a
o
.
~
'E
E ~ a s
H . H w
-'
'v. rJ'~ is , ~
. e~ .O
~ C
~ ~ ~ ~ O
_C . O ~ a X s '~
~j 0
a E ~ '~-'' E
~ .
5 ~
O ~ _ N 3 ~ E
V
C G ~ v V n E
'~ r t
d o~ ~ a ~ . Z su vu
s_~
R = ~ ~ s ~n ~ '~ ~ o
~ ~ ~ E
' ~ H a~ ~ a a
a a ~ ~ a
~
a
v a ~ ~
0a ti N O H
oL. a' a a U
-79-
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
LAL, Preeti
YUE, Henry
TANG, Y. Tom
HILLMAN, Jennifer L.
BANDMAN, Olga
CORLEY, Neil C.
GUEGLER, Karl J.
GORGONE, Gina A.
BAUGHN, Mariah R.
PATTERSON, Chandra
LU, Dyung Aina M.
<120> PROTEINS REGULATING GENE EXPRESSION
<130> PF-0539 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/089,029; 60/094,575; 60/104,624
<151> 1998-06-12; 1998-07-29; 1998-10-14
<160> 62
<170> PERL Program
<210> 1
<211> 379
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 591290
<400> 1
Met Val Phe Phe Thr Cys Asn Ala Cys Gly Glu Ser Val Lys Lys
1 5 10 15
Ile Gln Val Glu Lys His Val Ser Val Cys Arg Asn Cys Glu Cys
20 25 30
Leu Ser Cys Ile Asp Cys Gly Lys Asp Phe Trp Gly Asp Asp Tyr
35 40 45
Lys Asn His Val Lys Cys Ile Ser Glu Asp Gln Lys Tyr Gly Gly
50 55 60
Lys Gly Tyr Glu Gly Lys Thr His Lys Gly Asp Ile Lys Gln Gln
65 70 75
Ala Trp Ile Gln Lys Ile Ser Glu Leu Ile Lys Arg Pro Asn Val
80 85 90
Ser Pro Lys Val Arg Glu Leu Leu Glu Gln Ile Ser Ala Phe Asp
95 100 105
Asn Val Pro Arg Lys Lys Ala Lys Phe Gln Asn Trp Met Lys Asn
110 115 120
Ser Leu Lys Val His Asn Glu Ser Ile Leu Asp Gln Val Trp Asn
1/66
CA 02329685 2000-12-11
WO 99164596 PCT/US99/13281
125 130 135
Ile Phe Ser Glu Ala Ser Asn Ser Glu Pro Val Aan Lys Glu Gln
140 145 150
Asp Gln Arg Pro Leu His Pro Val Ala Asn Pro His Ala Glu Ile
155 160 165
Ser Thr Lys Val Pro Ala Ser Lys Val Lys Asp Ala Val Glu Gln
170 175 180
Gln Gly Glu Val Lys Lys Asn Lys Arg Glu Lys Lys Glu Glu Arg
185 190 195
Gln Lys Lys Arg Lys Arg Glu Lys Lys Glu Leu Lys Leu Glu Asn
200 205 210
His Gln Glu Asn Ser Arg Asn Gln Lys Pro Lys Lys Arg Lys Lys
215 220 225
Gly Gln Glu Ala Asp Leu Glu Ala Gly Gly Glu Glu Val Pro Glu
230 235 240
Ala Asn Gly Ser Ala Gly Lys Arg Ser Lys Lys Lys Lys Gln Arg
245 250 255
Lys Asp Ser Ala Ser Glu Glu Glu Ala Arg Val Gly Ala Gly Lys
260 265 270
Arg Lys Arg Arg His Ser Glu Val Glu Thr Asp Ser Lys Lys Lys
275 280 285
Lys Met Lys Leu Pro Glu His Pro Glu Gly Gly Glu Pro Glu Asp
290 295 300
Asp Glu Ala Pro Ala Lys Gly Lys Phe Asn Trp Lys Gly Thr Ile
305 310 315
Lys Ala Ile Leu Lys Gln Ala Pro Asp Asn Glu Ile Thr Ile Lys
320 325 330
Lys Leu Arg Lys Lys Val Leu Ala Gln Tyr Tyr Thr Val Thr Asp
335 340 345
Glu His His Arg Ser Glu Glu Glu Leu Leu Val Ile Phe Asn Lys
350 355 360
Lys Ile Ser Lys Asn Pro Thr Phe Lys Leu Leu Lys Asp Lys Val
365 370 375
Lys Leu Val Lys
<210> 2
<211> 136
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 815856
<400> 2
Met Phe Gly Thr Pro Gln Glu His Arg Asn Met Pro Gln Ala Asp
1 5 10 15
AIa Met Val Leu VaI Ala Arg Asn Tyr Glu Arg Tyr Lys Asn Glu
20 25 30
Cys Arg Glu Lys Glu Arg Glu Glu Ile Ala Arg Gln Ala Ala Lys
35 40 45
Met Ala Asp Glu Ala Ile Leu Gln Glu Arg Glu Arg Gly Gly Pro
50 55 60
Glu Glu Gly Val Arg Gly Gly His Pro Pro Ala Ile Gln Ser Leu
2/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
65 70 75
Ile Asn Leu Leu Ala Asp Asn Arg Tyr Leu Thr Ala Glu Glu Thr
80 85 90
Asp Lys Ile Ile Asn Tyr Leu Arg Glu Arg Lys Glu Arg Leu Met
95 100 105
Arg Ser Ser Thr Asp Ser Leu Pro Gly Glu Leu Arg Gly Arg Ala
110 115 120
Glu Ala Arg Phe Pro Ala Asn His Ser Gly Arg Pro Arg Val Pro
125 130 135
Arg
<210> 3
<211> 230
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 996352
<400> 3
Met Ser Ser Ser Tyr Tyr Val Asn Ala Leu Phe Ser Lys Tyr Thr
1 5 10 15
Ala Gly Ala Ser Leu Phe Gln Asn Ala Glu Pro Thr Ser Cys Ser
20 25 30
Phe Ala Pro Asn Ser Gln Arg Ser Gly Tyr Gly Ala Gly Ala Gly
35 40 45
Ala Phe Ala Ser Thr Val Pro Gly Leu Tyr Asn Val Asn Ser Pro
50 55 60
Leu Tyr Gln Ser Pro Phe Ala Ser Gly Tyr Gly Leu Gly Ala Asp
65 70 75
Ala Tyr Gly Asn Leu Pro Cys Ala Ser Tyr Asp Gln Asn Ile Pro
80 85 90
Gly Leu Cys Ser Asp Leu Ala Lys Gly Ala Cys Asp Lys Thr Asp
95 100 105
Glu Gly Ala Leu His Gly Ala Ala Glu Ala Asn Phe Arg Ile Tyr
110 115 120
Pro Trp Met Arg Ser Ser Gly Pro Asp Arg Lys Arg Gly Arg Gln
125 130 135
Thr Tyr Thr Arg Tyr Gln Thr Leu Glu Leu Glu Lys Glu Phe His
140 145 150
Phe Asn Arg Tyr Leu Ile Arg Arg Arg Arg Ile Glu Ile Ala His
155 160 165
Ala Leu Cys Leu Thr Glu Arg Gln Ile Lys Ile Trp Phe Gln Asn
170 175 180
Arg Arg Met Lys Trp Lys Lys Glu His Lys Asp Glu Gly Pro Thr
185 190 195
Ala Ala Ala Ala Pro Glu Gly Ala Val Pro Ser Ala Ala Ala Thr
200 205 210
Ala Ala Ala Asp Lys Ala Asp Glu Glu Asp Asp Asp Glu Glu Glu
215 220 225
Glu Asp Glu Glu Glu
230
3/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
<210> 4
<211> 131
<212> PRT
<2I3> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1273778
<400> 4
Met Ser Gln Val Thr Phe Ser Asp Val Ala Ile Asp Phe Ser His
1 5 10 15
Glu Glu Trp Ala Cys Leu Asp Ser Ala Gln Arg Asp Leu Tyr Lys
20 25 30
Asp Val Met Val Gln Asn Tyr Glu Asn Leu Val Ser Val Gly Leu
35 40 45
Ser Val Thr Lys Pro Tyr Val Ile Met Leu Leu Glu Asp Gly Lys
SO 55 60
Glu Pro Trp Met Met Glu Lys Lys Leu Ser Lys Ala Tyr Pro Phe
65 70 75
Pro Leu Ser His Ser Val Pro Ala Ser Val Asn Phe GIy Phe Ser
BO 85 90
Ala Leu Phe Glu His Cys Ser Glu Val Thr Glu Ile Phe Glu Leu
g5 100 105
Ser Glu Leu Cys Val Phe Trp Val Leu His Phe Leu Ser Asn Ser
110 115 120
Pro Asn Ser Thr Val Glu Ala Phe Phe Lys Lys
125 130
<210> 5
<211> 411
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1509715
<400> 5
Met Ser Lys Arg Pro Ser Tyr Ala Pro Pro Pro Thr Pro Ala Pro
1 5 10 15
Ala Thr Gln Met Pro Ser Thr Pro Gly Phe Val Gly Tyr Asn Pro
20 25 30
Tyr Ser His Leu Ala Tyr Asn Asn Tyr Arg Leu Gly Gly Asn Pro
35 40 45 ,
Gly Thr Asn Ser Arg Val Thr Ala Ser Ser Gly Ile Thr Ile Pro
50 55 60
Lys Pro Pro Lys Pro Pro Asp Lys Pro Leu Met Pro Tyr Met Arg
65 70 75
Tyr Ser Arg Lys Val Trp Asp Gln Val Lys Ala Ser Asn Pro Asp
80 85 90
Leu Lys Leu Trp Glu Ile Gly Lys Ile Ile Gly Gly Met Trp Arg
4/66
CA 02329685 2000-12-11
WO 99!64596 PCT/US99/13281
g5 100 105
Asp Leu Thr Asp Glu Glu Lys Gln Glu Tyr Leu Asn Glu Tyr Glu
110 115 120
Ala Glu Lys Ile Glu Tyr Asn Glu Ser Met Lys Ala Tyr His Asn
125 130 135
Ser Pro Ala Tyr Leu Ala Tyr Ile Asn Ala Lys Ser Arg Ala Glu
140 145 150
Ala Ala Leu Glu Glu Glu Ser Arg Gln Arg Gln Ser Arg Met Glu
155 160 165
Lys Gly Glu Pro Tyr Met Ser Ile Gln Pro Ala Glu Asp Pro Asp
170 175 180
Asp Tyr Asp Asp Gly Phe Ser Met Lys His Thr Ala Thr Ala Arg
185 190 195
Phe Gln Arg Asn His Arg Leu Ile Ser Glu Ile Leu Ser Glu Ser
200 205 210
Val Val Pro Asp Val Arg Ser Val Val Thr Thr Ala Arg Met Gln
215 220 225
Val Leu Lys Arg Gln Val Gln Ser Leu Met Val His Gln Arg Lys
230 235 240
Leu Glu Ala Glu Leu Leu Gln Ile Glu Glu Arg His Gln Glu Lys
245 250 255
Lys Arg Lys Phe Leu Glu Ser Thr Asp Ser Phe Asn Asn Glu Leu
260 265 270
Lys Arg Leu Cys Gly Leu Lys Val Glu Val Asp Met Glu Lys Ile
275 280 285
Ala Ala Glu Ile Ala Gln Ala Glu Glu Gln Ala Arg Lys Arg Gln
290 295 300
Glu Glu Arg Glu Lys Glu Ala Ala Glu Gln Ala Glu Arg Ser Gln
305 310 315
Ser Ser Ile Val Pro Glu Glu Glu Gln Ala Ala Asn Lys Gly Glu
320 325 330
Glu Lys Lys Asp Asp Glu Asn Ile Pro Met Glu Thr Glu Glu Thr
335 340 345
His Leu Glu Glu Thr Thr Glu Ser Gln Gln Asn Gly Glu Glu Gly
350 355 360
Thr Ser Thr Pro Glu Asp Lys Glu Ser Gly Gln Glu Gly Val Asp
365 370 375
Ser Met Ala Glu Glu Gly Thr Ser Asp Ser Asn Thr Gly Ser Glu
380 385 390
Ser Asn Ser Ala Thr Val Glu Glu Pro Pro Thr Asp Pro Ile Pro
395 400 405
Glu Asp Glu Lys Lys Glu
410
<210> 6
<211> 226
<212> PRT
<213> Homo Sapiens
<220>
<221> unsure
<222> 221, 228
<223> unknown or other
<220>
5/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
<221> misc_feature
<223> Incyte Clone No.: 1676367
<400> 6
Met Ala Ala Lys Val Asp Leu Ser Thr Ser Thr Asp Trp Lys Glu
1 5 10 15
Ala Lys Ser Phe Leu Lys Gly Leu Ser Asp Lys Gln Arg Glu Glu
20 25 30
His Tyr Phe Cys Lys Asp Phe Val Arg Leu Lys Lys Ile Pro Thr
35 40 45
Trp Lys Glu Met Ala Lys Gly Val Ala Val Lys Val Glu Glu Pro
50 55 60
Arg Tyr Lys Lys Asp Lys Gln Leu Asn Glu Lys Ile Ser Leu Leu
65 70 75
Arg Ser Asp Ile Thr Lys Leu Glu Val Asp Ala Ile Val Asn Ala
80 85 90
Ala Asn Ser Ser Leu Leu Gly Gly Gly Gly Val Asp Gly Cys Ile
95 100 105
His Arg Ala Ala Gly Pro Leu Leu Thr Asp Glu Cys Arg Thr Leu
110 115 120
Gln Ser Cys Lys Thr Gly Lys Ala Lys Ile Thr Gly Gly Tyr Arg
125 130 I35
Leu Pro Ala Lys Tyr Val Ile His Thr Val Gly Pro Ile Ala Tyr
140 145 150
Gly Glu Pro Ser Ala Ser Gln Ala Ala Glu Leu Arg Ser Cys Tyr
155 160 165
Leu Ser Ser Leu Asp Leu Leu Leu Glu His Arg Leu Arg Ser Val
170 175 180
Ala Phe Pro Cys Zle Ser Thr Gly Val Phe Gly Tyr Pro Cys Glu
185 190 195
Ala Ala Ala Glu Ile Val Leu Ala Thr Leu Arg Glu Trp Leu Gly
200 205 210
Ser Ser Thr Arg Glu Pro Arg Xaa Asn Leu Asn Phe Xaa Glu Pro
215 220 225
Gly
<210> 7
<211> I83
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1734119
<400> 7
Met Gly Arg Leu Cys Cys Leu Arg Pro Pro Pro His Arg Asp Pro
1 5 10 15
Ala Arg Leu Leu Leu Ala Ser Thr Asp Asp Lys Arg Asn Ser Pro
20 25 30
Lys Ile Arg Pro Leu Gln Pro Ala Val Pro Ala Cys Leu Pro Ala
35 40 45
Thr Val Arg Pro Ala Leu Ala Ser Ser Ser Ala Gly Leu Ser Ala
50 55 60
6/66
CA 02329685 2000-12-11
WO 99/64596 PCT/U599/13281
Gly Phe Trp Gly Gln Lys Ser Gly Glu Pro Arg Gly Arg Val Arg
65 70 75
Gly Asp Gln Val Arg Ala Ala Thr Phe Leu Val Ile Ser Pro Met
80 85 90
Gly Arg Arg Gly Trp Arg Asp Thr Ala Pro Pro Gly Phe Pro Thr
95 100 105
Pro Leu Leu Ser His Pro Glu Ala Ser Phe Phe Cys Ala Arg Cys
110 115 120
Leu Pro Lys Arg Val Gly Ala Arg Ser Pro Pro Trp Arg Val Leu
125 130 135
Gly Pro Gly Gly Ala Leu Gly Glu Gln Met Gly Pro Pro Leu Ala
140 145 150
Gly Pro Leu Gln Leu Phe Pro Ala Ala Glu Pro Ser Gly Gly Pro
155 160 165
Val Leu Val Ala Ser Leu Arg Ala Gln Ile Ala Gln Gly Asp Leu
170 175 180
Ala Val Ala
<210> 8
<211> 317
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1944813
<400> 8
Met Lys Ser Asp Cys Met Gln Thr Thr Ile Cys Gln Glu Arg Lys
1 5 10 15
Lys Asp Pro Ile Glu Met Phe His Ser Gly Gln Leu Val Lys Val
20 25 30
Cys Ala Pro Met Val Arg Tyr Ser Lys Leu Ala Phe Arg Thr Leu
35 40 45
Val Arg Lys Tyr Ser Cys Asp Leu Cys Tyr Thr Pro Met Ile Vas
50 55 60
Ala Ala Asp Phe Val Lys Ser Ile Lys Ala Arg Asp Ser Glu Phe
65 70 75
Thr Thr Asn Gln Gly Asp Cys Pro Leu Ile Val Gln Phe Ala Ala
80 85 90
Asn Asp Ala Arg Leu Leu Ser Asp Ala Ala Arg Ile Val Cys Pro
95 100 105
Tyr Ala Asn Gly Ile Asp Ile Asn Cys Gly Cys Pro Gln Arg Trp
110 115 120
AIa Met Ala Glu Gly Tyr Gly Ala Cys Leu Ile Asn Lys Pro Glu
125 130 135
Leu Val Gln Asp Met Val Lys Gln Val Arg Asn Gln Val Glu Thr
140 145 150
Pro Gly Phe Ser Val Ser Ile Lys Ile Arg Ile His Asp Asp Leu
155 160 165
Lys Arg Thr Val Asp Leu Cys Gln Lys Ala Glu Ala Thr Gly Val
170 175 180
Ser Trp Ile Thr Val His Gly Arg Thr Ala Glu Glu Arg His Gln
185 190 195
7/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 ,
Pro Val His Tyr Asp Ser Ile Lys Ile Ile Lys Glu Asn Met Ser
200 205 210
Ile Pro Val Ile Ala Asn Gly Asp Ile Arg Ser Leu Lys Glu Ala
215 220 225
Glu Asn Val Trp Arg Ile Thr Gly Thr Asp Gly Val Met Val Ala
230 235 240
Arg Gly Leu Leu Ala Asn Pro Ala Met Phe Ala Gly Tyr Glu Glu
245 250 255
Thr Pro Leu Lys Cys Ile Trp Asp Trp Val Asp Ile Ala Leu Glu
260 265 270
Leu Gly Thr Pro Tyr Met Cys Phe His Gln His Leu Met Tyr Met
275 280 285
Met Glu Lys ile Thr Ser Arg Gln Glu Lys Arg Val Phe Asn Ala
290 295 300
Leu Ser Ser Thr Ser Ala Ile Ile Asp Tyr Leu Thr Asp His Tyr
305 310 315
Gly Ile
<210> 9
<211> 479
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2683322
<400> 9
Met Ala Thr Asp Ser Trp Ala Leu Ala Val Asp Glu Gln Glu Ala
1 5 10 15
Ala Ala Glu Ser Leu Ser Asn Leu His Leu Lys Glu Glu Lys Ile
20 25 30
Lys Pro Asp Thr Asn Gly Ala Val Val Lys Thr Asn Ala Asn Ala
35 40 45
Glu Lys Thr Asp Glu Glu Glu Lys Glu Asp Arg Ala Ala Gln Ser
50 55 60
Leu Leu Asn Lys Leu Ile Arg Ser Asn Leu Val Asp Asn Thr Asn
65 70 75
Gln Val Glu Val Leu Gln Arg Asp Pro Asn Ser Pro Leu Tyr Ser
80 85 90
Val Lys Ser Phe Glu Glu Leu Arg Leu Lys Pro Gln Leu Leu Gln
95 100 105
Gly Val Tyr Ala Met Gly Phe Asn Arg Pro Ser Lys Ile Gln Glu
110 115 120
Asn Ala Leu Pro Met Met Leu Ala Glu Pro Pro Gln Asn Leu Ile
125 130 135
Ala Gln Ser Gln Ser Gly Thr Gly Lys Thr Ala Ala Phe Val Leu
140 145 150
Ala Met Leu Ser Arg Val Glu Pro Ser Asp Arg Tyr Pro Gln Cys
155 160 165
Leu Cys Leu Ser Pro Thr Tyr Glu Leu Ala Leu Gln Thr Gly Lys
170 175 180
Val Ile Glu Gln Met Gly Lys Phe Tyr Pro Glu Leu Lys Leu Ala
185 190 195
8/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
Tyr Ala Val Arg Gly Asn Lys Leu Glu Arg Gly Gln Lys Ile Ser
200 205 210
Glu Gln Ile Val Ile Gly Thr Pro Gly Thr Val Leu Asp Trp Cys
215 220 225
Ser Lys Leu Lys Phe Ile Asp Pro Lys Lys Ile Lys Val Phe Val
230 235 240
Leu Asp Glu Ala Asp Val Met Ile Ala Thr Gln Gly His Gln Asp
245 250 255
Gln Ser Ile Arg Ile Gln Arg Met Leu Pro Arg Asn Cys Gln Met
260 265 270
Leu Leu Phe Ser Ala Thr Phe Glu Asp Ser Val Trp Lys Phe Ala
275 280 285
Gln Lys Val Val Pro Asp Pro Asn Val Ile Lys Leu Lys Arg Glu
2gp 295 300
Glu Glu Thr Leu Asp Thr Ile Lys Gln Tyr Tyr Val Leu Cys Ser
305 310 315
Ser Arg Asp Glu Lys Phe Gln Ala Leu Cys Asn Leu Tyr Gly Ala
320 325 330
Ile Thr Ile Ala Gln Ala Met Ile Phe Cys His Thr Arg Lys Thr
335 340 345
Ala Ser Trp Leu Ala Ala Glu Leu Ser Lys Glu Gly His Gln Val
350 355 360
Ala Leu Leu Ser Gly Glu Met Met Val Glu Gln Arg Ala Ala Val
365 370 375
Ile Glu Arg Phe Arg Glu Gly Lys Glu Lys Val Leu Val Thr Thr
380 385 390
Asn Val Cys Ala Arg Gly Ile Asp Val Glu Gln Val Ser Val Val
395 400 405
Ile Asn Phe Asp Leu Pro Val Asp Lys Asp Gly Asn Pro Asp Asn
410 415 420
Glu Thr Tyr Leu His Arg Ile Gly Arg Thr Gly Arg Phe Gly Lys
425 430 435
Arg Gly Leu Ala Val Asn Met Val Asp Ser Lys His Ser Met Asn
440 445 450
Ile Leu Asn Arg Ile Gln Glu His Phe Asn Lys Lys Ile Glu Arg
455 460 465
Leu Asp Thr Asp Asp Leu Asp Glu Ile Glu Lys Ile Ala Asn
470 475
<210> 10
<211> 582
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2684552
<400> 10
Met Ala Glu Phe Leu Asp Asp Gln Glu Thr Arg Leu Cys Asp Asn
1 5 10 15
Cys Lys Lys Glu Ile Pro Val Phe Asn Phe Thr Ile His Glu Ile
20 25 30
His Cys Gln Arg Asn Ile Gly Met Cys Pro Thr Cys Lys Glu Pro
9/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/I3281 _
35 40 45
Phe Pro Lys Ser Asp Met Glu Thr His Met Ala Ala Glu His Cys
50 55 60
Gln Val Thr Cys Lys Cys Asn Lys Lys Leu Glu Lys Arg Leu Leu
65 70 75
Lys Lys His Glu Glu Thr Glu Cys Pro Leu Arg Leu Ala Val Cys
80 85 90
Gln His Cys Asp Leu Glu Leu Ser Ile Leu Lys Leu Lys Glu His
95 100 105
Glu Asp Tyr Cys Gly Ala Arg Thr Glu Leu Cys Gly Asn Cys Gly
110 115 120
Arg Asn Val Leu Val Lys Asp Leu Lys Thr His Pro Glu Val Cys
125 130 135
Gly Arg Glu Gly Glu Glu Lys Arg Asn Glu Val Ala Ile Pro Pro
140 145 150
Asn Ala Tyr Asp Glu Ser Trp Gly Gln Asp Gly ZIe Trp Ile Ala
155 160 165
Ser Gln Leu Leu Arg Gln Ile Glu Ala Leu Asp Pro Pro Met Arg
1?0 175 180
Leu Pro Arg Arg Pro Leu Arg Ala Phe Glu Ser Asp Val Phe His
185 190 195
Asn Arg Thr Thr Asn Gln Arg Asn Ile Thr Ala Gln Val Ser Ile
200 205 210
Gln Asn Asn Leu Phe Glu Glu Gln Glu Arg Gln Glu Arg Asn Arg
215 220 225
Gly Gln Gln Pro Pro Lys Glu Gly Gly Glu Glu Ser Ala Asn Leu
230 235 240
Asp Phe Met Leu Ala Leu Ser Leu Gln Asn Glu Gly Gln Ala Ser
245 250 255
Ser Val Ala Glu Gln Asp Phe Trp Arg Ala Val Cys Glu Ala Asp
260 265 270
Gln Ser His Gly Gly Pro Arg Ser Leu Ser Asp Ile Lys Gly Ala
275 280 285
Ala Asp Glu Ile Met Leu Pro Cys Glu Phe Cys Glu Glu Leu Tyr
290 295 300
Pro Glu Glu Leu Leu Ile Asp His Gln Thr Ser Cys Asn Pro Ser
305 310 315
Arg Ala Leu Pro Ser Leu Asn Thr Gly Ser Ser Ser Pro Arg Gly
320 325 330
Val Glu Glu Pro Asp Val Ile Phe Gln Asn Phe Leu Gln Gln Ala
335 340 345
Ala Ser Asn Gln Leu Asp Ser Leu Met Gly Leu Ser Asn Ser His
350 355 360
Pro Val Glu Glu Ser Ile Ile Ile Pro Cys Glu Phe Cys Gly Val
365 370 375
Gln Leu Glu Glu Glu Val Leu Phe His His Gln Asp Gln Cys Asp
380 3B5 390
Gln Arg Pro Ala Thr Ala Thr Asn His Val Thr Glu Gly Ile Pro
395 400 405
Arg Leu Asp Ser Gln Pro Gln Glu Thr Ser Pro Glu Leu Pro Arg
410 415 420
Arg Arg Val Arg His Gln Gly Asp Leu Ser Ser Gly Tyr Leu Asp
425 430 435
Asp Thr Lys Gln Glu Thr Ala Asn Gly Pro Thr Ser Cys Leu Pro
440 445 450
Pro Ser Arg Pro Ile Asn Asn Met Thr Ala Thr Tyr Asn Gln Leu
455 460 465
10/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
Ser Arg Ser Thr Ser Gly Pro Arg Pro Gly Cys Gln Pro Ser Ser
470 475 480
Pro Cys Val Pro Lys Leu Ser Asn Ser Asp Ser Gln Asp Ile Gln
485 490 495
Gly Arg Asn Arg Asp Ser Gln Asn Gly Ala Ile Ala Pro Gly His
500 505 510
Val Ser Val Ile Arg Pro Pro Gln Asn Leu Tyr Pro Glu Asn Ile
515 520 525
Val Pro Ser Phe Ser Pro Gly Pro Ser Gly Arg Tyr Gly Ala Ser
530 535 540
Gly Arg Ser Glu Gly Gly Arg Asn Ser Arg Val Thr Pro Ala Ala
545 550 555
Ala Asn Tyr Arg Ser Arg Thr Ala Lys Ala Lys Pro Ser Lys Gln
560 565 570
Gln Gly Ala GIy Asp Ala Glu Glu Glu Glu Glu Glu
575 580
<210> 11
<211> 327
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2830310
<400> 11
Met Arg Trp Pro Gly His Tyr Ser Arg Ala Pro Tyr Pro Tyr Phe
1 5 10 15
Ser Ser Arg His Phe Ser Leu Asn Trp Arg Pro Pro Cys Leu Phe
20 25 30
Glu Ser Arg Thr Gln Phe Gln Tyr Cys Asn Trp Arg Pro Asp Asn
35 40 45
Leu Ser Gln Thr Ser Leu Ile His Leu Ser Ser Tyr Val Met Asn
50 55 60
Ala Glu Gly Asp Glu Pro Ser Ser Lys Arg Arg Lys His Gln Gly
65 70 75
Val Ile Lys Arg Asn Trp Glu Tyr Ile Cys Ser His Asp Lys Glu
80 85 90
Lys Thr Lys Ile Leu Gly Asp Lys Asn Val Asp Pro Lys Cys Glu
95 100 105
Asp Ser Glu Asn Lys Phe Asp Phe Ser Val Met Ser Tyr Asn Ile
110 115 120
Leu Ser Gln Asp Leu Leu Glu Asp Asn Ser His Leu Tyr Arg His
125 130 135
Cys Arg Arg Pro Val Leu His Trp Ser Phe Arg Phe Pro Asn Ile
140 145 150
Leu Lys Glu Ile Lys His Phe Asp Ala Asp Val Leu Cys Leu Gln
155 160 165
Glu Val Gln Glu Asp His Tyr Gly Ala Glu Ile Arg Pro Ser Leu
170 175 180
Glu Ser Leu Gly Tyr His Cys Glu Tyr Lys Met Arg Thr Gly Arg
185 190 195
Lys Pro Asp Gly Cys Ala Ile Cys Phe Lys His Ser Lys Phe Ser
11 /66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
200 205 2I0
Leu Leu Ser Val Asn Pro Val Glu Phe Phe Arg Pro Asp Ile Ser
215 220 225
Leu Leu Asp Arg Asp Asn Val Gly Leu Val Leu Leu Leu Gln Pro
230 235 240
Lys Ile Pro Tyr Ala Ala Cys Pro Ala Ile Cys Val Ala Asn Thr
245 250 255
His Leu Leu Tyr Asn Pro Arg Arg Gly Asp Ile Lys Leu Thr Gln
260 265 270
Leu Ala Met Leu Leu Ala Glu Ile Ser Ser Val Ala His Gln Lys
275 280 285
Asp Gly Ser Phe Cys Pro Ile Val Met Cys Gly Asp Phe Asn Ser
290 295 300
Val Pro Gly Ser Pro Leu Tyr Ser Phe Ile Lys Glu Gly Lys Leu
305 310 315
Asn Tyr Glu Gly Leu Pro Ile Gly Lys Thr Val Ile
320 325
<210> 12
<211> 502
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2963346
<400> 12
Met Ala Ser Lys Lys Leu Gly Ala Asp Phe His Gly Thr Phe Ser
1 5 10 15
Tyr Leu Asp Asp Val Pro Phe Lys Thr Gly Asp Lys Phe Lys Thr
20 25 30
Pro Ala Lys Val Gly Leu Pro Ile Gly Phe Ser Leu Pro Asp Cys
35 40 45
Leu Gln Val Val Arg Glu Val Gln Tyr Asp Phe Ser Leu Glu Lys
50 55 60
Lys Thr Ile Glu Trp Ala Glu Glu Ile Lys Lys Ile Glu Glu Ala
65 70 75
Glu Arg Glu Ala Glu Cys Lys Ile Ala Glu Ala Glu Ala Lys Val
80 85 90
Asn Ser Lys Ser Gly Pro Glu Gly Asp Ser Lys Met Ser Phe Ser
95 100 105
Lys Thr His Ser Thr Ala Thr Met Pro Pro Pro Ile Asn Pro Ile
lI0 115 120
Leu Ala Ser Leu Gln His Asn Ser Ile Leu Thr Pro Thr Arg Val
125 130 135
Ser Ser Ser Ala Thr Lys Gln Lys Val Leu Ser Pro Pro His Ile
140 145 150
Lys Ala Asp Phe Asn Leu Ala Asp Phe Glu Cys Glu Glu Asp Pro
155 160 165
Phe Asp Asn Leu Glu Leu Lys Thr Ile Asp Glu Lys Glu Glu Leu
170 175 180
Arg Asn Ile Leu Val Gly Thr Thr Gly Pro Ile Met Ala Gln Leu
185 190 195
12/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
Leu Asp Asn Asn Leu Pro Arg Gly Gly Ser Gly Ser Val Leu Gln
200 205 210
Asp Glu Glu Val Leu Ala Ser Leu Glu Arg Ala Thr Leu Asp Phe
215 220 225
Lys Pro Leu His Lys Pro Asn Gly Phe Ile Thr Leu Pro Gln Leu
230 235 240
Gly Asn Cys Glu Lys Met Ser Leu Ser Ser Lys Val Ser Leu Pro
245 250 255
Pro Ile Pro Ala Val Ser Asn Ile Lys Ser Leu Ser Phe Pro Lys
260 265 270
Leu Asp Ser Asp Asp Ser Asn Gln Lys Thr Ala Lys Leu Ala Ser
275 280 285
Thr Phe His Ser Thr Ser Cys Leu Arg Asn Gly Thr Phe Gln Asn
290 295 300
Ser Leu Lys Pro Ser Thr Gln Ser Ser Ala Ser Glu Leu Asn Gly
305 310 315
His His Thr Leu Gly Leu Ser Ala Leu Asn Leu Asp Ser Gly Thr
320 325 330
Glu Met Pro Ala Leu Thr Ser Ser Gln Met Pro Ser Leu Ser Val
335 340 345
Leu Ser Val Cys Thr Glu Glu Ser Ser Pro Pro Asn Thr Gly Pro
350 355 360
Thr Val Thr Pro Pro Asn Phe Ser Val Ser Gln Val Pro Asn Met
365 370 375
Pro Ser Cys Pro Gln Ala Tyr Ser Glu Leu Gln Met Leu Ser Pro
380 385 390
Ser Glu Arg Gln Cys Val Glu Thr Val Val Asn Met Gly Tyr Ser
395 400 405
Tyr Glu Cys Val Leu Arg Ala Met Lys Lys Lys Gly Glu Asn Ile
410 415 420
Glu Gln Ile Leu Asp Tyr Leu Phe Ala His Gly Gln Leu Cys Glu
425 430 435
Lys Gly Phe Asp Pro Leu Leu Val Glu Glu Ala Leu Glu Met His
440 445 450
Gln Cys Ser Glu Glu Lys Met Met Glu Phe Leu Gln Leu Met Ser
455 460 465
Lys Phe Lys Glu Met Gly Phe Glu Leu Lys Asp Ile Lys Glu Val
470 475 480
Leu Leu Leu His Asn Asn Asp Gln Asp Asn Ala Leu Glu Asp Leu
485 490 495
Met Ala Arg Ala Gly Ala Ser
500
<210> 13
<211> 375
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2994234
<400> 13
Met Leu Trp Lys Leu Leu Leu Arg Ser Gln Ser Cys Arg Leu Cys
13/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
1 5 10 15
Ser Phe Arg Lys Met Arg Ser Pro Pro Lys Tyr Arg Pro Phe Leu
20 25 30
Ala Cys Phe Thr Tyr Thr Thr Aep Lys Gln Ser Ser Lys Glu Asn
35 40 45
Thr Arg Thr Val Glu Lys Leu Tyr Lys Cys Ser Val Asp IIe Arg
50 55 60
Lys Ile Arg Arg Leu Lys Gly Trp Val Leu Leu Glu Asp Glu Thr
65 70 75
Tyr Val Glu Glu Ile Ala Asn IIe Leu Gln Glu Leu Gly Ala Asp
80 85 90
Glu Thr Ala Val Ala Ser Ile Leu Glu Arg Cys Pro Glu Ala Ile
95 100 105
Val Cys Ser Pro Thr Ala Val Asn Thr Gln Arg Lys Leu Trp Gln
110 115 120
Leu Val Cys Lys Asn Glu Glu Glu Leu Ile Lys Leu IIe Glu Gln
125 130 135
Phe Pro Glu Ser Phe Phe Thr Ile Lys Asp Gln Glu Asn Gln Lys
140 145 150
Leu Asn Val Gln Phe Phe Gln Glu Leu Gly Leu Lys Asn Val Val
155 160 165
Ile Ser Arg Leu Leu Thr Ala Ala Pro Asn Val Phe His Asn Pro
170 175 180
Val Glu Lys Asn Lys Gln Met Val Arg Ile Leu Gln Glu Ser Tyr
185 190 I95
Leu Asp Val Gly Gly Ser Glu Ala Asn Met Lys Val Trp Leu Leu
200 205 210
Lys Leu Leu Ser Gln Asn Pro Phe Ile Leu Leu Asn Ser Pro Thr
215 220 225
Ala Ile Lys Glu Thr Leu Glu Phe Leu Gln Glu Gln Gly Phe Thr
230 235 240
Ser Phe Glu Ile Leu Gln Leu Leu Ser Lys Leu Lys Gly Phe Leu
245 250 255
Phe Gln Leu Cys Pro Arg Ser Ile Gln Asn Ser Ile Ser Phe Ser
260 265 270
Lys Asn Ala Phe Lys Cys Thr Asp His Asp Leu Lys Gln Leu Val
275 280 285
Leu Lys Cys Pro Ala Leu Leu Tyr Tyr Ser Val Pro Val Leu Glu
290 295 300
Glu Arg Met Gln Gly Leu Leu Arg Glu Gly Ile Ser Ile Ala Gln
305 310 315
Ile Arg Glu Thr Pro Met Val Leu Glu Leu Thr Pro Gln Ile Val
320 325 330
Gln Tyr Arg Ile Arg Lys Leu Asn Ser Ser Gly Tyr Arg Ile Lys
335 340 345
Asp Gly His Leu Ala Asn Leu Asn Gly Ser Lys Lys Glu Phe Glu
350 355 360
Ala Asn Phe Gly Lys Ile Gln Ala Lys Lys Ser Lys Ala Ile Ile
365 370 375
<210> 14
<211> 341
<212> PRT
<213> Homo sapiens
14/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
<220>
<221> misc_feature
<223> Incyte Clone No.: 4115958
<400> 14
Met His Asp Ser Ser Ser Val Ala Ser Lys Val Phe Arg Ser Ser
1 5 10 15
Tyr Glu Asp Lys Asn Leu Leu Lys Lys Asn Lys Asp Glu Ser Ser
20 25 30
Val Ser Ile Ser His Thr Lys Cys Ser Leu Leu Gly Asp Ile Ser
35 40 45
Asp Gly Lys Asn Leu Ile Pro Asn Lys Cys Phe Thr Ser Phe Lys
50 55 60
Asn Asn Ser Lys Glu Lys Cys Ser Leu Lys His Gln Thr Arg Asn
65 70 75
Gln Cys Gln Asn Asn Pro Ser Glu Ile Ile Gln Ser Thr Tyr Gln
80 85 90
Glu Thr Gln Asn Lys Ser Ser Ser Leu Ser Thr Ser Ser Ile Leu
95 100 105
Ser Gln His Lys Glu Asn Asn Leu Asp Leu Thr Ser Arg Phe Lys
110 115 120
Glu Gln Glu Met Ser Asn Gly Ile Asp Lys Gln Tyr Ser Asn Cys
125 130 135
Thr Thr Ile Asp Lys Gln Ile Cys Thr Asn Lys Tyr Lys Glu Lys
140 145 150
Ile Ile Asn Glu Asn Tyr Asn Pro Lys Phe Phe Gly Asn Leu Gln
155 160 165
Ser Asp Asp Ser Lys Lys Asn Asp Ser Lys Ile Lys Val Thr Val
170 175 180
Leu Glu Met Ser Glu Tyr Leu Asn Lys Tyr Glu Ser Met Ser Ser
185 190 195
Asn Lys Asp Ser Lys Arg Pro Lys Thr Cys Glu Gln Asn Thr Gln
200 205 210
Leu Asn Ser Ile Glu Asn Tyr Leu Asn Lys Asp Asn Glu Gly Phe
215 220 225
Lys Cys Lys Lys Ser Asp Gln Leu Lys Asn Glu Gln Asp Lys Gln
230 235 240
Glu Asp Pro Thr Asn Glu Lys Ser Gln Asn Tyr Ser Gln Arg Arg
245 250 255
Ser Ile Lys Asp Cys Leu Ser Thr Cys Glu Gln Pro Lys Asn Thr
260 265 270
Glu Val Leu Arg Thr Thr Leu Lys His Ser Asn Val Trp Arg Lys
275 280 285
His Asn Phe His Ser Leu Asp Gly Thr Ser Thr Arg Ala Phe His
290 295 300
Pro Gln Thr Gly Leu Pro Leu Leu Ser Ser Pro Val Pro Gln Arg
305 310 315
Lys Thr Gln Ser Gly Cys Phe Asp Leu Asp Ser Ser Leu Leu His
320 325 330
Leu Lys Ser Phe Ser Ser Arg Arg Asn Leu Ser
335 340
<210> 15
<211> 269
15/66
CA 02329685 2000-12-11
WO 99/64596 PCTNS99/13281
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 779255
<400> 15
Met His Thr Glu Thr Ile Lys Pro His Lys Cys Pro His Cys Ser
1 5 10 15
Lys Thr Phe Ala Asn Thr Ser Tyr Leu Ala Gln His Leu Arg Ile
20 25 30
His Ser Gly Ala Lys Pro Tyr Asn Cys Ser Tyr Cys Gln Lys Ala
35 40 45
Phe Arg Gln Leu Ser His Leu Gln Gln His Thr Arg Ile His Thr
50 55 60
Gly Asp Arg Pro Tyr Lys Cys Ala His Pro Gly Cys Glu Lys Ala
65 70 75
Phe Thr Gln Leu Ser Asn Leu Gln Ser His Arg Arg Gln His Asn
80 85 90
Lys Asp Lys Pro Phe Lys Cys His Asn Cys His Arg Ala Tyr Thr
95 100 105
Asp Ala Ala Ser Leu Glu Val His Leu Ser Thr His Thr Val Lys
110 115 120
His Ala Lys Val Tyr Thr Cys Thr Ile Cys Ser Arg Ala Tyr Thr
125 130 135
Ser Glu Thr Tyr Leu Met Lys His Met Arg Lys His Asn Pro Pro
140 145 150
Asp Leu Gln Gln Gln Val Gln Ala Ala Ala Ala Ala Ala Ala Val
155 160 165
Ala Gln Ala Gln Ala Gln Ala Gln Ala Gln Ala Gln Ala Gln Ala
170 175 180
Gln Ala Gln Ala Gln Ala Gln Ala Ser Gln Ala Ser Gln Gln Gln
185 190 195
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Pro Pro
200 205 210
Pro His Phe Gln Ser Pro Gly Ala Ala Pro Gln Gly Gly Gly Gly
215 220 225
Gly Asp Ser Asn Pro Asn Pro Pro Pro Gln Cys Ser Phe Asp Leu
230 235 240
Thr Pro Tyr Lys Thr Ala Glu His His Lys Asp Ile Cys Leu Thr
245 250 255
Val Thr Thr Ser Thr Ile Gln Val Glu His Leu Ala Ser Ser
260 265
<210> 16
<211> 264
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1303605
16/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
<400> is
Met Glu Asn Tyr Gly Ile Glu Trp His Ser Val Arg Asp Ser Glu
1 5 10 15
Gly Gln Lys Leu Leu Ile Gly Val Gly Pro Glu Gly Ile Ser Ile
20 25 30
Cys Lys Asp Asp Phe Ser Pro Ile Asn Arg Ile Ala Tyr Pro Val
35 40 45
Val Gln Met Ala Thr Gln Ser Gly Lys Asn Val Tyr Leu Thr Val
50 55 60
Thr Lys Glu Ser Gly Asn Ser Ile Val Leu Leu Phe Lys Met Ile
65 70 75
Ser Thr Arg Ala Ala Ser Gly Leu Tyr Arg Ala Ile Thr Glu Thr
80 85 90
His Ala Phe Tyr Arg Cys Asp Thr Val Thr Ser Ala Val Met Met
95 100 105
Gln Tyr Ser Arg Asp Leu Lys Gly His Leu Ala Ser Leu Phe Leu
110 115 120
Asn Glu Asn Ile Asn Leu Gly Lys Lys Tyr Val Phe Asp Ile Lys
125 130 135
Arg Thr Ser Lys Glu Val Tyr Asp His Ala Arg Arg Ala Leu Tyr
140 145 150
Asn Ala Gly Val Val Asp Leu Val Ser Arg Ser Asn Gln Ser Pro
155 160 165
Ser His Ser Pro Leu Lys Ser Ser Glu Ser Ser Met Asn Cys Ser
170 175 180
Ser Cys Glu Gly Leu Ser Cys Gln Gln Thr Arg Val Leu Gln Glu
185 190 195
Lys Leu Arg Lys Leu Lys Glu Ala Met Leu Cys Met Val Cys Cys
200 205 210
Glu Glu Glu Ile Asn Ser Thr Phe Cys Pro Cys Gly His Thr Val
215 220 225
Cys Cys Glu Ser Cys Ala Ala Gln Leu Gln Ser Cys Pro Val Cys
230 235 240
Arg Ser Arg Val Glu His Val Gln His Val Tyr Leu Pro Thr His
245 250 255
Thr Ser Leu Leu Asn Leu Thr Val Ile
260
<210> 17
<211> 605
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1611167
<400> 17
Met Ala Ala Gly Gln Arg Glu Ala Arg Pro Gln Val Ser Leu Thr
1 5 10 15
Phe Glu Asp Val Ala Val Leu Phe Thr Arg Asp Glu Trp Arg Lys
20 25 30
Leu Ala Pro Ser Gln Arg Asn Leu Tyr Arg Asp Val Met Leu Glu
35 40 45
17/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
Asn Tyr Arg Asn Leu Val Ser Leu Gly Leu Pro Phe Thr Lys Pro
50 55 60
Lys Val Ile Ser Leu Leu Gln Gln Gly Glu Asp Pro Trp Glu Val
65 70 75
Glu Lys Asp Gly Ser Gly Val Ser Ser Leu Gly Ser Lys Ser Ser
80 85 90
His Lys Thr Thr Lys Ser Thr Gln Thr Gln Asp Ser Ser Phe Gln
95 100 105
Gly Leu Ile Leu Lys Arg Ser Asn Arg Asn Val Pro Trp Asp Leu
110 115 120
Lys Leu Glu Lys Pro Tyr Ile Tyr Glu Gly Arg Leu Glu Lys Lys
125 130 135
Gln Asp Lys Lys Gly Ser Phe Gln Ile Val Ser Ala Thr His Lys
140 145 150
Lys Ile Pro Thr Ile Glu Arg Ser His Lys Asn Thr Glu Leu Ser
155 160 165
Gln Asn Phe Ser Pro Lys Ser Val Leu Ile Arg Gln Gln Ile Leu
170 175 180
Pro Arg Glu Lys Thr Pro Pro Lys Cys Glu Ile Gln Gly Asn Ser
185 190 195
Leu Lys Gln Asn Ser Gln Leu Leu Asn Gln Pro Lys Ile Thr Ala
200 205 210
Asp Lys Arg Tyr Lys Cys Ser Leu Cys Glu Lys Thr Phe Ile Asn
215 220 225
Thr Ser Ser Leu Arg Lys His Glu Lys Asn His Ser Gly Glu Lys
230 235 240
Leu Phe Lys Cys Lys Glu Cys Ser Lys Ala Phe Ser Gln Ser Ser
245 250 255
Ala Leu Ile Gln His Gln Ile Thr His Thr Gly Glu Lys Pro Tyr
260 265 270
Ile Cys Lys Glu Cys Gly Lys Ala Phe Thr Leu Ser Thr Ser Leu
275 280 285
Tyr Lys His Leu Arg Thr His Thr Val Glu Lys Ser Tyr Arg Cys
290 295 300
Lys Glu Cys Gly Lys Ser Phe Ser Arg Arg Ser Gly Leu Phe Ile
305 310 315
His Gln Lys Ile His Ala Glu Glu Asn Pro Cys Lys Tyr Asn Pro
320 325 330
Gly Arg Lys Ala Ser Ser Cys Ser Thr Ser Leu Ser Gly Cys Gln
335 340 345
Arg Ile His Ser Arg Lys Lys Ser Tyr Leu Cys Asn Glu Cys Gly
350 355 360
Asn Thr Phe Lys Ser Ser Ser Ser Leu Arg Tyr His Gln Arg Ile
365 370 375
His Thr Gly Glu Lys Pro Phe Lys Cys Ser Glu Cys Gly Arg Ala
380 385 390
Phe Ser Gln Ser Ala Ser Leu Ile Gln His Glu Arg Ile His Thr
395 400 405
Gly Glu Lys Pro Tyr Arg Cys Asn Glu Cys Gly Lys Gly Phe Thr
410 415 420
Ser Ile Ser Arg Leu Asn Arg His Arg Ile Ile His Thr Gly Glu
425 430 435
Lys Phe Tyr Asn Cys Asn Glu Cys Gly Lys Ala Leu Ser Ser His
440 445 450
Ser Thr Leu Ile Ile His Glu Arg Ile His Thr Gly Glu Lys Pro
455 460 465
Cys Lys Cys Lys Val Cys Gly Lys Ala Phe Arg Gln Ser Ser Ala
18/66
CA 02329685 2000-12-11
WO 99!64596 PCT/ITS99/13281 _
470 475 480
Leu Ile Gln His Gln Arg Met His Thr Gly Glu Arg Pro Tyr Lys
485 490 495
Cys Asn Glu Cys Gly Lys Thr Phe Arg Cys Asn Ser Ser Leu Ser
500 505 510
Asn His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Arg Cys Glu
515 520 525
Glu Cys Gly Ile Ser Phe Gly Gln Ser Ser Ala Leu Ile Gln His
530 535 540
Arg Arg Ile His Thr Gly Glu Lys Pro Phe Lys Cys Asn Thr Cys
545 550 555
Gly Lys Thr Phe Arg Gln Ser Ser Ser Arg Ile Ala His Gln Arg
560 565 570
Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Asn Thr Cys Gly Lys
575 580 585
Leu Phe Asn His Arg Ser Ser Leu Thr Asn His Tyr Lys Ile His
590 595 600
Ile Glu Glu Asp Pro
605
<210> 18
<211> 757
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1907472
<400> 18
Met Gln Ser Ser Pro Asn Gly Gln Phe Val Ala Pro Ser Asp Ile
1 5 10 15
Gln Leu Lys Cys Asn Tyr Cys Lys Asn Ser Phe Cys Ser Lys Pro
20 25 30
Glu Ile Leu Glu Trp Glu Asn Lys Val His Gln Phe Cys Ser Lys
35 40 45
Thr Cys Ser Asp Asp Tyr Lys Lys Leu His Cys Ile Val Thr Tyr
50 55 60
Cys Glu Tyr Cys Gln Glu Glu Lys Thr Leu His Glu Thr Val Asn
65 70 75
Phe Ser Gly Val Lys Arg Pro Phe Cys Ser Glu Gly Cys Lys Leu
80 85 90
Leu Tyr Lys Gln Asp Phe Ala Arg Arg Leu Gly Leu Arg Cys Val
95 100 105
Thr Cys Asn Tyr Cys Ser Gln Leu Cys Lys Lys Gly Ala Thr Lys
110 115 120
Glu Leu Asp Gly Val Val Arg Asp Phe Cys Ser Glu Asp Cys Cys
125 130 135
Lys Lys Phe Gln Asp Trp Tyr Tyr Lys Ala Ala Arg Cys Asp Cys
140 145 150
Cys Lys Ser Gln Gly Thr Leu Lys Glu Arg Val Gln Trp Arg Gly
155 160 165
Glu Met Lys His Phe Cys Asp Gln His Cys Leu Leu Arg Phe Tyr
170 175 180
19/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
Cys Gln Gln Asn Glu Pro Asn Met Thr Thr Gln Lys Gly Pro Glu
185 190 195
Asn Leu His Tyr Asp Gln Gly Cys Gln Thr Ser Arg Thr Lys Met
200 205 210
Thr Gly Ser Ala Pro Pro Pro Ser Pro Thr Pro Asn Lys Glu Met
215 220 225
Lys Asn Lys Ala Val Leu Cys Lys Pro Leu Thr Met Thr Lys Ala
230 235 240
Thr Tyr Cys Lys Pro His Met Gln Thr Lys Ser Cys Gln Thr Asp
245 250 255
Asp Thr Trp Arg Thr Glu Tyr Val Pro Val Pro Ile Pro Val Pro
260 265 270
Val Tyr Ile Pro Val Pro Met His Met Tyr Ser Gln Asn Ile Pro
275 280 285
Val Pro Thr Thr Val Pro Val Pro Val Pro Val Pro Val Phe Leu
290 295 300
Pro Ala Pro Leu Asp Ser Ser Glu Lys Ile Pro Ala Ala Ile Glu
305 310 315
Glu Leu Lys Ser Lys Val Ser Ser Asp Ala Leu Asp Thr Glu Leu
320 325 330
Leu Thr Met Thr Asp Met Met Ser Glu Asp Glu Gly Lys Thr Glu
335 340 345
Thr Thr Asn Ile Asn Ser Val Ile Ile Glu Thr Asp Ile Ile Gly
350 355 360
Ser Asp Leu Leu Lys Asn Ser Asp Pro Glu Thr Gln Ser Ser Met
365 370 375
Pro Asp Val Pro Tyr Glu Pro Asp Leu Asp Ile Glu Ile Asp Phe
380 385 390
Pro Arg Ala Ala Glu Glu Leu Asp Met Glu Asn Glu Phe Leu Leu
395 400 405
Pro Pro Val Phe Gly Glu Glu Tyr Glu Glu Gln Pro Arg Pro Arg
410 415 420
Ser Lys Lys Lys Gly Ala Lys Arg Lys Ala Val Ser Gly Tyr Gln
425 430 435
Ser His Asp Asp Ser Ser Asp Asn Ser Glu Cys Ser Phe Pro Phe
440 445 450
Lys Tyr Thr Tyr Gly Val Asn Ala Trp Lys His Trp Val Lys Thr
455 460 465
Arg Gln Leu Asp Glu Asp Leu Leu Val Leu Asp Glu Leu Lys Ser
470 475 480
Ser Lys Ser Val Lys Leu Lys Glu Asp Leu Leu Ser His Thr Thr
485 490 495
Ala Glu Leu Asn Tyr Gly Leu Ala His Phe Val Asn Glu Ile Arg
500 505 510
Arg Pro Asn Gly Glu Asn Tyr Ala Pro Asp Ser Ile Tyr Tyr Leu
515 520 525
Cys Leu Gly Ile Gln Glu Tyr Leu Cys Gly Ser Asn Arg Lys Asp
530 535 540
Asn Ile Phe Ile Asp Pro Gly Tyr Gln Thr Phe Glu Gln Glu Leu
545 550 555
Asn Lys Ile Leu Arg Ser Trp Gln Pro Ser Ile Leu Pro Asp Gly
560 565 570
Ser Ile Phe Ser Arg Val Glu Glu Asp Tyr Leu Trp Arg Ile Lys
575 580 585
Gln Leu Gly Ser His Ser Pro Val Ala Leu Leu Asn Thr Leu Phe
590 595 600
Tyr Phe Asn Thr Lys Tyr Phe Gly Leu Lys Thr Val Glu Gln His
20/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
605 610 615
Leu Arg Leu Ser Phe Gly Thr Val Phe Arg His Trp Lys Lys Asn
620 625 630
Pro Leu Thr Met Glu Asn Lys Ala Cys Leu Arg Tyr Gln Val Ser
635 640 645
Ser Leu Cys Gly Thr Asp Asn Glu Asp Lys Ile Thr Thr Gly Lys
650 655 660
Arg Lys His Glu Asp Asp Glu Pro Val Phe Glu Gln Ile Glu Asn
665 670 675
Thr Ala Asn Pro Ser Arg Cys Pro Val Lys Met Phe Glu Cys Tyr
680 685 690
Leu Ser Lys Ser Pro Gln Asn Leu Asn Gln Arg Met Asp Val Phe
695 700 705
Tyr Leu Gln Pro Glu Cys Ser Ser Ser Thr Asp Ser Pro Val Trp
710 715 720
Tyr Thr Ser Thr Ser Leu Asp Arg Asn Thr Leu Glu Asn Met Leu
725 730 735
Val Arg Val Leu Leu Val Lys Asp Ile Tyr Asp Lys Asp Asn Tyr
740 745 750
Glu Leu Asp Glu Asp Thr Asp
755
<210> 19
<211> 154
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1985458
<400> 19
Met Val Glu Lys Lys Thr Ser Val Arg Ser Gln Asp Pro Gly Gln
1 5 10 15
Arg Arg Val Leu Asp Arg Ala Ala Arg Gln Arg Arg Ile Asn Arg
20 25 30
Gln Leu Glu Ala Leu Glu Asn Asp Asn Phe Gln Asp Asp Pro His
35 40 45
Ala Gly Leu Pro Gln Leu Gly Lys Arg Leu Pro Gln Phe Asp Asp
50 55 60
Asp Ala Asp Thr Gly Lys Lys Lys Lys Lys Thr Arg Gly Asp His
65 70 75
Phe Lys Leu Arg Phe Arg Lys Asn Phe Gln Ala Leu Leu Glu Glu
80 85 90
Gln Asn Leu Ser Val Ala Glu Gly Pro Asn Tyr Leu Thr Ala Cys
95 100 105
Ala Gly Pro Pro Ser Arg Pro Gln Arg Pro Phe Cys Ala Val Cys
110 115 120
Gly Phe Pro Ser Pro Tyr Thr Cys Val Ser Cys Gly Ala Arg Tyr
125 130 135
Cys Thr Val Arg Cys Leu Gly Thr His Gln Glu Thr Arg Cys Leu
140 145 150
Lys Trp Thr Val
21 /66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
<220> 20
<211> 587
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2726431
<400> 20
Met Asp Ser Val Val Phe Glu Asp Val Ala Val Asp Phe Thr Leu
1 5 10 15
Glu Glu Trp Ala Leu Leu Asp Ser Ala Gln Arg Asp Leu Tyr Arg
20 25 30
Asp Val Met Leu Glu Thr Phe Gln Asn Leu Ala Ser Val Gly Lys
35 40 45
Ile Trp Asp Ser Leu Ser Ile Glu Asp Gln Thr Thr Asn Gln Gly
50 55 60
Arg Asn Leu Ser Arg Asn His Gly Leu Glu Arg Leu Cys Glu Ser
65 70 75
Asn Asp Gln Cys Gly Glu Ala Leu Ser Gln Ile Pro His Leu Asn
80 85 90
Leu Tyr Lys Lys Ile Pro Pro Gly Val Lys Gln Tyr Glu Tyr Asn
95 100 105
Thr Tyr Gly Lys Val Phe Met His Arg Arg Thr Ser Leu Lys Ser
110 115 120
Pro Ile Thr Val His Thr Gly His Lys Pro Tyr Gln Cys Gln Glu
125 130 135
Cys Gly Gln Ala Tyr Ser Cys Arg Ser His Leu Arg Met His Val
140 145 150
Arg Thr His Asn Gly Glu Arg Pro Tyr Val Cys Lys Leu Cys Gly
155 160 165
Lys Thr Phe Pro Arg Thr Ser Ser Leu Asn Arg His Val Arg Ile
170 175 180
His Thr Ala Glu Lys Thr Tyr Glu Cys Lys Gln Cys Gly Lys Ala
185 190 195
Phe Ile Asp Phe Ser Ser Leu Thr Ser His Leu Arg Ser His Thr
200 205 210
Gly Glu Lys Pro Tyr Lys Cys Lys Glu Cys Gly Lys Ala Phe Ser
215 220 225
Tyr Ser Ser Thr Phe Arg Arg His Thr Ile Thr His Thr Gly Glu
230 235 240
Lys Pro Tyr Lys Cys Lys Glu Cys Ala Glu Ala Phe Ser Tyr Ser
245 250 255
Ser Thr Phe Arg Arg His Met Ile Ser His Thr Gly Glu Lys Pro
260 265 270
His Lys Cys Lys Glu Cys Gly Glu Ala Phe Ser Tyr Ser Ser Ala
275 280 285
Phe Arg Arg His Met Ile Thr His Thr Gly Glu Lys Pro Tyr Glu
290 295 300
Cys Lys Gln Cys Gly Lys Thr Phe Ile Tyr Leu Gln Ser Phe Arg
305 310 315
Arg His Glu Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Lys
320 325 330
Gln Cys Gly Lys Thr Phe Ile Tyr Pro Gln Ser Phe Arg Arg His
?2/66
CA 02329685 2000-12-11
WO 99164596 PCT/US99/13281 _
335 340 345
Glu Arg Thr His Gly Gly Glu Lys Pro Tyr Glu Cys Asn Gln Cys
350 355 360
Gly Lys Ala Phe Ser His Pro Ser Ser Phe Arg Gly His Met Arg
365 370 375
Val His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys
380 385 390
Thr Phe Asn Trp Pro Ile Ser Leu Arg Lys His Met Arg Thr His
395 400 405
Thr Arg Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Ala Phe
420 415 420
Ser Leu Ser Ala Cys Phe Arg Glu His Val Arg Met His Pro Glu
425 430 435
Asp Lys Ser Tyr Glu Cys Lys Leu Cys Gly Lys Ala Phe Tyr Cys
440 445 450
His Ile Ser Leu Gln Lys His Met Arg Arg His Thr Ala Glu Lys
455 460 465
Leu Tyr Lys Cys Lys Gln Cys Gly Lys Ala Phe Ser Trp Pro Glu
470 475 480
Leu Leu Gln Gln His Val Arg Thr His Thr Val Glu Lys Pro Tyr
485 490 495
Glu Cys Lys Glu Cys Gly Lys Val Phe Lys Trp Pro Ser Ser Leu
500 505 510
Pro Ile His Met Arg Leu His Thr Gly Glu Lys Pro Tyr Gln Cys
515 520 525
Lys His Cys Gly Lys Ala Phe Asn Cys Ser Ser Ser Leu Arg Arg
530 535 540
His Val Arg Ile His Thr Thr Glu Lys Gln Tyr Lys Cys Asn Val
545 550 555
Gly His Pro Pro Ala Asn Glu Phe Met Cys Ser Ala Ser Glu Lys
560 565 570
Ser His Gln Glu Arg Asp Leu Ile Lys Val Val Asn Met Val Leu
575 580 585
Pro Leu
<210> 21
<211> 346
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2743828
<400> 21
Met Ser Lys Pro Arg Ala Val Glu Ala Ala Ala Ala Ala Ala Ala
1 5 10 15
Val Ala Ala Thr Ala Pro Gly Pro Glu Met Val Glu Arg Arg Gly
20 25 30
Pro Gly Arg Pro Arg Thr Asp Gly Glu Asn Val Phe Thr Gly Gln
35 40 45
Ser Lys Ile Tyr Ser Tyr Met Ser Pro Asn Lys Cys Ser Gly Met
50 55 60
Arg Phe Pro Leu Gln Glu Glu Asn Ser Val Thr His His Glu Val
23/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
65 70 75'
Lys Cys Gln Gly Lys Pro Leu Ala Gly Ile Tyr Arg Lys Arg Glu
g0 85 90
Glu Lys Arg Asn Ala Gly Asn Ala Val Arg Ser Ala Met Lys Ser
g5 100 105
Glu Glu Gln Lys Ile Lys Asp Ala Arg Lys Gly Pro Leu Val Pro
110 115 120
Phe Pro Asn Gln Lys Ser Glu Ala Ala Glu Pro Pro Lys Thr Pro
125 130 135
Pro Ser Ser Cys Asp Ser Thr Asn Ala Ala Ile Ala Lys Gln Ala
140 145 150
Leu Lys Lys Pro Ile Lys Gly Lys Gln Ala Pro Arg Lys Lys Ala
155 160 165
Gln Gly Lys Thr Gln Gln Asn Arg Lys Leu Thr Asp Phe Tyr Pro
170 175 180
Val Arg Arg Ser Ser Arg Lys Ser Lys Ala Glu Leu Gln Ser Glu
185 190 195
Glu Arg Lys Arg Ile Asp Glu Leu Ile Glu Ser Gly Lys Glu Glu
200 205 210
Gly Met Lys Ile Asp Leu Ile Asp Gly Lys Gly Arg Gly Val Ile
215 220 225
Ala Thr Lys Gln Phe Ser Arg Gly Asp Phe Val Val Glu Tyr His
230 235 240
Gly Asp Leu Ile Glu Ile Thr Asp Ala Lys Lys Arg Glu Ala Leu
245 250 255
Tyr Ala Gln Asp Pro Ser Thr Gly Cys Tyr Met Tyr Tyr Phe Gln
260 265 270
Tyr Leu Ser Lys Thr Tyr Cys Val Asp Ala Thr Arg Glu Thr Asn
275 280 285
Arg Leu Gly Arg Leu Ile Asn His Ser Lys Cys Gly Asn Cys Gln
290 295 300
Thr Lys Leu His Asp Ile Asp Gly Val Pro His Leu Ile Leu Ile
305 310 315
Ala Ser Arg Asp Ile Ala Ala Gly Glu Glu Leu Leu Tyr Asp Tyr
320 325 330
Gly Asp Arg Ser Lys Ala Ser Ile Glu Ala His Pro Trp Leu Lys
335 340 345
His
<210> 22
<211> 481
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2998209
<400> 22
Met Asp Phe Gln Arg Ile Glu Leu Ala Gly Ala Val Gly Ser Lys
1 5 10 15
Glu Glu Leu Glu Val Asp Phe Lys Lys Leu Lys Gln Ile Lys Asn
20 25 30
Arg Met Lys Lys Thr Asp Trp Leu Phe Leu Asn Ala Cys Val Gly
24/66
CA 02329685 2000-12-11
WO 99/64596 PCTNS99/13281 _
35 40 45
Val VaI Glu Gly Asp Leu Ala Ala Ile Glu Ala Tyr Lys Ser Ser
50 55 60
Gly Gly Asp Ile Ala Arg Gln Leu Thr Ala Asp Glu Val Arg Leu
65 70 75
Leu Asn Arg Pro Ser Ala Phe Asp Val Gly Tyr Thr Leu Val His
80 85 90
Leu Ala Ile Arg Phe Gln Arg Gln Asp Met Leu Ala Ile Leu Leu
95 100 105
Thr Glu Val Ser Gln Gln Ala Ala Lys Cys Ile Pro Ala Met Val
110 115 120
Cys Pro Glu Leu Thr Glu Gln Ile Arg Arg Glu Ile Ala Ala Ser
125 130 135
Leu His Gln Arg Lys Gly Asp Phe Ala Cys Tyr Phe Leu Thr Asp
140 145 150
Leu Val Thr Phe Thr Leu Pro Ala Asp Ile Glu Asp Leu Pro Pro
155 160 165
Thr Val Gln Glu Lys Leu Phe Asp Glu Val Leu Asp Arg Asp Val
170 175 280
Gln Lys Glu Leu Glu Glu Glu Ser Pro Ile Ile Asn Trp Ser Leu
1B5 190 195
Glu Leu Ala Thr Arg Leu Asp Ser Arg Leu Tyr Ala Leu Trp Asn
200 205 210
Arg Thr Ala Gly Asp Cys Leu Leu Asp Ser Val Leu Gln Ala Thr
215 220 225
Trp Gly Ile Tyr Asp Lys Asp Ser Val Leu Arg Lys Ala Leu His
230 235 240
Asp Ser Leu His Asp Cys Ser His Trp Phe Tyr Thr Arg Trp Lys
245 250 255
Asp Trp Glu Ser Trp Tyr Ser Gln Ser Phe Gly Leu His Phe Ser
260 265 270
Leu Arg Glu Glu Gln Trp Gln Glu Asp Trp Ala Phe Ile Leu Ser
275 280 285
Leu Ala Ser Gln Pro Gly Ala Ser Leu Glu Gln Thr His Ile Phe
290 295 300
Val Leu Ala His Ile Leu Arg Arg Pro Ile Ile Val Tyr Gly Val
305 310 315
Lys Tyr Tyr Lys Ser Phe Arg Gly Glu Thr Leu Gly Tyr Thr Arg
320 325 330
Phe Gln Gly Val Tyr Leu Pro Leu Leu Trp Glu Gln Ser Phe Cys
335 340 345
Trp Lys Ser Pro Ile Ala Leu Gly Tyr Thr Arg Gly His Phe Ser
350 355 360
Ala Leu Val Ala Met Glu Asn Asp Gly Tyr Gly Asn Arg Gly Ala
365 370 375
Gly Ala Asn Leu Asn Thr Asp Asp Asp Val Thr Ile Thr Phe Leu
380 385 390
Pro Leu Val Asp Ser Glu Arg Lys Leu Leu His Val His Phe Leu
395 400 405
Ser Ala Gln Glu Leu Gly Asn Glu Glu Gln Gln Glu Lys Leu Leu
410 415 420
Arg Glu Trp Leu Asp Cys Cys Val Thr Glu Gly Gly Val Leu Val
425 430 435
Ala Met Gln Lys Ser Ser Arg Arg Arg Asn His Pro Leu Val Thr
440 445 450
Gln Met Val Glu Lys Trp Leu Asp Arg Tyr Arg Gln Ile Arg Pro
455 460 465
25/66
Lys Thr Phe Pro Arg Thr Ser Ser Leu Asn Arg His Val Ar
CA 02329685 2000-12-11
WO 99/64596 PG"T/ITS99/13281 _
Cys Thr Ser Leu Ser Asp Gly Glu Glu Asp Glu Asp Asp Glu Asp
470 475 480
Glu
<210> 23
<211> 179
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 3340296
<400> 23
Met Ser Thr Gly Ser Leu Ser Asp Val Glu Asp Leu Gln Glu Val
1 5 10 15
Glu Met Leu Glu Cys Asp Gly Leu Lys Met Asp Ser Asn Lys Glu
20 25 30
Phe Val Thr Ser Asn Glu Ser Thr Glu Glu Ser Ser Asn Cys Glu
35 40 45
Asn Gly Ser Pro Gln Lys Gly Arg Gly Gly Leu Gly Lys Arg Arg
50 55 60
Lys Ala Pro Thr Lys Lys Ser Pro Leu Ser Gly Val Ser Gln Glu
65 70 75
Gly Lys Gln Val Gln Arg Asn Ala Ala Asn Ala Arg Glu Arg Ala
80 85 90
Arg Met Arg Val Leu Ser Lys Ala Phe Ser Arg Leu Lys Thr Thr
95 100 105
Leu Pro Trp Val Pro Pro Asp Thr Lys Leu Ser Lys Leu Asp Thr
110 115 120
Leu Arg Leu Ala Ser Ser Tyr Ile Ala His Leu Arg Gln Ile Leu
125 130 135
Ala Asn Asp Lys Tyr Glu Asn Gly Tyr Ile His Pro Val Asri Leu
140 145 150
Thr Trp Pro Phe Met Val Ala Gly Lys Pro Glu Ser Asp Leu Lys
155 160 165
Glu Val Val Thr Ala Ser Arg Leu Cys Gly Thr Thr Ala Ser
170 175
<210> 24
<211> 254
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 3536740
<400> 24
Met Ile Asp Glu Ile Leu Ser Lys Glu Thr Cys Asp Tyr Phe Glu
1 5 10 15
26/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
Lys Leu Ser Leu Tyr Ser Val Cys Pro Ser Leu VaI Val Arg Pro
20 25 30
Lys Pro Leu His Ser Cys Thr GIy Ser Pro Ser Leu Arg Ala Tyr
35 40 45
Pro Leu Leu Ser Val Ile Thr Arg Gln Pro Thr Val Ile Ser His
50 55 60
Leu Val Pro Ala Thr Pro Gly Ile Ala Gln Ala Leu Ser Cys His
65 70 75
Gln Val Thr Glu Ala Val Ser Ala Glu Ala Pro Gly Gly Glu Ala
80 85 90
Leu Ala Ser Ser Glu Ser Glu Thr Glu Gln Pro Thr Pro Arg Gln
95 100 105
Lys Lys Pro Arg Arg Ser Arg Thr Ile Phe Thr Glu Leu Gln Leu
110 115 120
Met Gly Leu Glu Lys Lys Phe Gln Lys Gln Lys Tyr Leu Ser Thr
125 130 135
Pro Asp Arg Leu Asp Leu Ala Gln Ser Leu Gly Leu Thr Gln Leu
140 145 150
Gln Val Lys Thr Trp Tyr Gln Asn Arg Arg Met Lys Trp Lys Lys
155 160 165
Met Val Leu Lys Gly Gly Gln Glu Ala Pro Thr Lys Pro Lys Gly
170 175 180
Arg Pro Lys Lys Asn Ser Ile Pro Thr Ser Glu Glu Ile Glu Ala
I85 190 195
Glu Glu Lys Met Asn Ser Gln Ala Gln Gly Gln Glu Gln Leu Glu
200 205 210
Pro Ser Gln Gly Gln Glu Glu Leu Cys Glu Ala Gln Glu Pro Lys
215 220 225
Ala Arg Asp Val Pro Leu Glu Met Ala Glu Pro Pro Asp Pro Pro
230 235 240
Gln Glu Leu Pro Ile Pro Ser Ser Glu Pro Pro Pro Leu Ser
245 250
<210> 25
<211> 498
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 082155
<400> 25
Met Asp Phe Ser Val Lys Val Asp Ile Glu Lys Glu Val Thr Cys
1 5 10 15
Pro Ile Cys Leu Glu Leu Leu Thr Glu Pro Leu Ser Leu Asp Cys
20 25 30
Gly His Ser Phe Cys Gln Ala Cys Ile Thr Ala Lys Ile Lys Glu
35 40 45
Ser Val Ile Ile Ser Arg Gly Glu Ser Ser Cys Pro Val Cys Gln
50 55 60
Thr Arg Phe Gln Pro Gly Asn Leu Arg Pro Asn Arg His Leu Ala
65 70 75
Asn Ile Val Glu Arg Val Lys Glu Val Lys Met Ser Pro Gln Glu
27/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
80 85 90
Gly Gln Lys Arg Asp Val Cys Glu His His Gly Lys Lys Leu Gln
95 I00 105
Ile Phe Cys Lys Glu Asp Gly Lys Val Ile Cys Trp Val Cys Glu
110 115 120
Leu Ser Gln Glu His Gln Gly His Gln Thr Phe Arg Ile Asn Glu
125 130 135
Val Val Lys Glu Cys Gln Glu Lys Leu Gln Val Ala Leu Gln Arg
240 145 150
Leu Ile Lys Glu Asp Gln Glu Ala Glu Lys Leu Glu Asp Asp Ile
155 160 165
Arg Gln Glu Arg Thr Ala Trp Lys Asn Tyr Ile Gln Ile Glu Arg
170 I75 180
Gln Lys Ile Leu Lys Gly Phe Asn Glu Met Arg Val Ile Leu Asp
185 190 195
Asn Glu Glu Gln Arg Glu Leu Gln Lys Leu Glu Glu Gly Glu Val
200 205 210
Asn Val Leu Asp Asn Leu Ala Ala Ala Thr Asp Gln Leu Val Gln
215 220 225
Gln Arg Gln Asp Ala Ser Thr Leu Ile Ser Asp Leu Gln Arg Arg
230 235 240
Leu Thr Gly Ser Ser Val Glu Met Leu Gln Asp Val Ile Asp Val
245 250 255
Met Lys Arg Ser Glu Ser Trp Thr Leu Lys Lys Pro Lys Ser Val
260 265 270
Ser Lys Lys Leu Lys Ser Val Phe Arg Val Pro Asp Leu Ser Gly
275 280 285
Met Leu Gln Val Leu Lys Glu Leu Thr Asp Val Gln Tyr Tyr Trp
290 295 300
Val Asp Val Met Leu Asn Pro Gly Ser Ala Thr Ser Asn Val Ala
305 310 315
Ile Ser Val Asp Gln Arg Gln Val Lys Thr Val Arg Thr Cys Thr
320 325 330
Phe Lys Asn Ser Asn Pro Cys Asp Phe Ser Ala Phe Gly Val Phe
335 340 345
Gly Cys Gln Tyr Phe Ser Ser Gly Lys Tyr Tyr Trp Glu Val Asp
350 355 360
Val Ser Gly Lys Ile Ala Trp Ile Leu Gly Val His Ser Lys Ile
365 370 375
Ser Ser Leu Asn Lys Arg Lys Ser Ser Gly Phe Ala Phe Asp Pro
380 385 390
Ser Val Asn Tyr Ser Lys Val Tyr Ser Arg Tyr Arg Pro Gln Tyr
395 400 405
Gly Tyr Trp Val Ile Gly Leu Gln Asn Thr Cys Glu Tyr Asn Ala
410 415 420
Phe Glu Asp Ser Ser Ser Ser Asp Pro Lys Val Leu Thr Leu Phe
425 430 435
Met Ala Val Pro Pro Cys Arg Ile Gly Val Phe Leu Asp Tyr Glu
440 445 450
Ala Gly Ile Val Ser Phe Phe Asn Val Thr Asn His Gly Ala Leu
455 460 465
Ile Tyr Lys Phe Ser Gly Cys Arg Phe Ser Arg Pro Ala Tyr Pro
470 475 480
Tyr Phe Asn Pro Trp Asn Cys Leu Val Pro Met Thr Val Cys Pro
485 490 495
Pro Ser Ser
28/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
<210> 26
<211> 1299
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 095477
<400> 26
Met Ala Ala Glu Thr Gln Thr Leu Asn Phe Gly Pro Glu Trp Leu
1 5 10 15
Arg Ala Leu Ser Ser Gly Gly Ser Ile Thr Ser Pro Pro Leu Ser
20 25 30
Pro Ala Leu Pro Lys Tyr Lys Leu Ala Asp Tyr Arg Tyr Gly Arg
35 40 45
Glu Glu Met Leu Ala Leu Phe Leu Lys Asp Asn Lys Ile Pro Ser
50 55 60
Asp Leu Leu Asp Lys Glu Phe Leu Pro Ile Leu Gln Glu Glu Pro
65 70 75
Leu Pro Pro Leu Ala Leu Val Pro Phe Thr Glu Glu Glu Gln Arg
80 85 90
Asn Phe Ser Met Ser Val Asn Ser Ala Ala Val Leu Arg Leu Thr
95 100 105
Gly Arg Gly Gly Gly Gly Thr Val Val Gly Ala Pro Arg Gly Arg
110 115 120
Ser Ser Ser Arg Gly Arg Gly Arg Gly Arg Gly Glu Cys Gly Phe
125 130 i35
Tyr Gln Arg Ser Phe Asp Glu Val Glu Gly Val Phe Gly Arg Gly
140 145 150
Gly Gly Arg Glu Met His Arg Ser Gln Ser Trp Glu Glu Arg Gly
155 160 165
Asp Arg Arg Phe Glu Lys Pro Gly Arg Lys Asp Val Gly Arg Pro
170 175 180
Asn Phe Glu Glu Gly Gly Pro Thr Ser Val Gly Arg Lys His Glu
185 190 195
Phe Ile Arg Ser Glu Ser Glu Asn Trp Arg Ile Phe Arg Glu Glu
200 205 210
Gln Asn Gly Glu Asp Glu Asp Gly Gly Trp Arg Leu Ala Gly Ser
215 220 225
Arg Arg Asp Gly Glu Arg Trp Arg Pro His Ser Pro Asp Gly Pro
230 235 240
Arg Ser Ala Gly,Trp Arg Glu His Met Glu Arg Arg Arg Arg Phe
245 250 255
Glu Phe Asp Phe Arg Asp Arg Asp Asp Glu Arg Gly Tyr Arg Arg
260 265 270
Val Arg Ser Gly Ser Gly Ser Ile Asp Asp Asp Arg Asp Ser Leu
275 280 285
Pro Glu Trp Cys Leu Glu Asp Ala Glu Glu Glu Met Gly Thr Phe
290 295 300
Asp Ser Ser Gly Ala Phe Leu Ser Leu Lys Lys Val Gln Lys Glu
305 310 315
Pro Ile Pro Glu Glu Gln Glu Met Asp Phe Arg Pro Val Asp Glu
320 325 330
Gly Glu Glu Cys Ser Asp Ser Glu Gly Ser His Asn Glu Glu Ala
29/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
335 340 345
Lys Glu Pro Asp Lys Thr Asn Lys Lys Glu Gly Glu Lys Thr Asp
350 355 360
Arg Val Gly Val Glu Ala Ser Glu Glu Thr Pro Gln Thr Ser Ser
365 370 375
Ser Ser Ala Arg Pro Gly Thr Pro Ser Asp His Gln Ser Gln Glu
380 385 390
Ala Ser Gln Phe Glu Arg Lys Asp Glu Pro Lys Thr Glu Gln Thr
395 400 405
Glu Lys Ala Glu Glu Glu Thr Arg Met Glu Asn Ser Leu Pro Ala
410 415 420
Lys Val Pro Ser Arg Gly Asp Glu Met Val Ala Asp Val Gln Gln
425 430 435
Pro Leu Ser Gln Ile Pro Ser Aap Thr Ala Ser Pro Leu Leu Ile
440 445 450
Leu Pro Pro Pro Val Pro Asn Pro Ser Pro Thr Leu Arg Pro Val
455 460 465
Glu Thr Pro Val Val Gly Ala Pro Gly Met Gly Ser Val Ser Thr
470 475 480
Glu Pro Asp Asp Glu Glu Gly Leu Lys His Leu Glu Gln Gln Ala
485 490 495
Glu Lys Met Val Ala Tyr Leu Gln Asp Ser Ala Leu Asp Asp Glu
500 505 510
Arg Leu Ala Ser Lys Leu Gln Glu His Arg Ala Lys Gly Val Ser
515 520 525
Ile Pro Leu Met His Glu Ala Met Gln Lys Trp Tyr Tyr Lys Asp
530 535 540
Pro Gln Gly Glu Ile Gln Gly Pro Phe Asn Asn Gln Glu Met Ala
545 550 555
Glu Trp Phe Gln Ala Gly Tyr Phe Thr Met Ser Leu Leu Val Lys
560 565 570
Arg Ala Cys Asp Glu Ser Phe Gln Pro Leu Gly Asp Ile Met Lys
575 580 585
Met Trp Gly Arg Val Pro Phe Ser Pro Gly Pro Ala Pro Pro Pro
590 595 600
His Met Gly Glu Leu Asp Gln Glu Arg Leu Thr Arg Gln Gln Glu
605 610 615
Leu Thr Ala Leu Tyr Gln Met Gln His Leu Gln Tyr Gln Gln Phe
620 625 630
Leu Ile Gln Gln Gln Tyr Ala Gln Val Leu Ala Gln Gln Gln Lys
635 640 645
Ala Ala Leu Ser Ser Gln Gln Gln Gln Gln Leu Ala Leu Leu Leu
650 655 660
Gln Gln Phe Gln Thr Leu Lys Met Arg Ile Ser Asp Gln Asn Ile
665 670 675
Ile Pro Ser Val Thr Arg Ser Val Ser Val Pro Asp Thr Gly Ser
680 6B5 690
Ile Trp Glu Leu Gln Pro Thr Ala Ser Gln Pro Thr Val Trp Glu
695 700 705
Gly Gly Ser Val Trp Asp Leu Pro Leu Asp Thr Thr Thr Pro Gly
710 715 720
Pro Ala Leu Glu Gln Leu Gln Gln Leu Glu Lys Ala Lys Ala Ala
725 730 735
Lys Leu Glu Gln Glu Arg Arg Glu Ala Glu Met Arg Ala Lys Arg
740 745 750
Glu Glu Glu Glu Arg Lys Arg Gln Glu Glu Leu Arg Arg Gln Gln
755 760 765
30/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
Glu Glu Ile Leu Arg Arg Gln Gln Glu Glu Glu Arg Lys Arg Arg
770 775 780
Glu Glu Glu Glu Leu Ala Arg Arg Lys Gln Glu Glu Ala Leu Arg
785 790 795
Arg Gln Arg Glu Gln Glu Ile Ala Leu Arg Arg Gln Arg Glu Glu
800 805 810
Glu Glu Arg Gln Gln Gln Glu Glu Ala Leu Arg Arg Leu Glu Glu
815 820 825
Arg Arg Arg Glu Glu Glu Glu Arg Arg Lys Gln Glu Glu Leu Leu
830 835 840
Arg Lys Gln Glu Glu Glu Ala Ala Lys Trp Ala Arg Glu Glu Glu
845 850 855
Glu Ala.Gln Arg Arg Leu Glu Glu Asn Arg Leu Arg Met Glu Glu
860 865 870
Glu Ala Ala Arg Leu Arg His Glu Glu Glu Glu Arg Lys Arg Lys
875 880 885
Glu Leu Glu Val Gln Arg Gln Lys Glu Leu Met Arg Gln Arg Gln
890 895 900
Gln Gln Gln Glu Ala Leu Arg Arg Leu Gln Gln Gln Gln Gln Gln
905 910 915
Gln Gln Leu Ala Gln Met Lys Leu Pro Ser Ser Ser Thr Trp Gly
920 925 930
Gln Gln Ser Asn Thr Thr Ala Cys Gln Ser Gln Ala Thr Leu Ser
935 940 945
Leu Ala Glu Ile Gln Lys Leu Glu Glu Glu Arg Glu Arg Gln Leu
950 955 960
Arg Glu Glu Gln Arg Arg Gln Gln Arg Glu Leu Met Lys Ala Leu
965 970 975
Gln Gln Gln Gln Gln Gln Gln Gln Gln Lys Leu Ser Gly Trp Gly
980 985 990
Asn Val Ser Lys Pro Ser Gly Thr Thr Lys Ser Leu Leu Glu Ile
995 1000 1005
Gln Gln Glu Glu Ala Arg Gln Met Gln Lys Gln Gln Gln Gln Gln
1010 1015 1020
Gln Gln His Gln Gln Pro Asn Arg Ala Arg Asn Asn Thr His Ser
1025 1030 1035
Asn Leu His Thr Ser Ile Gly Asn Ser Val Trp Gly Ser Ile Asn
1040 1045 1050
Thr Gly Pro Pro Asn Gln Trp Ala Ser Asp Leu Val Ser Ser Ile
1055 1060 1065
Trp Ser Asn Ala Asp Thr Lys Asn Ser Asn Met Gly Phe Trp Asp
1070 1075 1080
Asp Ala Val Lys Glu Val Gly Pro Arg Asn Ser Thr Asn Lys Asn
1085 1090 1095
Lys Asn Asn Ala Ser Leu Ser Lys Ser Val Gly Val Ser Asn Arg
1100 1105 1110
Gln Asn Lys Lys Val Glu Glu Glu Glu Lys Leu Leu Lys Leu Phe
1115 1120 1125
Gln Gly Val Asn Lys Ala Gln Asp Gly Phe Thr Gln Trp Cys Glu
1130 1135 1140
Gln Met Leu His Ala Leu Asn Thr Ala Asn Asn Leu Asp Val Pro
1145 1150 1155
Thr Phe Val Ser Phe Leu Lys Glu Val Glu Ser Pro Tyr Glu Val
1160 1165 1170
His Asp Tyr Ile Arg Ala Tyr Leu Gly Asp Thr Ser Glu Ala Lys
1175 1180 1185
Glu Phe Ala Lys Gln Phe Leu Glu Arg Arg Ala Lys Gln Lys Ala
31 /66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
1190 1195 1200
Asn Gln Gln Arg Gln Gln Gln Gln Leu Pro Gln Gln Gln Gln Gln
1205 1210 1215
Gln Pro Pro Gln Gln Pro Pro Gln Gln Pro Gln Gln Gln Asp Ser
1220 1225 1230
Val Trp Gly Met Asn His Ser Thr Leu His Ser Val Phe Gln Thr
1235 1240 1245
Asn Gln Ser Asn Asn Gln Gln Ser Asn Phe Glu Ala Val Gln Ser
1250 1255 1260
Gly Lys Lys Lys Lys Lys Gln Lys Met Val Arg Ala Asp Pro Ser
1265 1270 1275
Leu Leu Gly Phe Ser Val Asn Ala Ser Ser Glu Arg Leu Asn Met
1280 1285 1290
Gly Glu Ile Glu Thr Leu Asp Asp Tyr
1295
<210> 27
<211> 95I
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1399169
<400> 27
Met Ala Thr Gly Thr Gly Lys His Lys Leu Leu Ser Thr Gly Pro
1 5 10 15
Thr Glu Pro Trp Ser Ile Arg Glu Lys Leu Cys Leu Ala Ser Ser
20 25 30
Val Met Arg Ser Gly Asp Gln Asn Trp Val Ser Val Ser Arg Ala
35 40 45
Ile Lys Pro Phe Ala Glu Pro Gly Arg Pro Pro Asp Trp Phe Ser
50 55 60
Gln Lys His Cys Ala Ser Gln Tyr Ser Glu Leu Leu Glu Thr Thr
65 70 75
Glu Thr Pro Lys Arg Lys Arg Gly Glu Lys Gly Glu Val Val Glu
80 85 90
Thr Val Glu Asp Val Ile Val Arg Lys Leu Thr Ala Glu Arg Val
95 100 105
Glu Glu Leu Lys Lys Val Ile Lys Glu Thr Gln Glu Arg Tyr Arg
110 115 120
Arg Leu Lys Arg Asp Ala Glu Leu Ile Gln Ala Gly His Met Asp
125 130 135
Ser Arg Leu Asp Glu Leu Cys Asn Asp Ile Ala Thr Lys Lys Lys
140 145 150
Leu Glu Glu Glu Glu Ala Glu Val Lys Arg Lys Ala Thr Asp Ala
155 160 165
Ala Tyr Gln Ala Arg Gln Ala Val Lys Thr Pro Pro Arg Arg Leu
170 175 180
Pro Thr Val Met Val Arg Ser Pro Ile Asp Ser Ala Ser Pro Gly
185 190 195
Gly Asp Tyr Pro Leu Gly Asp Leu Thr Pro Thr Thr Met Glu Glu
200 205 210
32/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
Ala Thr Ser Gly Val Thr Pro Gly Thr Leu Pro Ser Thr Pro Val
215 220 225
Thr Ser Phe Pro Gly Ile Pro Asp Thr Leu Pro Pro Gly Ser Ala
230 235 240
Pro Leu Glu Ala Pro Met Thr Pro Val Thr Asp Asp Ser Pro Gln
245 250 255
Lys Lys Met Leu Gly Gln Lys Ala Thr Pro Pro Pro Ser Pro Leu
260 265 270
Leu Ser Glu Leu Leu Lys Lys Gly Ser Leu Leu Pro Thr Ser Pro
275 280 285
Arg Leu Val Asn Glu Ser Glu Met Ala Val Ala Ser Gly His Leu
290 295 300
Asn Ser Thr Gly Val Leu Leu Glu Val Gly Gly Val Leu Pro Met
305 310 315
Ile His Gly Gly Glu Ile Gln Gln Thr Pro Asn Thr Val Ala Ala
320 325 330
Ser Pro Ala Ala Ser Gly Ala Pro Thr Leu Ser Arg Leu Leu Glu
335 340 345
Ala Gly Pro Thr Gln Phe Thr Thr Pro Leu Ala Ser Phe Thr Thr
350 355 360
Val Ala Ser Glu Pro Pro Val Lys Leu Val Pro Pro Pro Val Glu
365 370 375
Ser Val Ser Gln Ala Thr Ile Val Met Met Pro Ala Leu Pro Ala
380 385 390
Pro Ser Ser Ala Pro Ala Val Ser Thr Thr Glu Ser Val Ala Pro
395 400 405
Val Ser Gln Pro Asp Asn Cys Val Pro Met Glu Ala Val Gly Asp
410 415 420
Pro His Thr Val Thr Val Ser Met Asp Ser Ser Glu Ile Ser Met
425 430 435
Ile Ile Asn Ser Ile Lys Glu Glu Cys Phe Arg Ser Gly Val Ala
440 445 450
Glu Ala Pro Val Gly Ser Lys Ala Pro Ser Ile Asp Gly Lys Glu
455 460 465
Glu Leu Asp Leu Ala Glu Lys Met Asp Ile Ala Val Ser Tyr Thr
470 475 480
Gly Glu Glu Leu Asp Phe Glu Thr Val Gly Asp Ile Ile Ala Ile
485 490 495
Ile Glu Asp Lys Val Asp Asp His Pro Glu Val Leu Asp Val Ala
500 505 510
Ala Val Glu Ala Ala Leu Ser Phe Cys Glu Glu Asn Asp Asp Pro
515 520 525
Gln Ser Leu Pro Gly Pro Trp Glu His Pro Ile Gln Gln Glu Arg
530 535 540
Asp Lys Pro Val Pro Leu Pro Ala Pro Glu Met Thr Val Lys Gln
545 550 555
Glu Arg Leu Asp Phe Glu Glu Thr Glu Asn Lys Gly Ile His Glu
560 565 570
Leu Val Asp Ile Arg Glu Pro Ser Ala Glu Ile Lys Val Glu Pro
575 580 585
Ala Glu Pro Glu Pro Val Ile Ser Gly Ala Glu Ile Val Ala Gly
590 595 600
Val Val Pro Ala Thr Ser Met Glu Pro Pro Glu Leu Arg Ser Gln
605 610 615
Asp Leu Asp Glu Glu Leu Gly Ser Thr Ala Ala Gly Glu Ile Val
620 625 630
Glu Ala Asp Val Ala Ile Gly Lys Gly Asp Glu Thr Pro Leu Thr
33/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
635 640 645
Asn Val Lys Thr Glu Ala Ser Pro Glu Ser Met Leu Ser Pro Ser
650 655 660
His Gly Ser Asn Pro Ile Glu Asp Pro Leu Glu Ala Glu Thr Gln
665 670 675
His Lys Phe Glu Met Ser Asp Ser Leu Lys Glu Glu Ser Gly Thr
680 685 690
Ile Phe Gly Ser Gln Ile Lys Asp Ala Pro Gly Glu Asp Glu Glu
695 700 705
Glu Asp Gly Val Ser Glu Ala Ala Ser Leu Glu Glu Pro Lys Glu
710 715 720
Glu Asp Gln Gly Glu Gly Tyr Leu Ser Glu Met Asp Asn Glu Pro
725 730 735
Pro Val Ser Glu Ser Asp Asp Gly Phe Ser Ile His Asn Ala Thr
740 745 750
Leu Gln Ser His Thr Leu Ala Asp Ser Ile Pro Ser Ser Pro Ala
755 760 765
Ser Ser Gln Phe Ser Val Cys Ser Glu Asp Gln Glu Ala Ile Gln
770 775 780
Ala Gln Lys Ile Trp Lys Lys Ala Ile Met Leu Val Trp Arg Ala
785 790 795
Ala Ala Asn His Arg Tyr Ala Asn Val Phe Leu Gln Pro Val Thr
800 B05 B10
Asp Asp Ile Ala Pro Gly Tyr His Ser Ile Val Gln Arg Pro Met
815 820 825
Asp Leu Ser Thr Ile Lys Lys Asn Ile Glu Asn Gly Leu Ile Arg
830 835 840
Ser Thr Ala Glu Phe Gln Arg Asp Ile Met Leu Met Phe Gln Asn
845 850 855
Ala Val Met Tyr Asn Ser Ser Asp His Asp Val Tyr His Met Ala
860 865 870
Val Glu Met Gln Arg Asp Val Leu Glu Gln Ile Gln Gln Phe Leu
875 B80 885
Ala Thr Gln Leu Ile Met Gln Thr Ser Glu Ser Gly Ile Ser Ala
890 895 900
Lys Ser Leu Arg Gly Arg Asp Ser Thr Arg Lys Gln Asp Ala Ser
905 910 915
Glu Lys Asp Ser Val Pro Met Gly Ser Pro Ala Phe Leu Leu Ser
920 925 930
Leu Phe Asp Gly Gly Thr Arg Gly Arg Arg Cys Ala Ile Glu Ala
935 940 945
Asp Met Lys Met Lys Lys
950
<210> 28
<211> 282
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1442069
<400> 28
34/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13Z81 _
Met Pro Lys Arg Lys Ala Ala Gly Gln Gly Asp Met Arg Gln Glu
1 5 10 15
Pro Lys Arg Arg Ser Ala Arg Leu Ser Ala Met Leu Val Pro Val
20 25 30
Thr Pro Glu Val Lys Pro Lys Arg Thr Ser Ser Ser Arg Lys Met
35 40 45
Lys Thr Lys Ser Asp Met Met Glu Glu Asn Ile Asp Thr Ser Ala
50 55 60
Gln Ala Val Ala Glu Thr Lys Gln Glu Ala Val Val Glu Glu Asp
65 70 75
Tyr Asn Glu Asn Ala Lys Asn Gly Glu Ala Lys Ile Thr Glu Ala
80 85 90
Pro Ala Ser Glu Lys Glu Ile Val Glu Val Lys Glu Glu Asn Ile
95 100 105
Glu Asp Ala Thr Glu Lys Gly Gly Glu Lys Lys Glu Ala Val Ala
110 115 120
Ala Glu Val Lys Asn Glu Glu Glu Asp Gln Lys Glu Asp Glu Glu
125 130 135
Asp Gln Asn Glu Glu Lys Gly Glu Ala Gly Lys Glu Asp Lys Asp
140 145 150
Glu Lys Gly Glu Glu Asp Gly Lys Glu Asp Lys Asn Gly Asn Glu
155 160 165
Lys Gly Glu Asp Ala Lys Glu Lys Glu Asp Gly Lys Lys Gly Glu
170 175 180
Asp Gly Lys Gly Asn Gly Glu Asp Gly Lys Glu Lys Gly Glu Asp
185 190 195
Glu Lys Glu Glu Glu Asp Arg Lys Glu Thr Gly Asp Gly Lys Glu
200 205 210
Asn Glu Asp Gly Lys Glu Lys Gly Asp Lys Lys Glu Gly Lys Asp
215 220 225
Val Lys Val Lys Glu Asp Glu Lys Glu Arg Glu Asp Gly Lys Glu
230 235 240
Asp Glu Gly Gly Asn Glu Glu Glu Ala Gly Lys Glu Lys Glu Asp
245 250 255
Leu Lys Glu Glu Glu Glu Gly Lys Glu Glu Asp Glu Ile Lys Glu
260 265 270
Asp Asp Gly Lys Lys Glu Glu Pro Gln Ser Ile Val
275 280
<210> 29
<211> 186
< 212 > Pi2T
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1596668
<400> 29
Met Asp Ala Asp Ser Asp Val Ala Leu Asp Ile Leu Ile Thr Asn
1 5 10 15
Val Val Cys Val Phe Arg Thr Arg Cys His Leu Asn Leu Arg Lys
20 25 30
Ile Ala Leu Glu Gly Ala Asn Val Ile Tyr Lys Arg Asp Val Gly
35/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
35 40 45
Lys Val Leu Met Lys Leu Arg Lys Pro Arg Ile Thr Ala Thr Ile
50 55 60
Trp Ser Ser Gly Lys Ile Ile Cys Thr Gly Ala Thr Ser Glu Glu
65 70 75
Glu Ala Lys Phe Gly Ala Arg Arg Leu Ala Arg Ser Leu Gln Lys
80 85 90
Leu Gly Phe Gln Val Ile Phe Thr Asp Phe Lys Val Val Asn Val
95 100 105
Leu Ala Val Cys Asn Met Pro Phe Glu Ile Arg Leu Pro Glu Phe
110 115 120
Thr Lys Asn Asn Arg Pro His Ala Ser Tyr Glu Pro Glu Leu His
125 130 135
Pro Ala Val Cys Tyr Arg Ile Lys Ser Leu Arg Ala Thr Leu Gln
140 145 150
Ile Phe Ser Thr Gly Ser Ile Thr Val Thr Gly Pro Asn Val Lys
155 160 165
Ala Val Ala Thr Ala Val Glu Gln Ile Tyr Pro Phe Val Phe Glu
170 175 180
Ser Arg Lys Glu Ile Leu
185
<210> 30
<211> 917
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1977214
<400> 30
Met Ala Glu Thr Leu Ser Gly Leu Gly Asp Ser Gly Ala Ala Gly
1 5 10 15
Ala Ala Ala Leu Sex Ser Ala Ser Ser Glu Thr Gly Thr Arg Arg
20 25 30
Leu Ser Asp Leu Arg Val Ile Asp Leu Arg Ala Glu Leu Arg Lys
35 40 45
Arg Asn Val Asp Ser Ser Gly Asn Lys Ser Val Leu Met Glu Arg
50 55 60
Leu Lys Lys Ala Ile Glu Asp Glu Gly Gly Asn Pro Asp Glu Ile
65 70 75
Glu Ile Thr Ser Glu Gly Asn Lys Lys Thr Ser Lys Arg Ser Ser
80 85 90
Lys Gly Arg Lys Pro Glu Glu Glu Gly Val Glu Asp Asn Gly Leu
95 100 105
Glu Glu Asn Ser Gly Asp Gly Gln Glu Asp Val Glu Thr Ser Leu
110 115 120
Glu Asn Leu Gln Asp Ile Asp Ile Met Asp Ile Ser Val Leu Asp
125 130 135
Glu Ala Glu Ile Asp Asn Gly Ser Val Ala Asp Cys Val Glu Asp
140 145 150
Asp Asp Ala Asp Asn Leu Gln Glu Ser Leu Ser Asp Ser Arg Glu
155 160 165
36/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
Leu Val Glu Gly Glu Met Lys Glu Leu Pro Glu Gln Leu Gln Glu
170 175 180
His Ala Ile Glu Asp Lys Glu Thr Ile Asn Asn Leu Asp Thr Ser
185 190 195
Ser Ser Asp Phe Thr Ile Leu Gln Glu Ile Glu Glu Pro Ser Leu
200 205 210
Glu Pro Glu Asn Glu Lys Ile Leu Asp Ile Leu Gly Glu Thr Cys
215 220 225
Lys Ser Glu Pro Val Lys Glu Glu Ser Ser Glu Leu Glu Gln Pro
230 235 240
Phe Ala Gln Asp Thr Ser Ser Val Gly Pro Asp Arg Lys Leu Ala
245 250 255
Glu Glu Glu Asp Leu Phe Asp Ser Ala His Pro Glu Glu Gly Asp
260 265 270
Leu Asp Leu Ala Ser Glu Ser Thr Ala His Ala Gln Ser Ser Lys
275 280 285
Ala Asp Ser Leu Leu Ala Val Val Lys Arg Glu Pro Ala Glu Gln
290 295 300
Pro Gly Asp Gly Glu Arg Thr Asp Cys Glu Pro Val Gly Leu Glu
305 310 315
Pro Ala Val Glu Gln Ser Ser Ala Ala Ser Glu Leu Ala Glu Ala
320 325 330
Ser Ser Glu Glu Leu Ala Glu Ala Pro Thr Glu Ala Pro Ser Pro
335 340 345
Glu Ala Arg Asp Ser Lys Glu Asp Gly Arg Lys Phe Asp Phe Asp
350 355 360
Ala Cys Asn Glu Val Pro Pro Ala Pro Lys Glu Ser Ser Thr Ser
365 370 375
Glu Gly Ala Asp Gln Lys Met Ser Ser Pro Glu Asp Asp Ser Asp
380 385 390
Thr Lys Arg Leu Ser Lys Glu Glu Lys Gly Arg Ser Ser Cys Gly
395 400 405
Arg Asn Phe Trp Val Ser Gly Leu Ser Ser Thr Thr Arg Ala Thr
410 415 420
Asp Leu Lys Asn Leu Phe Ser Lys Tyr Gly Lys Val Val Gly Ala
425 430 435
Lys Val Val Thr Asn Ala Arg Ser Pro Gly Ala Arg Cys Tyr Gly
440 445 450
Phe Val Thr Met Ser Thr Ala Glu Glu Ala Thr Lys Cys Ile Asn
455 460 465
His Leu His Lys Thr Glu Leu His Gly Lys Met Ile Ser Val Glu
470 475 480
Lys Ala Lys Asn Glu Pro VaI Gly Lys Lys Thr Ser Asp Lys Arg
485 490 495
Asp Ser Asp Gly Lys Lys Glu Lys Ser Ser Asn Ser Asp Arg Ser
500 505 510
Thr Asn Leu Lys Arg Asp Asp Lys Cys Asp Arg Lys Asp Asp Ala
515 520 525
Lys Lys Gly Asp Asp Gly Ser Gly Glu Lys Ser Lys Asp Gln Asp
530 535 540
Asp Gln Lys Pro Gly Pro Ser Glu Arg Ser Arg Ala Thr Lys Ser
545 550 555
Gly Ser Arg Gly Thr Glu Arg Thr Val Val Met Asp Lys Ser Lys
560 565 570
Gly Val Pro Val Ile Ser Val Lys Thr Ser Gly Ser Lys Glu Arg
575 580 585
Ala Ser Lys Ser Gln Asp Arg Lys Ser Ala Ser Arg Glu Lys Arg
37/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
590 595 600
Ser Val Val Ser Phe Asp Lys Val Lys Glu Pro Arg Lys Ser Arg
605 610 615
Asp Ser Glu Ser His Ser Arg Val Arg Glu Arg Ser Glu Arg Glu
620 625 630
Gln Arg Met Gln Ala Gln Trp Glu Arg Glu Glu Arg Glu Arg Leu
635 640 645
Glu Ile Ala Arg Glu Arg Leu Ala Phe Gln Arg Gln Arg Leu Glu
650 655 660
Arg Glu Arg Met Glu Arg Glu Arg Leu Glu Arg Glu Arg Met His
665 670 675
Val Glu His Asp Gly Arg Arg Glu Gln Glu Arg Ile His Arg Glu
680 685 690
Arg Glu Glu Leu Arg Arg Gln Gln Glu Leu Arg Tyr Glu Gln Glu
695 700 705
Arg Arg Pro Ala Val Arg Arg Pro Tyr Asp Leu Asp Arg Arg Asp
710 715 720
Asp Ala Tyr Trp Pro Glu Ala Lys Arg Ala Ala Leu Asp Glu Arg
725 730 735
Tyr His Ser Asp Phe Asn Arg Gln Asp Arg Phe His Asp Phe Asp
740 745 750
His Arg Asp Arg Gly Arg Tyr Pro Asp His Ser Val Asp Arg Arg
755 760 765
Glu Gly Ser Arg Ser Met Met Gly Glu Arg Glu Gly Gln His Tyr
770 775 780
Pro Glu Arg His Gly Gly Pro Glu Arg His Gly Gly Ala Ser Arg
785 790 795
Asp Gly Trp Gly Gly Tyr Gly Ser Asp Lys Arg Met Ser Glu Gly
800 805 810
Arg Gly Leu Pro Pro Pro Pro Arg Gly Arg Arg Asp Trp Gly Asp
815 820 825
His Gly Arg Arg Glu Asp Asp Arg Ser Trp Gln Gly Thr Ala Asp
830 835 840
Gly Gly Met Met Asp Arg Asp His Lys Arg Trp Gln Gly Gly Glu
845 850 855
Arg Ser Met Ser Gly His Ser Gly Pro Gly His Met Met Asn Arg
860 865 870
Gly Gly Met Ser Gly Arg Gly Ser Phe Ala Pro Gly Gly Ala Ser
875 880 885
Arg Gly His Pro Ile Pro His Gly Gly Met Gln Gly Gly Phe Gly
890 895 900
Gly Gln Ser Arg Gly Ser Arg Pro Ser Asp Ala Arg Phe Thr Arg
905 910 915
Arg Tyr
<210> 31
<211> 392
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2181282
38/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
<400> 31
Met Pro Lys Pro Ile Met Val Ile Pro Thr Leu Ala Ser Leu Ala
1 5 10 15
Ser Pro Thr Thr Leu Gln Ser Gln Met Leu Gly Gly Leu Gly Gln
20 25 30
Asp Val Leu Leu Asn Asn Ser Leu Thr Pro Lys Tyr Leu Gly Cys
35 40 45
Lys Gln Asp Asn Ser Ser Ser Pro Lys Pro Ser Ser Val Phe Arg
50 55 60
Asn Gly Phe Ser Gly Ile Lys Lys Pro Trp His Arg Cys His Val
65 70 75
Cys Asn His His Phe Gln Phe Lys Gln His Leu Arg Asp His Met
80 85 90
Asn Thr His Thr Asn Arg Arg Pro Tyr Ser Cys Arg Ile Cys Arg
95 100 105
Lys Ser Tyr Val Arg Pro Gly Ser Leu Ser Thr His Met Lys Leu
110 115 120
His His Gly Glu Asn Arg Leu Lys Lys Leu Met Cys Cys Glu Phe
125 130 135
Cys Ala Lys Val Phe Gly His Ile Arg Val Tyr Phe Gly His Leu
140 145 150
Lys Glu Val His Arg Val Val Ile Ser Thr Glu Pro Ala Pro Ser
155 160 165
Glu Leu Gln Pro Gly Asp Ile Pro Lys Asn Arg Asp Met Ser Val
170 175 180
Arg Gly Met Glu Gly Ser Leu Glu Arg Glu Asn Lys Ser Asn Leu
185 190 195
Glu Glu Asp Phe Leu Leu Asn Gln Ala Asp Glu Val Lys Leu Gin
200 205 210
Ile Lys Cys Gly Arg Cys Gln Ile Thr Ala Gln Ser Phe Ala Glu
215 220 225
Ile Lys Phe His Leu Leu Asp Val His Gly Glu Glu Ile Glu Gly
230 235 240
Arg Leu Gln Glu Gly Thr Phe Pro Gly Ser Lys Gly Thr Gln Glu
245 250 255
Glu Leu Val Gln His Ala Ser Pro Asp Trp Lys Arg His Pro Glu
260 265 270
Arg Gly Lys Pro Glu Lys Val His Ser Ser Ser Glu Glu Ser His
275 280 285
Ala Cys Pro Arg Leu Lys Arg Gln Leu His Leu His Gln Asn Gly
290 295 300
Val Glu Met Leu Met Glu Asn Glu Gly Pro Gln Ser Gly Thr Asn
305 310 315
Lys Pro Arg Glu Thr Cys Gln Gly Pro Glu Cys Pro Gly Leu His
320 325 330
Thr Phe Leu Leu Trp Ser His Ser Gly Phe Asn Cys Leu Leu Cys
335 340 345
Ala Glu Met Leu Gly Arg Lys Glu Asp Leu Leu His His Trp Lys
350 355 360
His Gln His Asn Cys Glu Asp Pro Ser Lys Leu Trp Ala Ile Leu
365 370 375
Asn Thr Val Ser Asn Gln Gly Val Ile Glu Leu Ser Ser Glu Ala
380 385 390
Glu Lys
39/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
<210> 32
<211> 1566
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 591290
<400> 32
tggttcagaa gcgaattctg caccgcgaac ctgagaggca ggtagctgcg gcgctgggct 60
ggcggcggcg agtccacgtg ctccccgcgg ccggttgaaa ccgttggcgg gcgctggctg 120
agaggcaatg tttgctgtct tccattggag tgactgaatt tctacatgac ggctttttga 180
caagacttaa aacctgtctt ggatagagaa tatttagcca tttacctaaa aatggtattt 240
tttacatgca atgcatgtgg tgaatcagtg aagaaaatac aagtggaaaa gcatgtgtct 300
gtttgcagaa actgtgaatg cctttcttgc attgactgcg gtaaagattt ctggggcgat 360
gactataaaa accacgtgaa atgcataagt gaagatcaga agtatggtgg caaaggctat 420
gaaggtaaaa cccacaaagg cgacatcaaa cagcaggcgt ggattcagaa aattagtgaa 480
ttaataaaga gacccaatgt cagccccaaa gtgagagaac ttttagagca aattagtgct 540
tttgacaacg ttcccaggaa aaaggcaaaa tttcagaatt ggatgaagaa cagtttaaaa 600
gttcataatg aatccattct ggaccaggtg tggaatatct tttctgaagc ttccaacagc 660
gaaccagtca ataaggaaca ggatcaacgg ccactccacc cagtggcaaa tccacatgca 720
gaaatctcca ccaaggttcc agcctccaaa gtgaaagacg ccgtggaaca gcaaggggag 780
gtgaagaaga ataaaagaga aaaaaaggaa gaacggcaga agaaaaggaa aagagaaaag 840
aaagaactaa agttagaaaa ccaccaggaa aactcaagga atcagaagcc taagaagcgc 900
aaaaagggac aggaggctga ccttgaggct ggtggggagg aagtccctga ggccaatggc 960
tctgcaggga agaggagcaa gaagaagaag cagcgcaagg acagcgccag tgaggaagag 1020
gcacgcgtgg gcgcagggaa gaggaagcgg aggcactcgg aagttgaaac agattctaag 1080
aagaaaaaga tgaagctccc agagcatcct gagggcggag aaccagaaga cgatgaggct 1140
cctgcaaaag gtaaattcaa ctggaaggga actattaaag caattctgaa acaggcccca 1200
gacaatgaaa taaccatcaa aaagctaagg aaaaaggttt tagctcagta ctacacagtg 1260
acagatgagc atcacagatc cgaagaggaa ctcctggtca tctttaacaa gaaaatcagc 1320
aagaacccta cctttaagtt attaaaggac aaagtcaagc ttgtgaaatg aacatttgtg 1380
tatttaaaaa ttgaatccat tctgctgact tcttcctttc actgctgttt ataaaatgtg 1440
taatgaattc taacaactca aattttgctt tttgaagctg tatttttaag ttaagaaaat 1500
atatttttgg tataactttt atgagaaaaa taaaatatat tctggtccaa acttctaaaa 1560
aaaaaa 1566
<210> 33
<211> 2338
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 815856
<400> 33
aggggaggtt ctccttttgc tattgtcatc acccagcaac accagattca ccgctcctgc 60
acagtcaaca tcatgtttgg aaccccgcaa gagcatcgca acatgcccca agcagatgcc 120
atggtgctgg tggccagaaa ttatgagcgt tacaagaatg agtgccggga gaaggaacgt 180
gaggagattg ccagacaggc agccaagatg gccgatgaag ccatcctgca ggaaagagag 240
agaggaggcc ctgaggaggg agtgcgtggg ggccaccctc cagccatcca gagcctcatc 300
aacctgctgg cagacaacag gtacctcact gctgaagaga ctgacaagat catcaactac 360
ctgcgagagc ggaaggagcg gctgatgagg agcagcaccg actctctgcc tggtgagcta 420
cgtggcaggg ccgaggcccg atttcccgcc aaccactcgg ggcgacctcg ggtgcctcgc 480
40/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99113281 _
tgaagacaca gccaagctcc caaccgctcc agagcggcca agtgctcccc tctgctacac 540
ccactccatc tgcacccccc acctcccagc aagagcttca ggccaaaatc ctcagcctct 600
tcaatagtgg cacagtgacg gccaatagca gctctgcatc cccctcggtt gctgccggaa 660
acaccccaaa ccagaatttt tccacagcag caaacagcca gc~tcaacaa agatcacagg 720
cttctggcaa tcagcctcca agcattttgg gacagggagg atctgctcag aacatgggcc 780
ccagacctgg ggctccttcc caagggcttt ttggccagcc ttccagtcgc ctggcacctg 840
ctagcaacat gactagccag aggcctgtgt cttccacagg tatcaacttt gacaatccaa 900
gtgtacagaa ggctctggat accctgatcc agagtggccc tgctctctcc cacctggtta 960
gccagaccac agcacagatg gggcagccac aggcccccat gggatcttac cagaggcatt 1020
actgaagcta aatctttcaa ctctccccag tcccctcatc ccctggcctc ctcccactta 1080
cttgttctaa atagagctgt ttggaggatg ttctctgcgc tcccaggccg gcatcgagtg 1140
tcatcaattt ctaccacctg ctctctcttc tgcccaaggc tgtgttgctt attccttaca 1200
aagtttatac tgcatttggg gctgtatctt tttttgtttt ttgttttgta gaaaataaat 1260
atctccgggg gcagtacagg tgtctgggct tgtatttgat ggggtttctc cggtccctgt 1320
ttctactgga tttggagcca ggcccagcta gccaagtttg gaatggcatt tgtcatgtca 1380
gtagccacca cctttgttca ttgtgaacct accaaggctt tccagcttca tacacattga 1440
ccagagctca agctcctgcc tgcaactcct gcctagagtt gaagaaaagc aaactggcct 1500
tggcaggcac agtgtcatca taccctcacc ccatatgttt ggggtctgct tgaggattca 1560
taaatcagcc actctggatt gttgaggaat ggccatggca gccacagaaa aaagaatttt 1620
tctctctgag ccaaggttgt tttttgtttt tttctctttt cttttttgtt ttcatttcat 1680
tggaagatct ccaatggact gaacagctcc agtcagcagc agttaccaca aactgtgaat 1740
ctgggcccca ccactcttcc ctgttaacca gttctgtcag catccccctc tccagcagca 1800
cttccatgaa gttggttctg agactctggc cgtgaacacc cgtttcttca gtgatttgtt 1860
ttgggctttt ggctcaaaac cccaggctct tgtttttgtc tagactctta ttctgtttcc 1920
tgagcagcag gaggtaggga ccactttgat gtcagacttc tggtagctgg acatgttctc 1980
gagatgggtg gctgttcgcg acttttgtac cagagtgaaa ttgttagaag gagggtttct 2040
ggctgtggtt ctaaatggag ccccaggaag ctgccctctc cccagggttt gtgctcagta 2100
gagccctgtg gatcacagtc ttgaggtcct ctagcagggg tgagggagag cagcgacttc 2160
agctgagtcc ctgccagtgg ttaagcaaac aatggtttca aaattcaagg tccccaaatg 2220
gcagcatttt atgttctgac ctgtttgtgt tatatagtgg tttttttttt cctctttgga 2280
actcttgtgt tgttaataaa atgaaatgat tactttttaa ttaaaatgaa aaaaaaaa 2338
<210> 34
<211> 870
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 996352
<400> 34
aaaaccgaca ctgaaagctg ccggacaaca aatcacaggt caaaattatg agttcttcgt 60
attatgtgaa cgcgcttttt agcaaatata cggcgggggc ttctctgttc caaaatgccg 120
agccgacttc ttgctccttt gctcccaact cacagagaag cggctacggg gcgggcgccg 180
gcgccttcgc ctcgaccgtt ccgggcttat acaatgtcaa cagccccctt tatcagagcc 240
cctttgcgtc cggctacggc ctgggcgccg acgcctacgg caacctgccc tgcgcctcct 300
acgaccaaaa catccccggg ctctgcagtg acctcgccaa aggcgcctgc gacaagacgg 360
acgagggcgc gctgcatggc gcggctgagg ccaatttccg catctacccc tggatgcggt 420
cttcaggacc tgacaggaag cggggccgcc agacctacac gcgctaccag acgctggagc 480
tggagaagga gttccacttc aaccgctacc tgatccggcg ccgccgcatt gaaatcgccc 540
acgcgctctg cctcaccgag cgccagatta agatctggtt ccagaaccgc cgcatgaagt 600
ggaagaaaga gcataaggac gaaggtccga ctgccgccgc agctcccgag ggcgccgtgc 660
cctctgccgc cgccactgct gccgcggaca aggccgacga ggaggacgat gatgaagaag 720
aggaagacga ggaggaatga ggggccgatc cggggccctc tctgcaccgg acagtcggaa 780
aagcgtcttt aagagactca ctggttttac ttacaaaaat gggaaaaata aaagaaaatg 840
41 /66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
taaaaaacaa aaacaaaaac aaaaaagcat 870
c210> 35
<211> 1365
<212> DNA
<213> Homo sapiens
c220>
<221> unsure
<222> 6, 61, 882, 883, 884, 885. 886, 887, 888, 889, 890, 891, 892, 893,
894, 895, <221> unsure
<222> 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 912,
914, 916, <221> unsure
<222> 920, 922, 929, 932, 964, 967, 971, 975, 986, 1000, 1028, 1033, 1039,
1052, <221> unsure
<222> 1053, 1063, 1065, 1071, 1080, 1090, 1100, 1102, 1110, 1113, 1123,
1128, <221> unsure
<222> 1134, 1139, 1142, 1143, 1144, 1153, 1158, 1164, 1169, 1177, 1180,
1181, <221> unsure
<222> 1182, 1184, 1189, 1191, 1192, 1211, 1224, 1239, 1246, 1247, 1248,
1263, c221> unsure
c222> 1266, 1274, 1284, 1291, 1314, 1321, 1322, 1339, 1341, 1343, 1347,
1351, <221> unsure
<222> 1353, 1359, 1362, 1363, 1364
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No.: 1273778
<400> 35
cttcangcct ggcggcggct tccaagctaa ggaacggttt ggggcagtgt cgttcccgga 60
ngtcggccgc cgttacccgc tcaccagcta cgcggcgcgt caggtccgcg gaggcgcggg 120
ctcggggcgc ctgcgggacg gtgaggccct gctgaggact ccggcaagtg tgggtcgcgg 180
cgacggcggg gctaaggccc tgggtccgcg cgcggtttga ccacggccgg ggccttgggc 240
atttcctggc cttcctgttg agccgtgtaa acgcggggtg atgacggcgc cgacctcttg 300
gcactgttgt gagagcgaag tgggcgcgag agcagacgcc agctacagtt ttttttgggt 360
tatgtcgtca tgaagccggc gctttcagtt gtgcaacctt gaacaaatgg gacactgccc 420
atctctaaga taagaacctg gaaaggggac tctgttggcc attggaaatt gcagaataat 480
gtctcaggtg acatttagtg atgtggctat agacttctct catgaagagt gggcatgcct 540
agattctgct cagagggact tatacaagga tgtgatggtc cagaattatg agaacctggt 600
ctctgtaggt ctttccgtaa ctaagccata tgtgatcatg ttattggagg atggaaaaga 660
gccctggatg atggagaaaa aactgtcaaa agcttaccca tttcctttat cacactctgt 720
tcctgcttct gtgaactttg gattctctgc tctatttgag cattgttcag aagtcactga 780
aatatttgag ttgtcagaac tatgtgtttt ctgggtgctt catttcttat ccaattctcc 840
taattccact gtagaagctt ttttcaagaa gtaaaaaaaa annnnnnnnn nnnnnnnnnn 900
nnnnnnnngg cngngnttgn cnttcttgnt anatttcaga ttgggaatca agatgggaaa 960
acanggnatt ntcancaaag aagganattt gtgatgaagn ttcaccccaa ccagtaacaa 1020
tggaaaangt tgnaaaacna agttttgaat tnncaaaatt ctnantaagg ntttggaatn 1080
tccagaatgn gacacgtttn gnaagagccn ggnattccaa agngtacnct ttcntgggnc 1140
gnnngaaaca aanttctncc tgtnggggna tttcccnccn nnantatgnt nnccaggttg 1200
agaggaattt nttccaaaag ggtnccggtc ataaaaggng gggggnnnaa tggggggaaa 1260
aanacntttg aaantcgaag aaangggggg ncggccttcc aacccggggc caancccgta 1320
nnccttcccc ggtctgttnt ngngggnaag ntntttacng gnnng 1365
42/66
CA 02329685 2000-12-11
WO 99/64596 PCTNS99/13281
<210> 36
<211> 2402
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1509715
<400> 36
agcgcgggac aaagggaagc gaagccggag ctgcgggcgc tttttctgcc cgcggtgtct 60
cagattcatt cttaaggaac tgagaactta atcttccaaa atgtcaaaaa gaccatctta 120
tgccccacct cccaccccag ctcctgcaac acaaatgccc agcacaccag ggtttgtggg 180
atacaatcca tacagtcatc tcgcctacaa caactacagg ctgggagggg aacccgggca 240
ccaacagccg ggtcacggca tcctctggta tcacgattcc aaaaccccca aagccaccag 300
ataagccgct gatgccctac atgaggtaca gcagaaaggt ctgggaccaa gtaaaggctt 360
ccaaccctga cctaaagttg tgggagattg gcaagattat tggtggcatg tggcgagatc 420
tcactgatga agaaaaacaa gaatatttaa acgaatacga agcagaaaag atagagtaca 480
atgaatctat gaaggcctat cataattccc ccgcgtacct tgcttacata aatgcaaaaa 540
gtcgtgcaga agctgcttta gaggaagaaa gtcgacagag acaatctcgc atggagaaag 600
gagaaccgta catgagcatt cagcctgctg aagatccaga tgattatgat gatggctttt 660
caatgaagca tacagccacc gcccgtttcc agagaaacca ccgcctcatc agtgaaattc 720
ttagtgagag tgtggtgcca gacgttcggt cagttgtcac aacagctaga atgcaggtcc 780
tcaaacggca ggtccagtcc ttaatggttc atcagcgaaa actagaagct gaacttcttc 840
aaatagagga acgacaccag gagaagaaga ggaaattcct ggaaagcaca gattcattta 900
acaatgaact taaaaggttg tgcggtctga aagtagaagt ggatatggag aaaattgcag 960
ctgagattgc acaggcagag gaacaggccc gcaaaaggca ggaggaaagg gagaaggagg 1020
ccgcagagca agctgagcgc agtcagagca gcatcgttcc tgaggaagaa caagcagcta 1080
acaaaggcga ggagaagaaa gacgacgaga acattccgat ggagacagag gagacacacc 1140
ttgaagaaac aacagagagc caacagaatg gtgaagaagg cacgtctact cctgaggaca 1200
aggagagtgg gcaggagggg gtcgacagta tggcagagga aggaaccagt gatagtaaca 1260
ctggctcgga gagcaacagt gcaacagtgg aggagccacc aacagatccc ataccagaag 1320
atgagaaaaa agaataagtg ttgccttgtt ttgtgtgttc taaatacttt ttttaatgaa 1380
aaaatgtttt ttggttttaa tggtgttacg tggtttgtgt attaattttt tttcttgtcc 1440
atatcacacc accaaaggct tttggaccat ttagcatcat gagcctaatg gctcagtcag 1500
tcacctttct taagtgttgt gaagatggct cttttctttg gatcttgttt ctagccctca 1560
actgctgaaa gcctcagaat ttagattaat tgagaaaaca cccacctctt ttagagaatt 1620
atcctttgat gctgcagaat ctactcttac aatgccttcc tacagctcac tggggtgctt 1680
accaaagcca tagctttaaa ccttcccagt ccccatcagt agcttcctga aagtctcctc 1740
tcttgtttac ttctgcaaag ggtagcttct taaaaacgtg atcatgtatg agtatgtatt 1800
tgttcactta ccctttttta cttttaatca atgtcagata ccaagagttg tgttaagctg 1860
agtgtagtgt gtaactaact acacttggat cttactgatc cagaaatagt ccccatagtt 1920
agagtagtta cttatgaagt ggttattaaa gtgaacacag cacatataca ttatctatac 1980
tgctttttgt tatgattaat actgggtatg ttctggtaaa tccatcctta ttgtatagaa 2040
aaaaaattac ttttttacca ggttttccaa agacagaata gatcacaaag ctcaaggaat 2100
ttaatattct tgtaatggac tagataattc aaactgatta gcccattcca gaagaaaaac 2160
agctgggaat taagttaatc cacttgaaat tgttttacaa taatcagaac atccaaacct 2220
caaggctcag gatcccatag accagagccc acctttttga taaacttagt aaagtcttgg 2280
agactagaag caagatagtt tgtgacacat aagcttccca aaaactagaa tagattttta 2340
ctgaatagtg gtatatctga tggtatatgt ttcttaaagg tccaaatgta ataaaaaaaa 2400
aaaaaaaaaa aaaaaaaa
2418
<210> 37
<211> 866
<212> DNA
43166
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
<213> Homo sapiens
<220>
<221> unsure
<222> 684, 698, 740, 755, 757, 776, 796, 805, 8I1, 824, 850, 856
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No.: 1676367
<400> 37
gccggggtgc gcacttgggc ccccctggcc atggcggcga aggtggacct gagcacctcc 60
accgactgga aggaggcgaa atcctttctg aagggcctga gtgacaagca gcgggaggaa 120
cattacttct gcaaggactt tgtcaggctg aagaagatcc cgacatggaa ggagatggcg 180
aaaggggtgg ctgtgaaggt ggaggagccc aggtataaaa aggacaagca gctcaatgag 240
aaaatctccc tgctccgcag cgacatcacc aagctggagg tggacgccat cgtcaacgcc 300
gccaacagct ccctgctcgg aggcggtggc gtggacggct gcattcatcg ggccgccggc 360
cccctgctta ccgacgagtg ccggaccctg cagagctgta agactggcaa ggccaagatc 420
accggcggct atcggctccc ggccaagtac gtcatccaca cagtggggcc catcgcctac 480
ggggagccca gcgccagtca ggctgccgag ctccgcagct gctacctgag cagtctggac 540
ctgctgctgg agcaccggct ccgctcggtg gcgttcccct gcatctccac cggcgtgttt 600
ggctacccct gtgaggcggc cgccgagatc gtgctggcca cgctgcgaga gtggcttggg 660
agcagcacaa gggaaccccg gggnaattta aatttccngg aacccgggta actttgcaag 720
gcggttaacc aagctttttn cccctaataa attgnanttc cgtaatttaa gaagcntttg 780
gggggtaaaa tcaatngggt caatnaagct nggttttccc cggnggttga aaaatttggt 840
taatcccggn ttaaanaaat ttccca 866
<210> 38
<211> 1651
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1734119
<400> 38
ggaaggtggt ggcaggcacc ggcctgggca gcttccaggc ctggctggcc acgaccacgg 60
cccgaggggg agcccgccag gccacgccgc actgagccac agccccgggg gccgcctccc 120
gggggcccct tgaggcactg aggcaccgag actggttctc cccgagagac tcggaaggtg 180
gggaacgagg ggactgtgtt tggggaggtg gctttttcgt ctgctgttga ctgaacacta 240
cagcgccctg tggttccggg cttcgcacag ctgtcccagg gatgggtcgc ctgtgctgcc 300
ttcgcccgcc gccacaccgg gaccctgcac ggctgcttct ggcctcgaca gatgacaaaa 360
gaaacagccc caaaatacga ccactccaac cagcagttcc cgcctgcctg cccgccactg 420
tcaggcctgc cctggcctcc tcgtccgcag ggctgtctgc tggcttctgg gggcagaaga 480
gcggggagcc ccgtggaagg gtcaggggag accaggtcag ggcagctaca tttctggtga 540
tcagccccat ggggagacgg ggctggcggg ataccgcccc ccccggcttc cccacaccac 6o0
ttctgtctca cccggaagcg tccttttttt gtgccaggtg tctacctaag agggttggtg 660
ccagaagccc cccatggcga gtgctggggc ccggcggtgc cctgggggag cagatggggc 720
cacccctggc agggccgcta caactttttc cagcagcgga gccctctggg gggcctgtgc 780
ttgtggcatc tctgagggcc cagattgcac aaggtgacct ggccgtggcc tgagggtgga 84o
gtcgcccagc acgcaggccg gggcgctgcg gggctaagta ttaggccttc ccagggaggg 900
ggcgtgccaa gcatcccaga gccgggctgg gaccgccaaa acgtcgtggc ctggatcctc 960
tgggtctgag tgcctgatcc cctgcccccc aaaaaaagca gaggtaggtg ttgcaggccc 1020
agggcagggg tgcctgcccc aggagagtcc caggcagtgg ttctcgtgcc agtggcaccc 1080
44/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
aggggcaagg acagccaacc cccacccttg ccacgtgtgg ggccacgtgg gcatgtgggg 1140
tgtgtgtttt taccttggtg aatctcacct gccaacgatt tctcgtgagt gccgaccacc 1200
ttctccgacc atgttacgcc cgggcggcag cagcccccgg ccactgcaaa cccatgccct 1260
gggtccctcg gctcccccag ggaggcatcc ccgtgccaat gtcccccagt ggtggcagca 1320
gatcctgtgg ccggcctggc ggacgggacc cagtgatact tgtatattac acagtcctga 1380
tttcagacaa tttcaacctt aatctattta aaaaagaata ttctatacaa gctgttttta 1440
agccttttac catttgaaat gcatgtgttg tgcgcgttgg ggatgggagg aggggctgag 1500
gagcggctca gtgtcacctc ccacagccac cggccctgac ccttaatcca gacaccgatg 1560
gaagtcgact tttcatatct ttctcctgaa atgaactctg ttttaaattg gaataaattt 1620
tgttcctaaa aaaaaaaaaa aaaaaaaaaa a 1651
<210> 39
<211> 1032
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1944813
<400> 39
tattaatgaa gagtgactgc atgcaaacga caatatgtca ggaaagaaaa aaagatccca 60
tagaaatgtt tcattctgga cagctggtaa aagtctgtgc cccaatggtt cgatattcaa 120
agttggcttt taggacacta gtaagaaaat atagttgtga tctgtgttac acaccaatga 180
ttgttgccgc tgattttgtc aaatctataa aagccagaga cagcgaattt accacaaatc 240
aaggtgattg cccattgatt gttcagtttg ctgctaacga tgcaagactt ttatctgatg 300
ctgctcgtat agtctgtcct tatgcgaatg gaatagacat taactgtggt tgccctcaga 360
ggtgggcaat ggcagaaggt tatggggctt gcttaataaa caagccagag cttgttcaag 420
acatggtgaa acaagtaaga aatcaagtgg aaacccctgg attttcagtt tctattaaaa 480
taaggatcca tgatgacctt aaaagaactg tagatctttg tcaaaaggct gaagcaacag 540
gagtttcatg gattacagtc catggaagaa ctgctgaaga aagacatcag ccagtgcact 600
atgattccat taaaataatt aaggaaaata tgtctatacc tgtaattgct aatggagaca 660
tcagaagctt aaaggaagca gaaaatgtgt ggcggattac tgggacagat ggtgtgatgg 720
ttgcaagagg actcttagca aacccggcca tgtttgctgg atatgaggaa accccactga 780
aatgcatctg ggactgggtt gacattgctc ttgaactcgg gactccttac atgtgtttcc 840
atcaacattt aatgtacatg atggaaaaga taacttcaag gcaggaaaaa agggtattta 900
atgctctgtc aagcacatca gcaatcatag attaccttac agaccattat ggcatttgac 960
tagacttccc aaataatttt aatatacact tttagaccca cagtgaaacc acagaaggtc 1020
atattttgta cc 1032
<210> 40
<211> 1797
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2683322
<400> 40
ttgtagtggg gctggagcag agcctgccgc gaacccccgg agcccacgat ccctcgtgcc 60
atccctcgaa tccaccagca cgagcgtccc acccgcgcct gggaccatgg ccactgactc 120
atgggccctg gcggtggacg agcaggaagc tgcggctgag tcgttgagca acttgcatct 180
taaggaagag aaaatcaaac cagataccaa tggtgctgtt gtcaagacca atgccaatgc 240
agagaagaca gatgaagaag agaaagagga cagagctgcc cagtccttac tcaacaagct 300
45/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
gatcagaagc aaccttgttg ataacacaaa ccaagtggaa gtcctgcagc gggatccaaa 360
ctcccctctg tactcggtga agtcttttga agagcttcgg ctgaaaccac agcttctcca 420
gggagtctat gccatgggct tcaatcgacc ctccaagata caagagaacg cattacccat 480
gatgcttgct gaacccccac agaatctgat tgcccagtct cagtctggca ctggtaaaac 540
agctgccttt gtcttagcca tgctcagccg agtggagcca tcagacagat acccccagtg 600
tctgtgcctc tccccaacat atgagctggc gcttcaaaca ggaaaagtga ttgagcagat 660
gggcaaattt tacccagaac tgaagcttgc ctatgccgtt cgaggcaata aattggaaag 720
aggccagaag atcagtgagc agattgtcat tggcacccct gggactgtgc tggactggtg 780
ctccaagctc aagttcattg atcccaagaa aatcaaggtg tttgttctgg atgaggctga 840
tgtcatgata gccactcagg gccaccaaga tcagagcatc cgcatccaga ggatgctgcc 900
caggaactgc cagatgctgc ttttctccgc cacctttgaa gactctgtgt ggaagtttgc 960
ccagaaagtg gtcccagacc caaacgttat caaactgaag cgtgaggaag agaccctgga 1020
caccatcaag cagtactatg tcctgtgcag cagcagagac gagaagttcc aggccttgtg 1080
taacctctac ggggccatca ccattgctca agccatgatc ttctgccata ctcgcaaaac 1140
agctagttgg ctggcagcag agctctcaaa agaaggccac caggtggctc tgctgagtgg 1200
ggagatgatg gtggagcaga gggctgcagt gattgagcgc ttccgagagg gcaaagagaa 1260
ggttttggtg accaccaacg tgtgtgcccg cggcattgat gttgaacaag tgtctgtcgt 1320
catcaacttt gatcttcccg tggacaagga cgggaatcct gacaatgaga cctacctgca 1380
ccggatcggg cgcacgggcc gctttggcaa gaggggcctg gcagtgaaca tggtggacag 1440
caagcacagc atgaacatcc tgaacagaat ccaggagcat tttaataaga agatagaaag 1500
attggacaca gatgatttgg acgagattga gaaaatagcc aactgagaag ctccaccagc 1560
cactgatgcc agccctggca ctgcccctgc acaggagaca agtgcgttca gggcacaggc 1620
cccgacatca ccccaaggac aacggcacaa gtagagagaa actacctacc tcacttcaaa 1680
ttatgtttgg acttgacaaa aatgtatgca aatgatgggg gatggtagaa aaaaattatt 1740
tacacaacct tggaagatta ggcatgaata cacagagatt tacctttaaa aaaaaaa 1797
<210> 41
<211> 1987
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2684552
<400> 41
tgacagagga ggctccgtgt ctgcagctag tgtgtcaact cagcgtttct cctctcgtcc 60
ctggaagagc taaagatggc tgaatttcta gatgaccagg aaactcgact gtgtgacaac 120
tgcaaaaaag aaattcctgt gtttaacttt accatccatg agatccactg tcaaaggaac 180
attggtatgt gtcctacctg taaggaacca tttcccaaat ctgacatgga gactcacatg 240
gctgcagaac actgtcaggt gacctgcaaa tgtaacaaga agttggagaa gaggctgtta 300
aagaagcatg aggagactga gtgccctttg cggcttgctg tctgccagca ctgtgattta 360
gaactttcca ttctcaaact gaaggaacat gaagattatt gtggtgcccg gacggaacta 420
tgtggcaact gtggtcgcaa tgtccttgtg aaagatctga agactcaccc tgaagtttgt 480
gggagagagg gggaggaaaa gagaaatgag gttgccatac ctcctaatgc atatgatgaa 540
tcttggggtc aggatggaat ctggattgca tcccaactcc tcagacaaat tgaagctctg 600
gacccaccca tgaggctgcc gcgaaggccc ctgagagcct ttgaatcaga tgttttccac 660
aatagaacta ccaaccaaag gaacattaca gcccaggttt caattcagaa taatctgttt 720
gaagaacaag agaggcagga aaggaataga ggccaacagc cccccaaaga gggtggtgaa 780
gagagtgcaa acttggactt catgttggcc ctaagtctgc aaaatgaagg ccaagcctcc 840
agtgtggcag agcaggactt ctggagggcc gtatgtgagg ccgaccagtc tcatggcggt 900
cccaggtctc tcagtgacat aaagggtgca gctgacgaga tcatgttgcc ttgtgaattt 960
tgtgaggagc tctacccaga ggaactgctg attgaccatc agacaagctg taacccttca 1020
cgtgccttac cttcactcaa tactggcagc tcttccccca gaggggtgga ggaacctgat 1080
gtcatcttcc agaacttctt gcaacaggct gcaagtaacc agttagactc tttgatgggc 1140
ctgagcaatt cacaccctgt ggaggagagc atcattatcc catgtgaatt ctgtggggta 1200
46/66
CA 02329685 2000-12-11
WO 99!64596 PCT/US99/13Z81 _
cagctggaag aggaggtgct gttccatcac caggaccagt gtgaccaacg cccagccact 1260
gcaaccaacc atgtgacaga ggggattcct agactggatt cccagcctca agagacctca 1320
ccagagctgc ccaggaggcg tgtcagacac cagggagacc tgtcttctgg ttacctggat 1380
gatactaagc aggaaacagc taatgggccc acctcctgtc tgcctcccag ccgacccatt 1440
aacaatatga cagctaccta taaccagcta tcgagatcaa catcaggccc cagacctggg 1500
tgccagccca gctctccttg tgtgccgaag ctcagcaact cagacagcca ggacatccag 1560
gggcggaatc gagacagcca gaatggggcc atagcccctg ggcacgtttc agtgattcgc 1620
cctcctcaaa atctctaccc agaaaacatt gtgccctctt tctcccctgg gccttcaggg 1680
agatacggag ctagtggtag gagtgaaggt ggcaggaatt cccgggtcac ccctgcagct 1740
gccaactacc gcagcagaac tgcaaaggca aagccttcca agcaacaggg agctggggat 1800
gcagaagagg aagaggagga gtaatggtgt ctccagagac tttaacaatc gggtcccggc 1860
ttctttgtga ccggaagaac cttgtcgctg ttgcagggcc acctctcttg gccctttggg 1920
gggggagagt tttttccaaa gttataatat tttccaaagg tatggcccat tgtgggctct 1980
ttaaggg 1987
<210> 42
<211> 2295
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 2228
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No.: 2830310
<400> 42
gcgggatagg taaacccgcc gcgcactggc gggacgctgg gccaacgagg tctggggtct 60
tggcgcggag ttcatgggcc gccggccagg cttgtgccag tggctttgga tccctggcct 120
gcttccccca acccagcctc cagaccagcg ctggcctgct ccacacactc cgcagcaccg 180
tcccgggaac cgagcgtcgg cggggaggcc gcctctccgc aataccccat gtttccccat 240
cactcgagga gtctgggcag agactggact acacgtggga gaatctgcaa aggtgttgct 300
ggaacagaca tatttctagt tgtatgaggt ggcctggaca ttactctcga gctccttacc 360
catacttcag tagtaggcat ttttcactaa attggagacc accctgtttg tttgagtcta 420
gaactcagtt ccagtactgt aactggagac ctgacaacct gagccagaca tctttgattc 480
atctctctag ttacgtcatg aacgctgagg gagatgagcc ttcatcaaaa cgaagaaaac 540
accaaggtgt gataaagcgg aattgggaat atatatgtag ccatgataaa gaaaaaacga 600
agatcctagg agacaaaaat gttgatccca aatgtgaaga cagtgagaac aagtttgact 660
tttcagtgat gtcctataat atactttcac aagatttact ggaagataac tctcaccttt 720
atagacattg ccggcggcca gtattacact ggagttttag gtttcccaat attctgaaag 780
aaattaaaca ttttgatgca gacgtacttt gtttgcaaga agttcaagaa gatcattatg 840
gagcagagat caggccaagt ttggaatcac tgggttatca ctgtgaatat aagatgcgga 900
caggaaggaa acctgatggc tgtgctattt gcttcaaaca ttccaaattt tcactcttgt 960
cagtgaaccc agtggaattc ttccgccctg atatttctct gttggacaga gacaatgttg 1020
gattagtttt actcttacag cccaaaattc catatgctgc ctgccctgca atctgcgtag 1080
caaatacgca tctgttgtat aatccaaggc gaggtgatat taagctgacg caattggcaa 1140
tgctactggc agagatttcc agtgttgccc accagaaaga tggcagcttc tgccctattg 1200
ttatgtgtgg tgactttaat tctgttcctg gttctccact atatagtttc ataaaggaag 1260
gaaaattgaa ttatgaagga cttcccatag gaaagacagt gatctgacac aaacacagct 1320
gaagcaaaca gaggtcctag tgacagctga aaaattgtct tcaaatttac agcaccattt 1380
cagtttgtca tctgtttatt cacattactt tcctgacact ggaattccag aagtgaccac 1440
ctgtcattcc cgaagtgcca taactgtgga ttatattttc tactctgcag aaaaggaaga 1500
47/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
tgttgctggg cacccaggag ctgaagttgc tttggttggt ggcttgaaac ttctagctag 1560
actgtcactt cttacagaac aagacttatg gactgttaat ggacttccaa acgaaaataa 1620
ctcttcagat catctgcctt tattggcaaa gttcagactt gagctctgac tctctttgat 1680
cacatactaa ttttctttcc aatttgtatt gtttttcaaa gaatgtaaag ttcttaagtg 1740
tatgcatgtt gtttattttt gcactgtgga gattctgaag cggttatgtt agatgctttg 1800
aaactccata tcagaagaaa taactttata acaatttttt ttaataatga aaaatatttt 1860
cgtgacaagt gagctctaaa ttctctttat tgtaaaagag atgtaaaggt tttatattct 1920
aaatcctagt aaaattgaca gtgattttta aatataatgc atcttccttt gtctgcttag 1980
taaaaaattt catttcataa ttttggcaag ctctgtagtg gatccaaagt atctttgagt 2040
tcttgcaaac tacaagttgt ttcctttcca gaaggcttga tttcattagg agacccctct 2100
attgagttct aaatagttta tcttagaaag ccttgggtca ttcacaggta tccaaccagc 2160
cattgtttag tttgtttttg aaggggtttg ataatgcttt ttaagttgta cagaatgctt 2220
aatccacnta ttactgtcct gagccagtaa tatgcctgca tcgtggtggg gaatgtttgg 2280
gaaatataag ccagc 2295
<210> 43
<211> 2819
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2963346
<400> 43
taggacttca acatggcggc tgcggcactg gcggtggcta cggtgacggc ctggcccgga 60
gcgggcagag ttggaggtgg tggcgttcgc tctccctagg ggctgtcggg agctcagcgg 120
ggaccgagcc tgggaggccg gccggtgcca gcacctttcg gcttctgaga cggcggcagc 180
agcggcattc aggttctaaa tggcttctaa gaagttgggt gcagattttc atgggacttt 240
cagttacctt gatgatgtcc catttaagac aggagacaaa ttcaaaacac cagctaaagt 300
tggtctacct attggcttct ccttgcctga ttgtttgcag gttgtcagag aagtacagta 360
tgacttctct ttggaaaaga aaaccattga gtgggctgaa gagattaaga aaatcgaaga 420
agccgagcgg gaagcagagt gcaaaattgc ggaagcagaa gctaaagtga attctaagag 480
tggcccagag ggcgatagca aaatgagctt ctccaagact cacagtacag ccacaatgcc 540
acctcctatt aaccccatcc tcgccagctt gcagcacaac agcatcctca caccaactcg 600
ggtcagcagt agtgccacga aacagaaagt tctcagccca cctcacataa aggcggattt 660
caatcttgct gactttgagt gtgaagaaga cccatttgat aatctggagt taaaaactat 720
tgatgagaag gaagagctga gaaatattct ggtaggaacc actggaccca ttatggctca 780
gttattggac aataacttgc ccaggggagg ctctgggtct gtgttacagg atgaggaggt 840
cctggcatcc ttggaacggg caaccctaga tttcaagcct cttcataaac ccaatggctt 900
tataacctta ccacagttgg gcaactgtga aaagatgtca ctgtcttcca aagtgtccct 960
cccccctata cctgcagtaa gcaatatcaa atccctgtct ttccccaaac ttgactctga 1020
tgacagcaat cagaagacag ccaagctggc gagcactttc catagcacat cctgcctccg 1080
caatggcacg ttccagaatt ccctaaagcc ttccacccaa agcagtgcca gtgagctcaa 1140
tgggcatcac actcttgggc tttcagcttt gaacttggac agtggcacag agatgccagc 1200
cctgacatcc tcccagatgc cttccctctc tgttttgtct gtgtgcacag aggaatcatc 1260
acctccaaat actggtccca cggtcacccc tcctaatttc tcagtgtcac aagtgcccaa 1320
catgcccagc tgtccccagg cctattctga actgcagatg ctgtccccca gcgagcggca 1380
gtgtgtggag acggtggtca acatgggcta ctcgtacgag tgtgtcctca gagccatgaa 1440
gaagaaagga gagaatattg agcagattct cgactatctc tttgcacatg gacagctttg 1500
tgagaagggc ttcgaccctc ttttagtgga agaggctctg gaaatgcacc agtgttcaga 1560
agaaaagatg atggagtttc ttcagttaat gagcaaattt aaggagatgg gctttgagct 1620
gaaagacatt aaggaagttt tgctattaca caacaatgac caggacaatg ctttggaaga 1680
cctcatggct cgggcaggag ccagctgaga ccaggccctg cctaggccct gccgcagaac 1740
caccatccct gggaggccct gcagagccca cctgtgggga aagagaaggg gcagcttccg 1800
gattttcttt tgggggttag aaggtcaggt gtggagactg ctcgccagtc tctgtgagcc 1860
48/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
taggccctga gctggggagg tggggaagat tcgggcatgt gagtgccccc agaactgtcc 1920
tggctccttc cgtattaaac gcatttgcat tttgagaagt gtccttccca cttcagccct 1980
ccggagagac taccctagtc tttctggggt gtttatgtcc tcagctgaag cctggcctag 2040
ttgctgagag gggctgggga gatggggcgg gagggccaga ctcagtgctg ctgtggagct 2100
aggtgcttcc cccttcccct gagactggtg gactgaactc cagtcaagtt gagttcaagt 2160
gaaagattct tccagggttt tattttttcc cctcctaaca aagtctcata gtgttaacac 2220
tggttctgca atatctctga ggtgcaaaga atgcactttt ccctatgggg cccagagttt 2280
gccttttctg ccaggcagtc accacgcttc cctaccccag cctgtttctt ttggcttggt 2340
ttggaccaca gtcctctgct acccagggtt ttagagcccc tgctctagga aacagtttaa 2400
gaaatcattg gccccttccc agcacattga atgggtaagc agacaggcca tgatttagtt 2460
ggccagcact aactccacct ctgttctcct tgaacagctt cccctccagc ccactgcttt 2520
aggatgacac aatgaataac acctagtcat agaaatcagt ctctctggtt tgttttgtat 2580
tatgttgtac atcattaaag atctaaatac aaaggatata cagtcttgaa tctaaaataa 2640
tttgctaact aactattttg attcttcaga gagaactact aataaaaatc taaaaggtaa 2700
aaaaaaaaaa aaaaaaaaaa gggcggcaga tctagaggaa ccaagcttaa gtaagcgagc 2760
atgcgacatc atagatcatc tatagtagtc acctaaattc aatttcagtg ccaacaaag 2819
<210> 44
<211> 1459
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2994234
<400> 44
cttcttgaga ccgaggaccg aaatcccggc tccaggcctc ggggactgcg gactgtgggg 60
aggctggccg gagagagagg gaaggacggg gcctggcccc cgggactccc tgtgccttgc 120
ttggagctga cgccgacggt ttattgcagg gaactgacaa gatcacactt tgagaagaag 180
ttggaaagaa tcccaagtgg atgaactgaa tatctggatg aggacaagat ctgtggggag 240
agactgtaag atagaatgag tccatttaag tcccaggacg gtggaaacta gctagtagat 300
tgcagccatg ttgtggaagc tgctgctgag atcccagtcc tgcaggctgt gttctttcag 360
aaagatgcga tcacctccaa aatacagacc tttcttagca tgcttcacct atacaactga 420
taaacagtcg agcaaagaaa atacaagaac agtggaaaag ctctataaat gttcagttga 480
cattaggaaa attcgtagat taaaaggatg ggtactttta gaggatgaaa cctatgttga 540
agaaattgcg aatattttac aagaactagg tgccgatgag actgctgtag ccagtatttt 600
ggaacgctgc ccggaagcaa ttgtctgtag tccaaccgct gttaacaccc agagaaaact 660
ctggcagttg gtctgcaaaa atgaggaaga gttaatcaag ttaatagagc agtttccaga 720
atctttcttt actattaaag accaagagaa ccagaagctg aatgttcagt tctttcaaga 780
gttgggacta aaaaatgtgg tcattagcag acttttgaca gctgcaccta atgtttttca 840
taatcctgtt gagaagaata agcaaatggt aagaattctc caagagagtt atctagatgt 900
aggtggctct gaggccaaca tgaaagtttg gctactaaaa ttgttaagcc aaaacccatt 960
tattttgtta aattctccca cagctataaa ggaaacacta gaatttctcc aggagcaagg 1020
tttcaccagc tttgaaattc tccagcttct atccaaactc aaaggatttc tttttcaact 1080
ttgcccaaga agtatacaga atagtatttc cttctctaaa aatgctttta aatgcacaga 1140
tcatgacctg aagcaattag ttttgaaatg tcctgccctt ttatattatt ctgttccagt 1200
tttagaagag agaatgcaag gattattgag agaaggaatt tccatagctc agataagaga 1260
gacgccaatg gttcttgaat taacaccaca gatagtacag tacaggataa ggaaactgaa 1320
ttcctcaggc tacagaataa aggatggaca tctagcaaat ctaaatggat caaaaaaaga 1380
gtttgaagct aattttggca aaattcaggc caaaaaaagt aaggccatta tttaaccctg 1440
tggcaccatt aaatgttga 1459
<210> 45
<211> 2733
49!66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 61, 63, 69, 92, 100, 151, 165, I79, 182, 198, 218
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No.: 4115958
<400> 45
ccggaaaggg ggaaaacctt tgggaaattt cccaaaaggg ccttttttaa atccccccaa 60
nangccccnt ttttggggag gaggacctta ancccctttn gaataggggc tcttttaacc 120
cccaagggac ccggccaaag attgtttttt nccaagggat ttgcnccagc catttgggng 180
cnccattgat cccaaccngc atcacatatc cccccggncg gtcattcaac atgcatgact 240
ccagttcggt tgcatctaaa gtgtttagga gttcatatga agacaaaaat ttgttgaaga 300
aaaataagga tgagtcctca gtttccattt ctcacacaaa atgttccttg ttaggagaca 360
tcagtgatgg gaaaaactta atacctaata aatgttttac ttcttttaaa aataatagta 420
aagaaaagtg ttctttgaaa catcaaacaa gaaatcagtg tcagaacaat cctagtgaaa 480
tcatccaaag tacgtatcag gagacacaga acaaaagttc tagtttatca acttcctcaa 540
ttttgtctca gcacaaagaa aataacttag atttgacaag cagattcaag gagcaagaaa 600
tgagcaatgg aattgataaa cagtattcaa attgcaccac tattgacaaa cagatttgta 660
caaataagta taaggaaaaa ataataaatg agaactataa tccaaaattc tttggcaatc 720
ttcagtctga tgattccaaa aaaaatgact caaaaataaa agttactgtg ttggaaatgt 780
ctgaatattt gaacaaatat gaaagcatgt cctcaaataa agactcaaaa aggcctaaga 840
catgtgagca aaatactcaa cttaatagca tagagaatta tctcaataaa gataatgaag 900
gtttcaaatg taaaaagtca gaccaattaa aaaatgaaca agataagcaa gaagatccaa 960
ctaatgaaaa atcccaaaac tattctcaga gaagaagtat aaaagactgt ttgtctacat 1020
gtgagcaacc aaaaaataca gaggtattga ggactacact gaaacattca aatgtgtggc 1080
gaaaacataa ttttcattcc ttggatggaa cttcaaccag agcctttcat cctcaaactg 1140
gattgcctct tctttcaagc cctgttcctc aaagaaaaac acaatcaggt tgctttgatc 1200
tggattcttc attactacat ctgaaaagct tctcatctag aaggaatctg tcttgaacta 1260
tcgtttcgat cctctcggca ttgttgatgg ttttactgcc gaggtagggg caagtggtgc 1320
tttctgcccc acacatttga ctcttccagt tgaagtgtca ttctacagtg tttcagatga 1380
caatgctccc tctccttata tgggtgtgat tactttagag tcccttggta aaaggggtta 1440
tcgagtacct ccttcaggaa caatacaagt ggtatgtgtt ttatagccta caattgcaat 1500
aatcattctc tcacatacat atagaactta gccctttctc ctgctactgc tgctgcaaca 1560
actttgacct tatttaatcc taataagact gtggtgaaga tgtttgttgt gatatatgat 1620
ttacgagata tgccagccaa tcatcagaca ttcctacgac aaagaacttt ttctgtacct 1680
gttaaacaag aagtgaagag aagtgttaat aaagagaaca tccgacacac agaagaacgg 1740
ttattacgct acctcataca tctgaggttc cagagttcta aatctggaaa gatctacctc 1800
catagagacg tacggctcct gttctctaga aagtcaatgg aggttgatag cggtgctgca 1860
tatgaactca aatcttacac tgaatcacca acaaaccctc agttttcacc aagatgttga 1920
taaggagtga tgatttaaag tatttactca gtacccaagt ttgcaagtaa aaattagcat 1980
agaatggagt gtaccaaatt aacaatcagg agagtggatt ctctcctgtt atcctggacc 2040
agtttttatg aaaggattcc tgaaatgaaa tccatatatt ccatgtagac tggaaaaact 2100
catgtcctaa tcctttttgt actgttgaaa ccacttcatt ggacatgttg caatagcaaa 2160
acccccagtt agattagtgt ttacacattt tctcagttat ttaatattta atgttttcct 2220
taatactcaa gtgatgtttg tctctagtgt tctaatgtag cacaaatcct atgtaaaatc 2280
atactatgta tttttgacat taatgttgaa atcaaatata tgcacaagtc tttaattttg 2340
tgtaatgtgt taagtgctgt tcatttaagt tattgaaaat gagaataaaa tgttgagctt 2400
ctttaaaagt aacacactat gcaagcatgt gtacttttta tatctctcat gtttagtttt 2460
tataacacca tatccaggtt gctatctcac atagtagtcc tttaacatat tgtattagca 2520
gtgcaatgtg gactaagctg cttcactttc cctttgcaag ttcagatcat catgcccatt 2580
catagccagg attccttatc cccaaaacag ttctattttt ccttaatcac tactatagag 2640
50/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/1328I
tctttacatt aaattactgt cgtatgctag ataattttct caaattgtta aaagaatatg 2700
tactttggaa acaaattagt atttatattg tga 2733
<210> 46
<211> 2177
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 779255
<400> 46
attgctccaa acgacgccaa cttctactac ctggcccttc acatccgtat acactcaggg 60
gctaagccct agagttgcca cttctgtgaa aaatccttcc gccagctctc ccaccttcag 120
cagcacaccc ggatccactc caagatgcac acggagacca tcaagcccca caagtgcccg 180
cactgctcca agaccttcgc caacacctcc tacctggccc agcacctccg tatccactcg 240
ggggccaagc cctacaactg ttcctactgc cagaaggcct tccgccagct ctcccacctt 300
cagcagcaca cacgaatcca cactggtgat agaccataca aatgtgcaca cccaggctgt 360
gagaaagcct tcacacaact ctccaatctg cagtcccaca gacggcaaca caacaaagat 420
aaacccttca agtgccacaa ctgtcatcgg gcgtacacgg atgcagcctc actagaggtg 480
cacctgtcta cgcacacagt gaagcatgcc aaggtgtaca cctgcactat ctgcagtcgg 540
gcatacacat cagaaacata ccttatgaaa catatgcgca aacacaaccc gcctgatctt 600
cagcaacagg tgcaggcagc agcagcagcg gcagcagtgg cccaggccca ggctcaagct 660
caagcccagg ctcaggctca ggctcaagcc caggcccagg cccaggcctc ccaggcatca 720
cagcagcagc agcagcagca gcagcagcag cagcagcagc agcaacagcc accaccacac 780
ttccagtctc ctggggcagc cccccagggt gggggtggtg gggacagcaa tcccaaccct 840
ccaccccagt gttcctttga cctgaccccg tataagacgg cggagcatca taaggacatc 900
tgcctcactg tcaccaccag~caccatccag gtggagcacc tggccagctc ttagagatcc 960
gtgctgccac ccactgggaa gaggaagaag tagtcctggt gtcttctttc tccaactctt 1020
ggtgggaaaa gtccttttct tccttgacag gccttggctc catctccttg ggcctctgtc 1080
acggctttcc ttcacaggat accatccttt ttctgaactc ttcttcaaaa ggaacatcag 1140
ccctcctgat tgcaaaggaa tactgagctg atggtgtcat ccagcagcct cccctcccaa 1200
gcaaagcttc taaaactggg ggtcggtgct caagggaagg atttgctatg acctcataga 1260
accttgtcca gtgtggccac ttaccctatc cttaccctcc ttatcctcaa agtttgggct 1320
gatgtaagac tagaggctgg ccctcccaga taacagagaa aagggagccc caaatgcaac 1380
cagcctcttg ttctattctt gcctgcaaaa gaacagaggt ttctcaaatg cctcagtccc 1440
tgagagccat ttcttcccct acatcgtctc actttgcttc ctattgactg ctggtagaag 1500
gagatttggg gtaggggcta gacctccttt tatttgaagg gggcaagggc tgagatgtgg 1560
tccccaaggg gccagaaatt cccaagttgg tcacaggtgg cttagaagtg tgtgttatgg 1620
ttttacggat ttccttgaag cctctctcct tctctgccta caaagaccct atactctcag 1680
tctccccaac ccacccccaa ggagctgtgg gaggctttgt gttatctgtg aaactccaaa 1740
acaggggtgt tgcggagaag ggagagttca aggcaaacgc aaggactgga cttagctccc 1800
taggtgccac agtcagatgc cggacacgga tttatatata aatatatata tataaatata 1860
ttatacccac tcatcacggc catctttgtt gtaaccattt ctgtgtttat aaatgcatta 1920
tctctgagaa ttttcatatt tgatgttttg tttatttttg tccttttttt ccctctctcc 1980
acccctgtcc tctagccaca gcatttttct ttttgtcttt tttttttttt tttaaatcat 2040
ggcagatttc agaggaaagg aaattaaaaa aaaaatcagg aaaccagttg ttataaagta 2100
atttaaaaat gaagaaaaaa agaaaaaaac ttatgtacaa accaaggggt tttttagaac 2160
attgtataga aatagag 2177
<210> 47
<211> 2685
<212> DNA
<213> Homo Sapiens
51 /66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281
<220>
<221> misc_feature
<223> Incyte Clone No.: 1303605
<400> 47
gctctgtttg acttcatgca gaatttttaa agtttttagg tttcttgaaa atgtaatttc 60
atgaactctt ctaaggctac tgtaactgaa ttccaaccca cagatatgtt acacacgatt 120
ggcatatctt tttcttgcac atcaaggagg ccctcttggc aggccacctc ttgtgttccc 180
cagagcaggc agtggaactc agtgccctcc tggcccagac caagtttgga gactacaacc 240
agaacactgc caagtataac tatgaggagc tctgtgccaa ggagctctcc tctgccacct 300
tgaacagcat tgttgcaaaa cataaggagt tggaggggac cagccaggct tcagctgaat 360
accaagtttt gcagattgtg tcggcaatgg aaaactatgg catagaatgg cattctgtgc 420
gggatagcga agggcagaaa ctgctcattg gggttggacc tgaaggaatc tcaatttgta 480
aagatgactt tagcccaatt aataggatag cttatcctgt ggtgcagatg gccacccagt 540
caggaaagaa tgtatatttg acggtcacca aggaatctgg gaacagcatc gtgctcttgt 600
ttaaaatgat cagcaccagg gcggccagcg ggctctaccg agcgataaca gagacgcacg 660
cattctacag gtgtgacaca gtgaccagcg ccgtgatgat gcagtatagc cgtgacttga 720
agggccactt ggcatctctg tttctgaatg aaaacattaa ccttggcaag aaatatgtct 780
ttgatattaa aagaacatca aaggaggtgt atgaccatgc caggagggct ctgtacaatg 840
ctggcgttgt ggacctcgtt tcaagaagca accagagccc ttcacactcg cctctgaagt 900
cctcagaaag cagcatgaac tgcagcagct gcgagggcct cagctgccag cagacccggg 960
tgctgcagga gaagctacgc aagctgaagg aagccatgct gtgcatggtg tgctgcgagg 1020
aggagatcaa ctccaccttc tgtccctgtg gccacactgt gtgctgtgag agctgcgccg 1080
cccagctaca gtcatgtccc gtctgcaggt cgcgtgtgga gcatgtccag cacgtctatc 1140
tgccaacgca caccagtctt ctcaatctga ctgtaatcta atctgttgtg cttttgttgg 1200
acttggcatg tttccatgaa ctgcactatt ataaactatt aaaatgatag attgtggaga 1260
aagtaattat tccaacaccc atctgccatg cgatgttaaa aaaaaaaaaa aggaagaaaa 1320
ataacacagc tactcctcac tgcaaaaaca tatccatgcg tagaatcaac aactccagtc 1380
atgggaccag gaggagctct gggacgcaga cacattcctt ggatgttgat tttttttatg 1440
atctagtaaa ggaataggta aagtctttga tgtcagtgaa gtggcaacat agccaaaaag 1500
ttgggtacct tttaggaaat gatgttgtaa gtctccttaa tgtatcctga ggtaagtttc 1560
ctactggcag cagattttgt aagaattact tttaagaatt tcattctttt tgtatggtca 1620
tggagctcca accattttta ataggaaagt cttttgtaaa ttgttgtcgt tttaatgtca 1680
tttctgtctt tataacttga tcaagaatga ttggaaggca aacaggttta caaatcaatt 1740
ctgtgacttt taaaaagttg acaatgttgt cagatttaaa ccagtgtggc tagtaaaaag 1800
cagctcactc aatgtgggtg gctccctatt cctttacgct ccccctatcc ctaccccaca 1860
agcctttcga ttataaaata ctaccaatct tgttataaga ttactgtgga gtagtcaagt 1920
actccccggg ccttctgagc tggtggaata ttttatttca gactgaaaac agagagcact 1980
ctccttggga agggaaagcg gagcttgctg agtgagagat ggagcctcat ggtgtacaac 2040
tgagggtagt taactcatca cttctcccaa gcactcgatc ccagcttcac ccactggtgt 2100
tgctttgctt gaactgttca agccttttat agccttacca taagtattta gatatggtgt 2160
ccttttctgt ttttgggggg ggagttttgt tgtgtttttt taaagtaagt gcttaagtat 2220
taactttggg ttgtcccctc tgtatgtttc gaaggggttt tggttctttt tgcttctgtt 2280
ttcttaaaca tgttttccac tcccacttgg gcattttgga agctggtcag ctagcaggtt 2340
ttctgggatg tcgggagacc tagatgacct tatcgggtgc aatactagct aaggtaaagc 2400
tagaaaccta cactgtcact ttactgagat ttctgagtat acttttcata ttgccttaat 2460
gtagcagtaa tgtgtttatg catttgtttc tttgcacaga cattttgtca aatattaaaa 2520
ctctactttt ttatggcaca tattagcata taagccttta ttccaagagg tatttatttt 2580
ttcacttgta aaaaaataat gtttccacgt aaagaactct gttatatcct agaggactct 2640
gtcttttata ttcgggataa taaagacttt aaagcaaaaa aaaaa 2685
<210> 48
<211> 2408
<212> DNA
<213> Homo Sapiens
52/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
<220>
<221> misc_feature
<223> Incyte Clone No.: 1611167
<400> 48
ggctgcatcc cggccggcgg ttccggagcc tcgcgtctgg ggaggcgcgg gccggcacgc 60
tcgagcttag ggagatctgc cttctggaga ctgcgccgtc ctcccgggag agccagaaag 120
aggacatggc tgctgggcag cgggaagcga ggccccaggt gtcactgacg tttgaggatg 180
tggctgtgct gtttacccga gatgagtgga gaaagctggc cccttctcag agaaacttgt 240
accgggatgt gatgctggag aactatagga acctggtctc actggggctc ccatttacca 300
aaccaaaagt gatctccctg ttgcagcaag gagaagatcc ctgggaggtg gagaaagacg 360
gttctggcgt ctcctctcta ggatcgaaga gcagtcataa aaccacaaag tcaacgcaaa 420
cacaagactc ttcatttcag ggactgatac tgaaaagatc caacaggaat gtaccttggg 480
atttgaaatt agaaaagcct tacatatatg aaggcagatt agagaaaaag caggataaaa 540
agggaagttt tcagatagtt tcagccaccc acaaaaaaat ccccactata gaaagaagcc 600
ataaaaatac tgaattgagc caaaacttca gcccaaagtc agtgcttatt aggcaacaga 660
tacttcccag agaaaaaaca ccaccaaaat gtgaaataca aggaaacagc ctcaaacaga 720
attcacaatt acttaatcaa ccaaaaatta cagcagataa acgctataaa tgtagtctgt 780
gtgaaaaaac cttcattaac acttcatccc ttcgtaaaca tgagaaaaac catagtggag 840
agaaactatt taagtgtaaa gaatgttcaa aagcctttag ccaaagttca gctcttattc 900
aacatcaaat aacacatact ggagagaaac cctacatatg taaagaatgt gggaaagcct 960
ttactctcag tacatccctt tataaacatc taagaaccca tactgtggag aaatcctaca 1020
gatgtaaaga atgtggtaaa tccttcagcc gaaggtcagg cctttttata catcaaaaaa 1080
ttcatgctga agaaaaccct tgtaagtata atccgggtag gaaggcatct agttgcagca 1140
catccctttc tggatgtcaa agaattcatt ctagaaagaa gtcctactta tgtaatgaat 1200
gtggcaacac ctttaagtct agctcatccc ttcgttatca tcagagaat.t cacactggag 1260
agaagccttt taaatgtagt gaatgtggga gagccttcag ccagagtgcc tctcttattc 1320
aacatgaaag aattcacacc ggagaaaagc cctatagatg caatgaatgt gggaaaggct 1380
ttacttctat ttcacgactt aatagacacc gaatcattca tactggagag aagttttata 1440
attgtaatga atgtggtaaa gccttaagct cccactcaac acttattatt cacgagcgaa 1500
ttcatactgg agaaaaacca tgtaaatgta aagtatgtgg aaaagccttc agacagagtt 1560
cagctctcat tcaacatcag agaatgcata ctggagaaag accctataaa tgtaacgagt 1620
gtgggaaaac attcaggtgt aactcatcac ttagtaatca ccagagaatt catactggag 1680
agaaaccata tcgatgtgag gaatgtggga tatcttttgg ccaaagttca gctcttattc 1740
agcatcgaag gattcataca ggagaaaaac cctttaaatg taatacatgt ggaaaaactt 1800
ttagacaaag ctcatcacgt attgcacatc agagaattca tactggagag aaaccctatg 1860
aatgtaatac atgtgggaaa cttttcaacc ataggtcatc ccttactaat cattataaaa 1920
ttcatatcga agaggacccc tagaaagtag atttgtatgt gtgaaagcct taaaccaaag 1980
ctcatcgaag aatacatcct tgagagagat gtaataaatg taatggatgt gaaaaaaact 2040
gtaataattt agccctcatt aggtatttaa ttccatggat aaacctcagc tatataatag 2100
ataatgagga aagtgtttgt gcctgtcaga cacttaaaaa aataacctga gatgaagaat 2160
ttacaattga agacattgac tttagccatt tgtgaaatgg gtttgctttt tcccttttcc 2220
tacagacgta tatgctagat gtcacgtgat catcagaaac agatatccga gtgggtgggg 2280
aggtgtgctt tagatttctc attagaagac caccaaactg gtaatatttt tatagcattt 2340
taatagcata caaatgaatt gatcaaattg taccttttta gagaaaagga ccaaaataaa 2400
agaaaaat 2408
<210> 49
<211> 2990
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1907472
53/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
<400> 49
gtacaacttt tgcaattcca gttgtgtggc taaatttcag gctctaagta tgcagtcatc 60
tccaaatggc cagtttgtag cgccaagtga tattcagttg aaatgcaact actgcaaaaa 120
ttccttttgt tcaaaaccag aaatcctgga atgggagaac aaagtgcatc agttctgcag 180
caaaacttgt tcagatgact ataagaagtt gcattgcata gttacatatt gcgaatactg 240
tcaagaggag aagactcttc atgaaacagt aaatttctct ggcgttaaga gacctttctg 300
tagtgaaggc tgcaaattat tatacaaaca ggattttgcc agacgtttag gattgagatg 360
tgttacttgc aactattgtt ctcagctatg taagaaggga gcaactaaag aactcgatgg 420
tgttgtgaga gatttctgca gtgaagattg ctgtaaaaaa tttcaggatt ggtactacaa 480
ggctgcaagg tgtgactgtt gtaaatctca aggaactctt aaagagcgag ttcagtggcg 540
tggggaaatg aaacatttct gtgatcaaca ttgcttactg cgtttctact gtcaacaaaa 600
tgagcccaac atgacaactc agaaaggacc tgaaaactta cattatgatc agggttgtca 660
gacatctcga accaaaatga caggttcagc accaccccct tctccaacac ctaacaaaga 720
gatgaagaac aaagcagttc tttgcaaacc tttaacaatg acaaaagcta cttactgtaa 780
acctcacatg cagaccaaat cttgtcagac agatgatact tggaggacag aatatgttcc 840
agtgcctatc cctgtgcctg tgtatatccc agttcctatg cacatgtaca gtcagaatat 900
tcctgttcct actacagttc ctgttcctgt gccagttcct gtttttctgc ctgctccatt 960
ggacagcagt gagaagattc ctgcagcaat tgaggagcta aaaagcaagg tttcttcaga 1020
tgctcttgat acagagttgc ttacaatgac ggatatgatg agtgaagacg aggggaaaac 1080
agagacaacc aacatcaaca gtgtaattat tgaaacagat ataattggtt cagacctttt 1140
gaagaactct gacccagaga cacagtccag catgcctgat gtaccatatg aaccagattt 1200
ggatatcgaa atagattttc ccagagctgc tgaggagctt gatatggaaa atgaattttt 1260
attaccacct gtttttggcg aagaatatga ggaacagccc agacctcgat ctaaaaaaaa 1320
gggagccaag agaaaggctg tatcaggata ccagtctcat gatgatagtt ctgacaattc 1380
agaatgcagc tttcctttca aatatacgta tggcgtaaat gcatggaaac actgggtcaa 1440
aactaggcaa cttgatgaag atcttctggt attagatgag ttaaaatctt ctaaatcagt 1500
aaagttaaaa gaggatctac tctctcacac cacagctgag cttaactatg ggttagctca 1560
ttttgtcaat gagatccgac ggccaaatgg agagaattat gcacctgaca gcatctatta 1620
cctttgcctt ggaatacagg agtatttgtg tggaagtaat cgaaaagaca acatatttat 1680
tgatcctgga taccaaacat ttgagcaaga attgaataaa atactgcgaa gctggcaacc 1740
aagcatactt ccagatgggt caatattctc tcgagttgaa gaagactatc tctggaggat 1800
aaaacaacta ggatcacact ctccagtagc tcttctgaat acactgttct actttaacac 1860
taagtatttt ggcctgaaaa cagtggaaca acacttaaga ctttcctttg gcactgtgtt 1920
taggcattgg aaaaaaaatc ctttaacgat ggaaaacaaa gcgtgtcttc gataccaagt 1980
gtcttccttg tgtggaacag ataatgaaga taaaattact actggaaaaa gaaaacatga 2040
agatgatgag ccagtatttg aacaaattga aaacacagcc aatccttcca gatgtcctgt 2100
gaaaatgttt gaatgctact tgtctaaaag tccacagaat cttaatcaga ggatggatgt 2160
tttttatttg caaccagaat gctctagttc tacagatagc cctgtctggt atacgtctac 2220
ttcactggac cgaaacacct tggaaaatat gcttgtacgg gttcttctag taaaagatat 2280
ttatgataaa gacaattatg aactggatga agacacagac taaaaaggaa cgttgcagaa 2340
gcaatcggga taaaacagca ttagatagtc atgctgctag atctttatta tggaaaacat 2400
ttcaagttta ctccttctgt tttgagtttt gtagcagtgt acccacgctg ggtattacca 2460
tgtaaataat ctgtgagtga aagttgccat tattctatgt agtggtttta ggatacttaa 2520
caaatacatt caaattcttt ttttattatt atttatttga ttaggtatgt ttgtaacttt 2580
ttacattaca gaatatgaat gagaatgtgc catgtataat ttttttcttg tagtaagaaa 2640
catccatatt gcacaactct actgttgcaa agcttccttg gaagggggct cttttactgg 2700
gttcttaacc agatggttgt gtatgggtag cactactaaa agtttagaac ttgcagtgtc 2760
tttcggaatt tttaaaataa actgtaaact aataggctgg ggtttttgtt ttgttttggg 2820
gttttgtttt gtttggtttt acattttagt tactgaagcc ttacaaggtt atgtagagag 2880
ataccatctt ctgtaccaaa aatagacaag agaatgctgt caatattggt gtactgtaat 2940
gtgaatctat gctggtgaaa acaatttttt ttccccttat taaaacctta 2990
<210> 50
<211> 771
<212> DNA
<213> Homo Sapiens
54/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
<220>
<221> misc_feature
<223> Incyte Clone No.: 1985458
<400> 50
cccctactaa agccttgggg ttagtacgcg tgcgcacagt ttcttccgac agttgtgttg 60
tgccaatggt ggagaagaaa acttcggttc gctcccagga ccccgggcag cggcgggtgc 120
tggaccgggc tgcccggcag cgtcgcatca accggcagct ggaggccctg gagaatgaca 180
acttccagga tgacccccac gcgggactcc ctcagctcgg caagagactg cctcagtttg 240
atgacgatgc ggacactgga aagaaaaaga agaaaacccg aggtgatcat tttaaacttc 300
gcttccgaaa aaactttcag gccctgttgg aggagcagaa cttgagtgtg gccgagggcc 360
ctaactacct gacggcctgt gcgggacccc catcgcggcc ccagcgcccc ttctgtgctg 420
tctgtggctt cccatccccc tacacctgtg tcagctgcgg tgcccggtac tgcactgtgc 480
gctgtctggg gacccaccag gagaccaggt gtctgaagtg gactgtgtga gcctgggcat 540
tcccagagag gaagggccgc tgtgcactgc ccggccttca gaaagacaga atttcatcac 600
ccaatgcagg gggagctctt cctggaccaa gggaggagcc gctcattcac ccaacaaaac 660
tgtgtcttat ctgccaggaa agaccagcct cactcctggg aactgtctgg caggtaggct 720
gggcccccca gtgctgttag aataaaaagc ctcgtgccgg aaaaaaaaaa a 771
<210> 51
<211> 2076
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 1957
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No.: 2726431
<400> 51
cccgcctgcc cctagcggtc cctggcgtcc cggttcctgt cgcgctcacc tgcgccggta 60
gcgaagaaat cgccccggga catggactca gtggtctttg aggatgtggc tgtggacttc 120
accctggagg agtgggcttt gctggattct gctcagaggg acctctacag agatgtgatg 180
ctggagacct ttcagaacct ggcctcagta ggaaaaattt gggacagtct tagcatcgaa 240
gatcaaacca caaaccaggg gagaaatctc agtagaaatc atgggttgga gagactctgt 300
gaaagtaatg atcaatgtgg agaagccctc agccagattc cacatcttaa tctgtacaag 360
aaaattccac ctggagtaaa acagtatgaa tacaacacgt acggaaaagt cttcatgcat 420
cgccgcacat ccctcaagag tcccatcaca gttcacactg gacacaaacc atatcagtgc 480
caggaatgtg ggcaggccta cagttgtcgt tcacacctaa gaatgcatgt gagaacccac 540
aatggagaga gaccctatgt gtgtaaatta tgtgggaaaa cctttcctcg tacttcctcc 600
ctcaatcggc atgtaaggat tcacactgct gagaaaactt acgaatgtaa gcaatgtggg 660
aaagccttta ttgacttctc aagtcttact agtcatctca gaagtcacac cggagagaag 720
ccatataagt gtaaggaatg tgggaaagct ttcagttatt cctcaacgtt tcgaagacac 780
acaataacac acactggcga gaagccatat aaatgtaagg aatgtgcgga agcctttagt 840
tattcctcaa cttttcgaag acatatgatt tcacacactg gagagaagcc acataaatgt 900
aaagaatgtg gggaggcctt cagttattct tcggcttttc gaagacacat gataacacac 960
actggagaga aaccctacga atgcaaacaa tgtgggaaaa ccttcattta tctccagtcc 1020
tttcgaagac atgaaaggat tcacactgga gagaaaccct acgaatgcaa acagtgtggg 1080
aagaccttca tttatcccca gtcctttcga agacatgaaa ggactcatgg tggagagaaa 1140
ccctatgaat gcaaccagtg cgggaaagca ttcagtcacc cctcctcctt tcgaggacac 1200
atgagggtgc acactggaga gaaaccctat gagtgcaagc aatgtgggaa aactttcaat 1260
tggcccatat ctttacgaaa acatatgaga acacatacta gagagaaacc ctatgaatgt 1320
55/66
CA 02329685 2000-12-11
WO 99/64596 PCTIUS99/13281 _
aagcagtgtg ggaaagcctt cagcttgtct gcttgctttc gagaacatgt gagaatgcac 1380
cctgaagaca aatcctatga atgcaagcta tgtgggaaag ctttctattg ccacatatcc 1440
ttacaaaaac atatgagaag gcataccgca gagaaactct ataaatgcaa gcagtgtggg 1500
aaagctttca gttggcctga acttttgcaa caacatgtga gaacgcacac tgtagagaag 1560
ccctatgaat gtaaggaatg tgggaaggtc ttcaaatggc catcatcttt accaatacat 1620
atgagactgc acactggaga gaaaccttat caatgtaagc attgtgggaa agcattcaat 1680
tgttcctcat ccttaaggcg acatgtgaga atacacacta cagaaaaaca gtataagtgt 1740
aatgtaggac atcctcctgc aaatgaattc atgtgcagtg cttcagaaaa gtcacaccag 1800
gagagagatc tgatcaaagt tgtaaatatg gtgttgcctt tatgagttcc ttatcctgaa 1860
agtggacact caaggagtgt gtctgtagtt catttgcata gaaactatag cgaagaggcc 1920
cgcaccgatc gcccttccca acagttgcgc agctganatg gcgaatggga cgcgccctgt 1980
agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgaccgcta cacttgccag 2040
cgccctagcg cccgctcctt tcgctttctt cccttc 2076
<210> 52
<211> 1197
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2743828
<400> 52
cccgggtccc cttctccagg caggaagatg tccaagcccc gcgcggtgga ggcggcggcg 60
gcggcggcgg cggtggcagc gacggccccg ggcccggaga tggtggagcg gaggggcccg 120
gggaggcccc gcaccgacgg ggagaacgta tttaccgggc agtcaaagat ctattcctac 180
atgagcccga acaaatgctc tggaatgcgt ttcccccttc aggaagagaa ctcagttaca 240
catcacgaag tcaaatgcca ggggaaacca ttagccggaa tctacaggaa acgagaagag 300
aaaagaaatg ctgggaacgc agtacggagc gccatgaagt ccgaggaaca gaagatcaaa 360
gacgccagga aaggtcccct ggtacctttt ccaaaccaaa aatctgaagc agcagaacct 420
ccaaaaactc caccctcatc ttgtgattcc accaatgcag ccatcgccaa gcaagccctg 480
aaaaagccca tcaagggcaa acaggccccc cgaaaaaaag ctcaaggaaa aacgcaacag 540
aatcgcaaac ttacggattt ctaccctgtc cgaaggagct ccaggaagag caaagccgag 600
ctgcagtctg aagaaaggaa aagaatagat gaattgattg aaagtgggaa ggaagaagga 660
atgaagattg acctcatcga tggcaaaggc aggggtgtga ttgccaccaa gcagttctcc 720
cggggtgact ttgtggtgga ataccacggg gacctcatcg agatcaccga cgccaagaaa 780
cgggaggctc tgtacgcaca ggacccttcc acgggctgct acatgtacta ttttcagtat 840
ctgagcaaaa cctactgcgt ggatgcaact agagagacaa atcgcctagg aagactgatc 900
aatcacagca aatgtgggaa ctgccaaacc aaactgcacg acatcgacgg cgtacctcac 960
ctcatcctca tcgcctcccg agacatcgcg gctggggagg agctcctgta tgactatggg 1020
gaccgcagca aggcttccat tgaagcccac ccgtggctga agcattaacc ggtgggcccc 1080
gtgccctccc cgccccactt tcccttcttc aaaggacaaa gtgccctcaa agggaattga 1140
attttttttt tacacactta atcttagcgg attacttcag atgtttttaa aaagtat 1197
<210> 53
<211> 2843
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2998209
<400> 53
56/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
tcagatgtac ccagtgctta tcccaacgta ggaccaggag tcctacagaa tctcctcagc 60
ctcaggatct ggctcaagac cagttgcttt ttctgttgat ccttgtgagg aatacaatga 120
tagaaataaa ctgaacacta ggacacagca ctggacttgc tctgtttgca catatgaaaa 180
ctgggccaag gctaaaagat gtgttgtttg tgatcatccc agacctaata acattgaagc 240
aatagaattg gcagagactg aagaggcttc ttcaataata aatgagcaag acagagctcg 300
atggagggga agttgcagta gtggtaatag ccaaaggaga tcacctcctg ctacgaagcg 360
ggactctgaa gtgaaaatgg attttcagag gattgaattg gctggtgctg tgggaagcaa 420
ggaggaactt gaagtagact ttaaaaaact aaagcaaatt aaaaacagga tgaaaaagac 480
tgattggctc ttcctcaatg cttgtgtggg ggttgtagaa ggtgatttag ctgccataga 540
agcatacaag tcatcaggag gagacattgc acgtcagctc accgcagatg aagtacgctt 600
gctgaatcgt ccttctgcct ttgatgttgg ctatactctt gtacacttgg ctatacgttt 660
tcagaggcag gatatgctag caatattgct tacagaggtg tctcaacaag cagcaaagtg 720
tattccagca atggtgtgtc ctgaactgac agaacaaatc cggagagaga tagctgcctc 780
tcttcatcag agaaaggggg attttgcttg ctattttctg actgaccttg taacatttac 840
attgccagca gatattgaag atttgccccc aacagtccaa gaaaaattat ttgatgaggt 900
gcttgataga gacgttcaaa aagaattaga agaagaatct ccaattatta actggtcctt 960
ggaattggct acacgtttgg acagtcgact gtatgcactt tggaaccgga ctgcaggaga 1020
ctgcctactt gattcagttc tacaagctac ctggggcatc tatgacaagg actcagtgct 1080
tcggaaagcc ctgcatgaca gcctgcatga ctgttcacat tggttttaca cacgctggaa 1140
agattgggaa tcatggtatt ctcagagctt tggtttacat ttttccttga gagaagaaca 1200
gtggcaagaa gactgggcat ttatactctc tcttgctagt cagcctggag caagcttgga 1260
gcagacgcac atttttgtac tggcacatat tcttagacga ccaattatag tttatggagt 1320
aaaatattac aagagtttcc ggggagaaac tttaggatat actcggtttc aaggtgttta 1380
tctgcctttg ttgtgggaac agagtttttg ttggaaaagt ccgattgctc tgggttatac 1440
gaggggccac ttctctgctt tggttgccat ggaaaatgat ggctatggca accgaggtgc 1500
tggtgctaat ctcaataccg atgatgatgt caccatcaca tttttgcctc tggttgacag 1560
tgaaaggaag ctactccatg tgcacttcct ttctgctcag gagctaggta atgaggaaca 1620
gcaagaaaaa ctgctcaggg agtggctgga ctgctgtgtg acggaggggg gagttctggt 1680
tgccatgcag aagagttctc ggcggcgaaa tcaccccctg gtcactcaga tggtagaaaa 1740
atggcttgac cgctaccgac agatccggcc gtgtacatcc ctgtctgatg gagaggaaga 1800
tgaggatgat gaagatgaat gaaaaaaaaa atcaaacagc agaagaccaa ggcatcagat 1860
ctgtaatgac cctaaagtta gtgtggtgct ccaagcagag tcgacatcat ggaatgaacc 1920
aaatctggca ggatctgctc ggggaagtgt tttcctggac cacacacacc ttatggagat 1980
aatgcctctg ctgcgtgagg agacagagaa ctttagttgg actacagttt gtaaaaaaaa 2040
ctaattttat taagacagaa ctttttttcc ttccaaattg taaatctgtc tataaatgta 2100
acgcatgtgg ttgtgtaaga cattgtttaa taggaaaagt tgtaccagca tcttcatatt 2160
attgagaaaa ttttttccag catgggcact tagaaaaagc acatggcaaa tggctctttg 2220
ttcctttcag atattatttc agtagaacct ggcattctcc tttcacctta aaagatccat 2280
ctaagtctca gatctggaaa cgttttgtac cgattatcca cagcaaaaca aaaataagct 2340
tttattttat taataatttc gttcctcttg tgcccaatca aatcttttag gaacaaactg 2400
caagaaaagc taagaatgtt ttagagtgaa ctaaatacag acattgctta cttgttttga 2460
agagggtttt ggttttggtt attgtgtctt ttaagttttc tgatatgccc cctttcaata 2520
tttagatatt tatttgttgg gaagaatacc ttaaaatgag ggttcttatt ccagattctg 2580
ggcagtggtc tgtgagtagt ttttttcctg gatgaaaagg gagcaagccc acttgtcact 2640
aaatgaattg tgtgaaatgt gctcacttgg actccatcaa caatgtgctg ctcccagatt 2700
gccatgccag agggtcttcg gattcttcct tctatcacct ctgctctaag caaatcttgt 2760
tagaagggca tgcctttgct taggcagatt gggaatacca attcactaca gaataaagat 2820
tttaaaaatg caaaaaaaaa aaa 2843
<210> 54
<211> 1272
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
57/66
CA 02329685 2000-12-11
WO 99164596 PCT/US99/13281 _
<223> Incyte Clone No.: 3340296
<400> 54
tctacggcca cgactctggg agtggggaaa cagagagccg gttcctctgc tgcagaagtc 60
ctcggggttc cttctcacaa ctctgcgaag gggaaagggt tgtgagaccc aaccagaccc 120
caactccagc tcccagcagg aggtggctgc gccacactcg ggaggcctct tggtttcagg 180
gtctctctgt ctctctctca ccctcttcct cgctttctct gtctctctgt ctctctctct 240
ctctctccct cgtccactcc cccaaacatg tccaccggct ccctcagcga tgtggaggac 300
cttcaagagg tggagatgtt ggaatgtgac gggttgaaaa tggattcgaa caaggaattt 360
gtgacttcca acgagagcac cgaggagagc tccaactgcg agaatgggtc tccccagaag 420
ggccgcggcg gcctgggcaa gaggaggaag gcgcccacca agaagagccc cctgagcggg 480
gtcagccagg aggggaagca ggtccagcgc aacgccgcca acgcgcgaga gcgggcccgc 540
atgcgagtgc tgagcaaggc cttctccaga ctcaagacca ccctgccctg ggtgcccccc 600
gacaccaagc tctccaagct ggacacgctc aggctggcgt ccagctacat cgcccacttg 660
aggcagatcc tggctaacga caaatacgag aacgggtaca ttcacccggt caacctgacg 720
tggcccttta tggtggccgg gaaacccgag agtgacctga aagaagtggt gaccgcgagc 780
cgcttatgtg gaaccaccgc gtcctgacct tggaggtgcg agtctgggaa aggcgcgctc 840
ccggggggag cgggccccgg gaaggcgacc cctgccctca gtgctctctg tctctgcttc 900
cccctcgcaa tgctcctctc tctgtcccac cccgcgagaa cactttacaa cgacgaggag 960
attcgtttcc aaaccagagg agatcaattg tacttacaaa gattcccatc tatttaactt 1020
tattaacttc taccgtgaat gactctgcaa gccttgctgg tccaagtgca atatgtaatt 1080
ataaatatat aaatagataa gagcctatca atgtatcttt tgtacaatat gttgtaaaat 1140
gtagatcata ggatagctga ctttgacagt cacatttata aagtaattca cttaaagata 1200
tatatttttt tcaaacaagt tttgctactt ttgaaaataa atctttcttt atattgctaa 1260
aaaaaaaaaa as 1272
<210> 55
<211> 1117
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 3536740
<400> 55
tgcactgcca cgccgagctg aggctgagct cgcccggcca gctcaaagca gccaggcggc 60
gctacaagac tttcatgatc gacgagatcc tctccaagga gacctgcgat tactttgaga 120
aactttccct ctactccgtg tgcccgtcgc tggtcgtgcg acccaagccc ctgcattcct 180
gtacgggctc cccttccctg cgggcatatc cgctcctctc ggtgatcacc cgccagccca 240
ctgtcatctc ccacctggtc cctgccaccc cgggaatcgc ccaggcactg tcctgccacc 300
aggtcaccga ggcggtctct gctgaggccc cagggggcga ggccctagcc agcagcgagt 360
cagagacgga acagcccacg ccccgacaga agaagccccg ccggagtcgc accatcttca 420
ccgagctgca gctcatgggc ctggagaaga aattccagaa gcagaagtat ttgtcaaccc 480
cagacaggtt ggacttggct cagtctctgg gactcactca gctgcaggtg aagacctggt 540
atcagaatcg caggatgaaa tggaagaaaa tggttcttaa aggtggacag gaagcaccca 600
caaaacccaa aggtcgcccc aagaagaact ccatccccac atcagaagag attgaagctg 660
aagagaagat gaacagccag gcccagggtc aggagcagct ggagccctct caggggcagg 720
aggagctctg tgaagcacag gaaccgaaag cacgtgatgt ccccttagag atggcagagc 780
caccagaccc gccccaggag ttgccaatac cctcttcgga acccccacca ttaagctaaa 840
gtaaaaccct tttgagggaa gagggagact ggggagaagg gaaaagagag aaggcaggga 900
gagtagggag agaaaacctt ccagcagccc agtaaactgc gggcgaagag atctacccgt 960
ctccctccct cccacagtta ccattggcct tgtcatcgca agcatttgac aaagacttgc 1020
ttgtcttggg cctgtcacct cctgaaaggc tgctttagct gtggatgccc ttgattaagg 1080
gagagagcgc ctaggagctg cctgccccag ctggggt 1117
58/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/1328I _
<210> 56
<211> 3033
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 082155
<400> 56
gcagatgcta gaacataatg tagcattact ttccccaggg tttattgtta tgtaagttct 60
tgttcagctt cctttgtttt ctttcacttc tgagaattta actttcgttt ctcactcagc 120
tcctgtgggg aaactcattt gtggagacca gccctctggc ttggtgagtg aatctggttt 180
acaccggctc ctgccctgcc ttcactcttc tcccctgatt caagactcct ctgctttgga 240
ctgaagcact gcaggagttt gtgaccaaga acttcaagag tcaagacaga aggaagccaa 300
gggagcagtg caatggattt ctcagtaaag gtagacatag agaaggaggt gacctgcccc 360
atctgcctgg agctcctgac agaacctctg agcctagatt gtggccacag cttctgccaa 420
gcctgcatca ctgcaaagat caaggagtca gtgatcatct caagagggga aagcagctgt 480
cctgtgtgtc agaccagatt ccagcctggg aacctccgac'ctaatcggca tctggccaac 540
atagttgaga gagtcaaaga ggtcaagatg agcccacagg aggggcagaa gagagatgtc 600
tgtgagcacc atggaaaaaa actccagatc ttctgtaagg aggatggaaa agtcatttgc 660
tgggtttgtg aactgtctca ggaacaccaa ggtcaccaaa cattccgcat aaacgaggtg 720
gtcaaggaat gtcaggaaaa gctgcaggta gccctgcaga ggctgataaa ggaggatcaa 780
gaggctgaga agctggaaga tgacatcaga caagagagaa ccgcctggaa gaattatatc 840
cagatcgaga gacagaagat tctgaaaggg ttcaatgaaa tgagagtcat cttggacaat 900
gaggagcaga gagagctgca aaagctggag gaaggtgagg tgaatgtgct ggacaacctg 960
gcagcagcta cagaccagct ggtccagcag aggcaggatg ccagcacgct catctcagat 1020
ctccagcgga ggttgacggg atcgtcagta gagatgctgc aggatgtgat tgacgtcatg 1080
aaaaggagtg aaagctggac attgaagaag ccaaaatctg tttccaagaa actaaagagt 1140
gtattccgag taccagatct gagtgggatg ctgcaagttc ttaaagagct gacagatgtc 1200
cagtactact gggtggacgt gatgctgaat ccaggcagtg ccacttcgaa tgttgctatt 1260
tctgtggatc agagacaagt gaaaactgta cgcacctgca catttaagaa ttcaaatcca 1320
tgtgattttt ctgcttttgg tgtcttcggc tgccaatatt tctcttcggg gaaatattac 1380
tgggaagtag atgtgtctgg aaagattgcc tggatcctgg gcgtacacag taaaataagt 1440
agtctgaata aaaggaagag ctctgggttt gcttttgatc caagtgtaaa ttattcaaaa 1500
gtttactcca gatatagacc tcaatatggc tactgggtta taggattaca gaatacatgt 1560
gaatataatg cttttgagga ctcctcctct tctgatccca aggttttgac tctctttatg 1620
gctgtgcctc cctgtcgtat tggggttttc ctagactatg aggcaggcat tgtctcattt 1680
ttcaatgtca caaaccacgg agcactcatc tacaagttct ctggatgtcg cttttctcga 1740
cctgcttatc cgtatttcaa tccttggaac tgcctagtcc ccatgactgt gtgcccaccg 1800
agctcctgag tgttctcatt cctttaccca cttctgcata gtagcccttg tgctgagact 1860
cagattctgc acctgagttc atctctactg agaccatctc ttcctttctt tccccttctt 1920
ttacttagaa tgtctttgta ttcatttgct agggcttcca tagcaaagca tcatagattg 1980
ctgatttaaa ctgtaattgt attgccgtac tgtgggctgg aaatcccaaa tctagattcc 2040
agcagagttg gttctttctg aggtctgcaa ggaagggctc tgttccatgc ctctctcctt 2100
ggcttgtaga aggcatcttg tccctatgac tcttcacatt gtctttatgt acatctctgt 2160
gcccaagttt tcccttttta ttaagacacc agtcatactg gctcagggcc caccgctaat 2220
gccttaatga aatcatttta acattatatt ctctacaaag accttatttc caaataagat 2280
aatatttgga ggtattggga ataaaaactc caacatataa atttgaggaa ggcacgattt 2340
cactcataac aatcttaccc tttcttgcaa gagatgcttg tacattattt tcctaatacc 2400
ttggtttcac tagtagtaaa cattattatt ttttttatat ttgcaaagga aacatatcta 2460
atccttccta tagaaagaac agtattgctg taattccttt tettttcttc ctcatttcct 2520
ctgcccctta aaagattgaa gaaagagaaa cttgtcaact catatccacg ttatctagca 2580
aagtacataa gaatctatca ctaagtaatg tatccttcag aatgtgttgg tttaccagtg 2640
acaccccata ttcatcacaa aattaaagca agaagtccat agtaatttat ttgctaatag 2700
tggattttta atgctcagag tttctgaggt caaattttat cttttcactt acaagctcta 2760
tgatcttaaa taatttactt aatgtatttt ggtgtatttt cctcaaatta atattggtgt 2820
59/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
tcaagactat atctaattcc tctgatcact ttgagaaaca aacttttatt aaatgtaagg 2880
cacttttcta tgaattttaa atataaaaat aaatattgtt ctgattatta ctgaaaagat 2940
gtcagccatt tcaatgtctt gggaaacaat tttttgtttt tgttctgttt tctttttgct 3000
tcaataaaac aatagctggc tctaaaaaaa aaa 3033
<2I0> 57
<2I1> 6138
<212> DNA
<2I3> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 095477
<400> 57
ggctgaggac tgactggggt tctgagactc cctgtcccgg accgcagcgt taaaaggatc 60
tgaacaaagt ctgctcaaat ctcctgctgt gaaccagcag aatttttgaa caggtttctt 120
cacatataaa aatctattgt aaaaatacgg aaaagaatgg cagcggaaac gcagacactg 180
aactttgggc ctgaatggct ccgagctctg tccagtggtg ggagtattac atcccctcct 240
ctttctccag cattgccgaa gtataaatta gcagattatc gttacggcag agaagaaatg 300
ttagcacttt tccttaaaga caacaagata ccttcagacc ttctggataa agaatttctg 360
cctatcctcc aggaggaacc ccttccacca ttggctctgg taccctttac agaagaagaa 420
cagagaaact tttccatgtc tgtaaatagt gctgctgtcc tgcgattgac aggacgagga 480
ggaggaggaa cagtggtggg ggctcctaga ggtcgaagtt cttcaagagg gcgaggcaga 540
ggcagaggtg aatgtggttt ctaccaaaga agttttgatg aagtagaggg tgtttttggt 600
cgaggaggtg gcagagaaat gcatagatcg cagagctggg aggaaagggg tgacagacgt 660
tttgaaaaac caggacgaaa agatgtaggg agaccaaatt ttgaggaagg tggaccaaca 720
tcagtaggga gaaagcatga atttatacgc tcagaaagtg aaaattggcg catctttaga 780
gaggaacaaa atggagaaga tgaagatgga ggttggcgac tagctggatc aaggagggat 840
ggagagaggt ggcgacctca cagtcctgat ggccctcgtt ctgcaggctg gcgggaacac 900
atggaacgac gtcggaggtt tgagtttgat tttcgagata gagatgatga acggggttac 960
cgaagggttc gctctggcag tgggagcata gatgatgaca gggatagctt gcccgaatgg 1020
tgcttagagg atgctgaaga agaaatgggt acatttgact catctggagc attcctttct 1080
ctaaaaaaag tacagaaaga gcctattcca gaagagcagg agatggactt ccggcctgtg 1140
gacgaagggg aggagtgctc tgactctgag ggtagccata atgaagaggc caaagaaccc 1200
gataagacaa ataagaaaga aggagagaaa acagatagag taggagttga agctagtgag 1260
gaaactcccc agacctcatc atcatctgct agaccaggta ctccttcaga ccatcagtct 1320
caggaagcat cacagtttga gaggaaagat gaaccaaaaa ctgagcaaac ggaaaaagct 1380
gaagaggaga ctcggatgga aaatagtcta ccagccaaag tgcccagcag aggggatgaa 1440
atggttgctg atgtccagca gcccctgtcg cagattcctt cagatacagc ctctcctctt 1500
ctcatacttc cacctcctgt tcccaatcct agtcctactc tccggccagt tgaaacacca 1560
gttgtaggtg ctcctggtat gggcagtgtt tccacagaac ctgatgatga agaaggtctc 1620
aaacatttgg agcagcaagc tgagaaaatg gtggcttatc tccaagacag tgcactagat 1680
gatgaaagat tggcatcaaa actgcaagag cacagagcta aaggagtgtc gattccattg 1740
atgcatgaag caatgcagaa gtggtattac aaagatcctc agggagaaat tcaaggtccc 1800
ttcaataatc aggagatggc agaatggttt caggcgggct attttactat gtctttattg 1860
gtgaagagag cgtgtgatga aagcttccaa cctcttggcg atatcatgaa aatgtgggga 1920
agggttccct tttctccagg tccagctccc cctcctcata tgggagagct ggaccaggaa 1980
cgactgacca ggcagcaaga actcacagcc ttataccaga tgcagcacct gcagtaccag 2040
cagtttttaa tacaacaaca atatgcacag gttttggccc aacagcagaa agcagcactg 2100
tcttcccagc agcagcagca gttggcactt cttcttcaac agtttcagac cttgaagatg 2160
agaatatctg atcagaacat cattccctca gtaactaggt ctgtgtccgt gccagatact 2220
ggctctatct gggagcttca gccaacagct tcacagccta cagtttggga aggtggtagt 2280
gtatgggatc ttcctctgga caccacgaca ccaggccctg ccctggaaca gcttcagcag 2340
ctagagaagg ccaaagctgc aaagctagag caagagagaa gagaggcaga aatgagggca 2400
aaacgggaag aggaagagcg aaagaggcag gaagaactcc gaagacaaca ggaggaaatt 2460
60/66
CA 02329685 2000-12-11
WO 99/64596 PCT/CIS99/13281 _
cttcggcgac agcaggaaga agaaaggaaa aggcgagagg aagaagaact tgcccgaagg 2520
aaacaggaag aggctctgcg tcgccagcgg gagcaagaaa ttgcattaag gcgacagcga 2580
gaagaggaag aaagacagca gcaagaagaa gctcttagaa gactggaaga gaggagaaga 2640
gaagaggaag aaaggcggaa gcaggaagaa ttgttacgca aacaggaaga ggaggctgca 2700
aaatgggccc gggaagaaga agaagcccag cgtcgattag aggagaaccg gctgcggatg 2760
gaagaggagg cagccagact ccggcatgag gaagaagaac ggaagagaaa ggagctggag 2820
gtccagcggc agaaggagtt aatgcgccag aggcagcagc agcaagaggc tctccggagg 2880
ttgcagcagc agcagcagca acaacagctg gcgcagatga agcttccttc ttcttcaacg 2940
tggggccagc agtccaatac aacagcatgt cagtcccagg ccacgctgtc gttggctgaa 3000
atccaaaaac tagaggaaga acgagaacgg cagcttcgag aagagcaaag gcgccagcag 3060
agggagttga tgaaagctct tcagcagcag cagcaacagc aacagcagaa actctcaggt 3120
tgggggaatg tcagcaaacc ttcaggtacc acgaaatctc ttctggagat ccagcaggaa 3180
gaggccaggc aaatgcaaaa gcagcagcag cagcagcagc aacaccagca accaaacaga 3240
gctcgtaaca atacgcattc caacctgcac accagcattg ggaattctgt ttggggctct 3300
ataaatactg gtcctcctaa ccagtgggca tctgacctag tcagtagtat ttggagtaat 3360
gctgacacta aaaactccaa catgggattc tgggatgatg cagtgaaaga ggtgggacct 3420
aggaattcaa caaataaaaa taaaaacaac gccagtctca gtaaatctgt aggtgtgtct 3480
aaccggcaga ataagaaagt agaagaagaa gaaaagttgc tgaagctctt tcagggagta 3540
aataaagccc aagatggatt tacgcagtgg tgtgaacaga tgcttcatgc ccttaatacg 3600
gcaaataact tggatgttcc cacatttgtt tctttcctga aagaagtaga atctccttat 3660
gaggtccatg attatatcag ggcctattta ggagatactt ctgaggccaa ggagtttgcc 3720
aagcagttcc ttgagcgccg tgccaaacag aaagccaacc agcagcgtca gcagcagcag 3780
ctgccacagc agcagcagca gcagccgcca cagcagccgc cacagcagcc acaacagcag 3840
gactctgtgt gggggatgaa ccacagtaca ctccattcag tatttcagac caatcaaagc 3900
aacaaccaac aatccaattt tgaggctgtg cagagtggca agaagaagaa aaagcagaag 3960
atggtccgag cagatcccag tttattagga ttttcagtca atgcatcatc ggagcgac.tc 4020
aacatgggtg aaatcgagac gttggatgac tactgagcac ctgccagtgg actggccatc 4080
cctctcctgt ctgccgacta tggagtctcc acctttggac acaacactta ctcaccattt 4140
actctttatc actctgcaac aaatcacaga accgatcatc tcaggctttt tcttctggcc 4200
ctttgtgtcc aagattcttt aatccatttt tgttggtgaa catctcagac tatagataag 4260
tggactggac cctgtgtctt gggggtggca gttgggatta ctccccaaca aggctgattt 4320
taggcagcat gtgttcactg tgctgtgatt tcatctactg tctcccagaa agtgtgttgg 4380
gatcggccat tagcagcttg ctttctcttg tcactttttt tcttctattt tgttttttct 4440
tcttcttttt ccccccatca gggcaaatgg tctaactggt gcaatcatga agagagttaa 4500
tggttaacag acattggcca ataacaaaac accccatgga ctgtgactcg agtatccaac 4560
aggcagtcag agctctcccg gtctgaaagt tgcattgcca ctgctaactt tgggattgca 4620
tcagagaggc cctgagtggg gttgagatga ggttggtttg gtttgatgtt acacactcct 4680
cacctgttct ttctgagtgt cctttctctg aaaggattta tgtttttctt cgttagatag 4740
tgacttctga gcaagctgat ctcccctggc atgctccaac ctgattggac aaaggaagct 4800
ctatggcctg ggagagagac tattcttaat ttttctttct tacaaaaact gatttttccc 4860
ataaatattt ttacttcaga ggactaggac cattttgttt tgggcccttc tgctgaaaat 4920
ttgtctcgtt taagaggcag ctagaatctt taccatatgt atgaatttgt ataatttcat 4980
ttttggatag ggataaactt ttgcttctga taaaagcctg gaatttcatc tggtcctcag 5040
agcattgcgt gtgtgtcttg ctgtagcccg gaaaaggttt tgtgtaaaga ttctgggatg 5100
gcaagttgtt tgccttttct gaaaagagaa catacagaac ctgtccatct ttaagacctt 5160
catccatgga atctactata caggaggatg cagtgggctg gaggggatgg gcgaaaatgg 5220
gagcaggaag cctggcctgg cttctggtca tggcctccta aaaccttaaa cttcaagtag 5280
aaatgtactc aagccctatt tataaacaaa tacttttcct gcctccacca aacccctaca 5340
gaacatcacc tggaattgcc actcacactg ggttggagtc attgggcagc tgtgcctgtg 5400
cgagaggtgc tgtggtctgg gcagcccctg gaaaagcacc tttgctgcct gtcattgttg 5460
cctgaagaag gctggagttg ctctgagagc agtttgggtt tggagtatta tatttggctt 5520
ctatttttat tattttggat caccattctc cctatccctt cttgcctccc tcccttctaa 5580
acatgtgtaa taactataca gagactgcta caaaattgta tatagttttt ggatcaaata 5640
gcatgagggg agaggaaacc attaaaaatt ggggctccta ctctcctttg ctttgtaaat 5700
tcaaaagttg ggggtgggta agagggatag ttaaaatgtt tacaaaactt taggctccct 5760
cggaactttt gccagtgtgg aggaaaataa aaaagaactt aaataaaatc tgattgtatt 5820
ctatctgagt gcacctcttg tactcacctt tatggaggct gagttctgca ctaaactgtt 5880
61 /66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
cctcttggta ccatggaaaa gctccaagca cccaagacat ggaggcagcc atggcttctt 5940
tctctgccag accacgtagc actggctggt tctgtatttg agaatgtaga ggtcaaggca 6000
gatgtcggaa tgctgatacc tgattctagt tatggatatg gaggcaagga gaagttgtta 6060
ggttaaccag cgcagtcctc cgtgcgtccc gaccgccgct gccctcactc ccggccaaga 6120
tggcatcctg tctggcac 6138
<210> 58
<211> 3190
<212> DNA
<213> Homo sapiens
<220>
<221> miac_feature
<223> Incyte Clone No.: 1399169
<400> 58
cttcggggcc aagatggcga cgggaacggg caaacacaag ctgctaagca ctggccccac 60
agagccatgg tccatccgag agaagctatg tttagcatct tctgtcatga gaagtggcga 120
tcaaaattgg gtatcagtta gcagagcaat caagcccttt gcagaacctg gccgccctcc 180
agactggttc tctcaaaaac attgtgcttc ccagtactcg gagcttttag agaccactga 240
gacaccaaaa cggaaacgag gtgaaaaggg agaagtggtg gaaactgttg aagatgttat 300
tgttcggaaa ttgactgctg agcgagttga agaactaaag aaagtgataa aggaaaccca 360
ggagagatat agacggctaa agagagatgc agaactaatt caagctggac acatggacag 420
cagactggat gagctttgca atgacattgc aacgaaaaag aaattggaag aagaggaggc 480
tgaagtaaag aggaaggcta cagatgctgc ataccaggct cgtcaagcag taaaaacacc 540
cccccggagg ttacccactg tgatggttcg ctctcctata gattctgcct ccccaggagg 600
tgattatcca cttggggact tgactccaac cactatggaa gaggctacct ctggggtaac 660
ccccgggact ttgccgagta ccccagtcac ctcgtttcct gggattcctg acacccttcc 720
tccaggctct gcacccttag aagcccccat gaccccagta acagatgatt caccccagaa 780
aaagatgctt ggacagaaag caactccacc cccctcccct ctgctgtcag agctcttgaa 840
gaagggcagc ctcctgccta ctagccccag actggtcaat gagagtgaaa tggctgtggc 900
ttctggccac ctgaacagta caggtgtcct cctggaggta ggcggggtcc ttcccatgat 960
acatggtggg gagatacagc aaacacccaa tactgttgca gcctcccctg ctgcatcagg 1020
tgctcccact ctttcccggc ttttagaagc tggtcctaca cagttcacca cacctcttgc 1080
ttccttcact actgttgcca gtgagcctcc agttaaactt gtgccacccc ctgtagagtc 1140
tgtgtcccaa gctaccattg tcatgatgcc tgcgctgcca gcaccatcct ctgctccggc 1200
tgtctccact actgaaagtg tagctccagt gagtcaaccc gacaactgtg ttcccatgga 1260
ggctgtgggg gatccacata ctgtgactgt ttccatggac agcagtgaaa tatccatgat 1320
catcaattct atcaaagaag agtgttttcg atcaggggta gcagaggctc ctgttggatc 1380
aaaggctccc agcatagatg ggaaggaaga attagatctg gctgagaaga tggatattgc 1440
tgtgtcttac acaggtgaag agctggattt tgagactgtt ggagacatca ttgccatcat 1500
tgaggacaag gtagatgatc atcctgaagt gctggatgtg gcagcagtgg aagcagcact 1560
gtcattttgt gaagaaaatg atgatcctca gtccctgcct ggcccctggg agcatcctat 1620
ccagcaggag cgggacaagc cagtacctct ccctgcacca gaaatgacgg tcaagcaaga 1680
gagactggac tttgaggaaa cggaaaacaa gggaatacat gaactggtgg acatcaggga 1740
gcccagtgca gagatcaagg tggaacctgc agaaccagag ccagtcattt caggagccga 1800
aatagtagct ggagttgttc cagccacaag tatggagcca ccagaactca ggagtcagga 1860
cttagatgag gaactgggaa gtactgcagc tggagagatt gttgaagcag atgttgccat 1920
tgggaaaggc gatgagactc cacttacaaa tgtgaagaca gaggcatccc ctgaaagcat 1980
gttgtctcca tcacatggct caaatcccat tgaagatcct ttagaggcag agactcagca 2040
caagtttgaa atgtcagact cattgaaaga agaatcaggg actatttttg gaagccagat 2100
aaaggatgcc ccaggtgagg atgaggagga agatggtgtc agtgaagcgg ccagcctaga 2160
ggagcctaag gaagaggatc aaggagaagg ctacttgtca gaaatggata atgaacctcc 2220
tgtgagcgag agtgatgatg gcttcagcat acacaatgct acactgcagt cacacacact 2280
ggcagactcc atccccagca gccctgcttc ttcacagttc tctgtctgta gtgaggatca 2340
ggaagctatt caggcacaga aaatttggaa gaaagccatc atgcttgtat ggagagctgc 2400
62/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
agctaatcat aggtatgcca atgtcttcct gcagcctgtt acagatgaca tagcacctgg 2460
ctaccacagc attgtgcaga ggcctatgga tttgtcaact attaagaaaa acatagaaaa 2520
tggactgatc cgaagcacag ctgaatttca gcgtgacatt atgctgatgt ttcagaatgc 2580
tgtaatgtac aatagctcag accatgatgt ctatcacatg gcagtggaga tgcagcgaga 2640
tgtcttggaa cagatccagc aattcttggc cacgcagttg attatgcaaa catccgagtc 2700
tgggatcagt gctaaaagtc ttcgagggag agattctacc cgcaaacagg atgcttcaga 2760
gaaggacagt gtcccaatgg gctctcctgc cttccttctc tctctctttg atggaggaac 2820
caggggacgc cgctgtgcca ttgaagcaga tatgaagatg aaaaagtgaa gcctcagagt 2880
taccctcttt gagccgaacc taaaataaaa gtaaacaaga tagagcttgg gcttgcgggc 2940
ccagttccag aggtggaagt tacagaagag gaggtacctg ggccacacga catgagctgg 3000
aaaatctctc ttagagagtt ggagtagcac aattgcctgt tttagggcag aaaccatggg 3060
ctatgttaat gtcctaatgt gtagctagca gatcgtagct agtttgtatt gtcttgtcaa 3120
ttgtacagac tttttaaaaa aaacaaccac cagtgaaatg tgtgtgtata caataaactg 3180
aaaaaaaaaa 3190
<210> 59
<211> 1391
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 1203, 1204
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No.: 1442069
<400> 59
tttttttttt ccagaggaga gtacaggtcg tgctgcagtt agttcattga aaactcattt 60
gctcttggag cagtcaggca gtgactgcct tcggcttttt ttctgctgac taagatctcc 120
tatagagagc tacaacaatg cccaaaagaa aggctgcagg tcaaggtgat atgaggcagg 180
agccaaagag aagatctgcc aggttgtctg ctatgcttgt gccagttaca ccagaggtga 240
agcctaaaag aacatcaagt tcaaggaaaa tgaagacaaa aagtgatatg atggaagaaa 300
acatagatac aagtgcccaa gcagttgctg aaaccaagca agaagcagtt gttgaagaag 360
actacaatga aaatgctaaa aatggagaag ccaaaattac agaggcacca gcttctgaaa 420
aagaaattgt ggaagtaaaa gaagaaaata ttgaagatgc cacagaaaag ggaggagaaa 480
agaaagaagc agtggcagca gaagtaaaaa atgaagaaga agatcagaaa gaagatgaag 540
aagatcaaaa cgaagagaaa ggggaagctg gaaaagaaga caaagatgaa aaaggggaag 600
aagatggaaa agaggataaa aatggaaatg agaaaggaga agatgcaaaa gagaaagaag 660
atggaaaaaa aggtgaagac ggaaaaggaa atggagaaga tggaaaagag aaaggagaag 720
atgaaaaaga ggaagaagac agaaaagaaa caggagatgg aaaagagaat gaagatggaa 780
aagagaaggg agataaaaaa gaggggaaag atgtaaaagt caaagaagat gaaaaagaga 840
gagaagatgg aaaagaagat gaaggtggaa atgaggaaga agctggaaaa gagaaagaag 900
atttaaaaga agaggaagaa ggaaaagagg aagatgagat caaagaagat gatggaaaaa 960
aagaggagcc acagagtatt gtttaaaact gccctatgta gtttcataat ttggtaacat 1020
gtaccttcat gttgtaaagt taatagagat aaatattttt atcaaaaatt ttataaacac 1080
agcctttctt tagcattgat ttaatttcag aacatcttca tattgattat tagccataaa 1140
gtttctaaca tgaaacattt atctataaat tttgtgatta tagtagtgga atacatagaa 1200
aannatatgc tttcaacttt gtgagtggat ttcgtgatgt gtagttatat gtcaatcttt 1260
ggttttaatt tactctttta tacatgtgat agttcataag tgagggatcc aaaacaaggt~1320
tcatccacat tcctgtctgc aggtgcttta taagaggtga ctatttcagt aatgtagggt 1380
aactatcttc c 1391
63/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/13281 _
<210> 60
<211> 1125
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1596668
<400> 60
caacaaagca aggaagacgg agtccgagcc tcgggggctc ctagcaacgg gccggggcgg 60
gagttccatg gagactgggg agcgcgcccg tctcatcctc atccttgtcc tccagcttct 120
ccttcgcatc cgacgcaacc ggcagcagcg ctgccgccgc gtcctcagcc accgctccct 180
cttcccacgg atgtgatctt cgtggtggaa agctaaattt taaaaccacc ccaatggatg 240
cagacagtga tgttgcattg gacattctaa ttacaaatgt agtctgtgtt tttagaacaa 300
gatgtcattt aaacttaagg aagattgctt tggaaggagc aaatgtaatt tataaacgtg 360
atgttggaaa agtattaatg aagcttagaa aacctagaat tacagctaca atttggtcct 420
caggaaaaat tatttgcact ggagcaacaa gtgaagaaga agctaaattt ggtgccagac 480
gcttagcccg tagtctgcag aaactaggtt ttcaggtaat atttacagat tttaaggttg 540
ttaacgttct ggcagtgtgt aacatgccat ttgaaatccg tttgccagaa ttcacaaaga 600
acaatagacc tcatgccagt tacgaacctg aacttcatcc tgctgtgtgc tatcggataa 660
aatctctaag agctacatta cagatttttt caacaggaag tatcacagta acagggccca ?20
atgtaaaggc tgttgctact gctgtggaac agatttaccc atttgtgttt gaaagcagga 780
aagaaatttt ataattcacc acttaattgg ttagaatctc taactgagca ccttttaaac 840
ctgctgcaca ttggactcaa aaggaaaact ggaccaacaa taattgagga aatagactct 900
tttattcatt cacggctaca gtgtaagctc cagtcccttt ggattttatt ccaaaccttg 960
ctgtaatata aaaggaagtt tacaagacat gatattgctg cttttacaaa aggacattct 1020
atttattttc gcagtaattc tcatgtcccc ataagcagag ctgtcacagt gtgcactacc 1080
ttagattgtt ttattgtcgt cattgttatt tttttccatt ttgag 1125
<210> 61
<211> 3073
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 1977214
<400> 61
cggaattcgg ctcgagggcg actggagcgg ttccctcgca ggcggcgcca ttttgtgcta 60
ggagcctgat aaaaccggcc cggttctgtg gaaagtgggc ggcggagcca gggtccctgg 120
aatggcggag actctgtcag gcctaggtga ttctggagcg gcgggcgcgg cggctctgag 180
ctccgcctcg tcagagaccg ggacgcggcg cctcagcgac ctgcgagtga tcgatctgcg 240
ggcggagctg aggaaacgga atgtggactc gagcggcaac aagagcgttt tgatggagcg 300
gctgaagaag gcaattgaag atgaaggtgg taatcctgac gaaattgaaa ttacctccga 360
gggaaacaag aaaacatcaa agaggtctag caaagggcgc aaaccagaag aagagggtgt 420
ggaagataac gggctggagg aaaactctgg ggatggacag gaggatgttg agaccagtct 480
ggagaacttg caggacatcg acatcatgga tatcagtgtg ttggatgaag cagaaattga 540
taatggaagc gttgcagatt gtgtcgaaga cgatgatgct gataacctcc aggagtccct 600
gtcggatagt agagagctag tcgaggggga aatgaaagag cttccggagc agcttcagga 660
acatgctata gaggacaaag aaactataaa caatttagat acttcatcat ctgacttcac 720
tatattacag gaaattgaag agccatccct ggagccagaa aatgagaaaa tactcgacat 780
tttgggggaa acttgtaaat ctgagccagt aaaagaagaa agttccgagc tggagcagcc 840
atttgcacag gacacaagta gcgtggggcc agacagaaag cttgcggagg aagaggacct 900
atttgacagc gcccatccgg aagagggtga tttagatttg gccagcgagt caacagcaca 960
64/66
CA 02329685 2000-12-11
WO 99/64596 PCT/US99/I3281 -
cgctcagtcg agcaaggcag acagcctgtt agcggtagtg aaaagggagc ccgcggagca 1020
gccaggcgat ggcgagagga cggactgtga gcctgtaggg ctagagccgg cagttgagca 1080
gagtagtgcg gcctccgagc tcgcggaggc ctctagcgag gagctcgcag aagcacccac 1140
ggaagcccca agcccagaag ccagagatag caaagaagac gggaggaagt ttgattttga 1200
cgcttgtaat gaagtccctc cggctcctaa agagtcctca accagtgagg gcgctgatca 1260
gaaaatgagt tctcccgaag atgactcgga tacaaaaagg ctttccaaag aggaaaaggg 1320
tcgcagcagt tgtggtagaa atttctgggt tagtggactc tcttctacaa ccagagctac 1380
agatttgaag aatcttttca gcaaatatgg gaaggtggtg ggcgccaagg ttgtgacaaa 1440
tgcccggagt cctggagctc gctgttacgg ttttgtcacg atgtccacag cagaagaggc 1500
cacaaaatgc attaaccacc tgcacaagac ggagctccac ggaaagatga tctccgtgga 1560
gaaagccaaa aatgaacctg tgggaaagaa aacctctgac aaaagagaca gtgacgggaa 1620
aaaggagaag tcgagcaaca gtgacagatc tacaaacctt aagagggatg ataaatgtga 1680
cagaaaagat gatgctaaga agggtgacga cggaagtgga gaaaagagta aggaccaaga 1740
tgatcagaaa cctggcccct cagagcgatc tcgagccaca aagtcaggaa gtcgagggac 1800
cgaacggact gtagtaatgg ataaatccaa aggggtgcct gtgattagtg taaaaacgtc 1860
cgggtccaaa gagagagctt ccaaaagcca ggatcgcaaa tcagccagca gagagaagcg 1920
gtccgtcgtg tcctttgata aggtcaagga gcctcggaag tcaagagact cagagtccca 1980
tagcagggtg cgtgaacgca gtgaacgcga acaacgcatg caggcgcagt gggagcgcga 2040
ggagcgtgag cggctggaga ttgcccgaga gaggctggcc ttccagcgcc agcggctgga 2100
gcgggagcgc atggagcggg aacggctgga gcgcgaacgc atgcacgtgg agcacgacgg 2160
caggcgcgag caggagcgca tccaccgtga gcgcgaggag ctgaggcgcc agcaggaact 2220
gcgctatgag caggagcggc ggcccgcggt gcggcggccc tacgacctgg accggcgaga 2280
tgatgcctat tggccggaag ccaagcgggc cgccctggat gagcgctacc attctgactt 2340
taaccgccag gaccgcttcc acgactttga ccacagggac cgcggccgct accccgacca 2400
ctcggtggac aggagagaag gttcaaggtc aatgatggga gaacgagaag gacagcatta 2460
cccagaacgc catggaggac cagagcgcca cggcggggcc tcccgcgatg gctggggggg 2520
ctatggctct gacaagagga tgagcgaggg ccgggggctg cctcctcccc ccaggggcag 2580
acgtgactgg ggggaccatg gccgaagaga ggatgaccgg tcatggcagg gcacggccga 2640
cgggggcatg atggacaggg atcacaagag gtggcaaggt ggcgagagaa gcatgtccgg 2700
tcactccggg cctggccaca tgatgaaccg aggaggaatg tcagggcgcg gcagctttgc 2760
cccaggcggg gcctcccggg gccaccccat cccacacggt ggcatgcagg gcgggtttgg 2820
aggccagagc cgggggagca ggcccagcga tgcccgcttc actcgccgct actgagtact 2880
tggaatcctg tgtcctgtct cgtggcaaca aggctatgtt ctgttaggag ttaccttaaa 2940
ctgtgtaaaa atattttttt ttaatctgct gccatattgt agctcaatac aatgtgaatt 3000
tgtttttcgt tttggggttt tttttttttg taataaatgt gtttccgttc acataccctt 3060
taaaaaaaaa aaa 3073
<210> 62
<211> 1446
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No.: 2181282
<400> 62
agagtaaaaa atgatcccca agaattcaga gaccaaaagc tggggaccct gaaaaaatac 60
cgtagcatta tgcccaaacc tatcatggtc atacccactt tggcctccct ggcttctcca 120
actacactac agtcccagat gcttgggggc ctaggacagg atgttttgtt aaataattca 180
ctcactccta aatatcttgg ctgtaagcaa gacaacagct cttcccctaa gcccagctcc 240
gtgttcagaa atggattctc tggcattaag aagccttggc acagatgtca cgtctgcaac 300
caccacttcc agttcaaaca gcaccttcga gaccacatga atacacacac caacagacgc 360
ccttacagtt gtcggatttg tcgcaagtcc tatgtacgtc ctggcagcct gagcacacac 420
atgaaacttc atcatggtga gaaccgtctg aagaaactca tgtgttgtga gttttgtgca 480
aaagtgtttg gccacatccg agtctatttt ggccatctga aagaagtgca tagggttgtg 540
65/66
gatgtcattt aaacttaagg aagattgctt tggaaggagc aaatgtaatt
CA 02329685 2000-12-11
WO 99/64596 PGT/I1S99/13Z81 _
atcagcactg agcctgcgcc cagtgaactg cagccaggag acataccaaa gaacagagac 600
atgagtgtgc gaggcatgga gggatcattg gagagggaaa acaagtccaa cctggaagaa 660
gacttccttc taaaccaggc agacgaagtc aaattacaaa tcaaatgtgg tcgttgtcag 720
attactgctc agtcttttgc ggaaataaaa tttcatttac ttgatgttca tggagaggaa 780
attgagggca ggctacaaga agggaccttc ccaggaagca aggggactca ggaagagttg 840
gtgcagcacg ctagccccga ctggaaaagg catcctgaga gagggaagcc ggagaaggtt 900
cattcctcct ccgaggaatc acatgcatgt ccaagactga aaaggcagct ccaccttcat 960
cagaatggcg tggaaatgct catggaaaat gaaggacccc agtcaggaac caacaagcca 1020
agggaaacct gccagggccc tgagtgtcct ggcctccaca cgtttctctt gtggtcccat 1080
tcaggcttta actgcctgct ttgtgcagag atgctgggac ggaaagagga cctcctccac 1140
cactggaagc accagcataa ctgtgaggac ccttccaaac tgtgggctat tttaaatacg 1200
gtctccaacc agggagtgat cgaactttcc agtgaagctg agaaatgaga ccccaaggca 1260
gcctggggtt aaggagagag ctctgccgcc accttccttc agagcttcgt gctttatggt 1320
ggtgcttagt cacaaagatc aaacaacagg attggtgtga gtgaacggaa atgatttttg 1380
tacatggttt tattttctta acgaaataaa atataagctc tcgaagcata tttttctaaa 1440
aaaaaa 1446
66/66
