Note: Descriptions are shown in the official language in which they were submitted.
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PROTEASES AND ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of proteases
and
associated proteins and to the use of these sequences in the diagnosis,
treatment, and prevention of
cell proliferative and immune disorders.
BACKGROUND OF THE INVENTION
Proteolytic processing is an essential component of normal cell growth,
differentiation,
remodeling, and homeostasis. The cleavage of peptide bonds within cells is
necessary for the
maturation of precursor proteins to their active forms, the removal of signal
sequences from
targeted proteins, the degradation of incorrectly folded proteins, and the
controlled turnover of
peptides within the cell. Proteases participate in apoptosis, inflammation,
and tissue remodeling
during embryonic development, wound heating, and normal growth. They are
necessary
components of bacterial, parasitic, and viral invasion and replication within
a host. Four principal
categories of mammalian proteases have been identified based on active site
structure, mechanism
of action, and overall three-dimensional structure. (See Beynon, R.J. and J.S.
Bond ( 1994)
Proteolvtic Enzymes: A Practical Approach, Oxford University Press, New York
NY, pp. I-5.)
The serine proteases (SPs) are a large family of proteolytic enzymes that
include the
digestive enzymes, trypsin and chymotrypsin; components of the complement
cascade and of the
blood-clotting cascade; and enzymes that control the degradation and turnover
of macromolecules
of the extracellular matrix. SPs are so named because of the presence of a
serine residue in the
active site for catalysis of protein cleavage. The active site of an SP is
composed of a triad of
residues including the aforementioned serine, an aspartate, and a histidine
residue. SPs have a
wide range of substrate specificities and can be subdivided into subfamilies
on the basis of these
specificities. The main sub-families are trypases which cleave after arginine
or lysine; aspases
which cleave after aspartate; chymases which cleave after phenylalanine or
leucine; metases which
cleavage after methionine; and serases which cleave after serine. Clp protease
is a unique member
of the serine protease family as its activity is controlled by a regulatory
subunit that binds and
hydrolyzes ATP. Clp protease was originally found in plant chloroplasts but is
believed to be
widespread in both prokaryotic and eukaryotic cells (Maurizi, M.R. et al. (
1990) J. Biol. Chem.
265:12546-12552). SKD3, a mammalian homoiog of the bacterial Clp regulatory
subunit, has
recently been identified in mouse (Perier, F. et al. ( 1995) Gene 152:157-
163).
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Cysteine proteases are involved in diverse cellular processes ranging from the
processing
of precursor proteins to intracellular degradation. Mammalian cysteine
proteases include
lysosomal cathepsins and cytosolic calcium activated proteases, calpains. Of
particular note,
cysteine proteases are produced by monocytes, macrophages and other cells of
the immune system
S which migrate to sites of inflammation and, in their protective role,
secrete various molecules to
repair damaged tissue. These cells may overproduce the same molecules and
cause tissue
destruction in certain disorders. In autoimmune diseases such as rheumatoid
arthritis, the
secretion of the cysteine protease, cathepsin C, degrades collagen, laminin,
elastin and other
structural proteins found in the extracellular matrix of bones. The cathepsin
family of lysosomal
proteases includes the cysteine proteases: cathepsins B, H, K, L, 02, and S;
and the aspartyl
proteases: cathepsins D and G. Various members of this endosomal protease
family are
differentially expressed. Some, such as cathepsin D, have a ubiquitous tissue
distribution while
others, such as cathepsin L, are found only in monocytes, macrophages, and
other cells of the
immune system.
IS Aspartic proteases include bacterial penicillopepsin, mammalian pepsin,
renin, chymosin,
and certain fungal proteases. The characteristic active site residues of
aspartic proteases are a pair
of aspartic acid residues, for example, Asp33 and Asp213 in penicillopepsin.
Aspartic proteases
are also called acid proteases because the optimum pH for their activity is
between 2 and 3. In this
pH range, one of the aspartate residues is ionized and the other is neutral. A
potent inhibitor of
aspartic proteases is the hexapeptide pepstatin which, in the transition
state, resembles normal
substrates.
Carboxypeptidases A and B are the principal mammalian representatives of the
metalloprotease family. Both are exopeptidases of similar structure and active
site configuration.
Carboxypeptidase A, tike chymotrypsin, prefers C-terminal aromatic and
aliphatic side chains of
hydrophobic nature, whereas carboxypeptidase B is directed toward basic
arginine and lysine
residues. Active site components include zinc, which coordinates one histidine
and two glutamic
acid residues in the protein.
Proteasomes and ubiquitin proteases are both associated with the ubiquitin
conjugation
system (UCS), a major pathway for the degradation of cellular proteins in
eukaryotic cells and
some bacteria. Proteasomes are large (2000 kDa), multisubunit complexes
composed of a central
catalytic core containing a variety of proteases, and terminal subunits that
serve in substrate
recognition and regulation of proteasome activity. The UCS mediates the
elimination of abnormal
proteins and regulates the half lives of important regulatory proteins that
control cellular processes
such as gene transcription and cell cycle progression. In the UCS pathway, a
protein targeted for
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degradation is conjugated to ubiquitin, a small, heat-stable protein. The
ubiquitinated protein is
then recognized and degraded by a proteasome, and ubiquitin is released by
ubiquitin protease for
reutilization in the UCS. The UCS is implicated in the degradation of mitotic
cyclic kinases,
oncoproteins, tumor suppressor genes such as p53, viral proteins, cell surface
receptors associated
with signal transduction, transcriptional regulators, and mutated or damaged
proteins
(Ciechanover, A. ( 1994) Cell 79:13-21 ). A murine proto-oncogene, Unp,
encodes a nuclear
ubiquitin protease whose overexpression leads to oncogenic transformation
ofNIH 3T3 cells, and
the human homolog of this gene is consistently elevated in small cell tumors
and adenocarcinomas
ofthe lung (Gray, D.A. (1995) Oncogene 10:2179-2183).
Many other proteolytic enzymes do not fit any of the major categories
discussed above
because their mechanisms of action and/or active sites have not been
elucidated. These include
the aminopeptidases and signal peptidases. Aminopeptidases catalyze the
hydrolysis of amino
acid residues from the amino terminus of peptide substrates. Bovine leucine
aminopeptidase is a
zinc metalloenzyme that utilizes the sulfhydryl groups from at least three
reactive cysteine
residues at its active site in the binding of metal ions (Cuypers, H.T. et al.
( 1982) J. Biol. Chem.
257:7086-7091 ).
Signal peptidases are a specialized class of proteases found in all
prokaryotic and
eukaryotic cell types that serve in the processing of signal peptides. Signal
peptides are
amino-terminal sequences which direct the protein from its ribosomal assembly
site to a particular
cellular or extracellular location. Once the protein has been exported,
removal of the signal
sequence by a signal peptidase and posttranslational processing activate the
protein. Signal
peptidases exist as multi-subunit complexes in both yeast and mammals.
Protease inhibitors and other regulators of protease activity control the
activity and effects
of proteases. Protease inhibitors have been shown to control pathogenesis in
animal models of
proteolytic disorders {Murphy, G. ( 1991 ) Agents Actions Suppl. 35:69-76).
Low levels of the
cystatins, low molecular weight inhibitors of the cysteine proteases,
correlate with malignant
progression of tumors. (Catkins, C. et al. (1995) Biol. Biochem. Hoppe Seyler
376:71-80). Also,
increases in cysteine protease levels, when accompanied by reductions in
inhibitor activity, are
correlated with the pathology of arthritis and immunological diseases in
humans.
Serpins are inhibitors of mammalian plasma serine proteases. Many serpins
serve to
regulate the blood clotting cascade and/or the complement cascade in mammals.
Sp32 is a
positive regulator of the mammalian acrosomal protease, acrosin. Sp32 binds
the proenzyme,
proacrosin, and thereby aides in packaging the enzyme into the acrosomal
matrix (Baba, T. et al.
(1994) J. Biol. Chem. 269:10133-10140).
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The Kunitz family of serine protease inhibitors is characterized by one or
more "Kunitz
domains" containing a series of cysteine residues that are regularly spaced
over approximately 50
amino acid residues and form three intrachain disulfide bonds. Members of this
family include
aprotinin, tissue factor pathway inhibitor (TFPI-1 and TFPI-2), inter-a-
trypsin inhibitor, and
bikunin (Marlor, C.W. et al. ( 1997) J. Biol. Chem. 272:12202-12208). Members
of this family are
potent inhibitors (in the nanomolar range) against serine proteases such as
kallikrein and plasmin.
The discovery of new proteases and associated proteins and the polynucleotides
encoding
them satisfies a need in the art by providing new compositions which are
useful in the diagnosis,
prevention, and treatment of cell proliferative and immune disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, proteases and
associated
proteins referred to collectively as "PPRG" and individually as "PPRG-1,"
"PPRG-2," "PPRG-3,"
"PPRG-4," "pPRG-S," "pPRG-6," "PPRG-7," "pPRG-8," "PPRG-9," "PPRG-10," "PPRG-1
I,"
"PPRG-12," "PPRG-13," "PPRG-14," "PPRG-I5," "PPRG-16," "PPRG-17," "PPRG-18,"
"PPRG-
19," and "PPRG-20." In one aspect, the invention provides a substantially
purified polypeptide
comprising an amino acid sequence selected from the group consisting of SEQ ID
NO: I-20, 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:I-20 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:1-20 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:1-20 and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide
which
hybridizes under stringent conditions to the polynucIeotide encoding the
polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID NO:1-20
and fragments
thereof. The invention also provides an isolated and purified polynucleotide
having a sequence
which is complementary to the polynucleotide encoding the polypeptide
comprising the amino
acid sequence selected from the group consisting of SEQ ID NO:I-20 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
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polynucleotide sequence to at least one of the polynucleotides of the sample,
thereby forming a
hybridization complex; and (b) detecting the hybridization complex, wherein
the presence of the
hybridization complex correlates with the presence of a polynucleotide in the
sample. In one
aspect, the method further comprises amplifying the polynucleotide prior to
hybridization.
The invention also provides an isolated and purified polynucleotide comprising
a
polynucleotide sequence selected from the group consisting of SEQ ID N0:21-40,
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 N0:21-40 and fragments thereof. The invention also
provides an
isolated and purified polynucleotide having a sequence which is complementary
to the
poiynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ
ID N0:21-40 and fragments thereof.
The invention further provides an expression vector containing at least a
fragment of the
polynucleotide encoding the polypeptide comprising an amino acid sequence
selected from the
group consisting of SEQ ID NO: I-20 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: I-20 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:1-20 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 PPRG, the method comprising administering
to a subject in
need of such treatment an effective amount of a pharmaceutical composition
comprising a
substantially purified polypeptide having the amino acid sequence selected
from the group
consisting of SEQ ID NO:1-20 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 PPRG, the method comprising administering
to a subject in
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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-20 and fragments
thereof.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID
NOs), clone identification numbers (clone IDs), cDNA libraries, and cDNA
fragments used to
assemble full-length sequences encoding PPRG.
Table 2 shows features of each polypeptide sequence, including potential
motifs,
homologous sequences, and methods and algorithms used for identification of
PPRG.
Table 3 shows the tissue-specific expression patterns of each nucleic acid
sequence as
determined by northern analysis, diseases, disorders, or conditions associated
with these tissues,
and the vector into which each cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA
clones encoding PPRG were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze PPRG, along
with
applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is
understood that this invention is not limited to the particular machines,
materials and methods
described, as these may vary. It is also to be understood that the terminology
used herein is for the
purpose of describing particular embodiments only, and is not intended to
limit the scope of the
present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a,"
"an," and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such host cells,
and a reference to "an
antibody" is a reference to one or more antibodies and equivalents thereof
known to those skilled
in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although any machines, materials, and methods similar or equivalent
to those described
herein can be used to 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
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construed as an admission that the invention is not entitled to antedate such
disclosure by virtue of
prior invention.
DEFINITIONS
"PPRG" refers to the amino acid sequences of substantially purified PPRG
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 PPRG, increases
or
prolongs the duration of the effect of PPRG. Agonists may include proteins,
nucleic acids,
carbohydrates, or any other molecules which bind to and modulate the effect of
PPRG.
An "allelic variant" is an alternative form of the gene encoding PPRG. 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
I S 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 PPRG include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polynucleotide the same as PPRG
or a polypeptide with at least one functional characteristic of PPRG. Included
within this
definition are polymorphisms which may or may not be readily detectable using
a particular
oligonucleotide probe of the polynucleotide encoding PPRG, and improper or
unexpected
hybridization to allelic variants, with a locus other than the normal
chromosomal locus for the
polynucleotide sequence encoding PPRG. 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 PPRG. 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 immunologicai
activity of PPRG is
retained. For example, negatively charged amino acids may include aspartic
acid and glutamic
acid, positively charged amino acids may include lysine and arginine, and
amino acids with
uncharged polar head groups having similar hydrophilicity values may include
leucine, isoleucine,
and valine; glycine and alanine; asparagine and glutamine; serine and
threonine; and
phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
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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 PPRG 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 PPRG. 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 PPRG,
decreases the
amount or the duration of the effect of the biological or immunological
activity of PPRG.
Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or
any other molecules
I S which decrease the effect of PPRG.
The term "antibody" refers to intact molecules as well as to fragments
thereof, such as
Fab, F(ab'),, and Fv fragments, which are capable of binding the epitopic
determinant. Antibodies
that bind PPRG 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 irnmunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition containing a nucleic acid
sequence which
is complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules
may be produced by any method including synthesis or transcription. Once
introduced into a cell,
the complementary nucleotides combine with natural sequences produced by the
cell to form
duplexes and to block either transcription or translation. The designation
"negative" can refer to
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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 PPRG, or of
any oligopeptide
thereof, to induce a specific immune response in appropriate animals or cells
and to bind with
specific antibodies.
The terms "complementary" and "complementarily" 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'." Complementarily 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 complementarily exists between the single stranded molecules. The degree
of
complementarily 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
IS design and use of peptide nucleic acid (PNA) molecules.
A "composition comprising a given poiynucleotide sequence" and a "composition
comprising a given amino acid sequence" refer broadly to any composition
containing the given
polynucleotide or amino acid sequence. The composition may comprise a dry
formulation or an
aqueous solution. Compositions comprising polynucleotide sequences encoding
PPRG or
fragments of PPRG 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 the XL-PCR kit (Perkin-Etmer, Norwalk
CT) in the S'
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 PPRG, by
northern analysis is indicative of the presence of nucleic acids encoding PPRG
in a sample, and
thereby correlates with expression of the transcript from the polynucleotide
encoding PPRG.
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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
IS 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" and "% identity" refer to the percentage of
sequence
similarity found in a comparison of two or more amino acid or nucleic acid
sequences. Percent
identity can be determined electronically, e.g., by using the MEGALIGN 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.) Parameters for each method may be the default parameters
provided by
MEGALIGN or may be specified by the user. The clustal algorithm groups
sequences into
clusters by examining the distances between 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
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in sequence A, minus the number of gap residues in sequence B, into the sum of
the residue
matches between sequence A and sequence B, times one hundred. Gaps of low or
of no similarity
between the two amino acid sequences are not included in determining
percentage similarity.
Percent identity between nucleic acid sequences can also be counted or
calculated by other
methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (
1990) Methods
Enzymol. 183:626-645.) Identity between sequences can also be determined by
other methods
known in the art, e.g., by varying hybridization conditions.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of
the elements
required for stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules in which the amino
acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to any process by which a strand of nucleic acid binds
with a
I S 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 ~tot analysis)
or formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a
solid support (e.g., paper, membranes, filters, chips, pins or glass slides,
or any other appropriate
substrate to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively,
to the sequence found in the naturally occurring molecule.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by
expression of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which
may affect cellular and systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotides on
a substrate.
The terms "element" and ''array element" in a microarray context, refer to
hybridizable
polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of PPRG. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of PPRG.
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The phrases "nucleic acid" or "nucleic acid sequence," as used herein, refer
to a
nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These
phrases also refer to
DNA or RNA of genomic or synthetic origin which may be single-stranded or
double-stranded
and may represent the sense or the antisense strand, to peptide nucleic acid
(PNA), or to any
DNA-like or RNA-like material. In this context, "fragments" refers to those
nucleic acid
sequences which comprise a region of unique polynucleotide sequence that
specifically identifies
SEQ ID N0:21-40, for example, as distinct from any other sequence in the same
genome. For
example, a fragment of SEQ ID N0:21-40 is useful in hybridization and
amplification
technologies and in analogous methods that distinguish SEQ ID N0:21-40 from
related
polynucleotide sequences. A fragment of SEQ ID N0:21-40 is at least about IS-
20 nucleotides in
length. The precise length of the fragment of SEQ ID N0:21-40 and the region
of SEQ ID
N0:21-40 to which the fragment corresponds are routinely determinable by one
of ordinary skill
in the art based on the intended purpose for the fragment. In some cases, a
fragment, when
translated, would produce polypeptides retaining some functional
characteristic, e.g., antigenicity,
or structural domain characteristic, e.g., ATP-binding site, of the full-
length polypeptide.
The terms "operably associated" and "operably linked" refer to functionally
related
nucleic acid sequences. A promoter is operably associated or operably linked
with a coding
sequence if the promoter controls the translation of the encoded poiypeptide.
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 1 S to 30 nucleotides, and
most preferably about 20
to 25 nucleotides, which can be used in PCR amplification or in a
hybridization assay or
microarray. "Oligonucleotide" is substantially equivalent to the terms
"amplimer," "primer,"
"oligomer," and "probe," as these terms are commonly defined in the art.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in 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 PPRG, or fragments thereof, or PPRG itself, may comprise a
bodily fluid; an
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extract from a cell, chromosome, organelle, or membrane isolated from a cell;
a cell; genomic
DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue
print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, or an antagonist. The
interaction is dependent upon
the presence of a particular structure of the protein, e.g., the antigenic
determinant or epitope,
recognized by the binding molecule. For example, if an antibody is specific
for epitope "A," the
presence of a polypeptide containing the epitope A, or the presence of free
unlabeled A, in a
reaction containing free labeled A and the antibody will reduce the amount of
labeled A that binds
to the antibody.
The term "stringent conditions" refers to conditions which permit
hybridization between
polynucleotides and the claimed polynucleotides. Stringent conditions can be
defined by salt
concentration, the concentration of organic solvent, e.g., formamide,
temperature, and other
conditions well known in the art. In particular, stringency can be increased
by reducing the
concentration of salt, increasing the concentration of formamide, or raising
the hybridization
temperature.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least about 60%
free, preferably about 75% free, and most preferably about 90% free from other
components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or
nucleotides by
different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
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
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limited periods of time.
A "variant" of PPRG polypeptides refers to an amino acid sequence that is
altered by one
or more amino acid residues. The variant may have "conservative" changes,
wherein a substituted
amino acid has similar structural or chemical properties (e.g., replacement of
leucine with
isoleucine). More rarely, a variant may have "nonconservative" changes (e.g.,
replacement of
glycine with tryptophan). Analogous minor variations may also include amino
acid deletions or
insertions, or both. Guidance in determining which amino acid residues may be
substituted,
inserted, or deleted without abolishing biological or immunological activity
may be found using
computer programs well known in the art, for example, LASERGENE software
(DNASTAR).
t 0 The term "variant," when used in the context of a polynucleotide sequence,
may
encompass a polynucleotide sequence related to PPRG. 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 polynucieotide 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 proteases and associated
proteins
(PPRG), the polynucleotides encoding PPRG, and the use of these compositions
for the diagnosis,
treatment, or prevention of cell proliferative and immune disorders.
Table I lists the Incyte clones used to assemble full length nucleotide
sequences encoding
PPRG. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of
the
polypeptide and nucleotide sequences, respectively. Column 3 shows the clone
IDs of the Incyte
clones in which nucleic acids encoding each PPRG were identified, and column 4
shows the
cDNA libraries from which these clones were isolated. Column S shows Incyte
clones and their
corresponding cDNA libraries. Clones for which cDNA libraries are not
indicated were derived
from pooled cDNA libraries. The clones in column ~ were used to assemble the
consensus
nucleotide sequence of each PPRG and are useful as fragments in hybridization
technologies.
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The columns of Table 2 show various properties of each 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 shows potential phosphorylation sites;
column 4 shows
potential glycosylation sites; column S shows the amino acid residues
comprising signature
sequences and motifs; column 6 shows the identity of each polypeptide; and
column 7 shows
analytical methods used to identify each polypeptide 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 PPRG. The first column of Table
3 lists the
nucleotide SEQ ID NOs. Column 2 lists tissue categories which express PPRG as
a fraction of
total tissue categories expressing PPRG. Column 3 lists diseases, disorders,
or conditions
associated with those tissues expressing PPRG. Column 4 lists the vectors used
to subctone the
cDNA library. Of particular note is the kidney-specific expression of SEQ ID
N0:29 in S out of 7
libraries (71 %). Also of note is expression of SEQ ID N0:34 in cervical tumor
libraries (60%).
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA
libraries from which cDNA clones encoding PPRG were isolated. Column 1
references the
nucleotide SEQ ID NOs, column 2 shows the cDNA libraries from which these
clones were
isolated, and column 3 shows the tissue origins and other descriptive
information relevant to the
cDNA libraries in column 2.
The following fragments of the nucleotide sequences encoding PPRG are useful,
for
example, in hybridization or amplification technologies to identify SEQ ID
N0:21-40 and to
distinguish between SEQ ID N0:21-40 and related polynucleotide sequences. The
useful
fragments include the fragment of SEQ ID N0:21 from about nucleotide I to
about nucleotide 56;
the fragment of SEQ ID N0:22 from about nucleotide 161 to about nucleotide
213; the fragment
of SEQ ID N0:23 from about nucleotide 110 to about nucleotide 158; the
fragment of SEQ ID
N0:24 from about nucleotide 117 to about nucleotide 174; the fragment of SEQ
ID N0:25 from
about nucleotide 191 to about nucleotide 245; the fragment of SEQ 1D N0:26
from about
nucleotide 204 to about nucleotide 269; the fragment of SEQ 1D N0:27 from
about nucleotide 181
to about nucleotide 221; the fragments of SEQ ID N0:28 from about nucleotide
509 to about
nucleotide 553, and from about nucleotide 1751 to about nucleotide 1795; the
fragment of SEQ ID
N0:29 from about nucleotide 326 to about nucletide 370; the fragment of SEQ ID
N0:30 from
about nucleotide 1190 to about nucleotide 1234; the fragment of SEQ ID N0:31
from about
nucleotide 283 to about nucleotide 324; the fragment of SEQ ID N0:32 from
about nucleotide 380
to about nucleotide 424; the fragments of SEQ ID N0:33 from about nucleotide
272 to about
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nucleotide 316, and from about nucleotide 1784 to about nucleotide I 831; the
fragment of SEQ ID
N0:34 from about nucleotide 217 to about nucleotide 261; the fragment of SEQ
ID N0:35 from
about nucleotide 2397 to about nucleotide 2441; the fragment of SEQ ID N0:36
from about
nucleotide 218 to about nucleotide 262; the fragments of SEQ ID N0:37 from
about nucleotide
165 to about nucleotide 209, and from about nucleotide 651 to about nucleotide
695; the fragment
of SEQ ID N0:38 from about nucleotide 812 to about nucleotide 856; the
fragment of SEQ ID
N0:39 from about nucleotide 541 to about nucleotide 585; and the fragment of
SEQ ID N0:40
from about nucleotide 163 to about nucleotide 207. The polypeptides encoded by
these fragments
are useful, for example, as immunogenic peptides.
The invention also encompasses PPRG variants. A preferred PPRG 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 PPRG amino acid sequence, and which
contains at least one
functional or structural characteristic of PPRG.
The invention also encompasses polynucleotides which encode PPRG. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence
selected from the group consisting of SEQ ID N0:21-40, which encodes PPRG.
The invention also encompasses a variant of a polynucleotide sequence encoding
PPRG.
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 PPRG. A particular aspect of the invention
encompasses a
variant of a polynucieotide sequence comprising a sequence selected from the
group consisting of
SEQ ID N0:21-40 which has at least about 80%, more preferably at least about
90%, and most
preferably at least about 95% polynucleotide sequence identity to a nucleic
acid sequence selected
from the group consisting of SEQ ID N0:21-40. Any one ofthe polynucleotide
variants described
above can encode an amino acid sequence which contains at least one functional
or structural
characteristic of PPRG.
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 PPRG, 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 PPRG, and all such variations
are to be considered
as being specifically disclosed.
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Although nucleotide sequences which encode PPRG and its variants are
preferably
capable of hybridizing to the nucleotide sequence of the naturally occurring
PPRG under
appropriately selected conditions of stringency, it may be advantageous to
produce nucleotide
sequences encoding PPRG 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 PPRG 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 PPRG
and
PPRG 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 PPRG 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
N0:21-40 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.
152:507-5 I I.) For example, stringent salt concentration will ordinarily be
less than about 750 mM
NaCi and 75 mM trisodium citrate, preferably less than about 500 mM NaCI and
50 mM trisodium
citrate, and most preferably less than about 250 mM NaCI and 25 mM trisodium
citrate. Low
stringency hybridization can be obtained in the absence of organic solvent,
e.g., formamide, while
high stringency hybridization can be obtained in the presence of at least
about 35% formamide,
and most preferably at least about SO% 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
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most preferred embodiment, hybridization will occur at 42°C in 250 mM
NaCI, 25 mM trisodium
citrate, 1% SDS, 50 % formamide, and 200 ~cg/ml ssDNA. Useful variations on
these conditions
will be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash
stringency conditions can be defined by salt concentration and by temperature.
As above, wash
stringency can be increased by decreasing salt concentration or by increasing
temperature. For
example, stringent salt concentration for the wash steps will preferably be
less than about 30 mM
NaCI and 3 mM trisodium citrate, and most preferably less than about 15 mM
NaCI and 1.5 mM
trisodium citrate. Stringent temperature conditions for the wash steps will
ordinarily include
temperature of at least about 25°C, more preferably of at least about
42°C, and most preferably of
at least about 68°C. In a preferred embodiment, wash steps will occur
at 25°C in 30 mM NaCI, 3
mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps
will occur at
42°C in 15 mM NaCI, I.5 mM trisodium citrate, and 0.1% SDS. In a most
preferred embodiment,
wash steps will occur at 68°C in I S 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 polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq
polymerase
(Perkin-Elmer), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucieases such as those found
in the
ELONGASE amplification system (Life Technologies, Gaithersburg MD).
Preferably, sequence
preparation is automated with machines such as the Hamilton MICROLAB 2200
(Hamilton, Reno
NV), Peltier thermal cycler 200 (PTC200; MJ Research, Watertown MA) and the
ABI
CATALYST 800 (Perkin-Elmer). Sequencing is then carried out using either ABI
373 or 377
DNA sequencing systems (Perkin-Elmer), the MEGABACE 1000 DNA sequencing system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting
sequences are analyzed using a variety of algorithms which are well known in
the art. . (See, e.g.,
Ausubel, F.M. ( 1997) Short Protocols in Molecular Biolo y, John Wiley & Sons,
New York NY,
unit 7.7; Meyers, R.A. ( 1995) Molecular Biolo~y and BiotechnoloQV, Wiley VCH,
New York NY,
pp.856-853.)
The nucleic acid sequences encoding PPRG 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
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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
S 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.
I:I 11-I 19.) In
this method, multiple restriction enzyme digestions and ligations may be used
to insert an
engineered double-stranded sequence into a region of unknown sequence before
performing PCR.
Other methods which may be used to retrieve unknown sequences are known in the
art. (See, e.g.,
Parker, J.D. et al. ( 1991 ) Nucleic Acids Res. 19:3055-3060). Additionally,
one may use PCR,
nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk
genomic
DNA. This procedure avoids the need to screen libraries and is useful in
finding 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 SO% 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 oiigo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of
sequence into S' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to
analyze the size or confirm the nucleotide sequence of sequencing or PCR
products. In particular,
capillary sequencing may employ flowable polymers for electrophoretic
separation, four different
nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled
device camera for
detection of the emitted wavelengths. Output/light intensity may be converted
to electrical signal
using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, 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 PPRG may be cloned in recombinant DNA molecules that direct
expression of
PPRG, or fragments or functional equivalents thereof, in appropriate host
cells. Due to the
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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
PPRG.
The nucleotide sequences of the present invention can be engineered using
methods
generally known in the art in order to alter PPRG-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 giycosylation patterns, change codon preference,
produce splice
variants, and so forth.
In another embodiment, sequences encoding PPRG may be synthesized, in whole or
in
part, using chemical methods well known in the art. (See, e.g., Caruthers,
M.H. et al. (1980)
Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. ( 1980) Nucleic Acids
Symp. Ser. 7:225-232.)
Alternatively, PPRG 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 431 A peptide synthesizer (Perkin-Elmer). Additionally, the amino acid
sequence of
PPRG, 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
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 PPRG, the nucleotide sequences
encoding PPRG
or derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which
contains the necessary elements for transcriptional and translational control
of the inserted coding
sequence in a suitable host. These elements include regulatory sequences, such
as enhancers,
constitutive and inducible promoters, and 5' and 3' untranslated regions in
the vector and in
polynucleotide sequences encoding PPRG. Such elements may vary in their
strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding PPRG. Such signals include the ATG initiation codon and
adjacent
sequences, e.g. the Kozak sequence. In cases where sequences encoding PPRG and
its initiation
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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
translationat 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 PPRG and appropriate
transcriptional and
translational control elements. These methods include in vitro recombinant DNA
techniques,
synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook,
J. et al. (1989)
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview
NY, ch. 4, 8, and
16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolo y,
John Wiley & Sons,
IS New York NY, ch. 9, 13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express
sequences encoding PPRG. 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 PPRG.
For example,
routine cloning, subcloning, and propagation of polynucleotide sequences
encoding PPRG can be
achieved using a multifunctional E. coli vector such as PBLUESCRIPT
(Stratagene, La Jolla CA)
or pSPORTI plasmid (Life Technologies). Ligation of sequences encoding PPRG
into the
vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric
screening procedure
for identification of transformed bacteria containing recombinant molecules.
In addition, these
vectors may be useful for in vitro transcription, dideoxy sequencing, single
strand rescue with
helper phage, and creation of nested deletions in the cloned sequence. (See,
e.g., Van Heeke, G.
and S.M. Schuster ( 1989) J. Biol. Chem. 264:5503-5509.) When large quantities
of PPRG are
needed, e.g. for the production of antibodies, vectors which direct high level
expression of PPRG
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may be used. For example, vectors containing the strong, inducible T5 or T7
bacteriophage
promoter may be used.
Yeast expression systems may be used for production of PPRG. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition,
such vectors direct either the secretion or intracellular retention of
expressed proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Grant et ai. ( 1987) Methods Enzymol. 153:516-54; and Scorer, C.
A. et al. ( 1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of PPRG. Transcription of
sequences
encoding PPRG may be driven viral promoters, e.g., the 35S and 195 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;
Broglie, R. et al. ( 1984) Science 224:838-843; and Winter, J. et al. ( 1991 )
Results Probl. Cell
Differ. 17:85-105.) These constructs can be introduced into plant cells by
direct DNA
transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill
Yearbook of
Science and Technolosy (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 PPRG
may be ligated
into an adenovirus transcription/translation complex consisting of the late
promoter and tripartite
leader sequence. Insertion in a non-essential E1 or E3 region of the viral
genome may be used to
obtain infective virus which expresses PPRG in host cells. (See, e.g., Logan,
J. and T. Shenk
( 1984) Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659.) In addition,
transcription enhancers, such as
the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in
mammalian host
cells. SV40 or EBV-based vectors may also be used for high-level protein
expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments
of DNA than can be contained in and expressed from a plasmid. HACs of about 6
kb to 10 Mb
are constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat.
Genet. 15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of PPRG in cell lines is preferred. For example, sequences encoding
PPRG can be
transformed into cell lines using expression vectors which may contain viral
origins of replication
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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, 1, 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.
U.S.A. 77:3567-3570;
Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-I4.) 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. U.S.A. 85:8047-
8051.) Visible
markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), !3
glucuronidase and its
substrate Q-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. Bioi. SS:I2I-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 PPRG is inserted within a marker gene
sequence, transformed
cells containing sequences encoding PPRG can be identified by the absence of
marker gene
function. Alternatively, a marker gene can be placed in tandem with a sequence
encoding PPRG
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 PPRG
and that
express PPRG 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
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protein sequences.
Immunological methods for detecting and measuring the expression of PPRG 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
S fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on PPRG 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) Serolosical Methods a Laboratory
Manual, APS Press, St.
Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in
ImmunoloEV, Greene Pub.
Associates and Wiley-Interscience, New York NY; and Pound, J.D. ( 1998)
Immunochemical
Protocols, Humana Press, Totowa NJ).
A wide variety of labels and conjugation techniques are known by those skilled
in the art
and may be used in various nucleic acid and amino acid assays. Means for
producing labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding PPRG
IS include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled
nucleotide. Alternatively, the sequences encoding PPRG, or any fragments
thereof, may be cloned
into a vector for the production of an mRNA probe. Such vectors are known in
the art, are
commercially available, and may be used to synthesize RNA probes in vitro by
addition of an
appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
These procedures
may be conducted using a variety of commercially available kits, such as those
provided by
Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable
reporter
molecules or labels which may be used for ease of detection include
radionuclides, enzymes,
fluorescent, chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors,
magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding PPRG 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 PPRG may be designed to
contain signal
sequences which direct secretion of PPRG through a prokaryotic or eukaryotic
cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications
of the polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro"
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form of the protein may also be used to specify protein targeting, folding,
and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are
available from
the American Type Culture Collection (ATCC, Bethesda MD) and may be chosen to
ensure the
S correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding PPRG 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 PPRG protein
containing a heterologous moiety that can be recognized by a commercially
available antibody
may facilitate the screening of peptide libraries for inhibitors of PPRG
activity. Heterotogous
protein and peptide moieties may also facilitate purification of fusion
proteins using commercially
available affinity matrices. Such moieties include, but are not limited to,
glutathione S-transferase
(GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding
peptide (CBP), 6-
His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification
of their cognate fusion proteins on immobilized glutathione, maltose,
phenylarsine oxide,
calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and
hemagglutinin (HA) enable
immunoaffinity purification of fusion proteins using commercially available
monoclonal and
polyclonal antibodies that specifically recognize these epitope tags. A fusion
protein may also be
engineered to contain a proteolytic cleavage site located between the PPRG
encoding sequence
and the heteroiogous protein sequence, so that PPRG 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 PPRG 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 PPRG may be produced not only by recombinant production, but also
by
direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton,
supra, 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
fragments of PPRG may be synthesized separately and then combined to produce
the full length
molecule.
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THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of PPRG and proteases and associated proteins. In addition,
the expression of
PPRG is closely associated with cell proliferative conditions, including
cancer, and with
inflammation and the immune response. Therefore, PPRG appears to play a role
in cell
proliferative and immune disorders. In the treatment of cell proliferative and
immune disorders
associated with increased PPRG expression or activity, it is desirable to
decrease the expression or
activity of PPRG. In the treatment of the above conditions associated with
decreased PPRG
expression or activity, it is desirable to increase the expression or activity
of PPRG.
Therefore, in one embodiment, PPRG 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 PPRG. Examples of such disorders include, but are not limited to,
a cell proliferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis,
mixed connective tissue disease {MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria,
polycythemia vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers
of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall
bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas,
parathyroid, penis,
prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus;
and 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, autoimmune
polyenodocrinopathy-
candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact
dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
episodic lymphopenia
with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic
gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis,
Werner syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral,
bacterial, fungal, parasitic, protozoal, and helminthic infections, and
trauma.
In another embodiment, a vector capable of expressing PPRG or a fragment or
derivative
thereof may be administered to a subject to treat or prevent a disorder
associated with decreased
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expression or activity of PPRG including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially
purified PPRG 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 PPRG
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of PPRG
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of PPRG including, but not limited to, those listed above.
In a further embodiment, an antagonist of PPRG may be administered to a
subject to treat
or prevent a disorder associated with increased expression or activity of
PPRG. Examples of such
disorders include, but are not limited to, those described above. In one
aspect, an antibody which
specifically binds PPRG 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 PPRG.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding PPRG may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of PPRG 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 PPRG may be produced using methods which are generally known
in the
art. In particular, purified PPRG may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind PPRG.
Antibodies to PPRG may
also be generated using methods that are well known in the art. Such
antibodies may include, but
are not limited to, polyclonal, monoclonal, chimeric, and single chain
antibodies, Fab fragments,
and fragments produced by a Fab expression library. Neutralizing antibodies
(i.e., those which
inhibit dimer formation) are especially preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice,
humans, and others may be immunized by injection with PPRG or with any
fragment or
oiigopeptide thereof which has immunogenic properties. Depending on the host
species, various
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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 dinitropheno(.
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium~arvum
are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
PPRG have an amino acid sequence consisting of at least about 5 amino acids,
and, more
preferably, of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides,
or fragments are identical to a portion of the amino acid sequence of the
natural protein and
contain the entire amino acid sequence of a small, naturally occurring
molecule. Short stretches of
PPRG 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 PPRG 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. U.S.A.
80:2026-2030; and Cole, S.P. et al. (1984) Mol. Ceil 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. U.S.A. 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
PPRG-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. U.S.A. 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. U.S.A. 86:
3833-3837; Winter, G. et al. ( 1991 ) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for PPRG may also be
generated.
For example, such fragments include, but are not limited to, F(ab')2 fragments
produced by
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pepsin digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide
bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may
be constructed to
allow rapid and easy identification of monoclonal Fab fragments with the
desired specificity.
(See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the
desired specificity. Numerous protocols for competitive binding or
immunoradiometric assays
using either polyclonal or monoclonal antibodies with established
specificities are well known in
the art. Such immunoassays typically involve the measurement of complex
formation between
PPRG and its specific antibody. A two-site, monoclonal-based immunoassay
utilizing monoclonal
antibodies reactive to two non-interfering PPRG 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 PPRG. Affinity
is expressed as an
association constant, Ka, which is defined as the molar concentration of PPRG-
antibody complex
t 5 divided by the molar concentrations of free antigen and free antibody
under equilibrium
conditions. The Ka determined for a preparation of polyclonal antibodies,
which are
heterogeneous in their affinities for multiple PPRG epitopes, represents the
average affinity, or
avidity, of the antibodies for PPRG. The Ka determined for a preparation of
monoclonal
antibodies, which are monospecific for a particular PPRG epitope, represents a
true measure of
affinity. High-affinity antibody preparations with Ka ranging from about 109
to 10'z L/mole are
preferred for use in immunoassays in which the PPRG-antibody complex must
withstand rigorous
manipulations. Low-affinity antibody preparations with Ka ranging from about
106 to 10' L/mole
are preferred for use in immunopurification and similar procedures which
ultimately require
dissociation of PPRG, 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 PPRG-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity,
and guidelines for antibody quality and usage in various applications, are
generally available.
(See, e.g., Catty, supra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding PPRG, or
any
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fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, the
complement of the polynucleotide encoding PPRG 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 PPRG. Thus, complementary
molecules
or fragments may be used to modulate PPRG 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 PPRG.
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 PPRG. (See, e.g., Sambrook, supra; Ausubel, 1995,
supra.)
Genes encoding PPRG can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding PPRG. 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 PPRG. Oligonucleotides derived from
the transcription
initiation site, e.g., between about positions -10 and +1 O from the start
site, are preferred.
Similarly, inhibition can be achieved using triple helix base-pairing
methodology. Triple helix
pairing is useful because it causes inhibition of the ability of the double
helix to open sufficiently
for the binding of polymerases, transcription factors, or regulatory
molecules. Recent therapeutic
advances using triplex DNA have been described in the literature. (See, e.g.,
Gee, J.E. et al.
( 1994) in Huber, B.E. and B.1. Can, Molecular and Immunologic Approaches,
Futura Publishing,
Mt. Kisco NY, pp. 163-I77.) A complementary sequence or antisense molecule may
also be
designed to block translation of mRNA by preventing the transcript from
binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage
of RNA. The mechanism of ribozyme action involves sequence-specific
hybridization of the
ribozyme molecule to complementary target RNA, followed by endonucleolytic
cleavage. For
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example, engineered hammerhead motif ribozyme molecules may specifically and
efficiently
catalyze endonucleolytic cleavage of sequences encoding PPRG.
Specifc ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences:
GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides, corresponding to the region of the target gene containing the
cleavage site, may
be evaluated for secondary structural features which may render the
oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing
accessibility to hybridization
with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared by any method known in the art for the synthesis of nucleic acid
molecules. These
include techniques for chemically synthesizing oligonucleotides such as solid
phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules may be
generated by in vitro
and in vivo transcription of DNA sequences encoding PPRG. Such DNA sequences
may be
IS incorporated into a wide variety of vectors with suitable RNA polymerase
promoters such as T7 or
SP6. Alternatively, these cDNA constructs that synthesize complementary RNA,
constitutively or
inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as
inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and
similarly modified forms
of adenine, cytidine, guanine, thymine, and uridine which are not as easily
recognized by
endogenous endonucleases.
Many methods for introducing vectors into cells or tissues are available and
equally
suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors
may be introduced into
stem cells taken from the patient and clonally propagated for autologous
transplant back into that
same patient. Delivery by transfection, by liposome injections, or by
polycationic amino polymers
may be achieved using methods which are well known in the art. (See, e.g.,
Goldman, C.K. et al.
(1997) Nat. Biotech. 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.
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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 PPRG, antibodies to PPRG, and mimetics, agonists, antagonists, or
inhibitors of PPRG. The
compositions may be administered alone or in combination with at least one
other agent, such as a
stabilizing compound, which may be administered in any sterile, biocompatible
pharmaceutical
carrier including, but not limited to, saline, buffered saline, dextrose, and
water. The compositions
may be administered to a patient alone, or in combination with other agents,
drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered
by any
number of routes including, but not limited to, oral, intravenous,
intramuscular, intra-arterial,
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
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~ton'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 get,
polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable
organic solvents or
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solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee
coatings for
product identification or to characterize the quantity of active compound,
i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of
gelatin, as weft as soft, sealed capsules made of gelatin and a coating, such
as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or
binders, such as lactose or
starches, lubricants, such as talc or magnesium stearate, and, optionally,
stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in suitable
liquids, such as fatty
oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be
formulated in
aqueous solutions, preferably in physiologically compatible buffers such as
Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous injection
suspensions may contain
substances which increase the viscosity of the suspension, such as sodium
carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the active
compounds may be
prepared as appropriate oily injection suspensions. Suitable lipophilic
solvents or vehicles include
t5 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, encapsutating, entrapping, or
lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed
with many
acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic,
tartaric, malic, and
succinic acids. Salts tend to be more soluble in aqueous or other protonic
solvents than are the
corresponding free base forms. In other cases, the preferred preparation may
be a lyophilized
powder which may contain any or all of the following: i mM to 50 mM histidine,
0.1 % to 2%
sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined
with buffer prior to
use.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate container and labeled for treatment of an indicated condition. For
administration of
PPRG, such labeling would include amount, frequency, and method of
administration.
Pharmaceutical compositions suitable for use in the invention include
compositions
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wherein the active ingredients are contained in an effective amount to achieve
the intended
purpose. The determination of an effective dose is well within the capability
of those skilled in the
art.
For any compound, the therapeutically effective dose can be estimated
initially either in
cell culture assays, e.g., of neoplastic cells or in animal models such as
mice, rats, rabbits, dogs, or
pigs. An animal model may also be used to determine the appropriate
concentration range and
route of administration. Such information can then be used to determine useful
doses and routes
for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
PPRG or fragments thereof, antibodies of PPRG, and agonists, antagonists or
inhibitors of PPRG,
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
IS effects is the therapeutic index, which can be expressed as the
LDS°/EDS° ratio. Pharmaceutical
compositions which exhibit large therapeutic indices are preferred. The data
obtained from cell
culture assays and animal studies are used to formulate a range of dosage for
human use. The
dosage contained in such compositions is preferably within a range of
circulating concentrations
that includes the EDS° with little or no toxicity. The dosage varies
within this range depending
upon the dosage form employed, the sensitivity of the patient, and the route
of administration.
The exact dosage will be determined by the practitioner, in Light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of
the active moiety or to maintain the desired effect. Factors which may be
taken into account
include the severity of the disease state, the general health of the subject,
the age, weight, and
gender of the subject, time and frequency of administration, drug
combination(s), reaction
sensitivities, and response to therapy. Long-acting pharmaceutical
compositions may be
administered every 3 to 4 days, every week, or biweekly depending on the half
life and clearance
rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 fig, 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.
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DIAGNOSTICS
In another embodiment, antibodies which specifically bind PPRG may be used for
the
diagnosis of disorders characterized by expression of PPRG, or in assays to
monitor patients being
treated with PPRG or agonists, antagonists, or inhibitors of PPRG. Antibodies
useful for
diagnostic purposes may be prepared in the same manner as described above for
therapeutics.
Diagnostic assays for PPRG include methods which utilize the antibody and a
label to detect
PPRG 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 PPRG, including ELISAs, RIAs, and FACS,
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of PPRG
expression. Normal or standard values for PPRG expression are established by
combining body
fluids or cell extracts taken from normal mammalian subjects, preferably
human, with antibody to
I S PPRG under conditions suitable for complex formation. The amount of
standard complex
formation may be quantitated by various methods, preferably by photometric
means. Quantities of
PPRG 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 PPRG 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 PPRG
may be correlated with disease. The diagnostic assay may be used to determine
absence,
presence, and excess expression of PPRG, and to monitor regulation of PPRG
levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucieotide sequences, including genomic sequences, encoding PPRG ar
closely related
molecules may be used to identify nucleic acid sequences which encode PPRG.
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 PPRG, allelic variants, or related
sequences.
Probes may also be used for the detection of related sequences, and should
preferably
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have at least 50% sequence identity to any of the PPRG encoding sequences. The
hybridization
probes of the subject invention may be DNA or RNA and may be derived from the
sequence of
SEQ ID N0:21-40 or from genomic sequences including promoters, enhancers, and
introns of the
PPRG gene.
Means for producing specific hybridization probes for DNAs encoding PPRG
include the
cloning of polynucleotide sequences encoding PPRG or PPRG 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 PPRG may be used for the diagnosis of
disorders
associated with expression of PPRG. Examples of such disorders include, but
are not limited to, a
cell proliferative disorder such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis,
paroxysmal nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas,
parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus; and
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,
autoimmune
polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis,
contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,
diabetes mellitus,
emphysema, episodic lymphopenia with fymphocytotoxins, 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, poiymyositis, psoriasis, Reiter's syndrome, rheumatoid
arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Wemer syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and
helminthic infections, and trauma. The polynucleotide sequences encoding PPRG
may be used in
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Southern or northern analysis, dot blot, or other membrane-based technologies;
in PCR
technologies; in dipstick, pin, and multiformat ELISA-like assays; and in
microarrays utilizing
fluids or tissues from patients to detect altered PPRG expression. Such
qualitative or quantitative
methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding PPRG may be useful
in assays
that detect the presence of associated disorders, particularly those mentioned
above. The
nucleotide sequences encoding PPRG 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 PPRG 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
PPRG, 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 PPRG, 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.
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Additional diagnostic uses for oligonucleotides designed from the sequences
encoding
PPRG 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
polynucieotide encoding PPRG, or a fragment of a poiynucleotide complementary
to the
S polynucleotide encoding PPRG, and will be employed under optimized
conditions for
identification of a specific gene or condition. Oligomers may also be employed
under less
stringent conditions for detection or quantitation of closely related DNA or
RNA sequences.
Methods which may also be used to quantify the expression of PPRG include
radiolabeiing or biotinylating nucleotides, coamplification of a control
nucleic acid, and
interpolating results from standard curves. (See, e.g., Melby, P.C. et al.
(1993) J. Immunol.
Methods 159:235-244; Duplaa, C. et al. ( 1993) Anal. Biochem. 212:229-236.)
The speed of
quantitation of multiple samples may be accelerated by running the assay in an
ELISA format
where the oligomer of interest is presented in various dilutions and a
spectrophotometric or
colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucieotide 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. U.S.A. 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W09S/251116;
Shalom D. et al. ( 1995) PCT application W095/3SSOS; Heller, R.A. et al. (
1997) Proc. Natl. Acad.
2S Sci. U.S.A. 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 PPRG
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
PI constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997)
Nat. Genet. 1 S:34S-3SS; Price, C.M. ( 1993) Blood Rev. 7:127-134; and Trask,
B.J. ( 1991 ) Trends
Genet. 7:149-I S4.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
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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 PPRG on a physical chromosomal map and a
specific disorder, or a
S 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
positionai cloning or other gene discovery techniques. Once the disease or
syndrome has been
IS crudely Localized by genetic linkage to a particular genomic region, e.g.,
ataxia-telangiectasia to
I 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, PPRG, 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 PPRG 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 PPRG, or
fragments thereof, and washed. Bound PPRG is then detected by methods well
known in the art.
Purified PPRG 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 PPRG specifically compete with a
test compound for
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binding PPRG. In this manner, antibodies can be used to detect the presence of
any peptide which
shares one or more antigenic determinants with PPRG.
In additional embodiments, the nucleotide sequences which encode PPRG may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely
on properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the
preceding description, utilize the present invention to its fullest extent.
The following preferred
specific embodiments are, therefore, to be construed as merely illustrative,
and not limitative of
the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below,
in particular U.S. Ser. No. 60/096,114 and U.S. Ser. No. 60/119,768, 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 (ysed 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, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively,
RNA was isolated directly from tissue lysates using other RNA isolation kits,
e.g., the
POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding
cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were
constructed with the
UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life
Technologies),
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using the recommended procedures or similar methods known in the art. (See,
e.g., Ausubel,
1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oligo
d(T) or random
primers. Synthetic oligonucleotide adapters were ligated to double stranded
cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most
libraries, the cDNA
was size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or
SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or
preparative
agarose gel electrophoresis. cDNAs were ligated into compatible restriction
enzyme sites of the
polylinker of a suitable plasmid, e.g., PBLUESCRIPT piasmid (Stratagene),
pSPORTI plasmid
(Life Technologies), or pINCY (Incyte Pharmaceuticals, Palo Alto CA).
Recombinant plasmids
were transformed into competent E. coli cells including XL1-Blue, XL1-BIueMRF,
or SOLR from
Stratagene or DHSa, DH l OB, or ElectroMAX DH I OB from Life Technologies.
II. Isolation of cDNA Clones
Plasmids were recovered from host cells by in vivo excision, using the UNIZAP
vector
system (Stratagene) or cell lysis. Plasmids were purified using at least one
of the following: a
Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep
purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid,
QIAWELL 8
Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L.
PREP 96 plasmid
purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1 ml of
distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCB in
a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-I4). Host cell
lysis and
thermal cycling steps were carried out in a single reaction mixture. Samples
were processed and
stored in 384-well plates, and the concentration of amplified plasmid DNA was
quantified
fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a
Fluoroskan II
fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
cDNA sequencing reactions were processed using standard methods or high-
throughput
instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) thermal cycler or
the PTC-200
thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser
(Bobbins
Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA
sequencing
reactions were prepared using reagents provided by Amersham Pharmacia Biotech
or supplied in
ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing
ready
reaction kit (Perkin-Elmer). Electrophoretic separation of cDNA sequencing
reactions and
detection of labeled polynucleotides were carried out using the MEGABACE 1000
DNA
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sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing
systems
(Perkin-Elmer) in conjunction with standard ABI protocols and base calling
software; or other
sequence analysis systems known in the art. Reading frames within the cDNA
sequences were
identified using standard methods (reviewed in Ausubel, 1997, supra, unit
7.7). Some of the
cDNA sequences were selected for extension using the techniques disclosed in
Example V.
The polynuc(eotide sequences derived from cDNA sequencing were assembled and
analyzed using a combination of software programs which utilize algorithms
well known to those
skilled in the art. Table 5 summarizes the tools, programs, and algorithms
used and provides
applicable descriptions, references, and threshold parameters. The first
column of Table S shows
the tools, programs, and algorithms used, the second column provides brief
descriptions thereof,
the third column presents appropriate references, all of which are
incorporated by reference herein
in their entirety, and the fourth column presents, where applicable, the
scores, probability values,
and other parameters used to evaluate the strength of a match between two
sequences (the higher
the score, the greater the homology between two sequences). Sequences were
analyzed using
MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA)
and
LASERGENE software (DNASTAR).
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
were then
queried against a selection of public databases such as the GenBank primate,
rodent, mammalian,
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 Conned, and were screened
for open
reading frames using programs based on GeneMark, BLAST, and FASTA. The full
length
polynucleotide sequences were translated to derive the corresponding full
length amino acid
sequences, and these full length sequences were subsequently analyzed by
querying against
databases such as the GenBank databases (described above), SwissProt, BLOCKS,
PRINTS,
Prosite, and Hidden Markov Model (HMM)-based protein family databases such as
PFAM.
HMM is a probabilistic approach which analyzes consensus primary structures of
gene families.
(See, e.g., Eddy, S.R. ( 1996) Curr. Opin. Struct. 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 N0:21-40. Fragments from about 20 to about 4000 nucleotides which are
useful in
hybridization and amplification technologies were described in The Invention
section above.
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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 (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 defned as:
% seguence 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
IS 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 PPRG 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
2S libraries across all categories. Percentage values of tissue-specific and
disease- or condition-
specific expression are reported in Table 3
V. Extension of PPRG Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:21-27 were produced by
extension
of an appropriate fragment of the full length molecule using oligonucleotide
primers designed
from this fragment. 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
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interest. The initial primers were designed from the cDNA using OLIGO 4.06
(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 (Life Technologies) 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-PCR kit
(Perkin-Elmer) and thoroughly mixing the enzyme and reaction mix. PCR was
performed using
the PTC200 thermal cycier (M.J. Research) beginning with 40 pmol of each
primer and the
recommended concentrations of all other components of the kit, with the
following parameters:
Step 1 94C for 1 min (initial denaturation)
Step 2 65 C for 1 min
Step 3 68C for 6 min
Step 4 94C for 15 sec
Step 5 65C for 1 min
Step 6 68C for 7 min
Step 7 Repeat steps 4-6 for an additional
15 cycles
Step 8 94C for IS sec
Step 9 65C for 1 min
Step 10 68C for 7:15 min
Step 1 i Repeat steps 8-10 for an additional
12 cycles
Step 12 72C for 8 min
Step 13 4C (and holding)
A 5 ul to 10 ~cl 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 the QIAQUICK kit (QIAGEN), and trimmed of overhangs using
Klenow
enzyme to facilitate relegation and cloning.
After ethanol precipitation, the products were redissolved in 13 ~1 of
legation buffer, 1/.cl
T4-DNA ligase ( 15 units) and l~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
40 Icl of appropriate media) were transformed with 3 ~l of legation mixture
and cultured in 80 ~1
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, supra,
Appendix A, p. I) containing carbenicillin (2x carb). The following day,
several colonies were
randomly picked from each plate and cultured in i 50 ,ul of liquid LB/2x carb
medium placed in an
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individual well of an appropriate commercially-available sterile 96-well
microtiter plate. The
following day, 5 ~1 of each overnight culture was transferred into a non-
sterile 96-well plate and,
after dilution 1:10 with water, 5 ~I from each sample was transferred into a
PCR array.
For PCR amplification, 18 ul of concentrated PCR reaction mix (3.3x)
containing 4 units
of rTth DNA polymerase, 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 94C for 60 sec
Step 2 94C for 20 sec
Step 3 SSC for 30 sec
Step 4 72 C for 90 sec
Step 5 Repeat steps 2-4 for an additional
29 cycles
Step 6 72C for 180 sec
Step 7 4C (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 sequence of SEQ ID N0:21-27 are used to obtain
5'
regulatory sequences using the procedure above, oligonucleotides designed for
5' extension, and
an appropriate genomic library.
The full length nucleic acid sequences of SEQ ID N0:28-40 were produced by
extension
of an appropriate fragment of the full length molecule using oligonucleotide
primers designed
from this fragment. One primer was synthesized to initiate 5' extension of the
known fragment,
and the other primer to initiate 3' extension of the known fragment. The
initial primers were
designed using OLIGO 4.06 software (National Biosciences), or another
appropriate program, to
be about 22 to 30 nucleotides in length, to have a GC content of about 50% or
more, and to anneal
to the target sequence at temperatures of about 68°C to about
72°C. Any stretch of nucleotides
which would result in hairpin structures and primer-primer dimerizations was
avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art.
PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ
Research, Inc.). The
reaction mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+,
(NHQ)zS04, and (3-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia
Biotech),
ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with
the
following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3
min; Step 2: 94°C, 15 sec;
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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, S min;
Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing I00 p.l
PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes,
Eugene OR)
dissolved in 1 X TE and 0.5 pl of undiluted PCR product into each well of an
opaque fluorimeter
plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The
plate was
scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the
fluorescence of the
sample and to quantify the concentration of DNA. A 5 ~l to 10 ~l 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 W1), and
sonicated or sheared prior to religation into pUC I 8 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.$%)
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. toll 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 carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step I: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, I 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 ( I :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 Iike manner, the nucleotide sequences of SEQ 1D N0:28-40 are used to obtain
5'
regulatory sequences using the procedure above, oligonucleotides designed for
such extension,
and an appropriate genomic library.
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VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:21-40 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 uCi of
[y-'2PJ adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing I 0' 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
IS 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 dodeeyl
sulfate. Hybridization patterns are visualized using autoradiography and
compared.
VII. Microarrays
A chemical coupling procedure and an ink jet device can be used to synthesize
array
elements on the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An
array analogous to a
dot or slot blot may also be used to arrange and link elements to the surface
of a substrate using
thermal, UV, chemical, or mechanical bonding procedures. A typical array may
be produced by
hand or using available methods and machines and contain any appropriate
number of elements.
After hybridization, nonhybridized probes are removed and a scanner used to
determine the levels
and patterns of fluorescence. The degree of complementarity and the relative
abundance of each
probe which hybridizes to an element on the microarray may be assessed through
analysis of the
scanned images.
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may
comprise the elements of the microarray. Fragments suitable for hybridization
can be selected
using software well known in the art such as LASERGENE software (DNASTAR).
Full-length
cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide
sequences of the
present invention, or selected at random from a cDNA library relevant to the
present invention, are
arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed
to the slide using, e.g.,
UV cross-linking followed by thermal and chemical treatments and subsequent
drying. (See, e.g.,
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Schena, M. et ai. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome
Res. 6:639-645.)
Fluorescent probes are prepared and used for hybridization to the elements on
the substrate. The
substrate is analyzed by procedures described above.
VIII. Complementary Polynucleotides
Sequences complementary to the PPRG-encoding sequences, or any parts thereof,
are
used to detect, decrease, or inhibit expression of naturally occurring PPRG.
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
PPRG. 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
PPRG-encoding transcript.
IX. Expression of PPRG
I S Expression and purification of PPRG is achieved using bacterial or virus-
based
expression systems. For expression of PPRG 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 PPRG upon induction
with isopropyl beta-
D-thiogalactopyranoside (IPTG). Expression of PPRG in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autoeraphica californica
nuclear polyhedrosis
virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin
gene of
baculovirus is replaced with cDNA encoding PPRG by either homologous
recombination or
bacterial-mediated transposition involving transfer plasmid intermediates.
Viral infectivity is
maintained and the strong polyhedrin promoter drives high levels of cDNA
transcription.
Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect
cells in most cases,
or human hepatocytes, in some cases. Infection of the latter requires
additional genetic
modifications to baculovirus. (See Engelhard, E.K. et ai. ( 1994) Proc. Natl.
Acad. Sci. U.S.A.
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.)
In most expression systems, PPRG 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.
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GST, a 26-kilodalton enryme from Schistosoma iaponicum, enables the
purification of fusion
proteins on immobilized glutathione under conditions that maintain protein
activity and
antigenicity (Amersham Pharmacia Biotech). Following purification, the GST
moiety can be
proteolyticaily cleaved from PPRG at specifically engineered sites. FLAG, an 8-
amino acid
S peptide, enables immunoaffinity purification using commercially available
monoclonal and
polyelonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six
consecutive histidine
residues, enables purification on metal-chelate resins (QIAGEN). Methods for
protein expression
and purification are discussed in Ausubel ( 1995, supra, ch 10 and 16).
Purified PPRG obtained by
these methods can be used directly in the following activity assay.
X. Demonstration of PPRG Activity
Protease activity of PPRG is measured by the hydrolysis of appropriate
synthetic peptide
substrates conjugated with various chromogenic molecules in which the degree
of hydrolysis is
quantified by spectrophotometric (or fluorometric) absorption of the released
chromophore.
(Beynon, R.J. and J.S. Bond ( 1994) Proteolytic Enzymes: A Practical Approach
Oxford
I S University Press, New York NY, pp.25-55.) Peptide substrates are designed
according to the
category of protease activity as endopeptidase (serine, cysteine, aspartic
proteases),
animopeptidase (leucine aminopeptidase), or carboxypeptidase (Carboxypeptidase
A and B,
procollagen C-proteinase). Chromogens commonly used are 2-naphthylamine, 4-
nitroaniline, and
furylacrylic acid. Assays are performed atambient temperature and contain an
aliquot of the
enzyme and the appropriate substrate in a suitable buffer. Reactions are
carried out in an optical
cuvette and followed by measurement of the increase/decrease in absorbance of
the chromogen
released during hydrolysis of the peptide substrate. The change in absorbance
is proportional to
the enryme activity in the assay.
Regulation of protease activity (agonism or antagonism) by PPRG is measured
using an
appropriate protease assay as described above in the presence or absence of
PPRG as an agonist or
inhibitor of this activity. Protease activity is measured in the absence of
PPRG (control activity)
and in the presence of varying amounts of PPRG. The change in protease
activity compared to the
control is proportional to the amount of PPRG in the assay and is a measure of
the protease
regulatory activity of PPRG.
For example, for inhibitory activity of PPRG-2, the assay is carried out as
described
above for PPRG using a calcium activated protease, such as calpain, assayed in
the absence and in
the presence of various concentrations of PPRG-2. Inhibition of calpain
protease activity is
proportional to the activity of PPRG-2 in the assay. Similarly, for inhibitory
activity of PPRG-4
and PPRG-9, assays are carried out as described above for PPRG using
pancreatic trypsin assayed
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in the absence and in the presence of various concentrations of PPRG-4 or PPRG-
9. Inhibition of
pancreatic trypsin protease activity is proportional to the activity of PPRG-4
or PPRG-9 in the
assay.
XI. Functional Assays
PPRG function is assessed by expressing the sequences encoding PPRG 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 ~g of
recombinant vector are transiently transfected into a human cell line,
preferably of endothelial or
hematopoietic origin, using either liposome formulations or electroporation. I-
2 ~g of an
additional plasmid containing sequences encoding a marker protein are co-
transfected. Expression
of a marker protein provides a means to distinguish transfected cells from
nontransfected cells and
is a reliable predictor of cDNA expression from the recombinant vector. Marker
proteins of
choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a
CD64-GFP fusion
protein. Flow cytomeiry (FCM), an automated, laser optics-based technique, is
used to identify
transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic
state of the cells and
other cellular properties. FCM detects and quantifies the uptake of
fluorescent molecules that
diagnose events preceding or coincident with cell death. These events include
changes in nuclear
DNA content as measured by staining of DNA with propidium iodide; changes in
cell size and
granularity as measured by forward light scatter and 90 degree side light
scatter; down-regulation
of DNA synthesis as measured by decrease in bromodeoxyuridine uptake;
alterations in
expression of cell surface and intracellular proteins as measured by
reactivity with specific
antibodies; and alterations in plasma membrane composition as measured by the
binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. ( 1994) Flow Cytometry, Oxford, New York NY.
The influence of PPRG on gene expression can be assessed using highly purified
populations of cells transfected with sequences encoding PPRG 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 PPRG and other genes
of interest can
be analyzed by northern analysis or microarray techniques.
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XII. Production of PPRG Specific Antibodies
PPRG 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.
S Alternatively, the PPRG amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic cegions are
well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides 1S residues in length are synthesized using an ABI
431A peptide
synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH (Sigma-
Aldrich, St. Louis
MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to
increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with
the oligopeptide-
ICLH complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide activity
1S by, for example, binding the peptide to plastic, blocking with 1% BSA,
reacting with rabbit
antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XIII. Purification of Naturally Occurring PPRG Using Specific Antibodies
Naturally occurring or recombinant PPRG is substantially purified by
immunoaffinity
chromatography using antibodies specific for PPRG. An immunoaffinity column is
constructed by
covalently coupling anti-PPRG 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 PPRG are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of PPRG (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/PPRG 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 PPRG is collected.
XIV. Identification of Molecules Which Interact with PPRG
PPRG, or biologically active fragments thereof, are labeled with 'ZSI Bolton-
Hunter
reagent. (See, e.g., Bolton, A.E. and W.M. Hunter ( 1973) Biochem. J. 133:529-
539.) Candidate
molecules previously arrayed in the wells of a multi-well plate are incubated
with the labeled
PPRG, washed, and any wells with labeled PPRG complex are assayed. Data
obtained using
different concentrations of PPRG are used to calculate values for the number,
affinity, and
association of PPRG with the candidate molecules.
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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.
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CA 02338386 2001-02-02
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CA 02338386 2001-02-02
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CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
BANDMAN, Olga
HILLMAN, Jennifer L.
BAUGHN, Mariah R.
AZIMZAI, Yalda
GUEGLER, Karl J.
CORLEY, Neil C.
YUE, Henry
TANG, Y. Tom
REDDY, Roopa
PATTERSON, Chandra
AU-YOUNG, Janice
SHI, Leo L.
LU, Dyung Aina M.
<120> PROTEASES AND ASSOCIATED PROTEINS
<130> PF-OS69 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/096,114; 60/119,768
<151> 1998-08-10; 1999-02-11
<160> 40
<170> PERL Program
<210> 1
<211> 206
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1220330
<400> 1
Met Pro Ser Arg Arg Arg Asp Ala Ile Lys Val Met Gln Arg Phe
1 5 10 15
Ala Gly Leu Pro Glu Thr Gly Arg Met Asp Pro Gly Thr Val Ala
20 25 30
Thr Met Arg Lys Pro Arg Cys Ser Leu Pro Asp Val Leu Gly Val
35 40 45
Ala Gly Leu Val Arg Arg Arg Arg Arg Tyr Ala Leu Ser Gly Ser
50 55 60
Val Trp Lys Lys Arg Thr Leu Thr Trp Arg Val Arg Ser Phe Pro
65 70 75
Gln Ser Ser Gln Leu Ser Gln Glu Thr Val Arg Val Leu Met Ser
80 85 90
Tyr Ala Leu Met Ala Trp Gly Met Glu Ser Gly Leu Thr Phe His
95 100 105
Glu Val Asp Ser Pro Gln Gly Gln Glu Pro Asp Ile Leu Ile Asp
1 /42
CA 02338386 2001-02-02
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ll0 115 120
Phe Ala Arg Ala Phe His Gln Asp Ser Tyr Pro Phe Asp Gly Leu
125 130 I35
Gly Gly Thr Leu Ala His Ala Phe Phe Pro Gly Glu His Pro Ile
140 145 150
Ser Gly Asp Thr His Phe Asp Asp Glu Glu Thr Trp Thr Phe Gly
155 160 165
Ser Lys Ala Ser Gln Gln Leu Glu Gln Glu Leu Ala Gly Gly Ser
170 175 180
Pro Val Asp Glu Glu Leu Gly Phe Ser Arg Gly Trp Arg Val Asn
185 190 195
Pro Leu Gly Pro Gly Ser Pro Glu Arg Leu Ser
200 205
<210> 2
<211> 754
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1342493
<400> 2
Met Ala Phe Ala Ser Trp Trp Tyr Lys Thr His Val Ser Glu Lys
1 5 10 15
Thr Ser Glu Ser Pro Ser Lys Pro Gly Glu Lys Lys Gly Ser Asp
20 25 30
Glu Lys Lys Ala Ala Ser Leu Gly Ser Ser Gln Ser Ser Arg Thr
35 40 45
Tyr Ala Gly Gly Thr Ala Ser Ala Thr Lys Val Ser Ala Ser Ser
50 55 60
Gly Ala Thr Ser Lys Ser Ser Ser Met Asn Pro Thr Glu Thr Lys
65 70 75
Ala Val Lys Thr Glu Pro Glu Lys Lys Ser Gln Ser Thr Lys Leu
80 85 90
Ser Val Val His Glu Lys Lys Ser Gln Glu Gly Lys Pro Lys Glu
95 100 105
His Thr Glu Pro Lys Ser Leu Pro Lys Gln Ala Ser Asp Thr Gly
110 115 120
Ser Asn Asp Ala His Asn Lys Lys Ala Val Ser Arg Ser Ala Glu
125 130 135
Gln Gln Pro Ser Glu Lys Ser Thr Glu Pro Lys Thr Lys Pro Gln
140 145 150
Asp Met Ile Ser Ala Gly Gly Glu Ser Val Ala Gly Ile Thr Ala
155 160 165
Ile Ser Gly Lys Pro Gly Asp Lys Lys Lys Glu Lys Lys Ser Leu
170 175 180
Thr Pro Ala Val Pro Val Glu Ser Lys Pro Asp Lys Pro Ser Gly
185 190 195
Lys Ser Gly Met Asp Ala Ala Leu Asp Asp Leu Ile Asp Thr Leu
200 205 210
Gly Gly Pro Glu Glu Thr Glu Glu Glu Asn Thr Thr Tyr Thr Gly
215 220 225
Pro Glu Val Ser Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu
230 235 240
2/42
CA 02338386 2001-02-02
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Giy Lys Arg Glu Val Thr Ile Pro Pro Lys Tyr Arg Glu Leu Leu
245 250 255
Ala Lys Lys Glu Gly Ile Thr Gly Pro Pro Ala Asp Ser Ser Lys
260 265 270
Pro Ile Gly Pro Asp Asp Ala Ile Asp Ala Leu Ser Ser Asp Phe
275 280 285
Thr Cys Gly Ser Pro Thr Ala Ala Gly Lys Lys Thr Glu Lys Glu
290 295 300
Glu Ser Thr Glu Val Leu Lys Ala Gln Ser Ala Gly Thr Val Arg
305 310 315
Ser Ala Ala Pro Pro Gln Glu Lys Lys Arg Lys Val Glu Lys Asp
320 325 330
Thr Met Ser Asp Gin Ala Leu Glu Ala Leu Ser Ala Ser Leu Gly
335 340 345
Thr Arg Gln Ala Glu Pro GIu Leu Asp Leu Arg Ser Ile Lys Glu
350 355 360
Val Asp Glu Ala Lys Ala Lys Glu Glu Lys Leu Glu Lys Cys Gly
365 370 375
Glu Asp Asp Glu Thr Ile Pro Ser Glu Tyr Arg Leu Lys Pro Ala
380 385 390
Thr Asp Lys Asp Gly Lys Pro Leu Leu Pro Glu Pro Glu Glu Lys
395 400 405
Pro Lys Pro Arg Ser Glu Ser Glu Leu Ile Asp Glu Leu Ser Glu
410 415 420
Asp Phe Asp Arg Ser Glu Cys Lys Glu Lys Pro Ser Lys Pro Thr
425 430 435
Glu Lys Thr Glu Glu Ser Lys Ala Ala Ala Pro Ala Pro Val Ser
440 445 450
Glu Ala Val Cys Arg Thr Ser Met Cys Ser Ile Gln Ser Ala Pro
455 460 465
Pro Glu Pro Ala Thr Leu Lys Gly Thr Val Pro Asp Asp Ala Val
470 475 480
Glu Ala Leu Ala Asp Ser Leu Gly Lys Lys Glu Ala Asp Pro Glu
485 490 495
Asp Gly Lys Pro Val Met Asp Lys Val Lys Giu Lys Ala Lys Glu
500 505 510
Glu Asp Arg Glu Lys Leu Gly Glu Lys Glu Glu Thr Ile Pro Pro
515 520 525
Asp Tyr Arg Leu Glu Glu Val Lys Asp Lys Asp Gly Lys Pro Leu
530 535 540
Leu Pro Lys Glu Ser Lys Glu Gln Leu Pro Pro Met Ser Glu Asp
545 550 555
Phe Leu Leu Asp Ala Leu Ser Glu Asp Phe Ser Gly Pro Gln Asn
560 565 570
Ala Ser Ser Leu Lys Phe Glu Asp Ala Lys Leu Ala Ala Ala Ile
575 580 585
Ser Glu Val Val Ser Gln Thr Pro Ala Ser Thr Thr Gln Ala Gly
590 595 600
Ala Pro Pro Arg Asp Thr Ser Gln Ser Asp Lys Asp Leu Asp Asp
605 610 615
Aia Leu Asp Lys Leu Ser Asp Ser Leu Gly Gln Arg Gln Pro Asp
620 625 630
Pro Asp Glu Asn Lys Pro Met Glu Asp Lys Val Lys Glu Lys Ala
635 640 645
Lys Ala Glu His Arg Asp Lys Leu Gly Glu Arg Asp Asp Thr Ile
650 655 660
Pro Pro Glu Tyr Arg His Leu Leu Asp Asp Asn Gly Gln Asp Lys
3/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
665 670 675
Pro Val Lys Pro Pro Thr Lys Lys Ser Glu Asp Ser Lys Lys Pro
680 685 690
Ala Asp Asp Gln Asp Pro Ile Asp Ala Leu Ser Gly Asp Leu Asp
695 700 705
Ser Cys Pro Ser Thr Thr Glu Thr Ser Gln Asn Thr Ala Lys Asp
710 715 720
Lys Cys Lys Lys Ala Ala Ser Ser Ser Lys Ala Pro Lys Asn Gly
725 730 735
Gly Lys Ala Lys Asp Ser Ala Lys Thr Thr Glu Glu Thr Ser Lys
740 745 750
Pro Lys Asp Asp
<210> 3
<211> 308
<222> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1698270
<400> 3
Met Gly Glu Ile Lys Val Ser Pro Asp Tyr Asn Trp Phe Arg Gly
1 5 10 15
Thr Val Pro Leu Lys Lys Ile Ile Val Asp Asp Asp Asp Ser Lys
20 25 30
Ile Trp Ser Leu Tyr Asp Ala Gly Pro Arg Ser Ile Arg Cys Pro
35 40 45
Leu Ile Phe Leu Pro Pro Val Ser Gly Thr Ala Asp Val Phe Phe
50 55 60
Arg Gln Ile Leu Ala Leu Thr Gly Trp Gly Tyr Arg Val Ile Ala
65 70 75
Leu Gln Tyr Pro Val Tyr Trp Asp His Leu Glu Phe Cys Asp Gly
80 85 90
Phe Arg Lys Leu Leu Asp His Leu Gln Leu Asp Lys Val His Leu
95 I00 105
Phe Gly Ala Ser Leu Gly Gly Phe Leu Ala Gln Lys Phe Ala Glu
110 115 120
Tyr Thr His Lys Ser Pro Arg Val His Ser Leu ile Leu Cys Asn
125 130 135
Ser Phe Ser Asp Thr Ser Ile Phe Asn Gln Thr Trp Thr Ala Asn
140 145 150
Ser Phe Trp Leu Met Pro Ala Phe Met Leu Lys Lys Ile Val Leu
I55 160 I65
Gly Asn Phe Ser Ser Gly Pro Val Asp Pro Met Met Ala Asp Ala
170 175 180
Ile Asp Phe Met Val Asp Arg Leu Glu Ser Leu Gly Gln Ser Glu
185 190 195
Leu Ala Ser Arg Leu Thr Leu Asn Cys Gln Asn Ser Tyr Val Glu
200 205 210
Pro His Lys Ile Arg Asp Ile Pro Val Thr Ile Met Asp Val Phe
215 220 225
Asp Gln Ser Ala Leu Ser Thr Glu Ala Lys Glu Glu Met Tyr Lys
230 235 240
Leu Tyr Pro Asn Ala Arg Arg Ala His Leu Lys Thr Gly Gly Asn
4/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
245 250 255
Phe Pro Tyr Leu Cys Arg Ser Ala Glu Val Asn Leu Tyr Val Gln
260 265 270
Ile His Leu Leu Gln Phe His Gly Thr Lys Tyr Ala Ala Ile Asp
275 280 285
Pro Ser Met Val Ser Ala Glu Glu Leu Glu Val Gln Lys Gly Ser
290 295 300
Leu Gly Ile Ser Gln Glu Glu Gln
305
<210> 4
<211> 164
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2012492
<400> 4
Met Lys Thr Gln Asp Gly Gly Ile His Ser Glu Gly Ala Ala Ala
1 5 10 15
Glu His Ser Lys Phe Gly Asn His Gln Lys Gly Trp Pro Leu Phe
20 25 30
Asn Met Gly Ser Ser Gly Leu Leu Ser Leu Leu Val Leu Phe Val
35 40 45
Leu Leu Ala Asn Val Gln Gly Pro Gly Leu Thr Asp Trp Leu Phe
50 55 60
Pro Arg Arg Cys Pro Lys Ile Arg Glu Glu Cys Glu Phe Gln Glu
65 70 75
Arg Asp Val Cys Thr Lys Asp Arg Gln Cys Gln Asp Asn Lys Lys
80 85 90
Cys Cys Val Phe Ser Cys Gly Lys Lys Cys Leu Asp Leu Lys Gln
95 100 I05
Asp Val Cys Glu Met Pro Lys Glu Thr Gly Pro Cys Leu Ala Tyr
110 115 120
Phe Leu His Trp Trp Tyr Asp Lys Lys Asp Asn Thr Cys Ser Met
125 130 135
Phe Val Tyr Gly Gly Cys Gln Gly Asn Asn Asn Asn Phe Gln Ser
140 145 150
Lys Ala Asn Cys Leu Asn Thr Cys Lys Asn Lys Arg Phe Pro
155 160
<2i0> 5
<211> 565
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2309875
<400> 5
Met Pro Gln Ala Ser Glu His Arg Leu Gly Arg Thr Arg Glu Pro
1 5 10 15
Pro Val Asn Ile Gln Pro Arg Val Gly Ser Lys Leu Pro Phe Ala
5/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
20 25 30
Pro Arg Ala Arg Ser Lys Glu Arg Arg Asn Pro Ala Ser Gly Pro
35 40 45
Asn Pro Met Leu Arg Pro Leu Pro Pro Arg Pro Gly Leu Pro Asp
50 55 60
Glu Arg Leu Lys Lys Leu Glu Leu Gly Arg Gly Arg Thr Ser Gly
65 70 75
Pro Arg Pro Arg Gly Pro Leu Arg Ala Asp His Gly Val Pro Leu
80 85 90
Pro Gly Ser Pro Pro Pro Thr Val Ala Leu Pro Leu Pro Ser Arg
95 100 105
Thr Asn Leu Ala Arg Ser Lys Ser Val Ser Ser Gly Asp Leu Arg
110 115 120
Pro Met Gly Ile Ala Leu Gly Gly His Arg Gly Thr Gly Glu Leu
125 130 135
Gly Ala Ala Leu Ser Arg Leu Ala Leu Arg Pro Glu Pro Pro Thr
140 145 150
Leu Arg Arg Ser Thr Ser Leu Arg Arg Leu Gly Gly Phe Pro Gly
155 160 165
Pro Pro Thr Leu Phe Ser Ile Arg Thr Glu Pro Pro Ala Ser His
170 175 180
Gly Ser Phe His Met Ile Ser Ala Arg Ser Ser Glu Pro Phe Tyr
185 190 195
Ser Asp Asp Lys Met Ala His His Thr Leu Leu Leu Gly Ser Gly
200 205 220
His Val Gly Leu Arg Asn Leu Gly Asn Thr Cys Phe Leu Asn Ala
2I5 220 225
Val Leu Gln Cys Leu Ser Ser Thr Arg Pro Leu Arg Asp Phe Cys
230 235 240
Leu Arg Arg Asp Phe Arg Gln Glu Val Pro Gly Gly Gly Arg Ala
245 250 255
GIn Glu Leu Thr Glu Ala Phe Ala Asp Val Ile Gly Ala Leu Trp
260 265 270
His Pro Asp Ser Cys Glu Ala Val Asn Pro Thr Arg Phe Arg Ala
275 280 285
Val Phe Gln Lys Tyr Val Pro Ser Phe Ser Gly Tyr Ser Gln Gln
290 295 300
Asp Ala Gln Glu Phe Leu Lys Leu Leu Met Glu Arg Leu His Leu
305 310 315
Glu Ile Asn Arg Arg Gly Arg Arg Ala Pro Pro Ile Leu Ala Asn
320 325 330
Gly Pro VaI Pro Ser Pro Pro Arg Arg Gly Gly Ala Leu Leu Glu
335 340 345
Glu Pro Glu Leu Ser Asp Asp Asp Arg Ala Asn Leu Met Trp Lys
350 355 360
Arg Tyr Leu Glu Arg Glu Asp Ser Lys Ile Val Asp Leu Phe Val
365 370 375
Gly Gln Leu Lys Ser Cys Leu Lys Cys Gln Ala Cys Gly Tyr Arg
380 385 390
Ser Thr Thr Phe Glu Val Phe Cys Asp Leu Ser Leu Pro Ile Pro
395 400 405
Lys Lys Gly Phe Ala Gly Gly Lys Val Ser Leu Arg Asp Cys Phe
410 415 420
Asn Leu Phe Thr Lys Glu Glu Glu Leu Glu Ser Glu Asn Ala Pro
425 430 435
Val Cys Asp Arg Cys Arg Gln Lys Thr Arg Ser Thr Lys Lys Leu
440 445 450
6/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/1?818
Thr Val Gln Arg Phe Pro Arg Ile Leu Val Leu His Leu Asn Arg
455 460 465
Phe Ser Ala Ser Arg Gly Ser Ile Lys Lys Ser Ser Val Gly Val
470 475 480
Asp Phe Pro Leu Gln Arg Leu Ser Leu Gly Asp Phe Ala Ser Asp
485 490 495
Lys Ala Gly Ser Pro Val Tyr Gln Leu Tyr Ala Leu Cys Asn His
500 505 510
Ser Gly Ser Val His Tyr Gly His Tyr Thr Ala Leu Cys Arg Cys
515 520 525
Gln Thr Gly Trp His Val Tyr Asn Asp Ser Arg Val Ser Pro Val
530 535 540
Ser Glu Asn Gln Val Ala Ser Ser Glu Gly Tyr Val Leu Phe Tyr
545 550 555
Gln Leu Met Gln Glu Pro Pro Arg Cys Leu
560 565
<210> 6
<211> 421
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2479394
<400> 6
Met Arg Trp Ile Leu Phe Ile GIy Ala Leu Ile Gly Ser Ser Ile
1 5 10 15
Cys Gly Gln Glu Lys Phe Phe Gly Asp Gln Val Leu Arg Ile Asn
20 25 30
Val Arg Asn Gly Asp Glu Ile Ser Lys Leu Ser Gln Leu Val Asn
35 40 45
Ser Asn Asn Leu Lys Leu Asn Phe Trp Lys Ser Pro Ser Ser Phe
50 55 60
Asn Arg Pro Val Asp Val Leu Val Pro Ser Val Ser Leu Gln Ala
65 70 75
Phe Lys Ser Phe Leu Arg Ser Gln Gly Leu Glu Tyr Ala Val Thr
80 85 90
Ile Glu Asp Leu Gln Ala Leu Leu Asp Asn Glu Asp Asp Glu Met
95 100 105
Gln His Asn Glu Gly Gln Glu Arg Ser Ser Asn Asn Phe Asn Tyr
110 115 120
Gly Ala Tyr His Ser Leu Glu Ala Ile Tyr His Glu Met Asp Asn
125 130 135
Ile AIa Ala Asp Phe Pro Asp Leu Ala Arg Arg Val Lys Ile Gly
140 145 150
His Ser Phe Glu Asn Arg Pro Met Tyr Val Leu Lys Phe Ser Thr
155 160 165
GIy Lys Gly Val Arg Arg Pro Ala Val Trp Leu Asn Ala Gly Ile
170 175 1B0
His Ser Arg Glu Trp Ile Ser Gln Ala Thr Ala Ile Trp Thr Ala
185 190 195
Arg Lys Ile Val Ser Asp Tyr Gln Arg Asp Pro Ala Ile Thr Ser
200 205 210
Ile Leu Glu Lys Met Asp Ile Phe Leu Leu Pro Val Ala Asn Pro
7/42
CA 02338386 2001-02-02
- WO 00/09709 PCT/US99/17818
215 220 225
Asp Gly Tyr Val Tyr Thr Gln Thr Gln Asn Arg Leu Trp Arg Lys
230 235 240
Thr Arg Ser Arg Asn Pro Gly Ser Ser Cys Ile Gly Ala Asp Pro
245 250 255
Asn Arg Asn Trp Asn Ala Ser Phe Ala Gly Lys Gly Ala Ser Asp
260 265 270
Asn Pro Cys Ser Glu Val Tyr His Gly Pro His Ala Asn Ser Glu
275 280 285
Val Glu Val Lys Ser Val Val Asp Phe Ile Gln Lys His Gly Asn
290 295 300
Phe Lys GIy Phe Ile Asp Leu His Ser Tyr Ser Gln Leu Leu Met
305 310 315
Tyr Pro Tyr Gly Tyr Ser Val Lys Lys Ala Pro Asp Ala Glu Glu
320 325 330
Leu Asp Lys Val Ala Arg Leu Ala Ala Lys Ala Leu Ala Ser Val
335 340 345
Ser Gly Thr Glu Tyr Gln Val Gly Pro Thr Cys Thr Thr Val Tyr
350 355 360
Pro Ala Ser Gly Ser Ser Ile Asp Trp Ala Tyr Asp Asn Gly Ile
365 370 375
Lys Phe Ala Phe Thr Phe Glu Leu Arg Asp Thr Gly Thr Tyr Gly
380 385 390
Phe Leu Leu Pro Ala Asn Gln Ile Ile Pro Thr Ala Glu Glu Thr
395 400 405
Trp Leu Gly Leu Lys Thr Ile Met Glu His Val Arg Asp Asn Leu
410 415 420
Tyr
<210> 7
<211> 666
<2I2> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2613215
<400> 7
Met Ala Ala Ser Arg Lys Pro Pro Arg Val Arg Val Asn His Gln
1 5 10 15
Asp Phe Gln Leu Arg Asn Leu Arg Ile Ile Glu Pro Asn Glu Val
20 25 30
Thr His Ser Gly Asp Thr Gly Val Glu Thr Asp Gly Arg Met Pro
35 40 45
Pro Lys Val Thr Ser Glu Leu Leu Arg Gln Leu Arg Gln Ala Met
50 55 60
Arg Asn Ser Glu Tyr Val Thr Glu Pro Ile Gln Ala Tyr Ile Ile
65 70 75
Pro Ser Gly Asp Ala His Gln Ser Glu Tyr Ile Ala Pro Cys Asp
80 85 90
Cys Arg Arg Ala Phe Val Ser Gly Phe Asp Gly Ser Ala Gly Thr
95 100 105
Ala Ile Ile Thr Glu Glu His Ala Ala Met Trp Thr Asp Gly Arg
IIO 115 120
Tyr Phe Leu Gln Ala Ala Lys Gln Met Asp Ser Asn Trp Thr Leu
8/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
125 130 135
Met Lys Met Gly Leu Lys Asp Thr Pro Thr Gln Glu Asp Trp Leu
140 145 150
Val Ser Val Leu Pro Glu Gly Ser Arg Val Gly Val Asp Pro Leu
155 160 16S
Ile Ile Pro Thr Asp Tyr Trp Lys Lys Met Ala Lys Val Leu Arg
170 175 180
Ser Ala Gly His His Leu Ile Pro Val Lys Glu Asn Leu Val Asp
185 190 195
Lys Ile Trp Thr Asp Arg Pro Glu Arg Pro Cys Lys Pro Leu Leu
200 205 210
Thr Leu Gly Leu Asp Tyr Thr Gly Ile Ser Trp Lys Asp Lys Val
215 220 225
Ala Asp Leu Arg Leu Lys Met Ala Glu Arg Asn Val Met Trp Phe
230 235 240
Val Val Thr Ala Leu Asp Glu Ile Ala Trp Leu Phe Asn Leu Arg
245 2S0 255
Gly Ser Asp Val Glu His Asn Pro Val Phe Phe Ser Tyr Ala Ile
260 265 270
Ile Gly Leu Glu Thr Ile Met Leu Phe Ile Asp Gly Asp Arg Ile
275 280 285
Asp Ala Pro Ser Val Lys Glu His Leu Leu Leu Asp Leu Gly Leu
290 295 300
Glu Ala Glu Tyr Arg Ile Gln Val His Pro Tyr Lys Ser Ile Leu
305 310 315
Ser Glu Leu Lys Ala Leu Cys Ala Asp Leu Ser Pro Arg Glu Lys
320 325 330
Val Trp Val Ser Asp Lys Ala Ser Tyr Ala Val Ser Glu Thr Ile
335 340 345
Pro Lys Asp His Arg Cys Cys Met Pro Tyr Thr Pro Ile Cys Ile
350 355 360
Ala Lys Ala Val Lys Asn Ser Ala Glu Ser Glu Gly Met Arg Arg
365 370 375
Ala His Ile Lys Asp Ala Val Ala Leu Cys Glu Leu Phe Asn Trp
380 385 390
Leu Glu Lys Glu Val Pro Lys Gly Gly Val Thr Glu Ile Ser Ala
395 400 405
Ala Asp Lys Ala Glu Glu Phe Arg Arg Gln Gln Ala Asp Phe Val
410 415 420
Asp Leu Ser Phe Pro Thr Ile Ser Ser Thr Gly Pro Asn Gly Ala
425 430 435
Ile Ile His Tyr Ala Pro Val Pro Glu Thr Asn Arg Thr Leu Ser
440 445 450
Leu Asp Glu Val Tyr Leu Ile Asp Ser Gly Ala Gln Tyr Lys Asp
455 460 465
Gly Thr Thr Asp Val Thr Arg Thr Met His Phe Gly Thr Pro Thr
470 475 480
Ala Tyr Glu Lys Glu Cys Phe Thr Tyr Val Leu Lys Gly His Ile
485 490 495
Ala Val Ser Ala Ala Val Phe Pro Thr Gly Thr Lys Gly His Leu
500 505 510
Leu Asp Ser Phe Ala Arg Ser Ala Leu Trp Asp Ser Gly Leu Asp
515 520 525
Tyr Leu His Gly Thr Gly His Gly Vai Gly Ser Phe Leu Asn Val
530 535 540
His Glu Gly Pro Cys Gly Ile Ser Tyr Lys Thr Phe Ser Asp Glu
545 550 555
9/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
Pro Leu Glu Ala Gly Met Ile Val Thr Asp Glu Pro Gly Tyr Tyr
560 565 570
Glu Asp Gly Ala Phe Gly Ile Arg Ile Glu Asn Val Val Leu Val
575 580 585
Val Pro Val Lys Thr Lys Tyr Asn Phe Asn Asn Arg Gly Ser Leu
590 595 600
Thr Phe Glu Pro Leu Thr Leu Val Pro Ile Gln Thr Lys Met Ile
605 610 615
Asp Val Asp Ser Leu Thr Asp Lys Glu Cys Asp Trp Leu Asn Asn
620 625 630
Tyr His Leu Thr Cys Arg Asp Val Ile Gly Lys Glu Leu G1n Lys
635 640 645
Gln Gly Arg Gln Glu Ala Leu Glu Trp Leu Ile Arg Glu Thr Gln
650 655 660
Pro Ile Ser Lys Gln His
665
<210> 8
<211> 952
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 001528
<400> 8
Met Ala Glu Gly Gly Ala Ala Asp Leu Asp Thr Gln Arg Ser Asp
1 5 10 15
Ile Ala Thr Leu Leu Lys Thr Ser Leu Arg Lys Gly Asp Thr Trp
20 25 30
Tyr Leu Val Asp Ser Arg Trp Phe Lys Gln Trp Lys Lys Tyr Val
35 40 45
Gly Phe Asp Ser Trp Asp Lys Tyr Gln Met Gly Asp Gln Asn Val
50 55 60
Tyr Pro Gly Pro Ile Asp Asn Ser Gly Leu Leu Lys Asp Gly Asp
65 70 75
Ala Gln Ser Leu Lys Glu His Leu Ile Asp Glu Leu Asp Tyr Ile
80 85 90
Leu Leu Pro Thr Glu Gly Trp Asn Lys Leu Val Ser Trp Tyr Thr
95 100 105
Leu Met Glu Gly Gln Glu Pro Ile Ala Arg Lys Val Val Glu Gln
110 115 120
Gly Met Phe Val Lys Arg Cys Lys Val Glu Val Tyr Leu Thr Glu
125 130 135
Leu Lys Leu Cys Glu Asn Gly Asn Met Asn Asn Val Val Thr Arg
140 145 150
Arg Phe Ser Lys Ala Asp Thr Ile Asp Thr Ile Glu Lys Glu Ile
155 160 165
Arg Lys Ile Phe Ser Ile Pro Asp Glu Lys Glu Thr Arg Leu Trp
170 175 180
Asn Lys Tyr Met Ser Asn Thr Phe Glu Pro Leu Asn Lys Pro Asp
185 I90 195
Ser Thr Ile Gln Asp Ala Gly Leu Tyr Gln Gly Gln Val Leu Val
200 205 210
Ile Glu Gln Lys Asn Glu Asp Gly Thr Arg Pro Arg Gly Pro Ser
10/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/178I8
215 220 225
Thr Pro Asn Val Lys Asn Ser Asn Tyr Cys Leu Pro Ser Tyr Thr
230 235 240
Ala Tyr Lys Asn Tyr Asp Tyr Ser Glu Pro Gly Arg Asn Asn Glu
245 250 255
Gln Pro Gly Leu Cys Gly Leu Ser Asn Leu Gly Asn Thr Cys Phe
260 265 270
Met Asn Ser Ala Ile Gln Cys Leu Ser Asn Thr Pro Pro Leu Thr
275 280 285
Glu Tyr Phe Leu Asn Asp Lys Tyr Gln Glu Glu Leu Asn Phe Asp
290 295 300
Asn Pro Leu Gly Met Arg Gly GIu Ile Ala Lys Ser Tyr Ala Glu
305 310 315
Leu Ile Lys Gln Met Trp Ser Gly Lys Phe Ser Tyr Val Thr Pro
320 325 330
Arg Ala Phe Lys Thr Gln Val Gly Arg Phe Ala Pro Gln Phe Ser
335 340 345
Gly Tyr Gln Gln Gln Asp Cys Gln Glu Leu Leu Ala Phe Leu Leu
350 355 360
Asp Gly Leu His Glu Asp Leu Asn Arg Ile Arg Lys Lys Pro Tyr
365 370 375
Ile Gln Leu Lys Asp Ala Asp Gly Arg Pro Asp Lys Val Val Ala
380 385 390
Glu Glu Ala Trp Glu Asn His Leu Lys Arg Asn Asp Ser Ile Ile
395 400 405
Val Asp Ile Phe His Gly Leu Phe Lys Ser Thr Leu Val Cys Pro
410 415 420
Glu Cys Ala Lys Ile Ser Val Thr Phe Asp Pro Phe Cys Tyr Leu
425 430 435
Thr Leu Pro Leu Pro Met Lys Lys Glu Arg Thr Leu Glu Val Tyr
440 445 450
Leu Val Arg Met Asp Pro Leu Thr Lys Pro Met Gln Tyr Lys Val
455 460 465
Val Val Pro Lys Ile Gly Asn Ile Leu Asp Leu Cys Thr Ala Leu
470 475 480
Ser Ala Leu Ser Gly Ile Pro Ala Asp Lys Met Ile Val Thr Asp
485 490 495
Ile Tyr Asn His Arg Phe His Arg Ile Phe Ala Met Asp Glu Asn
500 505 510
Leu Ser Ser Ile Met Glu Arg Asp Asp Ile Tyr Val Phe Glu Ile
515 520 525
Asn Ile Asn Arg Thr Glu Asp Thr Glu His Val Ile Ile Pro Val
530 535 540
Cys Leu Arg Glu Lys Phe Arg His Ser Ser Tyr Thr His His Thr
545 550 555
Gly Ser Ser Leu Phe Gly Gln Pro Phe Leu Met Ala Val Pro Arg
560 565 570
Asn Asn Thr Glu Asp Lys Leu Tyr Asn Leu Leu Leu Leu Arg Met
575 580 585
Cys Arg Tyr Val Lys Ile Ser Thr Glu Thr Glu Glu Thr Glu Gly
590 595 600
Ser Leu His Cys Cys Lys Asp Gln Asn Ile Asn Gly Asn Gly Pro
605 610 615
Asn Gly Ile His Glu Glu Gly Ser Pro Ser Glu Met Glu Thr Asp
620 625 630
Glu Pro Asp Asp Glu Ser Ser Gln Asp Gln Glu Leu Pro Ser Glu
635 640 645
11 /42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
Asn Glu Asn Ser Gln Ser Glu Asp Ser Val Gly Gly Asp Asn Asp
650 655 660
Ser Glu Asn Gly Leu Cys Thr Glu Asp Thr Cys Lys Gly Gln Leu
665 670 675
Thr Gly His Lys Lys Arg Leu Phe Thr Phe Gln Phe Asn Asn Leu
680 685 690
Gly Asn Thr Asp Ile Asn Tyr Ile Lys Asp Asp Thr Arg His Ile
695 700 705
Arg Phe Asp Asp Arg Gln Leu Arg Leu Asp Glu Arg Ser Phe Leu
710 715 720
Ala Leu Asp Trp Asp Pro Asp Leu Lys Lys Arg Tyr Phe Asp Glu
725 730 735
Asn Ala Ala Glu Asp Phe Glu Lys His Glu Ser Val Glu Tyr Lys
740 745 750
Pro Pro Lys Lys Pro Phe Val Lys Leu Lys Asp Cys Ile Glu Leu
755 760 765
Phe Thr Thr Lys Glu Lys Leu Gly Ala Glu Asp Pro Trp Tyr Cys
770 775 780
Pro Asn Cys Lys Glu His Gln Gln Ala Thr Lys Lys Leu Asp Leu
785 790 795
Trp Ser Leu Pro Pro Val Leu Val Val His Leu Lys Arg Phe Ser
800 805 810
Tyr Ser Arg Tyr Met Arg Asp Lys Leu Asp Thr Leu Val Asp Phe
815 820 825
Pro Ile Asn Asp Leu Asp Met Ser Glu Phe Leu Ile Asn Pro Asn
830 835 840
Ala Gly Pro Cys Arg Tyr Asn Leu Ile Ala Val Ser Asn His Tyr
845 850 855
Gly Gly Met Gly Gly Gly His Tyr Thr Ala Phe Ala Lys Asn Lys
860 865 870
Asp Asp Gly Lys Trp Tyr Tyr Phe Asp Asp Ser Ser Val Ser Thr
875 880 885
Ala Ser Glu Asp Gln Ile Val Ser Lys Ala Ala Tyr Val Leu Phe
890 895 900
Tyr Gln Arg Gln Asp Thr Phe Ser Gly Thr Gly Phe Phe Pro Leu
905 910 915
Asp Arg Glu Thr Lys Gly Ala Ser Ala Ala Thr Gly Ile Pro Leu
920 925 930
Glu Ser Asp Glu Asp Ser Asn Asp Asn Asp Asn Asp Ile Glu Asn
935 940 945
Glu Asn Cys Met His Thr Asn
950
<210> 9
<211> 166
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 998626
<400> 9
Met Leu His Pro Glu Thr Ser Pro Gly Arg Gly His Leu Leu Ala
1 5 10 15
Val Leu Leu Ala Leu Leu Gly Thr Ala Trp Ala Glu Val Trp Pro
12/42
Ser Leu His Cys Cys Lys Asp Gln Asn Ile Asn
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
20 25 30
Pro Gln Leu Gln Glu Gln Ala Pro Met Ala Gly Ala Leu Asn Arg
35 40 45
Lys Glu Ser Phe Leu Leu Leu Ser Leu His Asn Arg Leu Arg Ser
50 55 60
Trp Val Gln Pro Pro Ala Ala Asp Met Arg Arg Leu Asp Trp Ser
65 70 75
Asp Ser Leu Ala Gln Leu Ala Gln Ala Arg Ala Ala Leu Cys Gly
80 85 90
Ile Pro Thr Pro Ser Leu Ala Ser Gly Leu Trp Arg Thr Leu Gln
95 100 105
Val Gly Trp Asn Met Gln Leu Leu Pro Ala Gly Leu Ala Ser Phe
110 115 120
Val Glu Val Val Ser Leu Trp Phe Ala Glu Gly Gln Arg Tyr Ser
125 130 135
His Ala Ala Gly Glu Cys Ala Arg Asn Ala Thr Cys Thr His Tyr
140 145 150
Thr Gln Leu Val Trp Ala Thr Ser Ser Gln Leu Gly Cys Gly Arg
155 160 165
His
<210> 10
<211> 543
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1393301
<400> 10
Met Arg Lys Pro Ala Ala Gly Phe Leu Pro Ser Leu Leu Lys VaI
1 5 10 15
Leu Leu Leu Pro Leu Ala Pro Ala Ala Ala Gln Asp Ser Thr Gln
20 25 30
Ala Ser Thr Pro Gly Ser Pro Leu Ser Pro Thr Glu Tyr Glu Arg
35 40 45
Phe Phe Ala Leu Leu Thr Pro Thr Trp Lys Ala Glu Thr Thr Cys
50 55 60
Arg Leu Arg Ala Thr His Gly Cys Arg Asn Pro Thr Leu Val Gln
65 70 75
Leu Asp Gln Tyr Glu Asn His Gly Leu Val Pro Asp Gly Ala Val
80 85 90
Cys Ser Asn Leu Pro Tyr Ala Ser Trp Phe Glu Ser Phe Cys Gln
95 100 105
Phe Thr His Tyr Arg Cys Ser Asn His Val Tyr Tyr Ala Lys Arg
110 115 120
VaI Leu Cys Ser Gln Pro Val Ser Ile Leu Ser Pro Asn Thr Leu
125 130 135
Lys Glu Ile Glu Ala Ser Ala Glu Val Ser Pro Thr Thr Met Thr
140 145 150
Ser Pro Ile Ser Pro His Phe Thr Val Thr Glu Arg Gln Thr Phe
155 160 165
Gln Pro Trp Pro Glu Arg Leu Ser Asn Asn Val Glu Glu Leu Leu
170 175 180
Gln Ser Ser Leu Ser Leu Gly Gly Gln Glu Gln Ala Pro Glu His
13/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
185 190 195
Lys Gln Glu Gln Gly Val Glu His Arg Gln Glu Pro Thr Gln Glu
200 205 210
His Lys Gln Glu Glu Gly Gln Lys Gln Glu Glu Gln Glu Glu Glu
215 220 225
Gln Glu Glu Glu Gly Lys Gln Glu Glu Gly Gln Gly Thr Lys Glu
230 235 240
Gly Arg Glu Ala Val Ser Gln Leu Gln Thr Asp Ser Glu Pro Lys
245 250 255
Phe His Ser Glu Ser Leu Ser Ser Asn Pro Ser Ser Phe Ala Pro
260 265 270
Arg Val Arg Glu Val Glu Ser Thr Pro Met Ile Met Glu Asn Ile
275 280 285
Gln Glu Leu Ile Arg Ser Ala Gln Glu Ile Asp Glu Met Asn Glu
290 295 300
Ile Tyr Asp Glu Asn Ser Tyr Trp Arg Asn Gln Asn Pro Gly Ser
305 310 315
Leu Leu Gln Leu Pro His Thr Glu Ala Leu Leu Val Leu Cys Tyr
320 325 330
Ser Ile Val Glu Asn Thr Cys Ile Ile Thr Pro Thr Ala Lys Ala
335 340 345
Trp Lys Tyr Met Glu Glu Glu Ile Leu Gly Phe Gly Lys Ser Val
350 355 360
Cys Asp Ser Leu Gly Arg Arg His Met Ser Thr Cys Ala Leu Cys
365 370 375
Asp Phe Cys Ser Leu Lys Leu Glu Gln Cys His Ser Glu Ala Ser
380 385 390
Leu Gln Arg Gln Gln Cys Asp Thr Ser His Lys Thr Pro Phe Val
395 400 405
Ser Pro Leu Leu Ala Ser Gln Ser Leu Ser Ile Gly Asn Gln Val
410 415 420
Gly Ser Pro Glu Ser Gly Arg Phe Tyr Gly Leu Asp Leu Tyr Gly
425 430 435
Gly Leu His Met Asp Phe Trp Cys Ala Arg Leu Ala Thr Lys Gly
440 445 450
Cys Glu Asp Val Arg Val Ser Gly Trp Leu Gln Thr Glu Phe Leu
455 460 465
Ser Phe Gln Asp Gly Asp Phe Pro Thr Lys Ile Cys Asp Thr Asp
470 475 480
Tyr Ile Gln Tyr Pro Asn Tyr Cys Ser Phe Lys Ser Gln Gln Cys
485 490 495
Leu Met Arg Asn Arg Asn Arg Lys Val Ser Arg Met Arg Cys Leu
500 505 510
Gln Asn Glu Thr Tyr Ser Ala Leu Ser Pro Gly Lys Ser Glu Asp
515 520 525
Val Val Leu Arg Trp Ser Gln Glu Phe Ser Thr Leu Thr Leu Gly
530 535 540
Gln Phe Gly
<210> lI
<211> 83
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
14/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
<223> Incyte Clone No: 1444055
<400> 11
Met Ile Gly Trp Asp Ser Leu Arg Leu IIe Leu Gly Asn Thr Asp
1 5 10 15
Asn Val Ser Arg Arg Asp Ser Thr Arg Gly Ser Ile Phe Ile Thr
20 25 30
Gln Leu Ile Ala Cys Phe Gln Arg Tyr Ser Trp Arg Cys His Leu
35 40 45
Glu Glu Val Phe Trp Lys Val Gln Gln Ala~ Phe Glu Ser Pro Glu
50 55 60
Ala Thr Val Gln Met Pro Thr Ile Glu Arg Val Ser Met Thr Arg
65 70 75
Tyr Phe Tyr Leu Phe Pro Gly Asn
<210> 12
<211> 648
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1650177
<400> 12
Met Leu Gly Ser Leu Val Leu Arg Arg Lys Ala Leu Ala Pro Arg
1 5 10 15
Leu Leu Leu Arg Leu Leu Arg Ser Pro Thr Leu Arg Gly His Gly
20 25 30
Gly Ala Ser Gly Arg Asn Val Thr Thr Gly Ser Leu Gly Glu Pro
35 40 45
Gln Trp Leu Arg Val Ala Thr Gly Gly Arg Pro Gly Thr Ser Pro
50 55 60
Ala Leu Phe Ser Gly Arg Gly Ala Ala Thr Gly Gly Arg Gln Gly
65 70 75
Gly Arg Phe Asp Thr Lys Cys Leu Ala Ala Ala Thr Trp Gly Arg
80 85 90
Leu Pro Gly Pro Glu Glu Thr Leu Pro Gly Gln Asp Ser Trp Asn
95 100 105
Gly Val Pro Ser Arg Ala Gly Leu Gly Met Cys Ala Leu AIa Ala
110 115 120
Ala Leu Val Val His Cys Tyr Ser Lys Ser Pro Ser Asn Lys Asp
125 130 135
Ala Ala Leu Leu Glu Ala Ala Arg Ala Asn Asn Met Gln Glu Val
140 145 150
Ser Ser Val Val Gln Val Leu Leu Ala Ala Gly Ala Asp Pro Asn
155 160 165
Leu Gly Asp Asp Phe Ser Ser Val Phe Lys Thr Ala Lys Glu Gln
170 175 180
Gly Ile His Ser Leu Glu Val Leu Ile Thr Arg Glu Asp Asp Phe
185 190 195
Asn Asn Arg Leu Asn Asn Arg Ala Ser Phe Lys Gly Cys Thr Ala
200 205 210
Leu His Tyr Ala Val Leu Ala Asp Asp Tyr Arg Thr Val Lys Glu
215 220 225
15/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
Leu Leu Asp Gly Gly Ala Asn Pro Leu Gln Arg Asn Glu Met Gly
230 23S 240
His Thr Pro Leu Asp Tyr Ala Arg Glu Gly Glu Val Met Lys Leu
245 250 2S5
Leu Arg Thr Ser Glu Ala Lys Tyr Gln Glu Lys Gln Arg Lys Arg
260 265 270
Glu Ala Glu Glu Arg Arg Arg Phe Pro Leu Glu Gln Arg Leu Lys
275 280 285
Glu His Ile Ile Gly Gln Glu Ser Ala Ile Ala Thr Val Gly Ala
290 29S 300
Ala Ile Arg Arg Lys Glu Asn Gly Trp Tyr Asp Glu Glu His Pro
305 310 315
Leu Val Phe Leu Phe Leu Gly Ser Ser Gly Ile Gly Lys Thr Glu
320 325 330
Leu Ala Lys Gln Thr Ala Lys Tyr Met His Lys Asp Ala Lys Lys
335 340 345
Gly Phe Ile Arg Leu Asp Met Ser Glu Phe Gln Glu Arg His Glu
350 355 360
Val Ala Lys Phe Ile Gly Ser Pro Pro Gly Tyr Val Gly His Glu
365 370 375
Glu Gly Gly Gln Leu Thr Lys Lys Leu Lys Gln Cys Pro Asn Ala
380 385 390
Val Val Leu Phe Asp Glu Val Asp Lys Ala His Pro Asp Val Leu
395 400 405
Thr Ile Met Leu Gln Leu Phe Asp Glu Gly Arg Leu Thr Asp Gly
410 415 420
Lys Gly Lys Thr Ile Asp Cys Lys Asp Ala Ile Phe Ile Met Thr
42S 430 435
Ser Asn Val Ala Ser Asp Glu Ile Ala Gln His Ala Leu Gln Leu
440 445 450
Arg Gln Glu Ala Leu Glu Met Ser Arg Asn Arg Ile Ala Glu Asn
455 460 465
Leu Gly Asp Val Gln Ile Ser Asp Lys Ile Thr Ile Ser Lys Asn
470 475 480
Phe Lys Glu Asn Val Ile Arg Pro Ile Leu Lys Ala His Phe Arg
485 490 495
Arg Asp Glu Phe Leu Gly Arg Ile Asn Glu Ile Val Tyr Phe Leu
500 505 510
Pro Phe Cys His Ser Glu Leu Ile Gln Leu Val Asn Lys Glu Leu
515 520 525
Asn Phe Trp Ala Lys Arg Ala Lys Gln Arg His Asn Ile Thr Leu
530 535 540
Leu Trp Asp Arg Glu Val Ala Asp Val Leu Val Asp Gly Tyr Asn
545 550 555
Val His Tyr Gly Ala Arg Ser Ile Lys His Glu Val Glu Arg Arg
560 565 570
Val Val Asn Gln Leu Ala Ala Ala Tyr Glu Gln Asp Leu Leu Pro
575 580 585
Gly Gly Cys Thr Leu Arg Ile Thr Val Glu Asp Ser Asp Lys Gln
590 595 600
Leu Leu Lys Ser Pro Glu Leu Pro Ser Pro Gln Ala Glu Lys Arg
605 610 615
Leu Pro Lys Leu Arg Leu Glu Ile Ile Asp Lys Asp Ser Lys Thr
620 625 630
Arg Arg Leu Asp Ile Arg Ala Pro Leu His Pro Glu Lys Val Cys
635 640 645
Asn Thr Ile
16/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
<210> 13
<211> 672
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1902576
<400> 13
Met Arg Ala Gly Arg Gly Ala Thr Pro Ala Arg Glu Leu Phe Arg
1 5 10 15
Asp Ala Ala Phe Pro Ala Ala Asp Ser Ser Leu Phe Cys Asp Leu
20 25 30
Ser Thr Pro Leu Ala Gln Phe Arg Glu Asp Ile Thr Trp Arg Arg
35 40 45
Pro Gln Glu Ile Cys Ala Thr Pro Arg Leu Phe Pro Asp Asp Pro
50 55 60
Arg Glu Gly Gln Val Lys Gln Gly Leu Leu Gly Asp Cys Trp Phe
65 70 75
Leu Cys Ala Cys Ala Ala Leu Gln Lys Ser Arg His Leu Leu Asp
80 85 90
Gln Val Ile Pro Pro Gly Gln Pro Ser Trp Ala Asp Gln Glu Tyr
95 100 105
Arg Gly Ser Phe Thr Cys Arg Ile Trp Gln Phe Gly Arg Trp Val
110 115 120
Glu Val Thr Thr Asp Asp Arg Leu Pro Cys Leu Ala Gly Arg Leu
125 130 135
Cys Phe Ser Arg Cys Gln Arg Glu Asp Val Phe Trp Leu Pro Leu
140 145 150
Leu Glu Lys Val Tyr Ala Lys Val His Gly Ser Tyr Glu His Leu
155 160 165
Trp Ala Gly Gln Val Ala Asp Ala Leu Val Asp Leu Thr Gly Gly
170 175 180
Leu Ala Glu Arg Trp Asn Leu Lys Gly Val Ala Gly Ser Gly Gly
185 190 195
Gln Gln Asp Arg Pro Gly Arg Trp Glu His Arg Thr Cys Arg Gln
200 205 210
Leu Leu His Leu Lys Asp Gln Cys Leu Ile Ser Cys Cys Val Leu
215 220 225
Ser Pro Arg Ala Gly Ala Arg Glu Leu Gly Glu Phe His Ala Phe
230 235 240
Ile Val Ser Asp Leu Arg Glu Leu Gln Gly Gln Ala Gly Gln Cys
245 250 255
Ile Leu Leu Leu Arg Ile Gln Asn Pro Trp Gly Arg Arg Cys Trp
260 265 270
Gln Gly Leu Trp Arg Glu Gly Gly Glu Gly Trp Ser Gln Val Asp
275 280 285
Ala Ala Val Ala Ser Glu Leu Leu Ser Gln Leu Gln Glu Gly Glu
290 295 300
Phe Trp Val Glu Glu Glu Glu Phe Leu Arg Glu Phe Asp Glu Leu
305 310 315
Thr Val Gly Tyr Pro Val Thr Glu Ala Gly His Leu Gln Ser Leu
320 325 330
Tyr Thr Glu Arg Leu Leu Cys His Thr Arg Ala Leu Pro Gly Ala
335 340 345
17/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
Trp Val Lys Gly Gln Ser Ala Gly Gly Cys Arg Asn Asn Ser Gly
350 355 360
Phe Pro Ser Asn Pro Lys Phe Trp Leu Arg Val Ser Glu Pro Ser
365 370 375
Glu Val Tyr Ile Ala Val Leu Gln Arg Ser Arg Leu His Ala Ala
380 385 390
Asp Trp Ala Gly Arg Ala Arg Ala Leu Val Gly Asp Ser His Thr
395 400 405
Ser Trp Ser Pro Ala Ser Ile Pro Gly Lys His Tyr Gln Ala Val
410 4I5 420
Gly Leu His Leu Trp Lys Val Glu Lys Arg Arg Val Asn Leu -Pro
425 430 435
Arg Val Leu Ser Met Pro Pro Val Ala Gly Thr Ala Cys His Ala
440 445 450
Tyr Asp Arg Glu Val His Leu Arg Cys Glu Leu Ser Pro Gly Tyr
455 460 465
Tyr Leu Ala VaI Pro Ser Thr Phe Leu Lys Asp Ala Pro Gly Glu
470 475 480
Phe Leu Leu Arg Val Phe Ser Thr Gly Arg Val Ser Leu Ser Ala
485 490 495
Ile Arg Ala Val Ala Lys Asn Thr Ala Pro Gly Ala Ala Leu Pro
500 505 510
Ala Gly Glu Trp Gly Thr Val Gln Leu Arg Gly Ser Trp Arg Val
515 520 525
Gly Gln Thr Ala Gly Gly Ser Arg Asn Phe Ala Ser Tyr Pro Thr
530 535 540
Asn Pro Cys Phe Pro Phe Ser Val Pro Glu Gly Pro Gly Pro Arg
545 550 555
Cys Val Arg Ile Thr Leu His Gln His Cys Arg Pro Ser Asp Thr
560 565 570
Glu Phe His Pro Ile Gly Phe His Ile Phe Gln Val Pro Glu Gly
575 580 5B5
Gly Arg Ser Gln Asp Ala Pro Pro Leu Leu Leu Gln Glu Pro Leu
590 595 600
Leu Ser Cys Val Pro His Arg Tyr Ala Gln Glu Val Ser Arg Leu
605 610 615
Cys Leu Leu Pro Ala Gly Thr Tyr Lys Val Val Pro Ser Thr Tyr
620 625 630
Leu Pro Asp Thr Glu Gly Ala Phe Thr Val Thr Ile Ala Thr Arg
635 640 645
Ile Asp Arg Pro Ser Ile His Ser Gln Glu Met Leu Gly Gln Phe
650 655 660
Leu Gln Glu Val Ser Val Met Ala Val Met Lys Thr
665 670
<210> 14
<211> 80
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2024210
<400> 14
Met Lys Leu Ser Gly Met Phe Leu Leu Leu Ser Leu Ala Leu Phe
18/42
CA 02338386 2001-02-02
WO 00/09709 PCTNS99/17818
1 5 10 15
Cys Phe Leu Thr Gly Val Phe Ser Gln Gly Gly Gln Val Asp Cys
20 25 30
Gly Glu Phe Gln Asp Pro Lys Val Tyr Cys Thr Arg Glu Ser Asn
35 40 45
Pro His Cys Gly Ser Asp Gly Gln Thr Tyr Gly Asn Lys Cys Ala
50 55 60
Phe Cys Lys Ala Ile Val Lys Ser Gly Gly Lys Ile Ser Leu Lys
65 70 75
His Pro Gly Lys Cys
<210> 15
<211> 795
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2523109
<400> 15
Met Ala Val Leu Leu Leu Leu Leu Arg Ala Leu Arg Arg Gly Pro
1 5 10 15
Gly Pro Gly Pro Arg Pro Leu Trp Gly Pro Gly Pro Ala Trp Ser
20 25 30
Pro Gly Phe Pro Ala Arg Pro Gly Arg Gly Arg Pro Tyr Met Ala
35 40 45
Ser Arg Pro Pro Gly Asp Leu Ala Glu Ala Gly Gly Arg Ala Leu
50 55 60
Gln Ser Leu Gln Leu Arg Leu Leu Thr Pro Thr Phe Glu Gly Ile
65 70 75
Asn Gly Leu Leu Leu Lys Gln His Leu Val Gln Asn Pro Val Arg
80 85 90
Leu Trp Gln Leu Leu Gly Gly Thr Phe Tyr Phe Asn Thr Ser Arg
95 100 105
Leu Lys G1n Lys Asn Lys Glu Lys Asp Lys Ser Lys Gly Lys Ala
110 115 220
Pro Glu Glu Asp Glu Glu Glu Arg Arg Arg Arg Glu Arg Asp Asp
125 130 135
Gln Met Tyr Arg Glu Arg Leu Arg Thr Leu Leu Val Ile Ala Val
140 145 150
Val Met Ser Leu Leu Asn Ala Leu Ser Thr Ser Gly Gly Ser Ile
155 160 165
Ser Trp Asn Asp Phe Val His Glu Met Leu Ala Lys Gly Glu Val
170 175 180
Gln Arg Val Gln Val Val Pro Glu Ser Asp Val Val Glu Val Tyr
185 190 195
Leu His Pro Gly Ala Val Val Phe Gly Arg Pro Arg Leu Ala Leu
200 205 210
Met Tyr Arg Met Gln Val Ala Asn Ile Asp Lys Phe Glu Glu Lys
215 220 225
Leu Arg Ala Ala Glu Asp Glu Leu Asn Ile Glu Ala Lys Asp Arg
230 235 240
Ile Pro Val Ser Tyr Lys Arg Thr Gly Phe Phe Gly Asn Ala Leu
245 250 255
19/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
Tyr Ser Val Gly Met Thr Ala Val Gly Leu Ala Ile Leu Trp Tyr
260 265 270
Val Phe Arg Leu Ala Gly Met Thr Gly Arg Glu Gly Gly Phe Ser
275 280 285
Ala Phe Asn Gln Leu Lys Met Ala Arg Phe Thr Ile Val Asp Gly
290 295 300
Lys Met Gly Lys Gly Val Ser Phe Lys Asp Val Ala Gly Met His
305 310 315
Glu Ala Lys Leu Glu Val Arg Glu Phe Val Asp Tyr Leu Lys Ser
320 325 330
Pro Lys Arg Phe Leu Gln Leu Gly Ala Lys Val Pro Lys Gly Ala
335 340 345
Leu Leu Leu Gly Pro Pro Gly Cys Gly Lys Thr Leu Leu Ala Lys
350 355 360
Ala Val Ala Thr Glu Ala Gln Val Pro Phe Leu Ala Met Ala Gly
365 370 375
Pro Glu Phe Val Glu Val Ile Gly Gly Leu Gly Ala Ala Arg Val
380 385 390
Arg Ser Leu Phe Lys Glu Ala Arg Ala Arg Ala Pro Cys Ile Val
395 400 405
Tyr Ile Asp Glu Ile Asp Ala Val Gly Lys Lys Arg Ser Thr Thr
410 415 420
Met Ser Gly Phe Ser Asn Thr Glu Glu Glu Gln Thr Leu Asn Gln
425 430 435
Leu Leu Val Glu Met Asp Gly Met Gly Thr Thr Asp His Val Ile
440 445 450
Val Leu Ala Ser Thr Asn Arg Ala Asp Ile Leu Asp Gly Ala Leu
455 460 465
Met Arg Pro Gly Arg Leu Asp Arg His Val Phe Ile Asp Leu Pro
470 475 480
Thr Leu Gln Glu Arg Arg Glu Ile Phe Glu Gln His Leu Lys Ser
485 490 495
Leu Lys Leu Thr Gln Ser Ser Thr Phe Tyr Ser Gln Arg Leu Ala
500 505 510
Glu Leu Thr Pro Gly Phe Ser Gly Ala Asp Ile Ala Asn Ile Cys
515 520 525
Asn Glu Ala Ala Leu His Ala Ala Arg Glu Gly His Thr Ser Val
530 535 540
His Thr Leu Asn Phe Glu Tyr Ala Val Glu Arg Val Leu Ala Gly
545 550 555
Thr Ala Lys Lys Ser Lys Ile Leu Ser Lys Glu Glu Gln Lys Val
560 565 570
Val Ala Phe His Glu Ser Gly His Ala Leu Val Gly Trp Met Leu
575 580 585
Glu His Thr Glu Ala Val Met Lys Val Ser Ile Thr Pro Arg Thr
590 595 600
Asn Ala Ala Leu Gly Phe Ala Gln Met Leu Pro Arg Asp Gln His
605 610 615
Leu Phe Thr Lys Glu Gln Leu Phe Glu Arg Met Cys Met Ala Leu
620 625 630
Gly Gly Arg Ala Ser Glu Ala Leu Ser Phe Asn Glu Val Thr Ser
635 640 645
Gly Ala Gln Asp Asp Leu Arg Lys Val Thr Arg Ile Ala Tyr Ser
650 655 660
Met Val Lys Gln Phe Gly Met Ala Pro Gly Ile Gly Pro Ile Ser
665 670 675
Phe Pro Glu Ala Gln Glu Gly Leu Met Gly Ile Gly Arg Arg Pro
20/42
CA 02338386 2001-02-02
WO 00!09709 PCT/US99/17818
680 685 690
Phe Ser Gln Gly Leu Gln Gln Met Met Asp His Glu Ala Arg Leu
695 700 705
Leu Val Ala Lys Ala Tyr Arg His Thr Glu Lys Val Leu Gln Asp
710 715 720
Asn Leu Asp Lys Leu Gln Ala Leu Ala Asn Ala Leu Leu Glu Lys
725 730 735
Glu Val Ile Asn Tyr Glu Asp Ile Glu Ala Leu Ile Gly Pro Pro
740 745 750
Pro His Gly Pro Lys Lys Met Ile Ala Pro Gln Arg Trp Ile Asp
755 760 765
Ala Gln Arg Glu Lys Gln Asp Leu Gly Glu Glu Glu Thr Glu Glu
770 775 780
Thr Gln Gln Pro Pro Leu Gly Gly Glu Glu Pro Thr Trp Pro Lys
785 790 795
<210> 16
<211> 193
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2588566
<400> 16
Met Pro Asp Ser Asp Arg His Leu Ser Ser His Phe Asn Leu Arg
1 5 10 15
Met Lys Gly Ser Pro Ser Glu His Gly Ser Gln Gln Ser Ile Phe
20 25 30
Asn Arg Tyr Ala Gln Gln Arg Leu Asp Ile Asp Ala Thr Gln Leu
35 40 45
Gln Gly Leu Leu Asn Gln Glu Leu Leu Thr Gly Pro Pro Gly Asp
50 55 60
Met Phe Ser Leu Asp Glu Cys Arg Ser Leu Val Ala Leu Met Glu
65 70 75
Leu Lys Val Asn Gly Arg Leu Asp Gln Glu Glu Phe Ala Arg Leu
BO 85 90
Trp Lys Arg Leu Val His Tyr Gln His Val Phe Gln Lys Val Gln
95 100 105
Thr Ser Pro Gly Val Leu Leu Ser Ser Asp Leu Trp Lys Ala Ile
110 115 120
Glu Asn Thr Asp Phe Leu Arg Gly Ile Phe Ile Ser Arg Glu Leu
125 130 135
Leu His Leu Val Thr Leu Arg Tyr Ser Asp Ser Val Gly Arg Val
140 145 150
Ser Phe Pro Ser Leu Val Cys Phe Leu Met Arg Leu Glu Ala Met
155 160 165
Ala Lys Thr Phe Arg Asn Leu Ser Lys Asp Gly Lys Gly Leu Tyr
170 175 180
Leu Thr Glu Met Glu Trp Met Ser Leu Val Met Tyr Asn
185 190
<210> 17
<211> 663
21 /42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
<212> P12T
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2740570
<400> 17
Met Asp Leu Leu His Glu Glu Leu Lys Glu Gln Val Met Glu Val
1 5 10 15
Glu Glu Asp Pro Gln Thr Ile Thr Thr Glu Glu Thr Met Glu Glu
20 25 30
Asp Lys Ser Gln Ser Asp Val Asp Phe Gln Ser Cys Glu Ser Cys
35 40 45
Ser Asn Ser Asp Arg Ala Glu Asn Glu Asn Gly Ser Arg Cys Phe
50 55 60
Ser Glu Asp Asn Asn Glu Thr Thr Met Leu Ile Gln Asp Asp Glu
65 70 75
Asn Asn Ser Glu Met Ser Lys Asp Trp Gln Lys Glu Lys Met Cys
80 85 90
Asn Lys Ile Asn Lys Val Asn Ser Glu Gly Glu Phe Asp Lys Asp
95 100 105
Arg Asp Ser Ile Ser Glu Thr Val Asp Leu Asn Asn G1n Glu Thr
110 115 120
Val Lys Val Gln Ile His Ser Arg Ala Ser Glu Tyr Ile Thr Asp
125 130 135
Val His Ser Asn Asp Leu Ser Thr Pro Gln Ile Leu Pro Ser Asn
140 145 150
Glu Gly Val Asn Pro Arg Leu Ser Ala Ser Pro Pro Lys Ser Gly
155 160 165
Asn Leu Trp Pro Gly Leu Ala Pro Pro His Lys Lys Ala Gln Ser
170 175 180
Ala Ser Pro Lys Arg Lys Lys Gln His Lys Lys Tyr Arg Ser Val
185 I90 195
Ile Ser Asp Ile Phe Asp Gly Thr Ile Ile Ser Ser Val Gln Cys
200 205 210
Leu Thr Cys Asp Arg Val Ser Val Thr Leu Glu Thr Phe Gln Asp
215 220 225
Leu Ser Leu Pro Ile Pro Gly Lys Glu Asp Leu Ala Lys Leu His
230 235 240
Ser Ser Ser His Pro Thr Ser Ile Val Lys Ala Gly Ser Cys Gly
245 250 255
Glu Ala Tyr Ala Pro Gln Gly Trp Ile Ala Phe Phe Met Glu Tyr
260 265 270
Val Lys Arg Phe Val Val Ser Cys Val Pro Ser Trp Phe Trp Gly
275 280 285
Pro Val Val Thr Leu Gin Asp Cys Leu Ala Ala Phe Phe Ala Arg
290 295 300
Asp Glu Leu Lys Gly Asp Asn Met Tyr Ser Cys Glu Lys Cys Lys
305 310 315
Lys Leu Arg Asn Gly Val Lys Phe Cys Lys Val Gln Asn Phe Pro
320 325 330
Glu Ile Leu Cys Ile His Leu Lys Arg Phe Arg His Glu Leu Met
335 340 345
Phe Ser Thr Lys Ile Ser Thr His Val Ser Phe Pro Leu Glu Gly
350 355 360
Leu Asp Leu Gln Pro Phe Leu Ala Lys Asp Ser Pro Ala Gln Ile
22/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
365 370 375
Val Thr Tyr Asp Leu Leu Ser Val Ile Cys His His Gly Thr Ala
380 385 390
Ser Ser Gly His Tyr Ile Ala Tyr Cys Arg Asn Asn Leu Asn Asn
395 400 405
Leu Trp Tyr Glu Phe Asp Asp Gln Ser Val Thr Glu Val Ser Glu
410 415 420
Ser Thr Val Gln Asn Ala Glu Ala Tyr Val Leu Phe Tyr Arg Lys
425 430 435
Ser Ser Glu Glu Ala Gln Lys Glu Arg Arg Arg Ile Ser Asn Leu
440 445 450
Leu Asn Ile Met Glu Pro Ser Leu Leu Gln Phe Tyr Ile Ser Arg
455 460 465
Gln Trp Leu Asn Lys Phe Lys Thr Phe Ala Glu Pro Gly Pro Ile
470 475 480
Ser Asn Asn Asp Phe Leu Cys Ile His Gly Gly Val Pro Pro Arg
485 490 495
Lys Ala Gly Tyr Ile Glu Asp Leu Val Leu Met Leu Pro Gln Asn
500 505 510
Ile Trp Asp Asn Leu Tyr Ser Arg Tyr Gly Gly Gly Pro Ala Val
515 520 525
Asn His Leu Tyr Ile Cys His Thr Cys Gln Ile Glu Ala Glu Lys
530 535 540
Ile Glu Lys Arg Arg Lys Thr Glu Leu Glu Ile Phe Ile Arg Leu
545 550 555
Asn Arg Ala Phe Gln Lys Glu Asp Ser Pro Ala Thr Phe Tyr Cys
560 565 570
Ile Ser Met Gln Trp Phe Arg Glu Trp Glu Ser Phe Val Lys Gly
575 580 5B5
Lys Asp Gly Asp Pro Pro Gly Pro Ile Asp Asn Thr Lys Ile Ala
590 595 600
Val Thr Lys Cys Gly Asn Val Met Leu Arg Gln Gly AIa Asp Ser
605 610 615
Gly Gln Ile Ser Glu Glu Thr Trp Asn Phe Leu Gln Ser Ile Tyr
620 625 630
Gly Gly Gly Pro Glu Val Ile Leu Arg Pro Pro Val Val His Val
635 640 645
Asp Pro Asp Ile Leu Gln Ala Glu Glu Lys Ile Glu VaI Glu Thr
650 655 660
Arg Ser Leu
<210> 18
<211> 362
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2820384
<400> 18
Met Tyr Ser Cys Glu Arg Cys Lys Lys Leu Arg Asn Gly Val Lys
1 5 10 15
Tyr Cys Lys Val Leu Arg Leu Pro Glu Ile Leu Cys Ile His Leu
20 25 30
Lys Arg Phe Arg His Glu Val Met Tyr Ser Phe Lys Ile Asn Ser
23/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
35 40 45
His Val Ser Phe Pro Leu Glu Gly Leu Asp Leu Arg Pro Phe Leu
50 55 60
Ala Lys Glu Cys Thr Ser Gln Ile Thr Thr Tyr Asp Leu Leu Ser
65 70 75
Val Ile Cys His His Gly Thr Ala Gly Ser Gly His Tyr Ile Ala
80 85 90
Tyr Cys Gln Asn Val Ile Asn Gly Gln Trp Tyr Glu Phe Asp Asp
95 100 105
Gln Tyr Val Thr Glu Val His Glu Thr Val Val Gln Asn Ala Glu
110 lI5 120
Gly Tyr Val Leu Phe Tyr Arg Lys Ser Ser Glu Glu Ala Met Arg
125 130 135
Glu Arg Gln Gln Val Val Ser Leu Ala Ala Met Arg Glu Pro Ser
140 145 150
Leu Leu Arg Phe Tyr Val Ser Arg Glu Trp Leu Asn Lys Phe Asn
155 160 165
Thr Phe Ala Glu Pro Gly Pro Ile Thr Asn Gln Thr Phe Leu Cys
170 175 180
Ser His Gly Gly Ile Pro Pro His Lys Tyr His Tyr Ile Asp Asp
185 190 195
Leu Val Val Ile Leu Pro Gln Asn Val Trp Glu His Leu Tyr Asn
200 205 210
Arg Phe Gly Gly Gly Pro Ala Val Asn His Leu Tyr Val Cys Ser
215 220 225
Ile Cys Gln Val Glu Ile Glu Ala Leu Ala Lys Arg Arg Arg Ile
230 235 240
Glu IIe Asp Thr Phe Ile Lys Leu Asn Lys Ala Phe Gln Ala Glu
245 250 255
Glu Ser Pro Gly Val Ile Tyr Cys Ile Ser Met Gln Trp Phe Arg
260 265 270
Glu Trp Glu Ala Phe Val Lys Gly Lys Asp Asn Glu Pro Pro Gly
275 280 285
Pro Ile Asp Asn Ser Arg Ile Ala Gln Val Lys Gly Ser Gly His
290 295 300
Val Gln Leu Lys Gln Gly Ala Asp Tyr Gly Gln Ile Ser Glu Glu
305 310 315
Thr Trp Thr Tyr Leu Asn Ser Leu Tyr Gly Gly Gly Pro Glu Ile
320 325 330
Ala Ile Arg Gln Ser Val Ala Gln Arg Trp Ala Gln Arg Thr Cys
335 340 345
Thr Gly Ser Arg Arg Ser Lys Pro Arg Arg Gly Pro Cys Asp Leu
350 355 360
Leu Gly
<210> 19
<211> 210
<212> PRT
<213> Homo Sapiens
<220>
<22I> misc_feature
<223> Incyte Clone No: 2990692
<400> 19
Met Val Ser Leu Leu Pro Gly Glu Pro Pro Gln Lys Ile Pro Arg
24/42
CA 02338386 2001-02-02
WO 00/09709 PCTNS99/17818
1 5 10 15
Gly Val Tyr Gly Pro Leu Pro Glu Gly Arg Val Gly Leu Ile Leu
20 25 30
Gly Arg Ser Ser Leu Asn Leu Lys Gly Val Gln Ile His Thr GIy
35 40 45
Val IIe Tyr Ser Asp Tyr Lys Gly Gly Ile Gln Leu Val Ile Ser
50 55 60
Ser Thr Val Pro Trp Ser Ala Asn Pro Gly Asp Arg Ile Ala Gln
65 70 75
Leu Leu Leu Leu Pro Tyr Val Lys Ile Gly Glu Asn Lys Thr Glu
80 85 90
Arg Thr Gly Gly Phe Gly Ser Thr Asn Pro Ala Gly Lys Ala Thr
95 I00 105
Tyr Trp Ala Asn Gln Val Ser Glu Asp Arg Pro Val Cys Thr Val
110 115 120
Thr Ile Pro Gly Lys Glu Phe Glu Gly Leu Val Asp Thr Gln Ala
125 130 135
Asp Val Ser Ile Ile Gly Ile Gly Thr Ala Ser Glu Val Tyr Gln
140 145 150
Ser Ala Met Ile Leu His Cys Leu Gly Ser Asp Asn Gln Glu Ser
155 160 165
Thr Val Gln Pro Met Ile Thr Ser Ile Pro Ile Asn Leu Trp Gly
170 175 180
Arg Asp Leu Leu Gln Gln Trp His Ala Glu Ile Thr Ile Pro Ala
185 190 195
Ser Leu Tyr Ser Pro Arg Asn Gln Lys Ile Met Thr Lys Met Gly
200 205 210
<210> 20
<211> 283
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 4590384
<400> 20
Met Gly Leu Gly Leu Arg Gly Trp Gly Arg Pro Leu Leu Thr Val
1 5 10 15
Ala Thr Ala Leu Met Leu Pro Val Lys Pro Pro Ala Gly Ser Trp
20 25 30
Gly Ala Gln Ile Ile Gly Gly His Glu Val Thr Pro His Ser Arg
35 40 45
Pro Tyr Met Ala Ser Val Arg Phe Gly Gly Gln His His Cys Gly
50 55 60
Gly Phe Leu Leu Arg Ala Arg Trp Val Val Ser Ala Ala His Cys
65 70 75
Phe Ser His Arg Asp Leu Arg Thr Gly Leu Val Val Leu Gly Ala
80 85 90
His VaI Leu Ser Thr Ala Glu Pro Thr Gln Gln Val Phe Gly Ile
95 100 105
Asp Ala Leu Thr Thr His Pro Asp Tyr His Pro Met Thr His Ala
110 115 120
Asn Asp Ile Cys Leu Leu Arg Leu Asn Gly Ser Ala Val Leu Gly
125 130 135
25/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
Pro Ala Val Gly Leu Leu Arg Leu Pro Gly Arg Arg Ala Arg Pro
140 145 150
Pro Thr Ala Gly Thr Arg Cys Arg Val Ala Gly Trp Gly Phe Val
155 160 165
Ser Asp Phe Glu Glu Leu Pro Pro Gly Leu Met Glu Ala Lys Val
170 175 180
Arg Val Leu Asp Pro Asp Val Cys Asn Ser Ser Trp Lys Gly His
185 190 195
Leu Thr Leu Thr Met Leu Cys Thr Arg Ser Gly Asp Ser His Arg
200 205 210
Arg Gly Phe Cys Ser Ala Asp Ser Gly Gly Pro Leu Val Cys Arg
215 220 225
Asn Arg Ala His Gly Leu Val Ser Phe Ser Gly Leu Trp Cys Gly
230 235 240
Asp Pro Lys Thr Pro Asp Val Tyr Thr Gln Val Ser Ala Phe Val
245 250 255
Ala Trp Ile Trp Asp Val Val Arg Arg Ser Ser Pro Gln Pro Gly
260 265 270
Pro Leu Pro Gly Thr Thr Arg Pro Pro Gly Glu Ala Ala
275 280
<210> 21
<211> 896
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 660
c223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 1220330
<400> 21
atgccatcgc gccggcgcga tgccatcaaa gtcatgcaga ggttcgcggg gctgccggag 60
accggccgca tggacccagg gacagtggcc accatgcgta agccccgctg ctccctgcct 120
gacgtgctgg gcgtggcggg gctggtcagg cggcgtcgtc ggtacgctct gagcggcagc 180
gtgtggaaga agcgaaccct gacatggagg gtacgttcct tcccccagag ctcccagctg 240
agccaggaga ccgtgcgggt cctcatgagc tatgccctga tggcctgggg catggagtca 300
ggcctcacat ttcatgaggt ggattccccc cagggccagg agcccgacat cctcatcgac 360
tttgcccgcg ccttccacca ggacagctac cccttcgacg ggttgggggg caccctagcc 420
catgccttct tccctgggga gcaccccatc tccggggaca ctcactttga cgatgaggag 480
acctggactt ttgggtcaaa ggcctctcag cagctggagc aggagctggc aggcggctca 540
ccggttgatg aggagctggg cttcagccgg ggctggcgtg tgaatcctct gggtcctggc 600
agtcctgagc gcctgagctg aatacagagg gaagaggctg ggagcaaggc cgggtgctgn 660
ggcccgcagg cctgtgttct gagagtgcct gctacgagga gctctgtggt tccccaagga 720
gatgggaggg aagacctggg ggttgggggg ttggtacagg gggtaggggc agaaggaagg 780
gggcaagaag gcttgttgaa ccaaggggaa gaattggggg gaaggggggg aattggaatg 840
gaccttcaaa gggggttggt taaagggggt taattgggcc ttttaaggga aggggg 896
c210> 22
<211> 4906
<212> DNA
c213> Homo sapiens
26/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
<220>
<221> misc_feature
<223> Incyte Clone No: 1342493
<400> 22
ttgggatgtg cggagtcagt gccagcccgg tcccggccaa gcggagtgtg agcccggcgc 60
ctccaacgca acaccccgcg ccctcgccgg ctcccccgcc gtgcggatcg gagccagccg 120
gttgttgcca tggcattcgc cagctggtgg tacaagacgc atgtcagtga aaaaaccagt 180
gaatcgcctt ccaaaccagg agaaaagaaa ggatcagatg agaaaaaagc agcaagcctc 240
ggcagcagtc aatcctccag aacctatgct ggtggaacag cctcggccac caaggtgtca 300
gcttcctctg gtgcaaccag caagtcttcc agtatgaatc ccacagaaac caaggctgta 360
aaaacagaac ctgagaagaa gtcacagtca accaagctgt ctgtggttca tgagaaaaaa 420
tcccaagaag gaaagccaaa agaacacaca gagccaaaaa gcctacccaa gcaggcatca 480
gatacaggaa gtaacgatgc tcacaataaa aaagcagttt ccagatcagc tgaacagcag 540
ccatcagaga aatcaacaga accaaagact aaaccacaag acatgatttc tgctggtgga 600
gagagtgttg ctggtatcac tgcaatatct ggcaagccgg gtgacaagaa aaaagaaaag 660
aaatcattaa ccccagctgt gccagttgaa tctaaaccgg ataaaccatc gggaaagtca 720
ggcatggatg ctgctttgga tgacttaata gatactttag gaggacctga agaaactgaa 780
gaagaaaata caacgtatac tggaccagaa gtttcagatc caatgagttc cacctacata 840
gaggaattgg gtaaaagaga agtcacaatt cctccaaaat atagggaact attggctaaa 900
aaggaaggga tcacagggcc tcctgcagac tcttcgaaac ccatagggcc agatgatgct 960
atagacgcct tgtcatctga cttcacctgt gggtcgccta cagctgctgg aaagaaaact 1020
gaaaaagagg aatctacaga agttttaaaa gctcagtcag cagggacagt cagaagtgct 1080
gctccacccc aagagaagaa aagaaaggtg gagaaggata caatgagtga tcaagcactc 1140
gaggctctgt cggcttcact gggcacccgg caagcagaac ctgagctcga cctccgctca 1200
attaaggaag tcgatgaggc aaaagctaaa gaagaaaaac tagagaagtg tggtgaggat 1260
gatgaaacaa tcccatctga gtacagatta aaaccagcca cggataaaga tggaaaacca 1320
ctattgccag agcctgaaga aaaacccaag cctcggagtg aatcagaact cattgatgaa 1380
ctttcagaag attttgaccg gtctgaatgt aaagagaaac catctaagcc aactgaaaag 1440
acagaagaat ctaaggccgc tgctccagct cctgtgtcgg aggctgtgtg tcggacctcc 1500
atgtgtagta tacagtcagc accccctgag ccggctacct tgaagggcac agtgccagat 1560
gatgctgtag aagccttggc tgatagcctg gggaaaaagg aagcagatcc agaagatgga 1620
aaacctgtga tggataaagt caaggagaag gccaaagaag aagaccgtga aaagcttggt 1680
gaaaaagaag aaacaattcc tcctgattat agattagaag aggtcaagga taaagatgga 1740
aagccactcc tgccaaaaga gtctaaggaa cagcttccac ccatgagtga agacttcctt 1800
ctggatgctt tgtctgagga cttctctggt ccacaaaatg cttcatctct taaatttgaa 1860
gatgctaaac ttgctgctgc catctctgaa gtggtttccc aaaccccagc ttcaacgacc 1920
caagctggag ccccaccccg tgatacctcg cagagtgaca aagacctcga tgatgccttg 1980
gataaactct ctgacagtct aggacaaagg cagcctgacc cagatgagaa caaaccaatg 2040
gaagataaag taaaggaaaa agctaaagct gaacatagag acaagcttgg agaaagagat 2100
gacactatcc cacctgaata cagacatctc ctggatgata atggacagga caaaccagtg 2160
aagccaccta caaagaaatc agaggattca aagaaacctg cagatgacca agaccccatt 2220
gatgctctct caggagatct ggacagctgt ccctccacta cagaaacctc acagaacaca 2280
gcaaaggata agtgcaagaa ggctgcttcc agctccaaag cacctaagaa tggaggtaaa 2340
gcgaaggatt cagcaaagac aacagaggaa acttccaagc caaaagatga ctaaagaaat 2400
acaagttaag gtatctggta tctgcatgta aaatcttcag ctggtggatg gtgacttttg 2460
aagaacaaaa ggctttggca acagaaaaca attgttctgg gtgatttcta gaatggtttt 2520
tgttgagtct ctgaacatcc taaatattgg tttgttattc ttttccagaa agaaaatgaa 2580
tttgactggt tcacctgtgt actgagtatt gataaacttt gaattttttt aattgccttc 2640
aattgggaga gaaagcttta tatttgtaag aaatatattt gataaagttt cttaaagcaa 2700
caccaaaaaa acaaaagaaa agctaagtga atttttgcac attctacaca cagtgcctgt 2760
aaatctcatt tgtattttca gtttgccctt aatttttttt gttagtgttt agaaaacaat 2820
gttttaaaca ttcttcagtg ttctgatttc ttattacccc ctttcctctt gggcttttga 2880
actgtatttg atgttgcttt gggataatgt ttataagtca aacataagat attgtacatt 2940
gggcacatat ctcctcttgg gctgctaata ataaattaat aacaggtaac ctggacaaac 3000
caggaagcac caaacccctt ttcagtttga actcttcttt gccaggtgtg aggacttctg 3060
catcttacag tcagcacaga acacactgag acttgaatca agtcagcaac agagcaaaat 3120
z~i4z
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
aaaggttaga taagtccttg tgtagcaaat ttcgagcata agaaataaaa tctaattaat 3180
tcttagggta ctcatctgac ttgaactctg ttggtttact gtgttagtaa actgtgcttt 3240
ctattatcta tacataaaac ctgagcagca actgtgtctt tagagctatt gccacattag 3300
cctttgcact gtatagcgtc tggctttatg gaacttaagt ttaccaaata taaaaagaaa 3360
cttctgcttt taaaaaaatt atatatatat atattaaatt tgaaacctgc atttctccca 3420
cagcaatgta agaagtaggc tctgatgtcc taccactttg aatggttttc taatatctta 3480
atgaatagtt cctgaacatt gcactgatat catcgattag aattttgata tttaatttca 3540
tctttatttc ctggtagaga atgcaggaaa agatgtcagg tacataacat aaaacagatt 3600
gggaatttat tgtttccaaa gggcatggcc ttccttagca tcagtttgaa gcttttgtta 3660
tgacttagct gacttgtggc agcggggcaa gcaaaaacaa taacactgct tataaatggc 3720
accacatctt gttaacctcc cccccaaata ctctctgaaa gtcatgcaca tacctatggg 3780
attttacaca ccaccagctt aaaatgctat gtctctatcc atcagaaata gtcattattc 3840
tatttttaag gcagcaacaa gaaaagaaaa aacacttttc ctgagggatt tctaaccatg 3900
tatctaatcc tcccattttg ggcagtatag gtgtttgctt ttttgttttc tttttttaag 3960
aaaaaccttg aaacctttga cactgacaga tgtgtttgca aggatacggc tgcagtatta 4020
ctaatttcca tgtgtatctg gaagtatttt taaatggcat accaaaatcc agaagtttaa 4080
agatgcctat aaaagtaaac aacatttatt taaaaagaac tctgaatatg ccttcttttt 4140
taattagaaa tatcttcgag acttgggtgt ttgttaataa ctaataactg gagtaagcta 4200
caggatctaa agcagccctt tttacagtct agttaggaga gagaaaataa ttgcaaatat 4260
ccacttagag gcaaagaaca attttttatt atcaaaaagg tttctgcaca ttgttgtggc 4320
aatattgtat ctgtttagaa aatgggcttt tccaaaagca aacaaagata ggttcctcag 4380
gtgaccaaaa ctgaaaatca atatttccat gtttcattaa tcaaggcata aaatacaatt 4440
aaagcaaaat attttacatt aaaatcttgg ttgtgtattt ttttaaaaga agggaaatag 4500
tttagtttgg ggtggaaatt accagtgatt tctattttta ttaaaccatt cactacaaca 4560
aataagtata aaaattccaa ttccactttt atacctattt atttgttgta gtgaatggtt 4620
taataaatgg cagatttatg tccagaagtc actctatttt gtcgtgtatt agaggaacac 4680
attttgacat ttttcgtatc aatcatcaat catattccct tatcttgaag tttttgcctt 4740
cttatattca aaaagttcag tttgaattct cctttgccag gtgtgaggat ttccgcacct 4800
tagagtcagc gcaaaacacg ctgcaacttg aatcaatcaa gtcagcaaca gagcacaccg 4860
gacgcgtggg cggacgcgtg ggcggacgcg tgggcggacg cgtggg 4906
<210> 23
<211> 1641
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1698270
<400> 23
gccgcctcca cggcccgcgc tcgtactgga gcgaagagcg gcctcctgaa ggaggggaag 60
ggacgtgggg gcggccacgg caggattaac ctccatttca gctaatcatg ggagagatta 120
aagtctctcc tgattataac tggtttagag gtacagttcc ccttaaaaag attattgtgg 180
atgatgatga cagtaagata tggtcgctct atgacgcggg cccccgaagt atcaggtgtc 240
ctctcatatt cctgccccct gtcagtggaa ctgcagatgt ctttttccgg cagattttgg 300
ctctgactgg atggggttac cgggttatcg ctttgcagta tccagtttat tgggaccatc 360
tcgagttctg tgatggattc agaaaacttt tagaccattt acaattggat aaagttcatc 420
tttttggcgc ttctttggga ggctttttgg cccagaaatt tgctgaatac actcacaaat 480
ctcctagagt ccattcccta atcctctgca attccttcag tgacacctct atcttcaacc 540
aaacttggac tgcaaacagc ttttggctga tgcctgcatt tatgctcaaa aaaatagttc 600
ttggaaattt ttcatctggc ccggtggacc ctatgatggc tgatgccatt gatttcatgg 660
tagacaggct agaaagtttg ggtcagagtg aactggcttc aagacttacc ttgaattgtc 720
aaaattctta tgtggaacct cataaaattc gggacatacc tgtaactatt atggatgtgt 780
ttgatcagag tgcgctttca actgaagcta aagaagaaat gtacaagctg tatcctaatg 840
cccgaagagc tcatctgaaa acaggaggca atttcccata cctgtgcaga agtgcagagg 900
tcaatcttta tgtacagata catttgctgc aattccatgg aaccaaatac gcggccattg 960
28/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
acccatcaat ggtcagtgcc gaggagcttg aggtgcagaa aggcagcctt ggcatcagcc 1020
aggaggagca gtagtgtgtc tctcgctgtc aatgatgagt tgacccggtg tgttcttgta 1080
tagtcagtgg catcagcacc cgtcagccgg ccttttcctt caggttcgtc aggctcaccg 1140
gttctcactg tgtctgggaa gtaggactga tggtcatctt catgacaggc ggcatctcca 1200
ctaagcctgt gtaactgttc cctctttggt tttcttagct tttgaatttg aagaagtact 1260
tttgaagact cccattttaa gaaccgtgca gattttgcta ccaaaagtct tcaccactgt 1320
gttcttaagt gaatgttaat ttctgaggtt tgggactttg tggtggtttt tttcttcttt 1380
tcttttccat tcttctttct ttctttttat gttgtttgct gtaaatgctg cacatccaga 1440
ttgcatatca ggacattggt tattttatgc tttcttggat ataaccatga tcagagtgcc 1500
atggccacta ccccactgtt tgctctcctg caaatcaact gcttttaatt tacacttaaa 1560
caaattgttt tgagtgttag ctactgcctt tctagatatt agtcatttgg aataaaaatt 1620
caatttcact gaaaaaaaaa a 1641
<210> 24
<211> 849
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2012492
<400> 24
aaaatgaatg aagaatgagg acattccacc actctccaag ttatcaagat gaagacccaa 60
gatggtggca ttcactctga aggtgcagca gctgagcatt ccaaattcgg gaaccaccag 120
aaaggctggc ctctcttcaa catgggatct tctggacttt tgagcctcct ggtgctattc 180
gtcctcttag cgaatgtcca gggacctggt ctgactgatt ggttatttcc caggagatgt 240
cccaaaatca gagaagaatg tgaattccaa gaaagggatg tgtgtacaaa ggacagacaa 300
tgccaggaca acaagaagtg ttgtgtcttc agctgcggaa aaaaatgttt agatctcaaa 360
caagatgtat gcgaaatgcc aaaagaaact ggcccctgcc tggcttattt tcttcattgg 420
tggtatgaca agaaagataa tacttgctcc atgtttgtct atggtggctg ccagggaaac 480
aataacaact tccaatccaa agccaactgc ctgaacacct gcaagaataa acgctttccc 540
tgattggata aggatgcact ggaagaactg ccagaatgtg gctcatgctc tgagtactgt 600
tcctgtacct gacggatgct ccagactggc ttccagtttc actctcagca ttccaagatc 660
ttagcccttc ccagaacaga acgcttgcat ctacctcctc ttcctccatc tttggctctt 720
ttgatgcaca atatccatcc gttttgattt catctttatg tcccctttat ctccaacttc 780
tagaactccc agtttatacc tgtgtcactc tcaatttttt ccagtaaagt acttgatgta 840
aaaaaaaaa 849
<210> 25
<211> 2166
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2309875
<400> 25
ggaccaacaa agatggcggc ggcccctgcg gcgggagcga tctgggcaac ggctgcggct 60
aaagctgcag ccgggcccac gggggggctg cacgggggta gtagggggtg gccctgaact 120
ggggcctggc cctggctggc ctctcccgcc gcctcactgg gggacaggtc cagcctgtgg 180
tgtccacaat gccccaggcc tctgagcacc gcctgggccg tacccgagag ccacctgtta 240
atatccagcc ccgagtggga tccaagctac catttgcccc cagggcccgc agcaaggagc 300
gcagaaaccc agcctctggg ccaaacccca tgttacgacc tctgcctccc cggccaggtc 360
tgcctgatga acggctcaag aaactggagc tgggacgggg acggacctca ggccctcgtc 420
ccagaggccc ccttcgagca gatcatgggg ttcccctgcc tggctcacca cccccaacag 480
29/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
tggctttgcc tctcccatct cggaccaact tagcccgttc caagtctgtg agcagtgggg 540
acttgcgtcc aatggggatt gccttgggag ggcaccgtgg caccggagag cttggggctg 600
cactgagccg cttggccctc cggcctgagc cacccacttt gagacgtagc acttctctcc 660
gccgcctagg gggctttcct ggacccccta ccctgttcag catacggaca gagccccctg 720
cttcccatgg ctccttccac atgatatccg cccggtcctc tgagcctttc tactctgatg 780
acaagatggc tcatcacaca ctccttctgg gctctggtca tgttggcctt cgaaacctgg 840
gaaacacgtg cttcctgaat gctgtgctgc agtgtctgag cagcactcga cctcttcggg 900
acttctgtct gagaagggac ttccggcaag aggtgcctgg aggaggccga gcccaagagc 960
tcactgaagc ctttgcagat gtgattggtg ccctctggca ccctgactcc tgcgaagctg 1020
tgaatcctac tcgattccga gctgtcttcc agaaatatgt tccctccttc tctggataca 1080
gccagcagga tgcccaagag ttcctgaagc tcctcatgga gcggctacac cttgaaatca 1140
accgccgagg ccgccgggct ccaccgatac ttgccaatgg tccagttccc tctccacccc 1200
gccgaggagg ggctctgcta gaagaacctg agttaagtga tgatgaccga gccaacctaa 1260
tgtggaaacg ttacctggag cgagaggaca gcaagattgt ggacctgttt gtgggccagt 1320
tgaaaagttg tctcaagtgc caggcctgtg ggtatcgctc cacgaccttc gaggtttttt 1380
gtgacctgtc cctgcccatc cccaagaaag gatttgctgg gggcaaggtg tctctgcggg 1440
attgtttcaa ccttttcact aaggaagaag agctagagtc ggagaatgcc ccagtgtgtg 1500
accgatgtcg gcagaaaact cgaagtacca aaaagttgac agtacaaaga ttccctcgaa 1560
tcctcgtgct ccatctgaat cgattttctg cctcccgagg ctccatcaaa aaaagttcag 1620
taggtgtaga ctttccactg cagcgactga gcctagggga ctttgccagt gacaaagccg 1680
gaagtcctgt ataccagctg tatgcccttt gcaaccactc aggcagcgtc cactatggcc 1740
actacacagc cctgtgccgg tgccagactg gttggcatgt ctacaatgac tctcgtgtct 1800
cccctgtcag tgaaaaccag gtggcatcca gcgagggcta cgtgctgttc taccaactga 1860
tgcaggagcc accccggtgc ctgtgacacc tctaagctct ggcacctgtg aagcccttta 1920
aacaccctta agccccaggc tccccgttta cctcagagac gtctattttt gtgtcttttt 1980
aatcggggag gggggagggg gtggttgtag ctccattatt ttttttatta aaaaataccc 2040
ttccacctgg aggctccctt gtctcccagc cccatgtaca aagctcacca agcccctgcc 2100
catgtacagc ccccagaccc tctgcaatat cactttttgt gaataaattt attaagaaaa 2160
aaaaaa
2166
<210> 26
<211> 2069
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2479394
<400> 26
gacttgagtc actctcagac tctttataaa tacagcttga ctcagccact gtatgactga 60
ctccccgggg acatgaggtg gatactgttc attggggccc ttattgggtc cagcatctgt 120
ggccaagaaa aattttttgg ggaccaagtt ttgaggatta atgtcagaaa tggagacgag 180
atcagcaaat tgagtcaact agtgaattca aacaacttga agctcaattt ctggaaatct 240
ccctcctcct tcaatcggcc tgtggatgtc ctggtcccat ctgtcagtct gcaggcattt 300
aaatccttcc tgagatccca gggcttagag tacgcagtga caattgagga cctgcaggcc 360
cttttagaca atgaagatga tgaaatgcaa cacaatgaag ggcaagaacg gagcagtaat 420
aacttcaact acggggctta ccattccctg gaagctattt accacgagat ggacaacatt 480
gccgcagact ttcctgacct ggcgaggagg gtgaagattg gacattcgtt tgaaaaccgg 540
ccgatgtatg tactgaagtt cagcactggg aaaggcgtga ggcggccggc cgtttggctg 600
aatgcaggca tccattcccg agagtggatc tcccaggcca ctgcaatctg gacggcaagg 660
aagattgtat ctgattacca gagggatcca gctatcacct ccatcttgga gaaaatggat 720
attttcttgt tgcctgtggc caatcctgat ggatatgtgt atactcaaac tcaaaaccga 780
ttatggagga agacgcggtc ccgaaatcct ggaagctcct gcattggtgc tgacccaaat 840
agaaactgga acgctagttt tgcaggaaag ggagccagcg acaacccttg ctccgaagtg 900
taccatggac cccacgccaa ttcggaagtg gaggtgaaat cagtggtaga tttcatccaa 960
aaacatggga atttcaaggg cttcatcgac ctgcacagct actcgcagct gctgatgtat 1020
30/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
ccatatgggt actcagtcaa aaaggcccca gatgccgagg aactcgacaa ggtggcgagg 1080
cttgcggcca aagctctggc ttctgtgtcg ggcactgagt accaagtggg tcccacctgc 1140
accactgtct atccagctag cgggagcagc atcgactggg cgtatgacaa cggcatcaaa 1200
tttgcattca catttgagtt gagagatacc gggacctatg gcttcctcct gccagctaac 1260
cagatcatcc ccactgcaga ggagacgtgg ctggggctga agaccatcat ggagcatgtg 1320
cgggacaacc tctactaggc gatggctctg ctctgtctac atttatttgt acccacacgt 1380
gcacgcactg aggccattgt taaaggagct ctttcctacc tgtgtgagtc agagccctct 1440
gggtttgtgg agcacacagg cctgcccctc tccagccagc tccctggagt cgtgtgtcct 1500
ggcggtgtcc ctgcaagaac tggttctgcc agcctgctca attttggtcc tgctgttttt 1560
gatgagcctt ttgtctgttt ctccttccac cctgctggct gggcggctgc actcagcatc 1620
accccttcct gggtggcatg tctctctcta cctcattttt agaaccaaag aacatctgag 1680
atgattctct accctcatcc acatctagcc aagccagtga ccttgctctg gtggcactgt 1740
gggagacacc acttgtcttt aggtgggtct caaagatgat gtagaatttc ctttaatttc 1800
tcgcagtctt cctggaaaat attttccttt gagcagcaaa tcttgtaggg atatcagtga 1860
aggtctctcc ctccctcctc tcctgttttt ttttttttga gacagagttt tgctcttgtt 1920
gcccaggctg gagtttaagg gcccgatctg ggctcaccaa aacctttgcc tccggggttc 1980
aagaaatttt cctgcctaag ccttttgagt accttggttt aaaggggcat tcaacaatgc 2040
ctgggaaatt ttgggttttt agaagaaaa 2069
<210> 27
<211> 2490
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2613215
<400> 27
ccgcgggtga tcagctggtc tgcgctcccc tgacgtgggc tggggcacgt caccgccgaa 60
tggcagcctc cagaaagcca ccgcgagtaa gggtgaatca ccaggatttt caactgagaa 120
atttaagaat aattgaacct aacgaggtga cacactcagg agacacaggt gtggaaacag 180
acggcagaat gcctccaaag gtgacttcag agctgcttcg gcagctgaga caagccatga 240
ggaactctga gtatgtgacc gaaccgatcc aggcctacat catcccatcg ggagatgctc 300
atcagagtga gtatattgct ccatgtgact gtcggcgggc ttttgtctct ggattcgatg 360
gctctgcggg cacagccatc atcacagaag agcatgcagc catgtggact gacgggcgct 420
actttctcca ggctgccaag caaatggaca gcaactggac acttatgaag atgggtctga 480
aggacacacc aactcaggaa gactggctgg tgagtgtgct tcctgaaggg tccagggttg 540
gtgtggaccc cttgatcatt cctacagatt attggaagaa aatggccaaa gttctgagaa 600
gtgccggcca tcacctcatt cctgtcaagg agaacctcgt tgacaaaatc tggacagacc 660
gtcctgagcg cccttgcaag cctctcctca cactgggcct ggattacaca ggcatctcct 720
ggaaggacaa ggttgcagac cttcggttga aaatggctga gaggaacgtc atgtggtttg 780
tggtcactgc cttggatgag attgcgtggc tatttaatct ccgaggatca gatgtggagc 840
acaatccagt atttttctcc tacgcaatca taggactaga gacgatcatg ctcttcattg 900
atggtgaccg catagacgcc cccagtgtga aggagcacct gcttcttgac ttgggtctgg 960
aagccgaata caggatccag gtgcatccct acaagtccat cctgagcgag ctcaaggccc 1020
tgtgtgctga cctctcccca agggagaagg tgtgggtcag tgacaaggcc agctatgctg 1080
tgagcgagac catccccaag gaccaccgct gctgtatgcc ttacaccccc atctgcatcg 1140
ccaaagctgt gaagaattca gctgagtcag aaggcatgag gcgggctcac attaaagatg 1200
ctgttgctct ctgtgaactc tttaactggc tggagaaaga ggttcccaaa ggtggtgtga 1260
cagagatctc agctgctgac aaagctgagg agtttcgcag gcaacaggca gactttgtgg 1320
acctgagctt cccaacaatt tccagtacgg gacccaacgg cgccatcatt cactacgcgc 1380
cagtccctga gacgaatagg accttgtccc tggatgaggt gtaccttatt gactcgggtg 1440
ctcaatacaa ggatggcacc acagatgtga cgcggacaat gcattttggg acccctacag 1500
cctacgagaa ggaatgcttc acatatgtcc tcaagggcca catagctgtg agtgcagccg 1560
ttttcccgac tggaaccaaa ggtcaccttc ttgactcctt tgcccgttca gctttatggg 1620
attcaggcct agattacttg cacgggactg gacatggtgt tgggtctttt ttgaatgtcc 1680
31 /42
CA 02338386 2001-02-02
WO OOJ09709 PCTNS99/17818
atgagggtcc ttgcggcatc agttacaaaa cattctctga tgagcccttg gaggcaggca 1740
tgattgtcac tgatgagccc gggtactatg aagatggggc ttttggaatt cgcattgaga 1800
atgttgtcct tgtggttcct gtgaagacca agtataattt taataaccgg ggaagcctga 1860
cctttgaacc tctaacattg gttccaattc agaccaaaat gatagatgtg gattctctta 1920
cagacaaaga gtgcgactgg ctcaacaatt accacctgac ctgcagggat gtgattggga 1980
aggaattgca gaaacagggc cgccaggaag ctctcgagtg gctcatcaga gagacgcaac 2040
ccatctccaa acagcattaa taaatacctc cccggttttg tttttgtaaa atgctctgga 2100
ggaaggaaga aacgtggcag atccctgaca tctttcccct ttcctttcct tcttccctac 2160
ctcccctttt tactttagac tttaagaaga acagaaaatc ttcttatcct ctttgatatt 2220
ttattgcaaa cactcagtct tttatgattt tttaattgtt gagaacaagc caagaataaa 2280
attgctgcac cagaaggagg gtccctccaa agttgaacac ttggtgaaag gaagatgccc 2340
cgacttcttt ggccagtgat ggggaatcag tgagtgctcc atgatggtca tgttccaggt 2400
gctagtacat cattcatgat caccttaatg ctcatgagac tatatttatg atcagtgaat 2460
aaaaatgtca gaactgtgaa aaaaaaaaaa 2490
<210> 2B
<211> 3148
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 001528
<400> 28
gaagatggcg gaaggcggag cggcggatct ggacacccag cggtctgaca tcgcgacgct 60
gctcaaaacc tcgctccgga aaggggacac ctggtaccta gtcgatagtc gctggttcaa 120
acagtggaaa aaatatgttg gctttgacag ttgggacaaa taccagatgg gagatcaaaa 180
tgtgtatcct ggacccattg ataactctgg acttctcaaa gatggtgatg cccagtcact 240
taaggaacac cttattgatg aattggatta catactgttg ccaactgaag gttggaataa 300
acttgtcagc tggtacacat tgatggaagg tcaagagcca atagcacgaa aggtggttga 360
acagggtatg tttgtaaagc actgcaaagt agaagtatat ctcacagaat tgaagctatg 420
tgaaaatgga aacatgaata atgttgtaac tcgaagattt agcaaagctg acacaataga 480
tacaattgaa aaggaaataa gaaaaatctt cagtattcca gatgaaaagg agaccagatt 540
gtggaacaaa tacatgagta acacatttga accactgaat aaaccagaca gcaccattca 600
ggatgctggt ttataccaag gacaggtatt agtgatagaa cagaaaaatg aagatggaac 660
acggccaagg ggtccttcta ctcctaatgt gaaaaactca aattactgtc ttccatcata 720
taccgcttat aagaactatg attattcgga acctggaaga aacaatgaac agccaggcct 780
ctgtggccta agtaacttgg gaaatacgtg tttcatgaac tcagctattc agtgtttgag 840
caacacacct ccacttactg agtatttcct caatgataag tatcaagaag aactgaattt 900
tgacaatccc ttaggaatga gaggtgaaat agctaaatct tatgccgaac tgatcaagca 960
aatgtggtct ggaaagttta gctacgtcac cccaagagcc tttaagacac aggtaggacg 1020
ttttgcacct cagttctctg gatatcagca gcaagactgt caagaactgt tagctttcct 1080
attagatgga ttacatgagg atttgaatag aattaggaaa aaaccatata tacaattaaa 1140
agatgcagat ggaaggccag ataaggtggt tgccgaagaa gcctgggaaa accatttaaa 1200
acgaaatgat tctatcatag tagatatatt tcatggcctt ttcaaatcaa ctttagtttg 1260
tcctgagtgt gctaagattt cagtaacatt tgatcctttt tgttacttga cacttccatt 1320
gcccatgaaa aaagaacgca ccttggaagt ttacttagtt agaatggatc cacttaccaa 1380
acctatgcag tacaaagtgg ttgtccccaa aattggaaac atattagatc tttgtacagc 1440
attgtctgct ttgtcaggaa tacctgcaga taagatgata gttactgata tatacaatca 1500
tagatttcac agaatattcg ctatggatga aaaccttagt agtattatgg aacgggatga 1560
tatttatgtg tttgaaatta acatcaatag gacagaagat acagagcacg tgattattcc 1620
tgtttgccta agagaaaaat tcagacactc gagttatacc caccatactg gttcttcact 1680
ttttggtcag ccctttctta tggctgtacc acgaaacaat actgaagaca aactttataa 1740
tctcctgctc ttgagaatgt gccgatatgt caaaatatct actgaaactg aagaaactga 1800
aggatcccta cactgctgta aggaccaaaa tattaatggg aatggcccaa atggcataca 1860
tgaagaaggc tcaccaagtg aaatggaaac agatgagcca gatgatgaat ccagccagga 1920
32/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
tcaagaactt ccctcagaga atgaaaacag tcagtctgaa gattcagttg gaggagataa 1980
tgattctgaa aatggattat gtactgagga tacttgcaaa ggtcaactca cgggacacaa 2040
aaaacgattg tttacattcc agttcaacaa cttaggcaat actgatatca actacatcaa 2100
agatgatacc aggcatataa gatttgatga taggcagctt aggctagatg aaagatcttt 2160
tcttgctttg gattgggatc ctgatttgaa aaaaagatat tttgatgaaa atgctgctga 2220
ggactttgaa aaacatgaaa gtgtggagta taaacctcct aaaaaaccct ttgtgaaatt 2280
aaaagattgc attgaacttt ttacaacaaa agaaaagcta ggtgctgaag atccctggta 2340
ttgtccgaat tgtaaagaac atcagcaagc cacaaagaaa ttggatttat ggtccctgcc 2400
tccagtactt gtagtacatc tcaagcgatt ttcttacagt cgatacatga gagacaagtt 2460
ggatacctta gttgattttc ctatcaatga cttggatatg tcggaattct taattaatcc 2520
aaatgcaggt ccttgccgct ataatctgat tgctgtttcc aaccactatg gagggatggg 2580
aggaggacac tatactgctt ttgcaaaaaa taaagatgat ggaaaatggt actattttga 2640
tgacagtagt gtctccactg catctgaaga ccaaattgtg tccaaagcag catatgtact 2700
cttctaccag agacaagaca ctttcagtgg aactggcttt tttcctcttg accgagaaac 2760
taaaggtgct tcagctgcca ctggcatccc attagaaagt gatgaagata gcaatgataa 2820
tgacaatgat atagaaaatg aaaactgtat gcacactaac taatgaaagt cctagaagcc 2880
ataaaagaga cactttcctg ctggtggtat ctatggaaat gatgaagtta cccaccacat 2940
taaaacaaaa gtctgagatg gggagtttca gataaccgaa tgtaaatcct ttatcagatt 3000
ttaacttgtg cagtacttga agtgaaacac aatgaaaact ttaacagaaa ttgtctctta 3060
atacatttac agtcttgtat ttacaagcta aatatatata ggaaatcaca aataaatccc 3120
ttttaagttt gctgctgttt tgattaaa 3148
<210> 29
<211> 855
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 747
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte Clone No: 998626
<400> 29
caggcaggca tcccccggag ttgtctcttt tcatgccagc gccaacagga ggctgtctgg 60
acacactgat tactcactca ccagcctcct tcttttgtcc accagccccc ctcttttgtc 120
caccagccca gcctgactcc tggagattgt gaatagctcc atccagcctg agaaacaagc 180
cgggtggctg agccaggctg tgcacggagc gcctgacggg cccaacagac ccatgctgca 240
tccagagacc tcccctggcc gggggcatct cctggctgtg ctcctggccc tccttggcac 300
cgcctgggca gaggtgtggc caccccagct gcaggagcag gctccgatgg ccggagccct 360
gaacaggaag gagagtttct tgctcctctc cctgcacaac cgcctgcgca gctgggtcca 420
gccccctgcg gctgacatgc ggaggctgga ctggagtgac agcctggccc aactggctca 480
agccagggca gccctctgtg gaatcccaac cccgagcctg gcgtccggcc tgtggcgcac 540
cctgcaagtg ggctggaaca tgcagctgct gcccgcgggc ttggcgtcct ttgttgaagt 600
ggtcagccta tggtttgcag aggggcagcg gtacagccac gcggcaggag agtgtgctcg 660
caacgccacc tgcacccact acacgcagct cgtgtgggcc acctcaagcc agctgggctg 720
tgggcggcac tagtgtctgc aggccangag catagaagct ttgtctgtgc tactccccgg 780
aggcactggg agtcacggga gacatcatcc tataagaggg tgctgtgtcg tctgacagca 840
tgttcagctg ctcaa 855
<210> 30
<211> 1912
<212> DNA
<213> Homo sapiens
33/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
<220>
<221> misc_feature
<223> Incyte Clone No: 1393301
<400> 30
agaattcggc acgagggtta gaggcggctt gtgtccacgg gacgcgggcg gatcttctcc 60
ggccatgagg aagccagccg ctggcttcct tccctcactc ctgaaggtgc tgctcctgcc 120
tctggcacct gccgcagccc aggattcgac tcaggcctcc actccaggca gccctctctc 180
tcctaccgaa tacgaacgct tcttcgcact gctgactcca acctggaagg cagagactac 240
ctgccgtctc cgtgcaaccc acggctgccg gaatcccaca ctcgtccagc tggaccaata 300
tgaaaaccac ggcttagtgc ccgatggtgc tgtctgctcc aacctccctt atgcctcctg 360
gtttgagtct ttctgccagt tcactcacta ccgttgctcc aaccacgtct actatgccaa 420
gagagtcctg tgttcccagc cagtctctat tctctcacct aacactctca aggagataga 480
agcttcagct gaagtctcac ccaccacgat gacctccccc atctcacccc acttcacagt 540
gacagaacgc cagaccttcc agccctggcc tgagaggctc agcaacaacg tggaagagct 600
cctacaatcc tccttgtccc tgggaggcca ggagcaagcg ccagagcaca agcaggagca 660
aggagtggag cacaggcagg agccgacaca agaacacaag caggaagagg ggcagaaaca 720
ggaagagcaa gaagaggaac aggaagagga gggaaagcag gaagaaggac aggggactaa 780
ggagggacgg gaggctgtgt ctcagctgca gacagactca gagcccaagt ttcactctga 840
atctctatct tctaaccctt cctcttttgc tccccgggta cgagaagtag agtctactcc 900
tatgataatg gagaacatcc aggagctcat tcgatcagcc caggaaatag atgaaatgaa 960
tgaaatatat gatgagaact cctactggag aaaccaaaac cctggcagcc tcctgcagct 1020
gccccacaca gaggccttgc tggtgctgtg ctattcgatc gtggagaata cctgcatcat 1080
aacccccaca gccaaggcct ggaagtacat ggaggaggag atccttggtt tcgggaagtc 1140
ggtctgtgac agccttgggc ggcgacacat gtctacctgt gccctctgtg acttctgctc 1200
cttgaagctg gagcagtgcc actcagaggc cagcctgcag cggcaacaat gcgacacctc 1260
ccacaagact ccctttgtca gccccttgct tgcctcccag agcctgtcca tcggcaacca 1320
ggtagggtcc ccagaatcag gccgctttta cgggctggat ttgtacggtg ggctccacat 1380
ggacttctgg tgtgcccggc ttgccacgaa aggctgtgaa gatgtccgag tctctgggtg 1440
gctccagact gagttcctta gcttccagga tggggatttc cctaccaaga tttgtgacac 1500
agactatatc cagtacccaa actactgttc cttcaaaagc cagcagtgtc tgatgagaaa 1560
ccgcaatcgg aaggtgtccc gcatgagatg tctgcagaat gagacttaca gtgcgctgag 1620
ccctggcaaa agtgaggacg ttgtgcttcg atggagccag gagttcagca ccttgactct 1680
aggccagttc ggatgagctg gcgtctattc tgcccacacc ccagcccaac ctgcccacgt 1740
tctctattgt tttgagaccc cattgctttc aggctgcccc ttctgggtct gttactcggc 1800
ccctactcac atttccttgg gttggagcaa cagtcccaga gagggccatg gtgggagctg 1860
cgccctcctt aaaagatgac tttacataaa atgttgatct tcaaaaaaaa as 1912
<210> 31
<211> 768
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1444055
<400> 31
taagtgttac aatttaaagc caaaggcata ccaaatatta aagcaacaca acataaggaa 60
tcccattcac cacacaccat ttaaacacaa atgatggatt ctgttattta tcaaacccat 120
ttaatgtttt gtgtagtgtt ttcatggatt ccaggaatgg cttccctttc tcaaatgatc 180
aatttcaagt cttattctcc atatttccca tctgattcca ctatgagcag tgccattgca 240
tgccctacta gaactgtgga tacgctttaa aacaagtgga agatgatagg gtgggactca 300
ttacgtctaa tcttggggaa cactgataac gtgtccagga gagacagcac aaggggctcc 360
atcttcatca cacaactcat cgcatgcttc cagagatatt cctggcgctg ccacctagag 420
gaagtatttt ggaaggttca gcaagcattt gaaagtccgg aggcaacagt ccaaatgccc 480
accatagaac gagtgtccat gacaagatat ttctacctct ttcctggcaa ctgaaaatgg 540
34/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
ttaagcattg agagttgttg gtggtgtatg aaataaatga aagtgtgata ttggagcagg 600
aaaccacaag cagcccagcc ctcctttatc aacttcaaga aacaccttta ctagtacaga 660
ttgaatgctt aacattttga atttcaataa aggtgaagac aaatgaaaaa aataaaaaaa 720
aaacaaaaac aaaaaacaaa caaaaaaaca aaaaaaaaat aaaaacgg 768
<210> 32
<211> 2069
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1650177
<400> 32
gcacaggggc cggcaccacg gggttatcga agcagctgtc aagatgctgg ggtccctggt 60
gttgaggaga aaagcactgg cgccacggct actcctccgg ctgctcaggt ccccaacgct 120
ccggggccat ggaggtgctt ccggccggaa tgtgactact gggagtctcg gggagccgca 180
gtggctgagg gtagccaccg gggggcgccc tggaacatcg ccggccttgt tctccggacg 240
tggggcagcc accggggggc gccagggagg acgcttcgat accaaatgcc tcgcggctgc 300
cacttgggga cgccttcctg gtcccgaaga aacactccca ggacaggaca gctggaacgg 360
ggtccccagc agggccggac tgggcatgtg cgccctggcc gcagcgctgg tggttcattg 420
ctacagcaag agtccgtcca acaaggatgc agccctgttg gaagctgccc gtgccaacaa 480
tatgcaagaa gtcagcagtg tggtacaggt cctgcttgct gctggggctg atccaaacct 540
tggagatgat ttcagcagtg ttttcaagac tgccaaggaa cagggaatcc attctttgga 600
agtcctgatc acccgagagg atgacttcaa caacaggctg aacaaccgcg ccagtttcaa 660
gggctgcacg gccttgcact atgctgttct tgctgatgac taccgcactg tcaaggagct 720
gcttgatgga ggagccaacc ccctgcagag gaatgaaatg ggacacacac ccttggatta 780
tgcccgagaa ggggaagtga tgaagcttct gaggacttct gaagccaagt accaagagaa 840
gcagcggaag cgtgaggctg aggagcggcg ccgcttcccc ctggagcagc gactaaagga 900
gcacatcatt ggccaggaga gcgccatcgc cacagtgggt gctgcgatcc ggaggaagga 960
gaatggctgg tacgatgaag aacaccctct ggtcttcctc ttcttgggat catctggaat 1020
aggaaaaaca gagctggcca agcagacagc caaatatatg cacaaagatg ctaaaaaggg 1080
cttcatcagg ctggacatgt ccgagttcca ggagcgacac gaggtggcca agtttattgg 1140
gtctccacca ggctacgttg gccatgagga gggtggccag ctgaccaaga agttgaagca 1200
gtgccccaat gctgtggtgc tctttgatga agtagacaag gcccatccag atgtgctcac 1260
catcatgctg cagctgtttg atgagggccg gctgacagat ggaaaaggga agaccattga 1320
ttgcaaggac gccatcttca tcatgacctc caatgtggcc agcgacgaga tcgcacagca 1380
cgcgctgcag ctgaggcagg aagctttgga gatgagccgt aaccgtattg ccgaaaacct 1440
gggggatgtc cagataagtg acaagatcac catctcaaag aacttcaagg agaatgtgat 1500
tcgccctatc ctgaaagctc acttccggag ggatgagttt ctgggacgga tcaatgagat 1560
cgtctacttc ctccccttct gccactcgga gctcatccaa ctcgtcaaca aggaactaaa 1620
cttctgggcc aagagagcca agcaaaggca caacatcacg ctgctctggg accgcgaggt 1680
ggcagatgtg ctggtcgacg gctacaatgt gcactatggc gcccgctcca tcaaacatga 1740
ggtagaacgc cgtgtggtga accagctggc agcagcctat gagcaggacc tgctgccagg 1800
gggctgtact ttgcgcatca cggtggagga ctcagacaag cagctactca aaagcccaga 1860
actgccctca ccccaggctg agaagcgcct ccccaagctg cgtctggaga tcatcgacaa 1920
ggacagcaag actcgcagac tggacatccg ggcaccactg caccctgaga aggtgtgcaa 1980
caccatctag cagccacctg cctgctccta tgtgccctca ccatccaata aaggcccctt 2040
ggctgtggca tggcaaaaaa aaaaaaaaa 2069
<210> 33
<211> 2594
<212> DNA
<213> Homo sapiens
<220>
35/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
<221> misc_feature
<223> Incyte Clone No: 1902576
<400> 33
ccagcacctg cggggccctc gggcttggaa ggctgggccg gacggtgaac ggtcggcgcg 60
ggccggatcg gcggcggctg actcgccttc tctccggggc tgcgaccccg aggcaaccgg 120
ctgcagatgg gagcccgcgg agccgcggat gcgggcgggc cggggcgcga cgccggcgag 180
ggagctgttc cgggacgccg ccttccccgc cgcggactcc tcgctcttct gcgacttgtc 240
tacgccgctg gcccagttcc gcgaggacat cacgtggagg cggccccagg agatttgtgc 300
cacaccccgg ctgtttccag atgacccacg ggaagggcag gtgaagcagg ggctgctggg 36D
ggattgctgg ttcctgtgtg cctgcgccgc gctgcagaag agcaggcacc tcctggacca 420
ggtcattcct ccgggacagc cgagctgggc cgaccaggag taccggggct ccttcacctg 480
tcgcatttgg cagtttggac gctgggtgga ggtgaccaca gatgaccgcc tgccgtgcct 540
tgcagggaga ctctgtttct cccgctgcca gagggaggat gtgttctggc tccccttact 600
ggaaaaggtc tacgccaagg tccatgggtc ctacgagcac ctgtgggccg ggcaggtggc 660
ggatgccctg gtggacctga ccggcggcct ggcagaaaga tggaacctga agggcgtagc 720
aggaagcgga ggccagcagg acaggccggg ccgctgggag cacaggactt gtcggcagct 780
gctccacctg aaggaccagt gtctgatcag ctgctgcgtg ctcagcccca gagcaggtgc 840
ccgggagctg ggggagttcc atgccttcat tgtctcggac ctgcgggagc tccagggtca 900
ggcgggccag tgcatcctgc tgctgcggat ccagaacccc tggggccggc ggtgctggca 960
ggggctctgg agagaggggg gtgaagggtg gagccaggta gatgcagcgg tagcatctga 1020
gctcctgtcc cagctccagg aaggggagtt ctgggtggag gaggaggagt tcctcaggga 1080
gtttgacgag ctcaccgttg gctacccggt cacggaggcc ggccacctgc agagcctcta 1140
cacagagagg ctgctctgcc atacgcgggc gctgcctggg gcctgggtca agggccagtc 1200
agcaggaggc tgccggaaca acagcggctt tcccagcaac cccaaattct ggctgcgggt 1260
ctcagaaccg agtgaggtgt acattgccgt cctgcagaga tccaggctgc acgcggcgga 1320
ctgggcaggc cgggcccggg cactggtggg tgacagtcat acttcgtgga gcccagcgag 1380
catcccgggc aagcactacc aggctgtggg tctgcacctc tggaaggtag agaagcggcg 1440
ggtcaatctg cctagggtcc tgtccatgcc ccccgtggct ggcaccgcgt gccatgcata 1500
cgaccgggag gtccacctgc gttgtgagct ctcaccgggc tactacctgg ctgtccccag 1560
caccttcctg aaggacgcgc caggggagtt cctgctccga gtcttctcta ccgggcgagt 1620
ctcccttagc gccatcaggg cagtggccaa gaacaccgcc cccggggcag ccctgcctgc 1680
gggggagtgg gggaccgtgc agctacgggg ttcttggaga gtcggccaga cggcgggggg 1740
cagcaggaac tttgcctcat accccaccaa cccctgcttc cccttctcgg tccccgaggg 1800
ccctggcccc cgctgcgtcc gcatcactct gcatcagcac tgccggccca gtgacaccga 1860
gttccacccc atcggcttcc atatcttcca ggtcccagag ggtggaagga gccaggacgc 1920
acccccactg ctgctgcagg agccgctgct gagctgcgtg ccacatcgct acgcccagga 1980
ggtgagccgg ctctgcctcc tgcctgcggg cacctacaag gttgtgccct ccacctacct 2040
gccggacaca gagggggcct tcacagtgac catcgcaacc aggattgaca ggccatccat 2100
tcacagccag gagatgctgg gccagttcct ccaagaggtc tccgtcatgg cagtgatgaa 2160
aacctaacag ggtggccccc tgtgccagct caggtgactg gagcccgagg gcctgacagg 2220
ttcccagcag ctgggccggc cagccttgca ctgtgggggc tggtcctgag tcttggcctg 2280
cctcccagcc ctgccagggg gctgcggcct aggggtccac gggaagcctc cgtcaggaga 2340
gacgcagccc tgggggccag ctggtgctgc aaggaagggt gggaagcttg ctggcttctg 2400
ttgcgccact gagacggcag agaccccagg atcccagagc ttcccaggat ccctcccaga 2460
tcctctgctg actccatatg gaggcctcac acccagaggg tagggcagca gatcttcttt 2520
ataactattt attgttcgaa tcacttttag gatgtaactt tataaataaa catgagcgct 2580
gaaaaaaaaa aggg 2594
<210> 34
<211> 481
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2024210
36/42
CA 02338386 2001-02-02
WO 00/09709
PCT/US99/17818
<400> 34
tgaggtgcga cacacataat tgtcccaatt tttaagattg atggggagca tgaagcattt 60
ttttaatgtg ttggcaggcc ccattaaatg cataaactgc ataggactca tgtggtctga 120
atgtatttta gggctttctg ggaattgtct tgacagagaa cctcagctgg acaaagcagc 180
cttgatctga gtgagctaac tgacacaatg aaactgtcag gcatgtttct gctcctctct 240
ctggctcttt tctgcttttt aacaggtgtc ttcagtcagg gaggacaggt tgactgtggt 300
gagttccagg accccaaggt ctactgcact cgggaatcta acccacactg tggctctgat 360
ggccagacat atggcaataa atgtgccttc tgtaaggcca tagtgaaaag tggtggaaag 420
attagcctaa agcatcctgg aaaatgctga gttaaagcca atgtttcttg gtgacttgcc 480
481
<210> 35
<211> 3080
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2523109
<400> 35
cgcagcgcgg ctttcaggcc aacatggccg tgctgctgct gctgctccgt gccctccgcc 60
ggggtccagg cccgggtcct cggccgctgt ggggcccagg cccggcctgg agtccagggt 120
tccccgccag gcccgggagg gggcggccgt acatggccag caggcctccg ggggacctcg 180
ccgaggctgg aggccgagct ctgcagagct tacaattgag actgctaacc cctacctttg 240
aagggatcaa cggattgttg ttgaaacaac atttagttca gaatccagtc agactctggc 300
aacttttagg tggtactttc tattttaaca cctcaaggtt gaagcagaag aataaggaga 360
aggataagtc gaaggggaag gcgcctgaag aggacgaaga ggagaggaga cgccgtgagc 420
gggacgacca gatgtaccga gagcggctgc gcaccttgct ggtcatcgcg gttgtcatga 480
gcctcctgaa tgctctcagc accagcggag gcagcatttc ctggaacgac tttgtccacg 540
agatgctggc caagggcgag gtgcagcgcg tccaggtggt gcctgagagc gacgtggtgg 600
aagtctacct gcaccctgga gccgtggtgt ttgggcggcc tcggctagcc ttgatgtacc 660
gaatgcaggt tgcaaatatt gacaagtttg aagagaagct tcgagcagct gaagatgagc 720
tgaatatcga ggccaaggac aggatcccag tttcctacaa gcgaacagga ttctttggaa 780
atgccctgta ctctgtgggg atgacggcag tgggcctggc catcctgtgg tatgttttcc 840
gtctggccgg gatgactgga agggaaggtg gattcagtgc ttttaatcag cttaaaatgg 900
ctcgtttcac cattgtggat gggaagatgg ggaaaggagt cagcttcaaa gacgtggcag 960
gaatgcacga agccaaactg gaagtccgcg agtttgtgga ttatctgaag agcccaaaac 1020
gcttcctcca gcttggcgcc aaggtcccaa agggcgcact gctgctcggc ccccccggct 1080
gtgggaagac gctgctggcc aaggcggtgg ccacggaggc tcaggtgccc ttcctggcga 1140
tggccggccc agagttcgtg gaggtcattg gaggcctcgg cgctgcccgt gtgcggagcc 1200
tctttaagga agcccgagcc cgggccccct gcatcgtcta catcgatgag atcgacgcgg 1260
tgggcaagaa gcgctccacc accatgtccg gcttctccaa cacggaggag gagcagacgc 1320
tcaaccagct tctggtagaa atggatggaa tgggtaccac agaccatgtc atcgtcctgg 1380
cgtccacgaa ccgagctgac attttggacg gtgctctgat gaggccaggc cgactggacc 1440
ggcacgtctt cattgatctc cccacgctgc aggagaggcg ggagattttt gagcagcacc 1500
tgaagagcct gaagctgacc cagtccagca ccttttactc ccagcgtctg gcagagctga 1560
caccaggatt cagtggggct gacatcgcca acatctgcaa tgaggctgcg ctgcacgcgg 1620
cgcgggaggg acacacttcc gtgcacactc tcaacttcga gtacgccgtg gagcgcgtcc 1680
tcgcagggac tgccaaaaag agcaagatcc tgtccaagga agaacagaaa gtggttgcgt 1740
ttcatgagtc gggccacgcc ttggtgggct ggatgctgga gcacacggag gccgtgatga 1800
aggtctccat aacccctcgg acaaacgccg ccctgggctt tgctcagatg ctccccagag 1860
accagcacct cttcaccaag gagcagctgt ttgagcggat gtgcatggcc ttgggaggac 1920
gggcctcgga agcactgtcc ttcaacgagg tcacttctgg ggcacaggac gacctgagga 1980
aggtcacccg catcgcctac tccatggtga agcagtttgg gatggcacct ggcatcgggc 2040
ccatctcctt ccctgaggcg caggagggcc tcatgggcat cgggcggcgc cccttcagcc 2100
aaggcctgca gcagatgatg gaccatgaag caagactgct ggtggccaag gcctacagac 2160
37/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
acaccgagaa ggtgctgcag gacaacctgg acaagttgca ggcgctggca aacgcccttc 2220
tggaaaagga agtgataaac tatgaggaca ttgaggctct cattggcccg ccgccccatg 2280
ggccgaagaa aatgatcgca ccgcagaggt ggatcgacgc ccagagggag aaacaggact 2340
tgggcgagga ggagaccgaa gagacccagc agcctccact tggaggcgaa gagccgactt 2400
ggcccaagta gttgggaggt gttggctgca cgtgcgggtg gtccgggaag tgagggctca 2460
ctcagccacc ctgagttgct tttcagctga ggtttgcact tcctctcgcg gccctcagta 2520
gtccctgcac agtgacttct gagatctgtt gattgatgac ccttttcatg attttaagtt 2580
tctctgcaga aactactgac ggagtcctgt gtttgtgagt cgtttcccct atggggaagg 2640
ttatcagtgc ttcccgagtg agcatggaac acttcgagtt cccagggtta tagacagtcg 2700
ttcccagtgt ggctgaggcc acccagaggc agcagagcat tcagactcca aacagacccc 2760
tgttcatgcc gacgcttgca cgaccgcccc agttcctgtg gctccctcgg aatgctaagg 2820
ggatcggaca tgaaaggacc ctgtgagccg attgtcctat ctccagcggc cctgtcatcc 2880
agctcactca tcaatggggc cacacagtca ggcccaggca ctgggctccg gaggactcac 2940
cactgccccc tgctgccatg tggactggtg caagttgagg acttcttgct ggtctagtca 3000
cgcatgcagt gttggggatg ccttggtttt tactgctctg agaattgttg agatacttta 3060
ctaataaact gtgtagttgg 3080
<210> 36
<211> 1154
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2588566
<400> 36
gaactatgtt gtggttgcac agacacggag aaaatcagcg gagttcttgc tccgaatctt 60
cctgtaaatg ccagacagtg acaggcacct gagcagccat ttcaacctca gaatgaaggg 120
aagcccttca gaacatggct cccaacaaag cattttcaac agatatgctc agcagaggct 180
ggacattgat gccacccagc ttcagggcct tctcaaccag gagcttctaa caggacctcc 240
aggggacatg ttctccttag atgagtgccg cagcttggtg gctctgatgg aactgaaagt 300
gaatgggcgg ctagaccaag aggagtttgc gcgactgtgg aagcgccttg ttcactacca 360
gcatgttttc cagaaggttc agacaagccc tggagtcctc ctgagctcgg acttgtggaa 420
ggccatagag aatacagact tcctcagagg gatcttcatc agccgtgagc tgctgcatct 480
ggtgaccctc aggtacagcg acagcgtcgg cagggtcagc ttccccagcc tggtctgctt 540
cctgatgcgg cttgaagcca tggcaaagac cttccgcaac ctctctaagg atggaaaagg 600
actctacctg acagaaatgg agtggatgag cctggtcatg tacaactgaa gcaaagagga 660
aagcagaccc atggctcagg acaagctccc agtgatcact caagaatctg gctctcattc 720
taagaggctg tgctgcccag tatggtggtt gtgataaatc taaaccagcc ctgcatgaaa 780
cagagtccaa gctgtctccc aacagcctgg gttcggtcct tggctggccc aggcccagtt 840
aagcctgtgg ccaccaagca gctcatctga gcactttggg atgtattcag cctacgttgc 900
cctggaaaag gaagcaggag atgtctccct gtgggaaagg agaagagaag ttgtctctga 960
gtcccctgtc accagttgga ttcatttctt ggaagagcca gaatgagcca ctttgaccac 1020
cctcgggtgc tatgggtgac acaagagctg tccactgggt gtttgcagaa taattacact 1080
atcttatgtc tggatcctga tgatttcaca gctaaatggc aaaaataaaa catgtttccc 1140
ataaaaaaaa aaaa 1154
<210> 37
<211> 2827
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 2811
<223> a or g or c or t, unknown, or other
3 8/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
<220>
<221> misc_feature
<223> Incyte Clone No: 2740570
<400> 37
catttcgggg gtattctcag caggatgctc aagaattcct tcgatgttta atggatttgc 60
ttcatgaaga attgaaagag caagtcatgg aagtagaaga agatccgcaa accataacca 120
ctgaggagac aatggaagaa gacaagagcc agtcggatgt agattttcag tcttgtgaat 180
cttgtagcaa cagtgataga gcagaaaatg aaaatggctc tagatgcttt tctgaagata 240
ataatgaaac aacaatgtta attcaggatg atgaaaacaa ttcagaaatg tcaaaggatt 300
ggcaaaaaga gaagatgtgc aataagatta ataaagtaaa ttctgaaggc gaatttgata 360
aagatagaga ctctatatct gaaacagtcg acttaaacaa ccaggaaact gtcaaagtgc 420
aaatacacag cagagcttca gaatatatca ctgatgtcca ttcgaatgac ctgtctacac 480
cacagatcct tccatcaaat gaaggtgtta atccacgttt atcggcaagc cctcctaaat 540
caggcaattt gtggccagga ttggcaccac cacacaaaaa agctcagtct gcatctccaa 600
agagaaaaaa acagcacaag aaatacagaa gtgttatttc agacatattt gatggaacaa 660
tcattagttc agtgcagtgt ctgacttgtg acagggtgtc tgtaaccctc gagacctttc 720
aagatctgtc cttgccaatt cctggcaagg aagaccttgc taagctgcat tcatcaagtc 780
atccaacttc tatagtcaaa gcaggatcat gtggcgaagc atatgctcca caagggtgga 840
tagctttttt catggaatat gtgaagaggt ttgttgtctc atgtgtccct agctggtttt 900
ggggtccagt agtaaccttg caagattgtc ttgctgcctt ctttgccaga gatgaactaa 960
aaggtgacaa tatgtacagt tgtgaaaaat gcaaaaagct gagaaatgga gtgaagtttt 1020
gtaaagtaca aaactttcct gagattttgt gcatccacct taaaagattc agacatgaac 1080
taatgttttc caccaaaatc agtacccatg tttcatttcc gctagaaggc ttggatcttc 1140
agccatttct tgctaaggat agtccagctc aaattgtgac atatgatctt ctgtcagtca 1200
tttgccatca tggaactgca agtagtggac actatatagc ctactgccga aacaatctaa 1260
ataatctctg gtatgaattt gatgatcaga gtgtcactga agtttcagaa tctactgtac 1320
aaaatgcaga agcttacgtt cttttctata ggaagagcag cgaagaggca caaaaagaga 1380
ggagaaggat atcaaattta ttgaacataa tggaaccaag cctccttcag ttttatattt 1440
ctcgacagtg gcttaataaa tttaagacct ttgccgaacc tggccctatt tcaaataatg 1500
actttctttg tattcatgga ggtgttcctc caagaaaagc tggttatatt gaagacctgg 1560
ttttgatgct gcctcagaac atttgggata acctatatag caggtatggt ggaggaccag 1620
ctgtcaacca tctgtacatt tgtcatactt gccaaattga ggcggagaaa attgaaaaaa 1680
gaagaaaaac tgaattggaa atttttattc ggcttaacag agcgttccaa aaagaggact 1740
ctccagctac tttttattgc atcagtatgc agtggtttag agaatgggaa agttttgtga 1800
agggtaaaga tggagatcct ccaggtccta ttgacaatac taagattgca gtcactaaat 1860
gtggtaatgt gatgcttagg caaggagcag attctggcca gatttctgaa gaaacatgga 1920
attttctgca gtctatttat ggtggagggc ctgaagttat cctgcgacct ccggttgttc 1980
atgttgatcc agatatactt caagcagaag aaaaaattga agtagaaact cggtctttgt 2040
aatttttagg atgtagagag ttctaatgag gaatcatttt catgtgccct gacatgtaca 2100
catgcgaaaa cattcctaaa agcgtgttta tttgctttat tttttttcat catttatccc 2160
atttatttct tcttagtggg cattatggaa gaatatatta aaatgtgtaa tataccacag 2220
gttggtatat ttagttttaa atacttacca taaagtcttt cagtgtaatt tttttttgag 2280
acagagtctt gctttgtcac ccaggctgga gtgctgtggt gttacctcag ctcactgcag 2340
cctccacctc ctgggttcaa gcgattctcc tgcctcagcc tctcgagtag ctgggattac 2400
aggcacctgc caccatgccc ggctaatttt tgtattttag tagagatggg gtttcaccat 2460
gttggccagg ctagtctcaa actcctgacc tcaggtgatc cacccacctg ggcctcccaa 2520
agtgctggga ttacaggtgt gagccacagc gcctggctcg tgttctaagg aaatagctac 2580
ctaaggtatc ttattaaaac aaatgaacaa aaagtttccc aaactgtgtt ctaaggaaat 2640
agctacctat cttattaaaa aaaaaacaaa aacaaagttt tatgctctaa taagtttggg 2700
aaatgctagg ttatacaaag ctaaacatga ttctttccag tgggacatct gagagtcttt 2760
aattagctaa cagtgcattg tgattctgca aaggagttca ataattcacc ngtacgcgtg 2820
gtcgacc 2827
<210> 38
<211> 2987
<212> DNA
39/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2820384
<400> 38
tgctcaccta cgcagctcct ctcgcccctg cagccccgtc caccaccacg agggccatgc 60
caagctgtct agcagccccc ctcgtgcaag ccccgtgagg gtggcaccgt cgtacgtgct 120
caagaaagcc caggtattga gtgctggcag ccggaggggt aaggagcagc gctaccgcag 180
cgtcatctca gacatctttg acggctccat tcttagcctc gtgcagtgtc tcacctgtga 240
ccgggtatcc accacagtgg aaacgttcca ggacttatca ctgcccattc ctggaaagga 300
ggacctggcc aagctccatt cagccatcta ccagaatgtg ccggccaagc caggcgcctg 360
tggggacagc tatgccgccc agggctggct ggccttcatt gtggagtaca tccgacggtt 420
tgtggtatcc tgtacaccca gctggttttg ggggcctgtc gtcaccctgg aagactgcct 480
tgctgccttc tttgccgctg atgagttaaa gggtgacaac atgtacagct gtgagcggtg 540
taagaagctg cggaacggag tgaagtactg caaagtcctg cggttgcccg agatcctgtg 600
cattcaccta aagcgctttc ggcacgaggt gatgtactca ttcaagatca acagccacgt 660
ctccttcccc ctcgaggggc tcgacctgcg ccccttcctt gccaaggagt gcacatccca 720
gatcaccacc tacgacctcc tctcggtcat ctgccaccac ggcacggcag gcagtgggca 780
ctacatcgcc tactgccaga acgtgatcaa tgggcagtgg tacgagtttg atgaccagta 840
cgtcacagaa gtccacgaga cggtggtgca gaacgccgag ggctacgtac tcttctacag 900
gaagagcagc gaggaggcca tgcgggagcg acagcaggtg gtgtccctgg ccgccatgcg 960
ggagcccagc ctgctgcggt tctacgtgtc ccgcgagtgg ctcaacaagt tcaacacctt 1020
cgcagagcca ggccccatca ccaaccagac cttcctctgc tcccacggag gcatcccgcc 1080
ccacaaatac cactacatcg acgacctggt ggtcatcctg ccccagaacg tctgggagca 1140
cctgtacaac agattcgggg gtggccccgc cgtgaaccac ctgtacgtgt gctccatctg 1200
ccaggtggag atcgaggcac tggccaagcg caggaggatc gagatcgaca ccttcatcaa 1260
gttgaacaag gccttccagg ccgaggagtc gccgggcgtc atctactgca tcagcatgca 1320
gtggttccgg gagtgggagg cgttcgtcaa ggggaaggac aacgagcccc ccgggcccat 1380
tgacaacagc aggattgcac aggtcaaagg aagcggccat gtccagctga agcagggagc 1440
tgactacggg cagatttcgg aggagacctg gacctacctg aacagcctgt atggaggtgg 1500
ccccgagatt gccatccgcc agagtgtggc gcagcgctgg gcccagagaa cctgcacggg 1560
gagcagaaga tcgaagccga gacgcgggcc gtgtgatctg ctgggctagt ctccccatgt 1620
gccccacccc gcggaaggcg tgtttgtgcc cagaagagag gccgggctgc tgcagaaccc 1680
cgccgtgtaa agaggcagaa aagttggttt ggtttgcagt aacgctgcaa ctagaaaata 1740
tatgcacttc aggcttgttg aaacgaccaa gactctgtga cgttaatttg ggtctttgtc 1800
ctggcagtgc ctctgccagt cactgtcatc gttgtgtccc ccacaactgt cctcttgcta 1860
gctcggccca gctttgtccc tggagcccga tgctacccct gtcagacaga ggctgcggcc 1920
tgggccagag tcagggagta gctgctgctt cacggcgtct ccactgtgcg attggcccgg 1980
agccccgaag actcggaggg agctgctcag ggccggtgag cgcaccagaa gccctggcca 2040
gtgaggagct cacaggtcct ccctggtggt cccgccgcac ctctgcatct cctgggcgtc 2100
accaggaagg ctctgaagtc ccgggctgct ctcagcactt ctcctgcaga ctgaagactc 2160
tggactcatt gctgattgga acaccaggag gaggttggat ttctgccagt gggggatgtt 2220
tctggaggca gctggtcccc cacaccgcgt cctgctgagc ctgccccctg gattggctgt 2280
aatttgcctc gaagttcagc agttcatctt catgggaaat ttgctgagcc cccaccaggg 2340
aaccggatga tgaaacaggg atacctcaca gcttggccat ttgaggcaaa ggcagcttcc 2400
cgagctgatg ctaaagaaga cagactttcc cttcctccca gcagcagcag tgcagagccc 2460
gcctggaggg atgtgggggc tgtgcagggt gcagcgctca ggtggatcct gggaagcagc 2520
ctctggatgc tgagtggagg gagccactga gcacagcaag gcaccaaagc ccctggagaa 2580
accgccaggg cgaggtgcga ccatcatcag gatcaaagca gacggggcgt gggtggggaa 2640
ggggctctgg gaccagaccc cccacactac tgcgtctttg tttctatcag tctttgtaga 2700
agcaggtggt ggtggaaatt ccagcaggtg ggtcccgcag aggccctgag gcctcacttt 2760
tcggatcttc tgtcccagat cctgctccct ccctgctgag cctggggttc ccctggcatt 2820
ggccccagcc ttctgaaagc cggcgctgca gccagaggcc gcacgctgca ctgtcgcgac 2880
gcagagaggc ttctgtgcag gctgggatcg ggccccatgt ctgtgctgtc tagtttgtgt 2940
tcaaaatgtc agaataaaca cagaataaat gttaaaaaaa aaaaaaa 2987
40/42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
<210> 39
<211> 1215
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2990692
<400> 39
agagcacctt agtaggccgg attcggctca gatgagtatg cataaggcaa tgctaatggc 60
tcaagcaatg agggggctca ctctaggagg acaagttaga acatttggga aaaaatgtta 120
taattgtggt caaatcggtc atctgaaaag gagttgccca gtcttaaata aacagaatat 180
aataaatcaa gctattacag caaaaaataa aaagccatct ggcctgtgtc caaaatgtgg 240
aaaaggaaaa cattgggcca atcaatgtca ttctaaattt gataaagatg ggcaaccatt 300
gtcgggaaac aggaagaggg gccagcctca ggccccccaa caaactgggg cattcccagt 360
tcaactgttt gttcctcagg gttttcaagg acaacaaccc ctacagaaaa taccaccact 420
tcagggagtc agccaattac aacaatccaa cagctgtccc gcgccacagc aggcagcgcc 480
acagtagatt tatgttccac ccaaatggtc tctttactcc ctggagagcc cccacaaaag 540
attcctagag gggtatatgg cccgctgcca gaagggaggg taggccttat tttagggaga 600
tcaagtctaa atttgaaggg agtccaaatt catactgggg taatttattc agattataaa 660
gggggaattc agttagtgat cagctccact gttccctgga gtgccaatcc aggtgataga 720
attgctcaat tactgctttt gccttatgtt aaaattgggg aaaacaaaac ggaaagaaca 780
ggagggtttg gaagtaccaa ccctgcagga aaagccactt attgggctaa tcaggtctca 840
gaggatagac ccgtgtgtac agtcactatt ccagggaaag agtttgaagg attagtggat 900
acccaggctg atgtttctat catcggcata ggcaccgcct cagaagtgta tcaaagtgcc 960
atgattttac attgtctagg atctgataat caagaaagta cggttcagcc tatgatcact 1020
tctattccaa tcaatttatg gggccgagac ttgttacaac aatggcatgc agagattact 1080
atcccagcct ccctatacag ccccaggaat caaaaaatca tgactaaaat gggatagctc 1140
cctaaaaagg gactaggaaa gaatgaagat ggcattaaag tcccaactga ggctgaaaaa 1200
aatcaaaaaa aaaaa 1215
<210> 40
<211> 1037
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 4590384
<400> 40
gccatggggc tcgggttgag gggctgggga cgtcctctgc tgactgtggc caccgccctg 60
atgctgcccg tgaagccccc cgcaggctcc tggggggccc agatcatcgg gggccacgag 120
gtgacccccc actccaggcc ctacatggca tccgtgcgct tcgggggcca acatcactgc 180
ggaggcttcc tgctgcgagc ccgctgggtg gtctcggccg cccactgctt cagccacaga 240
gacctccgca ctggcctggt ggtgctgggc gcccacgtcc tgagtactgc ggagcccacc 300
cagcaggtgt ttggcatcga tgctctcacc acgcaccctg actaccaccc catgacccac 360
gccaacgaca tctgcctgct gcggctgaac ggctctgctg tcctgggccc tgcagtgggg 420
ctgctgaggc tgccagggag aagggccagg ccccccacag cggggacacg gtgccgggtg 480
gctggctggg gcttcgtgtc tgactttgag gagctgccgc ctggactgat ggaggccaag 540
gtccgagtgc tggacccgga cgtctgcaac agctcctgga agggccacct gacacttacc 600
atgctctgca cccgcagtgg ggacagccac agacggggct tctgctcggc cgactccgga 660
gggcccctgg tgtgcaggaa ccgggctcac ggcctcgttt ccttctcggg cctctggtgc 720
ggcgacccca agacccccga cgtgtacacg caggtgtccg cctttgtggc ctggatctgg 780
gacgtggttc ggcggagcag tccccagccc ggccccctgc ctgggaccac caggccccca 840
ggagaagccg cctgagccac aaccttgcgg catgcaaatg agatggccgc tccaggcctg 900
41 /42
CA 02338386 2001-02-02
WO 00/09709 PCT/US99/17818
gaatgttccg tggctgggcc ccacgggaag cctgatgttc agggttgggg tgggacgggc 960
agcggtgggg cacacccatt ccacatgcaa agggcagaag caaacccagt aaaatgttaa 1020
ctgacgaaaa aaaaaaa 1037
42/42
