Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN CYTOSKELETAL PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human
cytoskeletal proteins
and to the use of these sequences in the diagnosis, treatment, and prevention
of cell proliferative,
immunological, vesicle trafficking, reproductive, smooth muscle,
developmental, and nervous disorders.
BACKGROUND OF THE INVENTION
The physical-biochemical processes of cell motility, organelle movement,
chromosome
movement, cytokinesis, and the generation of cell shape are all dependent on a
complex of protein Ethers
found in the cytoplasm. This protein complex is termed the cytoskeleton. The
cytoskeleton of eukaryotic
, cells has three major filamentous systems. These systems are the actin
filaments, intermediate filaments,
and microtubules. Each of these filamentous systems is assembled from
different proteins, including
actin, myosin, tubulins, and intermediate filament proteins. Different cell
types and tissues express
specific isoforms of the proteins which comprise these filaments. In some
cases distinct isoforms and
mRNA splice variants are associated with cell-type specific functions. (Lees-
Miller, 3.P. and Helfman,
D.M. (1991) BioEssays 13:429-437.)
The actin filamentous system largely regulates cell motility, in particular
generation of muscle
tissue contraction and relaxation. The actin filamentous system comprises the
thick filament and the thin
filament. The thick filament is composed of myosin and the thin filament
contains actin and a protein
complex of troponin and tropomyosin. Activation of myosin binding to actin is
initiated by Cd'-
dependent phosphorylation of myosin light chains by Ca2+-dependent protein
kinases. 'this mechanism is
termed the primary Caz'-dependent mechanism. The interaction between actin and
myosin which drives
muscle contraction is regulated by binding of Ca2' ions to the troponin-
tropomyosin complex and is
termed the secondary Caz+-dependent mechanism. The bound troponin-tropomyosin
complex inhibits the
interaction between actin and myosin at low cellular concentrations of Ca~+ (<
1 ItM). Following a nerve
generated signal, Caz+ is released from the sarcoplasmic reticulum (SR). At
high levels (> I p.M) Cad'
binds to four sites on troponin and affects the specific molecular
interactions between tropomyosin and
the actin filament. This reveals the myosin-binding sites on actin, allowing
ATP-generated movement of
the myosin along the actin filament (contraction). An SR membrane, Cad+-
activated ATPase pumps Ca2'
back into the SR thereby returning cytoplasmic Ca2+ ion levels to <I pM.
Depletion of cytosolic Ca~+
enables the actin-troponin-tropomyosin complex to reform and myosin-actin
interactions cease
(relaxation). (Reedy, M.K. et al. (1994) Curr. Biol. 4:624-626.)
Different isoforms of tropomyosin have been identified in muscle and non-
muscle tissue. The a
gene isoform splice variants are in striated and smooth muscle, brain, and
fibroblasts; the (ichain isoform
splice variants are in skeletal muscle and smooth muscle fibroblasts; the
hTMnm isoform splice variants
are in skeletal muscle and fibroblasts; and the TM-4 isoform splice variant is
in rat platelets. (Lees-
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Miller, sub; Pittenger, M.F., et al. (1994) Curr. Biol. 6:96-104.)
Secondary Ca2'-dependent mechanisms can modulate the contractile state of the
cell. These
secondary mechanisms include, but are not limited to, interactions between I)
actin, tropomyosin and
calponin; ii) actin, myosin, tropomyosin, and caldesmon; iii) actin,
tropomyosin, and titin; iv) actin,
tropomyosin, and tropomodulin; and v) protein kinase C-dependent chemical
modification of actin
filament complexes. (Walsh, M.P. (1991) Biochem. Cell. Biol. 69:771-800;
Fowler, V.M. (1997) Soc.
Gen. Physiol. Ser. 52:79-89.)
Neuronal development and maturation are accompanied by dynamic spatial sorting
of
tropomyosin isoforms into different cellular compartments. (Gunning, P. et al.
(1997) Anat. Embryol.
(Berl.) 195:311-315.) Analysis of the developmental changes in the protein
compositions of the brain has
identified novel developmentally regulated actin-binding proteins termed
debrins. Debrin and
tropomyosin competitively bind to actin filaments. The exclusion of
tropomyosin from actin filaments by
debrin results in the appearance of thick, curving bundles of actin filaments
and the formation of cell
processes in cultured cells. (Shirao, T. (1995) J. Biochem. (Tokyo) 117:231-
236.)
Myocardial performance is impaired in chronically diabetic rats. Malhotra and
Sanghi have
suggested (1997, Cardiovasc. Res. 34:34-40) that diabetes-associated
cardiovascular diseases may involve
proteins of the actin filamentous system, in particular myosin and troponin.
Phosphorylation of troponin
has been associated with altered calcium force in isolated muscle
preparations. This may be due to
changes in the troponin-tropomyosin-actin complex to prevent or reduce
interactions) with myosin. It
was suggested that phosphorylation of troponin could contribute to depressed
myocardial contractility in
experimental diabetes. (Malhotra and Sanghi, suTra.) Mutations in troponin and
tropomyosin are
associated with familial hypertrophic cardiomyopathy. (Palmiter, K.A. and
Solaro, R.J. (1997) Basic.
Res. Cardiol. 92 (suppl. l ):63-74.)
The term receptor describes proteins that specifically recognize other
molecules. The bulk of
receptors are cell surface proteins which bind extracellular ligands and lead
to cellular responses
including growth, differentiation, endocytosis, and immune response. Cell
surface receptors are typically
integral membrane proteins of the plasma membrane. These receptors recognize
compounds, e.g.,
catecholamine and peptide hormones, growth and differentiation factors,
cytokines, small peptide factors,
neurotransmitters, and circulatory system-borne signaling molecules. Cell
surface receptors on immune
system cells recognize antigens, antibodies, and major histocompatibility
complex (MHC)-bound peptide.
Other cell surface receptors bind ligands to be internalized by the cell.
(Lodish, H. et al. (1995) lecular
Cell Bioloev, Scientific American Books, New York, NY, p. 723; Mikhailenko, I.
et al. ( 1997) J. Biol.
Chem. 272:6784-6791.)
The discovery that the transforming oncogene, trk, is a chimeric protein
between tropomyosin and
the membrane domain and intracellular domain of nerve growth factor receptor
has linked mutation of
tropomyosin(s) and abnormal expression of tropomyosin(s) to childhood
malignancy neuroblastoma.
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(Pahlman, S. and Hoehner, J.C. (1996) Mol. Med. Today 2:432-438.) In addition,
experimental chimeric
proteins containing receptor extracellular and transmembrane domains linked to
the integrin (9
cytoplasmic domain can be induced by exogenous factors to cause cytoskeletal
reorganization.
(Smilenov, L. et al. (1994) Mol. Biol. Cell 5:1215-1223.) These results show
that naturally-occurring and
synthetic chimeric proteins which combine extracellular, transmembrane, and
cytosoiic elements of
otherwise distinct individual proteins have additional and synergistic roles
in tissue biology.
Cell motility is governed by the interaction between cytoskeletal and other
cellular proteins.
Cytoskeletal proteins which are involved in the generation of motive force
within the cell are termed
contractile proteins. The energy for this force is generated by ATP.
Two predominant contractile proteins in all animal cells are actin and myosin.
Actin is present in
both soluble and polymerized forms. For example, filamentous (polymerized)
actin interacts with myosin
to contract or relax muscle tissues, to transport cell organelles through the
intracellular medium, to cause
cell movement, and to separate daughter nuclei during cytokinesis.
Multiprotein complexes associate with actin and myosin inin vivo. Actin
polymerization can be
initiated, prevented, or reversed by post-translational protein modification
and changes in the constituent
proteins of the multiprotein complexes. Examples of multiprotein complex
constituent proteins include
trychohyalin, p16-Arc, and actinin. Trichohyalin is a cross-linking protein
that modulates actin
polymerization and functions in intermediate filament- nuclear matrix
anchoring (Lee, S.C. (1993) J.
Biol. Chem. 268:12164-12176). p16-Arc is a subunit of the human Arp2/3
multiprotein complex. The
Arp2/3 complex is localized to the actin-rich lamellipodial protrusions of
cells where it is proposed to
promote actin assembly and cellular locomotion (Welch, M.D. et al. (1997) J.
Cell Biol. 138:375-384).
Actinin functions in the linkage of actin to the cell membrane (Honda, K. et
al. (1998) J. Cell Biol.
140:1383-1393).
Cytoskeletal proteins are involved in the regulation of muscle contraction.
Vertebrate smooth
muscle contraction is dependent upon levels of cAMP and intracellular calcium
ions (CAF'). The
sarcoplasmic reticulum (SR) serves as an intracellular store of Cad;.
Following hormonal stimulation of
the second messenger molecule, inositoltrisphosphate, Cap' is briefly released
from the SR into the
surrounding cytoplasm. Ca2+ binds to calmodulin (CaM), which activates CaM-
dependent myosin light
chain protein kinase (MLCK), which then phosphorylates MLC. In relaxed
skeletal muscle, myosin is
prevented from interacting with actin by binding to tropomyosin. An increase
in C~+ causes a
conformational change in tropomyosin-actin binding that leads to the release
of actin. This allows actin to
interact with phosphorylated MLC forming actinomyosin and initiating the
contraction process. Muscle
relaxation is brought about by active transport of CaZ+ into the SR by a
calcium ATPase pump and
activation of MLCK by a cAMP-dependent protein kinase (PKA). Interactions
between MLCK and PKA
may be modulated by other proteins. In particular, telokin, a kinase-related
protein encoded by the 3'
region of the vertebrate smooth muscle MLCK gene, inhibits MLCK-dependent
phosphorylation of MLC
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by modulating both the oligomeric state of MLCK and MLCK's interaction with
dephosphorylated
myosin filaments (Nieznanski, K. and Sobieszek, A. (1997) Biochem. J. 322:65-
71). Caldesmon is a
protein involved in smooth muscle contraction that performs a role similar to
that of tropomyosin in
skeletal muscle. Caldesrnon forms a complex with tropomyosin and actin that
prevents binding of myosin
to actin. Phosphorylation of caldesmon by casein kinase II releases its
binding to tropomyosin and actin
permitting the cross-linking of myosin to actin and the initiation of smooth
muscle contraction
(Sutherland, C. et al. ( 1994) J. Muscle Res. Cell. Motil. 15:440-456).
Elevation of intracellular cGMP
and activation of protein kinase G (PKG) produces relaxation of smooth muscle
(Li, H. et al. (1996) J.
Vasc. Res. 33:99-110).
Cytoskeletal filament proteins which generate cellular movement are components
of flagella and
cilia. Flagella and cilia are the hair-like structures which protrude from
many cells and are composed of
proteinaceous cylinders known as axonemes. The major mass of the axoneme
consists of tubulins which
polymerize to form microtubules. Nine microtubular doublets typically
surround, and are linked to, a
central pair of microtubules. Intermediate filament proteins, such as tektins,
interact with microtubules to
regulate movement. Tektins are predicted to form extended a-helical rods
capable of forming coiled-coil
structures which are interrupted by short non-helical linkers (Norrander, J.M.
et al. (1996) J. Mol. Biol.
257:385-397). Microtubule-associated proteins (MAPS) regulate cell division
and cell motility by
modulation of microtubule formation.
Cytoskeletal proteins are implicated in several diseases. Pathologies such as
muscular dystrophy,
nephrotic syndrome, and dilated cardiomyopathy have been associated with
differential expression of
alpha-actinin-3 (Vainzof, M. et al. ( 1997) Neuropediatrics 28:223-228;
Smoyer, W.E. and Mundel, P.
(1998) J. Mol. Med. 76:172-183; and Sussman, M.A. et al. (1998) J. Clin.
Invest. 101:51-61). Alpha-
actinin and several MAPs are present in Hirano bodies, which are observed more
frequently in the elderly
and in patients with neurodegenerative diseases such as Alzheimer's disease
(Maciver, S.K. and
Harrington, C.R. (1995) Neuroreport. 6:1985-1988). Actinin-4, a novel actin-
bundling protein, appears to
be associated with the cell motility of metastatic cancer cells. Other disease
associations include
premature chromosome condensation which is frequently observed in dividing
cells from tumor tissue
(Honda et al. sub; Murnane, J.P. (I995) Cancer Metastasis Rev. 14:17-29) and
the significant roles of
axonemal and assembly MAPs in viral pathogenesis (Sodeik, B. et al. (1997) J.
Cell Biol. 136:1007-
1021).
The discovery of new human cytoskeletal 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, immunological, vesicle trafficking,
reproductive, smooth muscle,
developmental, and nervous disorders.
SUMMARY OF THE INVENTION
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The invention features substantially purified polypeptides, human cytoskeletal
proteins, referred
to collectively as "HCYT" and individually as "HCYT-1," "HCYT-2," "HCYT-3,"
"HCYT-4,"
"HCYT-5," "HCYT-6," "HCYT-7," and "HCYT-8." In one aspect, the invention
provides a
substantially purified polypeptide comprising an amino acid sequence selected
from the group consisting
of SEQ ID NO:1-8 and fragments thereof.
The invention further provides a substantially purified variant having at
least 90% amino acid
identity to at least one of the amino acid sequences selected from the group
consisting of SEQ ID NO:1-
8, and fragments thereof. The invention also provides an isolated and purified
polynucleotide encoding
the polypeptide comprising an amino acid selected from the group consisting of
SEQ ID NO:1-8 and
fragments thereof. The invention also includes an isolated and purified
polynucleotide variant having at
least 70% polynucleotide sequence identity to the polynucieotide encoding the
polypeptide comprising
an amino 'acid sequence selected from the group consisting of SEQ ID NO:1-8
and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide
which hybridizes
under stringent conditions to the polynucleotide encoding the polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-8 and fragments
thereof. The invention
also provides an isolated and purified polynucleotide having a sequence which
is complementary to the
polynucleotide encoding the polypeptide comprising the amino acid sequence
selected from the group
consisting of SEQ ID NO:1-8 and fragments thereof.
The invention also provides a method for detecting a polynucleotide in a
sample containing
nucleic acids, the method comprising the steps of (a) hybridizing the
complement of the polynucleotide
sequence to at least one of the polynucleotides of the sample, thereby forming
a hybridization complex;
and (b) detecting the hybridization complex, wherein the presence of the
hybridization complex
correlates with the presence of a polynucleotide in the sample. In one aspect,
the method further
comprises amplifying the polynucleotide prior to hybridization.
The invention also provides an isolated and purified polynucleotide comprising
a polynucleotide
sequence selected from the group consisting of SEQ ID N0:9-16 and fragments
thereof. The invention
further provides an isolated and purified polynucleotide variant having at
least 70% polynucleotide
sequence identity to the polynucleotide sequence selected from the group
consisting of SEQ ID N0:9-
16, and fragments thereof. The invention also provides an isolated and
purified polynucleotide having a
sequence which is complementary to the polynucleotide comprising a
polynucleotide sequence selected
from the group consisting of SEQ ID N0:9-16 and fragments thereof.
The invention further provides an expression vector containing at least a
fragment of the
polynucleotide encoding the polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-8 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
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steps of: (a) culturing the host cell containing an expression vector
containing at least a fragment of a
polynucleotide under conditions suitable for the expression of the
polypeptide; and (b) recovering the
polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a
substantially purified
polypeptide having the amino acid sequence selected from the group consisting
of SEQ ID NO:1-8 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:1-8 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 HCYT, 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:1-8 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 HCYT, the method comprising administering
to a subject in need of
such treatment an effective amount of an antagonist of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-8 and fragments thereof.
BRIEF DESCRIPTION OF THE TABLES AND FIGURES
Table 1 shows nucleotide and polypeptide sequence identification numbers (SEQ
ID NO), clone
identification numbers (clone ID), cDNA libraries, and cDNA fragments used to
assemble full-length
sequences encoding HCYT.
Table 2 shows features of each polypeptide sequence including potential
motifs, homologous
sequences, and methods and algorithms used for identification of HCYT.
Table 3 shows the tissue-specific expression patterns of each nucleic acid
sequence as determined
by northern analysis, diseases or disorders associated with these tissues, and
the vector into which each
cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
Incyte cDNA
clones encoding HCYT were isolated.
Table 5 shows the programs, their descriptions, references, and threshold
parameters used to
analyze HCYT.
Figure 1 shows the amino acid sequence alignments between HCYT-8 (2195418; SEQ
ID
N0:8) and human p16-Arc subunit (GI 2282042; SEQ ID N0:17) produced using the
multisequence
alignment program of LASERGENE software (DNASTAR, Madison WI).
DESCRIPTION OF THE INVENTION
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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,
i5 protocols, reagents and vectors which are reported in the publications and
which might be used in
connection with the invention. Nothing herein is to be construed as an
admission that the invention is not
entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"HCYT" refers to the amino acid sequences of substantially purified HCYT
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 HCYT, increases
or prolongs the
duration of the effect of HCYT. Agonists may inciude proteins, nucleic acids,
carbohydrates, or any other
molecules which bind to and modulate the effect of HCYT.
An "allelic variant" is an alternative form of the gene encoding HCYT. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. Any given
natural or recombinant
gene may have none, one, or many allelic forms. Common mutational changes
which give rise to allelic
variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides. Each of
these types of changes may occur alone, or in combination with the others, one
or more times in a given
sequence.
"Altered" nucleic acid sequences encoding HCYT include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polynucleotide the same as HCYT or a
polypeptide with at least one functional characteristic of HCYT. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
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the polynucleotide encoding HCYT, and improper or unexpected hybridization to
allelic variants, with a
locus other than the normal chromosomal locus for the polynucleotide sequence
encoding HCYT. 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 HCYT. Deliberate
amino acid substitutions may be made on the basis of similarity in polarity,
charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues,
as long as the biological or
immunological activity of HCYT 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" or "amino acid sequence" refer to an oligopeptide,
peptide, polypeptide,
or protein sequence, or a fragment of any of these, and to naturally occurring
or synthetic molecules. 1n
this context, "fragments," "immunogenic fragments," or "antigenic fragments"
refer to fragments of
IS HCYT 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
HCYT. 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 HCYT,
decreases the amount or
the duration of the effect of the biological or immunological activity of
HCYT. Antagonists may include
proteins, nucleic acids, carbohydrates, antibodies, or any other molecules
which decrease the effect of
HCYT.
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
HCYT 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
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a host animal, numerous regions of the protein may induce the production of
antibodies which bind
specifically to antigenic determinants (given regions or three-dimensional
structures on the protein). An
antigenic determinant may compete with the intact antigen (i.e., the immunogen
used to elicit the immune
response) for binding to an antibody.
The term "antisense" refers to any composition containing a nucleic acid
sequence which is
complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules may be
produced by any method including synthesis or transcription. Once introduced
into a cell, the
complementary nucleotides combine with natural sequences produced by the cell
to form duplexes and to
block.either transcription or translation. The designation "negative".can
refer to the antisense strand, and
the designation "positive" can refer to the sense strand.
The term "biologically active," refers to a protein having structural,
regulatory, or biochemical
.functions of a naturally occurring molecule. Likewise, "immunologically
active" refers to the capability
of the natural, recombinant, or synthetic HCYT, or of any oligopeptide
thereof, to induce a specific
immune response in appropriate animals or cells and to bind with specific
antibodies.
The terms "complementary" or "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 design and use of peptide nucleic acid (PNA) molecules.
A "composition comprising a given polynucleotide sequence" or a "composition
comprising a
given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucieotide sequences encoding HCYT or fragments of
HCYT may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be associated
with a stabilizing agent such as a carbohydrate. In hybridizations, the probe
may be deployed in an
aqueous solution containing salts (e.g., NaCI), detergents (e.g., sodium
dodecyl sulfate; SDS), and other
components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence"refers to a nucleic acid sequence which has been
resequenced to resolve
uncalled bases, extended using XL-PCR kit (Perkin-Elmer, Norwalk CT) in the 5'
and/or the 3' direction,
and resequenced, or which has been assembled from the overlapping sequences of
more than one Incyte
Clone using a computer program for fragment assembly, such as the GELVIEW
Fragment Assembly
system (GCG, Madison WI). Some sequences have been both extended and assembled
to produce the
consensus sequence.
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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 HCYT, by northern
analysis is indicative of the presence of nucleic acids encoding HCYT in a
sample, and thereby correlates
with expression of the transcript from the polynucleotide encoding HCYT.
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
I S complementary sequence that at least partially inhibits an identical
sequence from hybridizing to a target
nucleic acid is referred to as "substantially similar." The inhibition of
hybridization of the completely
complementary sequence to the target sequence may be examined using a
hybridization assay (Southern
or northern blot, solution hybridization, and the like) under conditions of
reduced stringency. A
substantially similar sequence or hybridization probe will compete for and
inhibit the binding of a
completely similar (identical) sequence to the target sequence under
conditions of reduced stringency.
This is not to say that conditions of reduced stringency are such that non-
specific binding is permitted, as
reduced stringency conditions require that the binding of two sequences to one
another be a specific (i.e.,
a selective) interaction. The absence of non-specific binding may be tested by
the use of a second target
sequence which lacks even a partial degree of complementarity (e.g., less than
about 30% similarity or
identity). In the absence of non-specific binding, the substantially similar
sequence or probe will not
hybridize to the second non-complementary target sequence.
The phrases "percent identity" or "% identity" refer to the percentage of
sequence similarity
found in a comparison of two or more amino acid or nucleic acid sequences.
Percent identity can be
determined electronically, e.g., by using the MEGALIGN program (DNASTAR,
Madison WI). The
MEGALIGN program can create alignments between two or more sequences according
to different
methods, e.g., the clustal method. (See, e.g., Higgins, D.G. and P.M. Sharp
(1988) Gene 73:237-244.)
The clustal algorithm groups sequences into clusters by examining the
distances between all pairs. The
clusters are aligned pairwise and then in groups. The percentage similarity
between two amino acid
sequences, e.g., sequence A and sequence B, is calculated by dividing the
length of sequence A, minus
the number of gap residues in sequence A, minus the 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
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similarity between the two amino acid sequences are not included in
detenmining 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, c.g., Hein, J. ( 1990)
Methods Enrymol. 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 tenor "humanized antibody" refers to antibody molecules in which the amino
acid sequence
in the non-antigen binding regions has been altered so that the antibody more
closely resembles a human
antibody, and still retains its original binding ability.
"Hybridization" refers to any process by which a strand of nucleic acid binds
with a
complementary strand through base pairing.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A hybridization
complex may be formed in solution (e.g., Cot or Rot analysis) or formed
between one nucleic acid
sequence present in solution and another nucleic acid sequence immobilized on
a solid support (e.g.,
paper, membranes, filters, chips, pins or glass slides, or any other
appropriate substrate to which cells or
their nucleic acids have been fixed).
The words "insertion" or "addition" refer to changes in an amino acid or
nucleotide sequence
resulting in the addition of one or more amino acid residues or nucleotides,
respectively, to the sequence
found in the naturally occurring molecule.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression of
various factors, e.g., cytokines, chemokines, and other signaling molecules,
which may affect cellular and
systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotides on
a substrate.
The terms "element" or "array element" in a microarray context, refer to
hybridizable
polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of HCYT. For example,
modulation may
cause an increase or a decrease in protein activity, binding characteristics,
or any other biological,
functional, or immunological properties of HCYT.
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
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context, "fragments" refers to those nucleic acid sequences which, comprise a
region of unique
polynucleotide sequence that specifically identifies SEQ ID N0:9-16, for
example, as distinct from any
other sequence in the same genome. For example, a fragment of SEQ ID N0:9-16
is useful in
hybridization and amplification technologies and in analogous methods that
distinguish SEQ ID N0:9-16
from related polynucleotide sequences. A fragment of SEQ ID N0:9-16 is at
least about IS-20
nucleotides in length. The precise length of the fragment of SEQ ID N0:9-t 6
and the region of SEQ ID
N0:9-16 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" or "operably linked" refer to functionally
related nucleic acid
sequences. A promoter is operably associated or operably linked with a coding
sequence if the promoter
controls the translation of the encoded polypeptide. While operably associated
or operably linked nucleic
acid sequences can be contiguous and in the same reading frame, certain
genetic elements, e.g., repressor
genes, are not contiguously linked to the sequence encoding the polypeptide
but still bind to operator
sequences that control expression of the polypeptide.
The term "oligonucleotide" refers to a nucleic acid sequence of at least about
6 nucleotides to 60
nucleotides, preferably about IS 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 tenors 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 HCYT, or fragments thereof, or HCYT itself, may comprise a bodily
fluid; an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or cDNA, in
solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" or "speciftcally 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.
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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
I S pores, to which polynucleotides or polypeptides are bound.
"Transformation" describes a process by which exogenous DNA enters and changes
a recipient
cell. Transfonmation may occur under natural or artificial conditions
according to various methods well
known in the art, and may rely on any known method for the insertion of
foreign nucleic acid sequences
into a prokaryotic or eukaryotic host cell. The method for transformation is
selected based on the type of
host cell being transformed and may include, but is not limited to, viral
infection, electroporation, heat
shock, lipofection, and particle bombardment. The term "transformed" cells
includes stably transformed
cells in which the inserted DNA is capable of replication either as an
autonomously replicating plasmid or
as part of the host chromosome, as well as transiently transformed cells which
express the inserted DNA
or RNA for limited periods of time.
A "variant" of HCYT 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).
The term "variant," when used in the context of a polynucleotide sequence, may
encompass a
polynucleotide sequence related to HCYT. 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
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to alternate splicing of exons during mRNA processing. The corresponding
polypeptide may possess
additional functional domains or an absence of domains. Species variants are
polynucleotide sequences
that vary from one species to another. The resulting poiypeptides 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 cytoskeletal proteins
(HCYT), the
polynucleotides encoding HCYT, and the use of these compositions for the
diagnosis, treatment, or
prevention of cell proliferative, immunological, vesicle tracking,
reproductive, smooth muscle,
developmental, and nervous disorders.
Table 1 lists the Incyte Clones used to derive full length nucleotide
sequences encoding HCYT.
Columns 1 and 2 show the sequence identification numbers (SEQ ID NO) of the
amino acid and nucleic
acid sequences, respectively. Column 3 shows the Clone ID of the lncyte Clone
in which nucleic acids
encoding each HCYT was identified, and column 4, the cDNA libraries from which
these clones were
isolated. Column 5 shows lncyte clones, their corresponding cDNA libraries,
and shotgun sequences
useful as fragments in hybridization technologies, and which are part of the
consensus nucleotide
sequence of each HCYT.
The columns of Table 2 show various properties of the polypeptides of the
invention: column 1
references the SEQ ID NO; column 2 shows the number of amino acid residues in
each polypeptide;
column 3, potential phosphorylation sites; column 4, potential glycosylation
sites; column 5, the amino
acid residues comprising signature sequences and motifs; column 6, the
identity of each protein; and
column 7, analytical methods used to identify each protein through sequence
homology and protein
motifs. SEQ ID NO:10, which encodes HCYT-2, is a splice variant of SEQ ID
N0:9, which encodes
HCYT-1. In particular, the nucleotide sequence of SEQ ID NO:10 from nt 190 to
nt 811 is identical to
the nucleotide sequence of SEQ ID N0:9 from nt 319 to nt 940. The N-terminus
of HCYT-1 has two
leucine zipper patterns and five additional potential phosphorylation sites.
HCYT-1, HCYT-2, HCYT-3,
HCYT-4, HCYT-5, and HCYT-6 have chemical and structural similarity with
tropomyosin isoforms. The
N-termini of HCYT-5 (SEQ ID NO:S) and HCYT-6 (SEQ ID N0:6) have homology to
catecholamine
receptors and tektins, respectively; the N-terminus of HCYT-4 (SEQ ID N0:4)
and the intervening
regions of HCYT-5 and HCYT-6 have homology to tropomyosin isoforms; the C-
terminus of HCYT-4
has homology to proteins which bind nucleotide di- or triphosphate molecules;
and the C-termini of
HCYT-5 and HCYT-6 have homology to receptors. SEQ ID N0:7 has various
properties that are
related to intermediate filament proteins including numerous potential
phosphorylation sites, several
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leucine zipper motifs, and a tektin signature sequence.
The columns of Table 3 show the tissue-specificity and disease-association of
nucleotide
sequences encoding HCYT. The first column of Table 3 lists the polynucleotide
sequence identifiers. The
second column lists tissue categories which express HCYT as a fraction of
total tissues expressing HCYT.
The third column lists the diseases, disorders, or conditions associated with
those tissues expressing
HCYT as a fraction of total tissues expressing HCYT. The fourth column lists
the vectors used to
subclone the cDNA library.
Figure 1 shows that chemical and structural homology, in the context of
sequences and motifs,
exists between HCYT-8 (SEQ ID N0:8) and human p16-Arc (GI 2282042: SEQ ID
N0:17). In
particular, the two proteins share 66°! identity, the potential
phosphorylation sites at S7 and T148, and
the potential glycosylation site at N 122 in HCYT-8.
The following represent selected fragments of the nucleotide sequences
encoding HCYT which
are useful as hybridization probes: the fragment of SEQ ID NO:15 from about
nucleotide 7052 to about
nucleotide 7111; and the fragment of SEQ ID N0:16 from about nucleotide 182 to
about nucleotide
IS 235.
The invention also encompasses HCYT variants. A preferred HCYT 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 HCYT amino acid sequence, and which contains at least
one functional or
structural characteristic of HCYT.
The invention also encompasses polynucleotides which encode HCYT. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected from
the group consisting of SEQ ID N0:9-16, which encodes HCYT.
The invention also encompasses a variant of a polynucleotide sequence encoding
HCYT. In
particular, such a variant polynucleotide sequence will have at least about
70%, more preferably at least
about 85 % , and most preferably at least about 95 °6 polynucleotide
sequence identity to the
polynucleotide sequence encoding HCYT. A particular aspect of the invention
encompasses a variant of
a polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:9-16
which has at least about 70%, more preferably at least about 85%, and most
preferably at least about 95%
polynucleotide sequence identity to a nucleic acid, sequence selected from the
group consisting of SEQ ID
N0:9-16. Any one of the polynucleotide variants described above can encode an
amino acid sequence
which contains at least one functional or structural characteristic of HCYT.
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 HCYT, 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
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with the standard triplet genetic code as applied to the polynucleotide
sequence of naturally occurring
HCYT, and all such variations are to be considered as being specifically
disclosed.
Although nucleotide sequences which encode HCYT and its variants are
preferably capable of
hybridizing to the nucleotide sequence of the naturally occurring HCYT under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding HCYT 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 HCYT
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 HCYT
and HCYT
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 HCYT 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 N0:9-16 and
fragments thereof under various conditions of stringency. (See, e.g., Wahl,
G.M. and S.L. Berger (1987)
Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-
511.) For example,
stringent salt concentration will ordinarily be less than about 750 mM NaCI
and 75 mM trisodium citrate,
preferably less than about 500 mM NaCI and 50 mM trisodium citrate, and most
preferably less than
about 250 mM NaCI and 25 mM trisodium citrate. Low stringency hybridization
can be obtained in the
absence of organic solvent, e.g., formamide, while high stringency
hybridization can be obtained in the
presence of at least about 35% formamide, and most preferably at least about
50% formamide. Stringent
temperature conditions will ordinarily include temperatures of at least about
30°C, more preferably of at
least about 37°C, and most preferably of at least about 42°C.
Varying additional parameters, such as
hybridization time, the concentration of detergent, e.g., sodium dodecyl
sulfate (SDS), and the inclusion
or exclusion of carrier DNA, are well known to those skilled in the art.
Various levels of stringency are
accomplished by combining these various conditions as needed. In a preferred
embodiment, hybridization
will occur at 30°C in 750 mM NaCI, 75 mM trisodium citrate, and 1% SDS.
In a more preferred
embodiment, hybridization will occur at 37°C in 500 mM NaCI, 50 mM
trisodium citrate, I% SDS, 35%
formamide, and 100 ~cg/ml denatured salmon sperm DNA (ssDNA). In a most
preferred embodiment,
hybridization will occur at 42°C in 250 mM NaCI, 25 mM trisodium
citrate, I% SDS, 50 % formamide,
and 200 ug/ml ssDNA. Useful variations on these conditions will be readily
apparent to those skilled in
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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 I .5 mM trisodium
citrate. Stringent
temperature conditions for the wash steps will ordinarily include temperature
of at least about 25°C, more
preferably of at least about 42°C, and most preferably of at least
about 68°C. In a preferred embodiment,
wash steps will occur at 25°C in 30 mM NaCI, 3 mM trisodium citrate,
and 0.1% SDS.. In a more
preferred embodiment, wash steps will occur at 42°C in 15 mM NaCI, 1.5
mM trisodium citrate, and
0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C
in 15 mM NaCI, 1.5 mM
trisodium citrate, and 0.1 % SDS. Additional variations on these conditions
will be readily apparent to
those skilled in the art.
Methods for DNA sequencing are well known in the art and may be used to
practice any of the
embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment of DNA
polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Perkin-
Elmer),
thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or
combinations of
polymerases and proofreading exonucleases such as those found in the ELONGASE
amplification system
(Life Technologies, Gaithersburg MD). Preferably, sequence preparation is
automated with machines
such as the Bobbins HYDRA microdispenser (Bobbins Scientific, Sunnyvale CA),
Hamilton
MICROLAB 2200 (Hamilton, Reno NV), Pettier 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) or the MEGABACE 1000 DNA
sequencing system
(Molecular Dynamics, Sunnyvale CA). The resulting sequences are analyzed using
a variety of
algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997)
Short Protocols in
Molecular Bioloev, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
(1995) Molecular Bioloev
and Biotechnology, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding HCYT may be extended utilizing a partial
nucleotide
sequence and employing various PCB-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
PCB, uses universal and nested primers to amplify unknown sequence from
genomic DNA within a
cloning vector. (See, e.g., Sarkar, G. (1993) PCB Methods Applic. 2:318-322.)
Another method, inverse
PCB, uses primers that extend in divergent directions to amplify unknown
sequence from a circularized
template. The template is derived from restriction fragments comprising a
known genomic locus and
surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids
Res. 16:8186.) A third method,
capture PCB, involves PCB amplification of DNA fragments adjacent to known
sequences in human and
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yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. ( 1991 )
PCR Methods Applic.
1:111-119.) In this method, multiple restriction enzyme digestions and
ligations may be used to insert an
engineered double-stranded sequence into a region of unknown sequence before
performing PCR. Other
methods which may be used to retrieve unknown sequences are known in the art.
(See, e.g., Parker, J.D.
et al. (1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR,
nested primers, and
PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This
procedure avoids
the need to screen libraries and is useful in finding intron/exon junctions.
For all PCR-based methods,
primers may be designed using commercially available software, such as OLIGO
4.06 Primer Analysis
software.(National Biosciences, Plymouth MN) or another appropriate program,
to be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the template at
temperatures of about 68°C to 72°C.
- When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T) library
does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence into 5'
non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze the
size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for 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 HCYT may be cloned in recombinant DNA molecules that direct expression
of HCYT, or
fragments or functional equivalents thereof, in appropriate host cells. Due to
the inherent degeneracy of
the genetic code, other DNA sequences which encode substantially the same or a
functionally equivalent
amino acid sequence may be produced and used to express HCYT.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter HCYT-encoding sequences for a variety of
purposes including, but not
limited to, modification of the cloning, processing, and/or expression of the
gene product. DNA shuffling
by random fragmentation and PCR reassembly of gene fragments and synthetic
oligonucleotides may be
used to engineer the nucleotide sequences. For example, oligonucleotide-
mediated site-directed
mutagenesis may be used to introduce mutations that create new restriction
sites; alter glycosylation
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patterns, change codon preference, produce splice variants, and so forth.
In another embodiment, sequences encoding HCYT may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucl. Acids Res.
Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-
232.) Alternatively,
HCYT itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide
synthesis can be performed using various solid-phase techniques. (See, e.g.,
Roberge, J.Y. et al. (1995)
Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A
Peptide Synthesizer
(Perkin-Elmer). Additionally, the amino acid sequence of HCYT, 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 ~~rties, WH
Freeman, New York NY.)
In order to express a biologically active HCYT, the nucleotide sequences
encoding HCYT 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
HCYT. Such elements may vary in their strength and specificity. Specific
initiation signals may also be
used to achieve more efficient translation of sequences encoding HCYT. Such
signals include the ATG
initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases
where sequences encoding
HCYT and its initiation codon and upstream regulatory sequences are inserted
into the appropriate
expression vector, no additional transcriptional or translational control
signals may be needed. However,
in cases where only coding sequence, or a fragment thereof, is inserted,
exogenous translational control
signals including an in-frame ATG initiation codon should be provided by the
vector. Exogenous
translational elements and initiation codons may be of various origins, both
natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of enhancers
appropriate for the particular host
cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell
Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding HCYT and appropriate transcriptional and
translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and inin 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 Bioloev, John Wiley & Sons, New York NY, ch. 9, 13, and
16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
19
CA 02335656 2001-O1-22
_ WO 00/06730 PCT/US99lI7167
encoding HCYT. 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 HCYT. For example,
routine cloning,
subcloning, arid propagation of polynucleotide sequences encoding HCYT can be
achieved using a
multifunctional E.E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or pSPORTI plasmid
(Life Technologies). Ligation of sequences encoding HCYT into the vector's
multiple cloning site
disrupts the lacZ gene, allowing a colorimetric screening procedure fox
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 HCYT are needed, e.g. for the
production of antibodies,
vectors which direct high level expression of HCYT may be used. For example,
vectors containing the
strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of HCYT. A number of
vectors containing
constitutive or inducible promoters, such as alpha factor, alcohol oxidase,
and PGH, may be used in the
yeast Saccharomyces cerevisiae or Pic~,ia~astoris. In addition, such vectors
direct either the secretion or
intracellular retention of expressed proteins and enable integration of
foreign sequences into the host
genome for stable propagation. (See, e.g., Ausubel, 1995, supra; Grant et al.
(1987) Methods Enzymol.
153:516-54; and Scorer, C. A. et al. (1994) Bio/Technology 12:181-184.)
Plant systems may also be used for expression of HCYT. Transcription of
sequences encoding
HCYT may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV
used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO
J. 6:307-311).
Alternatively, plant promoters such as the small subunit of RUBISCO or heat
shock promoters may be
used. (See, e.g., Coruzzi, G. et 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 Technoloev (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 HCYT may be
ligated into an
adenovirus transcription/translation complex consisting of the late promoter
and tripartite leader
CA 02335656 2001-O1-22
WO 00106730 PC'T/US99/17167
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses HCYT in host cells. (See, e.g., Logan, J. and
T. Shenk ( 1984) Proc. Natl.
Acad. Sci. 81:3655-3659.) In addition, transcription enhancers, such as the
Rous sarcoma virus (RSV)
enhancer, may be used to increase expression in mammalian host cells. SV40 or
EBV-based vectors may
also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino polymers, or
vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al. (
1997) Nat Genet. 15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
HCYT in cell lines is preferred. For example, sequences encoding HCYT can be
transformed into cell
lines using expression vectors which may contain viral origins of replication
and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue culture
techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include,
but are not limited to, the herpes simplex virus thymidine kinase and adenine
phosphoribosyltransferase
genes, for use in tk or apr~ cells, respectively. (See, e.g., Wigler, M. et
al. (1977) Cell I 1:223-232; Lowy,
I. et al. ( 1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or
herbicide resistance can be used as
the basis for selection. For example, dhfr confers resistance to methotrexate;
neo confers resistance to the
aminoglycosides neomycin and G-418; and als or pat confer resistance to
chlorsulfuron and
phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler; M. et al.
(1980) Proc. Natl. Acad. Sci.
77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14.)
Additional selectable genes
have been described, e.g., trpB and hisD, which alter cellular requirements
for metabolites. (See, e.g.,
Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.)
Visible markers, e.g.,
anthocyanins, green fluorescent proteins (GFP; Clontech), f3 glucuronidase and
its substrate 13-
glucuronide, or luciferase and its substrate luciferin may be used. These
markers can be used not only to
identify transformants, but also to quantify the amount of transient or stable
protein expression
attributable to a specific vector system. {See, e.g., Rhodes, C.A. (1995)
Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding HCYT is inserted within a marker gene sequence, transformed
cells containing
sequences encoding HCYT can be identified by the absence of marker gene
function. Alternatively, a
21
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WO 00/06730 PCT/US99/17167
marker gene can be placed in tandem with a sequence encoding HCYT 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 HCYT
and that express
HCYT may be identified by a variety of procedures known to those of skill in
the art. These procedures
include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR
amplification, and protein
bioassay or immunoassay techniques which include membrane, solution, or chip
based technologies for
the detection and/or quantification of nucleic acid or protein sequences.
Immunological methods for detecting and measuring the expression of HCYT using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques include
enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and
fluorescence activated
cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering epitopes on HCYT 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)
rol~i~al Methods a Laboratom Manual, APS Press, St Paul MN, Sect. IV; Coligan,
J. E. et al. (1997)
('nrrent 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 HCYT
include oligolabeling,
nick translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the
sequences encoding HCYT, 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 HCYT 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 HCYT may be designed to contain signal sequences
which direct secretion
of HCYT 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
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WO 00/06730 PCT/US99/17167
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, phosphorytation,
lipidation, and acylation. Post-translational processing which cleaves a
"prepro" form of the protein may
also be used to specify protein targeting, folding, and/or activity. Different
host cells which have specific
cellular machinery and characteristic mechanisms for post-translational
activities {e.g., CHO, HeLa,
MDCK, HEK293, and WI38), are available from the American Type Culture
Collection (ATCC,
Bethesda MD) and may be chosen to ensure the correct modification and
processing of the foreign
protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid sequences
encoding HCYT 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 HCYT protein
containing a
heterologous moiety that can be recognized by a commercially available
antibody may facilitate the
screening of peptide libraries for inhibitors of HCYT activity. Heterologous
protein and peptide moieties
may also facilitate purification of fusion proteins using commercially
available affinity matrices. Such
moieties include, but are not limited to, glutathione S-transferase (GST),
maltose binding protein (MBP),
thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and
hemagglutinin (HA).
GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion
proteins on immobilized
glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate
resins, respectively. FLAG, c-
myc, and hemagglutinin {HA) enable immunoaffinity purification of fusion
proteins using commercially
available monoclonal and polyclonal antibodies that specifically recognize
these epitope tags. A fusion
protein may also be engineered to contain a proteolytic cleavage site located
between the HCYT encoding
sequence and the heterologous protein sequence, so that HCYT 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 HCYT may
be achieved
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
"S-methionine.
Fragments of HCYT may be produced not only by recombinant production, but also
by direct
peptide synthesis using solid-phase techniques. (See, e.g., Creighton, sub pp.
55-60.) Protein synthesis
may be performed by manual techniques or by automation. Automated synthesis
may be achieved, for
example, using the ABI 431 A Peptide Synthesizer (Perkin-Elmer). Various
fragments of HCYT may be
synthesized separately and then combined to produce the full length molecule.
THERAPEUTICS
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Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists between
HCYT and human cytoskeletal proteins. In addition, HCYT is expressed in
tissues associated with
cancer, cell proliferation, fetal development, and inflammation and the immune
response, as well as in
reproductive, nervous, cardiovascular, developmental, and gastrointestinal
tissues. Therefore, HCYT
appears to be involved with cell proliferative, immunological, vesicle
trafficking; reproductive, smooth
muscle, developmental, and nervous disorders. 1n disorders associated with
decreased expression or
activity of HCYT, it is desirable to increase the expression or activity of
HCYT. In disorders associated
with increased expression or activity of HCYT, it is desirable to decrease the
expression or activity of
HCYT.
Therefore, in one embodiment, HCYT or a fragment or derivative thereof may be
administered to
a subject to treat or prevent a disorder in which the expression or activity
of HCYT is decreased.
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
IS thrombocythemia; cancers including adenocarcinoma, leukemia, lymphoma,
melanoma, myeloma,
sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland,
bladder, bone, bone marrow,
brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart,
kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis,
thymus, thyroid, and uterus; an
immunological disorder such as actinic keratosis, acquired immunodeficiency
syndrome (AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis,
anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic
anemia, autoimmune thyroiditis,
bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's
disease, atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis,
erythema nodosum, atrophic
gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis,
paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable
bowel syndrome, episodic
lymphopenia with lymphocytotoxins, mixed connective tissue disease (MCTD),
multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis,
osteoarthritis, osteoporosis,
pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis,
scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus
erythematosus, systemic
sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial, fungal,
parasitic, protozoal, and helminthic infections, and trauma; a vesicle
trafficking disorder such as cystic
fibrosis, glucose-galactose malabsorption syndrome, hyperchoiesterolemia,
diabetes mellitus, diabetes
insipidus, hyper- and hypoglycemia, Grave's disease, goiter, Cushing's
disease, and Addison's disease;
gastrointestinal disorders including ulcerative colitis, gastric and duodenal
ulcers; other conditions
associated with abnormal vesicle trafficking, including acquired
immunodeficiency syndrome (AIDS);
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allergies including hay fever, asthma, and urticaria (hives); autoimmune
hemolytic anemia; proliferative
glomerulonephritis; inflammatory bowel disease; multiple sclerosis; myasthenia
gravis; rheumatoid and
osteoarthritis; sclerodenma; Chediak-Higashi and Sjogren's syndromes; systemic
lupus erythematosus;
toxic shock syndrome; traumatic tissue damage; and viral, bacterial, fungal,
helminthic, and protozoal
infections; a reproductive disorder such as disorders of prolactin production;
infertility, including tubal
disease, ovulatory defects, and endometriosis; disruptions of the estrous
cycle, disruptions of the
menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome,
endometriai and
ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies,
and teratogenesis; cancer of
the breast, fibrocystic breast disease, and galactorrhea; disruptions of
spermatogenesis, abnormal sperm
physiology, cancer of the testis, cancer of the prostate, benign prostatic
hyperplasia, prostatitis, Peyronie's
disease, carcinoma of the male breast, and gynecomastia; a smooth muscle
disorder such as any
impairment or alteration in the normal action of smooth muscle including, but
not limited to, that of the
blood vessels, gastrointestinal tract, heart, and uterus, and including but
not limited to, angina,
anaphylactic shock, arrhythmias, asthma, cardiovascular shock, Cushing's
syndrome, hypertension,
hypoglycemia, myocardial infarction, migraine, and pheochromocytoma, and
myopathies including
cardiomyopathy, encephalopathy, epilepsy, Kearns-Sayre syndrome, lactic
acidosis, myoclonic disorder,
and ophthalmoplegia; a developmental disorder, such as any disorder associated
with development or
function of a tissue, organ, or system (such as the brain, adrenal gland,
kidney, skeletal or reproductive
system) of a subject, including but not limited to, renal tubular acidosis,
anemia, Cushing's syndrome,
achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy,
gonadal dysgenesis,
WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation), Smith-
Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia, hereditary
keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and
neurofibromatosis,
hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea
and cerebral palsy, spine
bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and
sensorineural hearing loss; a
nervous disorder such as akathesia, Alzheimer's disease, amnesia, amyotrophic
lateral sclerosis and other
motor neuron disorders, bipolar disorder, catatonia, cerebral neoplasms,
dementia, depression, diabetic
neuropathy, Down's syndrome, tardive dyskinesia, dystonias, epilepsy,
Huntington's disease, peripheral
neuropathy, multiple sclerosis, neurofibromatosis, Parkinson's disease and
other extrapyramidal
disorders, postherpetic neuralgia, epilepsy, ischemic cerebrovascular disease,
stroke, cerebral neoplasms,
Pick's disease, Huntington's disease, dementia, progressive neural muscular
atrophy, retinitis pigmentosa,
hereditary ataxias, multiple sclerosis and other demyelinating diseases,
bacterial and viral meningitis,
brain abscess, subdural empyema, epidural abscess, suppurative intracranial
thrombophlebitis, myelitis
and radiculitis, viral central nervous system disease; prion diseases
including kuru, Creutzfeldt-Jakob
disease, and Gerstmann-Straussler-Scheinker syndrome; fatal familial insomnia,
nutritional and metabolic
diseases of the nervous system, neuroftbromatosis, tuberous sclerosis,
cerebelloretinal
CA 02335656 2001-O1-22
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hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and
other developmental
disorders of the central nervous system, cerebral palsy, neuroskeletal
disorders, autonomic nervous
system disorders, cranial nerve disorders, spinal cord diseases, muscular
dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis;
inherited, metabolic, endocrine, and toxic myopathies; myasthenia gravis,
periodic paralysis; mental
disorders including mood, anxiety, paranoid psychoses, and schizophrenic
disorders; diabetic neuropathy,
tardive dyskinesia, dystonias, and Tourette's disorder.
In another embodiment, a vector capable of expressing HCYT 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 HCYT including, but not limited to, those described above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
HCYT 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 HCYT
including, but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of HCYT
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or activity of
HCYT including, but not limited to, those listed above.
In a further embodiment, an antagonist of HCYT may be administered to a
subject to treat or
prevent a disorder in which the expression or activity of HCYT is increased.
Examples of such disorders
include, but are not limited to, those cell proliferative, immunological,
vesicle tracking, reproductive,
smooth muscle, developmental, and nervous disorders listed above. In one
aspect, an antibody which
specifically binds NCYT 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
HCYT.
In an additional embodiment, a vector expressing the complement of the
polynucleotide encoding
HCYT may be administered to a subject to treat or prevent a disorder
associated with increased
expression or activity of HCYT 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 HCYT may be produced using methods which are generally known
in the art.
In particular, purified HCYT may be used to produce antibodies or to screen
libraries of pharmaceutical
agents to identify those which specifically bind HCYT. Antibodies to HCYT may
also be generated using
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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 HCYT or with any fragment or
oligopeptide thereof which
has immunogenic properties. Depending on the host species, various adjuvants
may be used to increase
immunological response. Such adjuvants include, but are not limited to,
Freund's, mineral gels such as
aluminum hydroxide, and surface active substances such as lysolecithin,
pluronic poIyols, polyanions,
peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in
humans, BCG (bacilli '
Calmette-Guerin) and Conmebacteriumap rvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to HCYT
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 HCYT 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 HCYT may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not limited
to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-
hybridoma technique.
(See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al.
{1985) J. Immunol. Methods
81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and
Cole, S.P. et al. (1984) Mol.
Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate antigen
specificity and biological activity, can be used. (See, e.g., Morrison, S.L.
et al. {1984) Proc. Natl. Acad.
Sci. 81:6851-6855; Neuberger, M.S. et aL (1984) Nature 312:604-608; and
Takeda, S, et al. (1985)
Nature 314:452-454.) Alternatively, techniques described for the production of
single chain antibodies
may be adapted, using methods known in the art, to produce HCYT-specific
single chain antibodies.
Antibodies with related specificity, but of distinct idiotypic composition,
may be generated by chain
shuffling from random combinatorial immunoglobulin libraries. (See, e.g.,
Burton D.R. (1991) Proc.
Natl. Acad. Sci. 88:10134-10137.)
Antibodies may also be produced by inducing loin vivo production in the
lymphocyte population or
by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the
literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:
3833-3837; Winter, G. et al.
( 1991 ) Nature 349:293-299.)
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Antibody fragments which contain specific binding sites for HCYT may also be
generated. For
example, such fragments include, but are not limited to, F(ab')2 fragments
produced by pepsin digestion
of the antibody molecule and Fab fragments generated by reducing the disulfide
bridges of the F(ab')2
fragments. Alternatively, Fab expression libraries may be constructed to allow
rapid and easy
identification of monoclonal Fab fragments with the desired specificity. (See,
e.g., Huse, W.D. et al.
(1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
HCYT and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two
non-interfering HCYT 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 HCYT. Affinity is
expressed as an association
constant, K" which is defined as the molar concentration of HCYT-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The I~ determined
for a preparation of polyclonal antibodies, which are heterogeneous in their
affinities for multiple HCYT
epitopes, represents the average affinity, or avidity, of the antibodies for
HCYT. The K, determined for a
preparation of monoclonal antibodies, which are monospecific for a particular
HCYT epitope, represents
a true measure of affinity. High-affinity antibody preparations with IC,
ranging from about 109 to 10'Z
L/mole are preferred for use in immunoassays in which the HCYT-antibody
complex must withstand
rigorous manipulations. Low-affinity antibody preparations with K, ranging
from about 106 to 10' L/mole
are preferred for use in immunopurification and similar procedures which
ultimately require dissociation
of HCYT, preferably in active form, from the antibody (Catty, D. (1988)
Antibodies. Volume i: A
glactical Approach, IRL Press, Washington DC; Liddell, J. E. and Cryer, A.
(1991) Practical GuidP_ 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 HCYT-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, SUDfa_ and Coligan et
al. supra.)
In another embodiment of the invention, the polynucleotides encoding HCYT, or
any fragment or
complement thereof, may be used for therapeutic purposes. In one aspect, the
complement of the
28
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polynucleotide encoding HCYT 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 HCYT. Thus, complementary molecules or fragments may
be used to modulate
HCYT 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 HCYT.
In another embodiment of the invention, the polynucleotides encoding HCYT, or
any fragment or
complement thereof, may be used for therapeutic purposes. In one aspect, the
complement of the
polynucleotide encoding HCYT may be used in situations in which it would bye
desirable to block the
transcription of the mRNA. In particular, cells may be transformed with
sequences complementary to
polynucleotides encoding HCYT. Thus, complementary molecules or fragments may
be used to modulate'
HCYT activity, or to achieve regulation of gene function. Such technology is
now well known in the art,
and sense or antisense oligonucieotides or larger fragments can be designed
from various locations along
the coding or control regions of sequences encoding HCYT.
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 HCYT. (See,
e.g., Sambrook, supra; Ausubel, 1995, supra.)
Genes encoding HCYT can be turned off by transforming a cell or tissue with
expression vectors
which express high levels of a poiynucieotide, or fragment thereof, encoding
HCYT. Such constructs
may be used to introduce untranslatable sense or antisense sequences into a
cell. Even in the absence of
integration into the DNA, such vectors may continue to transcribe RNA
molecules until they are disabled
by endogenous nucleases. Transient expression may last for a month or more
with a non-replicating
vector, and may last even longer if appropriate replication elements are part
of the vector system.
As mentioned above, modifications of gene expression can be obtained by
designing
complementary sequences or antisense molecules (DNA, RNA, or PNA) to the
control, S', or regulatory
regions of the gene encoding HCYT. Oligonucleotides derived from the
transcription initiation site, e.g.,
between about positions -10 and +10 from the start site, are preferred.
Similarly, inhibition can be
achieved using triple helix base-pairing methodology. Triple helix pairing is
useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerises,
transcription factors, or regulatory molecuies. Recent therapeutic advances
using triplex DNA have been
described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber,
B.E. and B.I. Carr, Molecular and
Immunologic Ap roaches, Futura Publishing, Mt. Kisco NY, pp. 163-177.) A
complementary sequence
or antisense molecule may also be designed to block translation of mRNA by
preventing the transcript
from binding to ribosomes.
29
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Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example, engineered
hammerhead motif ribozyme molecules may specifically and efficiently catalyze
endonucleolytic
cleavage of sequences encoding HCYT.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA, GUU,
and GUC. Once identified, short RNA sequences of between I S 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 lain vivo
transcription of DNA sequences
encoding HCYT. Such DNA sequences may be incorporated into a wide variety of
vectors with suitable
RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA
constructs that synthesize
complementary RNA, constitutively or inducibly, can be introduced into cell
lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends of
the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages within
the backbone of the molecule. This concept is inherent in the production of
PNAs and can be extended in
all of these molecules by the inclusion of nontraditional bases such as
inosine, queosine, and wybutosine,
as well as acetyl-, methyl-, thio-, and similarly modified foams 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 j~yjyQ, in vitro, and exex vivo. For v'v therapy, vectors may be
introduced into stem cells taken
from the patient and cionally propagated for autologous transplant back into
that same patient. Delivery
by transfection, by liposome injections; or by polycationic amino polymers may
be achieved using
methods which are well known in the art. (See, e.g., Goldman, C.K. et al.
(1997) Nature Biotechnology
15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of such
therapy, including, for example, mammals such as dogs, cats, cows, horses,
rabbits, monkeys, and most
preferably, humans.
An additional embodiment of the invention relates to the administration of a
pharmaceutical or
CA 02335656 2001-O1-22
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sterile composition, in conjunction with a pharmaceutically acceptable
carrier, for any of the therapeutic
effects discussed above. Such pharmaceutical compositions may consist of HCYT,
antibodies to HCYT,
and mimetics, agonists, antagonists, or inhibitors of HCYT. 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, transdernal, 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 Reminds
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 pyn;oiidone, 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, polyviny(pyrrolidone,
carbopol gel, polyethylene
glycol, and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for
product identification or to
characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of gelatin,
as well as soft, sealed capsules made of gelatin and a coating, such as
glycerol or sorbitol. Push-fit
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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 fatty oils, such
as sesame oil, or synthetic
fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-
lipid polycationic amino polymers
may also be used for delivery. Optionally, the suspension may also contain
suitable stabilizers or agents
to increase the solubility of the compounds and allow for the preparation of
highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art.
The pharmaceutical compositions of the present invention may be manufactured
in a manner that
is known in the art, e.g., by means of conventional mixing, dissolving,
granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed
with many acids,
including but not limited to, hydrochloric, sulfuric, acetic, lactic,
tartaric, malic, and succinic acid. Salts
tend to be more soluble in aqueous or other protonic solvents than are the
corresponding free base forms.
In other cases, the preferred preparation may be a lyophilized powder which
may contain any or all of the
following: 1 mM to 50 mM histidine, 0.1 % to 2% sucrose, and 2% to 7%
mannitol, at a pH range of 4.5 to
5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate
container and labeled for treatment of an indicated condition. For
administration of HCYT, such labeling
would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include
compositions wherein the
active ingredients are contained in an effective amount to achieve the
intended purpose. The
determination of an effective dose is well within the capability of those
skilled in the art.
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells or in animal models such as mice,
rats, rabbits, dogs, or pigs. An
animal model may also be used to determine the appropriate concentration range
and route of
administration. Such information can then be used to determine useful doses
and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example HCYT or
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fragments thereof, antibodies of HCYT, and agonists, antagonists or inhibitors
of HCYT, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by standard
phanmaceutical procedures in cell cultures or with experimental animals, such
as by calculating the EDT
(the dose therapeutically effective in 50% of the population) or LDP (the dose
lethal to 50% of the
population) statistics. The dose ratio of therapeutic to toxic effects is the
therapeutic index, and it can be
expressed as the EDs°/LDs° 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 humam use. The dosage contained in such compositions is
preferably within a range of
circulating concentrations.that includes the EI7~° 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 dbsage 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.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind HCYT may be used for
the diagnosis
of cell proliferative, immunological, vesicle trafficking, reproductive,
smooth muscle, developmental, and
nervous disorders characterized by expression of HCYT, or in assays to monitor
patients being treated
with HCYT or agonists, antagonists, or inhibitors of HCYT. Antibodies useful
for diagnostic purposes
may be prepared in the same manner as described above for therapeutics.
Diagnostic assays for HCYT
include methods which utilize the antibody and a label to detect HCYT 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 HCYT, including ELISAs, RIAs, and FACS,
are known in
the art and provide a basis for diagnosing altered or abnormal levels of HCYT
expression. Nonmal or
standard values for HCYT expression are established by combining body fluids
or cell extracts taken
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from normal mammalian subjects, preferably human, with antibody to HCYT under
conditions suitable
for complex formation. The amount of standard complex formation may be
quantitated by various
methods, preferably by photometric means. Quantities of HCYT 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 HCYT 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 HCYT may
be correlated with
l0 disease. The diagnostic assay may be used to determine absence, presence,
and excess expression of
HCYT, and to monitor regulation of HCYT levels during therapeutic
intervention.
a In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding HCYT or closely related
molecules may be used to
identify nucleic acid sequences which encode HCYT. The specificity of the
probe, whether it is made
15 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 HCYT,
allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and should
preferably have at
20 least 50% sequence identity to any of the HCYT encoding sequences. The
hybridization probes of the
subject invention may be DNA or RNA and may be derived from the sequence of
HCYT or from
genomic sequences including promoters, enhancers, and introns ofthe HCYT gene.
Means for producing specific hybridization probes for DNAs encoding HCYT
include the cloning
of polynucleotide sequences encoding HCYT or HCYT derivatives into vectors for
the production of
25 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 33S, or by enzymatic labels, such as
alkaline phosphatase coupled
to the probe via avidin/biotin coupling systems, and the like.
30 Polynucleotide sequences encoding HCYT may be used for the diagnosis of
cell proliferative,
immunological, vesicle trafficking, reproductive, smooth muscle,
developmental, and nervous disorders
associated with expression of HCYT. 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,
35 polycythemia vera, psoriasis, primary thrombocythemia; cancers including
adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular,
cancers of the adrenal
34
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gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal tract, heart,
kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen,
testis, thymus, thyroid, and uterus; an immunological disorder such as actinic
keratosis, acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory
distress syndrome, allergies,
ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma,
atherosclerosis, autoimmune
hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis,
cirrhosis, contact dermatitis,
Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
emphysema, erythroblastosis
fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,
Goodpasture's syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria,
hepatitis, hypereosinophilia,
irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins; mixed
connective tissue disease
(MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial
inflammation, myelofibrosis,
osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis;
psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis,
systemic lupus
erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenic
purpura, ulcerative
colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and
extracorporeal circulation,
viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and
trauma; a vesicle trafficking
disorder such as cystic fibrosis, glucose-galactose malabsorption syndrome,
hypercholesterolemia,
diabetes mellitus, diabetes insipidus, hyper- and hypoglycemia, Grave's
disease, goiter, Cushing's
disease, and Addison's disease; gastrointestinal disorders including
ulcerative colitis, gastric and duodenal
ulcers; other conditions associated with abnormal vesicle trafficking,
including acquired
immunodeficiency syndrome (AIDS); allergies including hay fever, asthma, and
urticaria (hives);
autoimmune hemolytic anemia; proliferative glomerulonephritis; inflammatory
bowel disease; multiple
sclerosis; myasthenia gravis; rheumatoid and osteoarthritis; scleroderma;
Chediak-Higashi and Sjogren's
syndromes; systemic lupus erythematosus; toxic shock syndrome; traumatic
tissue damage; and viral,
bacterial, fungal, helminthic, and protozoal infections; a reproductive
disorder such as disorders of
prolactin production; infertility, including tubal disease, ovulatory defects,
and endometriosis; disruptions
of the estrous cycle, disruptions of the menstrual cycle, polycystic ovary
syndrome, ovarian
hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids,
autoimmune disorders,
ectopic pregnancies, and teratogenesis; cancer of the breast, fibrocystic
breast disease, and galactorrhea;
disruptions of spermatogenesis, abnormal sperm physiology, cancer of the
testis, cancer of the prostate,
benign prostatic hyperplasia, prostatitis, Peyronie's disease, carcinoma of
the mate breast, and
gynecomastia; a smooth muscle disorder such as any impairment or alteration in
the normal action of
smooth muscle including, but not limited to, that of the blood vessels,
gastrointestinal tract, heart, and
uterus, and including but not limited to, angina, anaphylactic shock,
arrhythmias, asthma, cardiovascular
shock, Cushing's syndrome, hypertension, hypoglycemia, myocardial infarction,
migraine, and
pheochromocytoma, and myopathies including cardiomyopathy, encephalopathy,
epilepsy, Kearns-Sayre
CA 02335656 2001-O1-22
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syndrome, lactic acidosis, myoclonic disorder, and ophthalmoplegia; a
developmental disorder, such as
any disorder associated with development or function of a tissue, organ, or
system (such as the brain,
adrenal gland, kidney, skeletal or reproductive system) of a subject,
including but not limited to, renal
tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism,
Duchenne and Becker
muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor,
aniridia,
genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome,
myelodysplastic
syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas,
hereditary neuropathies such as
Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,
hydrocephalus, seizure disorders
such as Syndenharn's chorea and cerebral palsy, spina bifida, anencephaly,
craniorachischisis, congenital
glaucoma, cataract, and sensorineural hearing loss; a nervous disorder such as
akathesia, Alzheimer's
disease, amnesia, amyotrophic lateral sclerosis and other motor neuron
disorders, bipolar disorder,
. catatonia, cerebral neoplasms, dementia, depression, diabetic neuropathy,
Down's syndrome, tardive
dyskinesia, dystonias, epilepsy, Huntington's disease, peripheral neuropathy,
multiple sclerosis,
neurofibromatosis, Parkinson's disease and other extrapyramidal disorders,
postherpetic neuralgia,
epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Pick's
disease, Huntington's
disease, dementia, progressive neural muscular atrophy, retinitis pigmentosa,
hereditary ataxias, multiple
sclerosis and other demyelinating diseases, bacterial and viral meningitis,
brain abscess, subdural
empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis
and radiculitis, viral
central nervous system disease; prion diseases including kuru, Creutzfeldt-
Jakob disease, and Gerstmann-
Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and
metabolic diseases of the nervous
system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous
system disorders, cranial
nerve disorders, spinal cord diseases, muscular dystrophy and other
neuromuscular disorders, peripheral
nervous system disorders, dermatomyositis and polymyositis; inherited,
metabolic, endocrine, and toxic
myopathies; myasthenia gravis, periodic paralysis; mental disorders including
mood, anxiety, paranoid
psychoses, and schizophrenic disorders; diabetic neuropathy, tardive
dyskinesia, dystonias, and Tourette's
disorder. The polynucleotide sequences encoding HCYT may be used in Southern
or northern analysis,
dot blot, or other membrane-based technologies; in PCR technologies; in
dipstick, pin, and ELISA assays;
and in microarrays utilizing fluids or tissues from patients to detect altered
HCYT expression. Such
qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding HCYT may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding HCYT 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.
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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 HCYT 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 HCYT, 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 HCYT, 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.
IS Once the presence of a disorder is established and a treatment protocol is
initiated, hybridization
assays may be repeated on a regular basis to determine if the level of
expression in the patient begins to
approximate that which is observed in the normal subject. The results obtained
from successive assays
may be used to show the efficacy of treatment over a period ranging from
several days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development of
the disease, or may provide a means for detecting the disease prior to the
appearance of actual clinical
symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding HCYT
may involve the use of PCR. These oligomers may be chemically synthesized,
generated enzymatically,
or produced in vitro. Oligomers will preferably contain a fragment of a
polynucleotide encoding HCYT,
or a fragment of a polynucleotide complementary to the polynucleotide encoding
HCYT, and will be
employed under optimized conditions for identification of a specific gene or
condition. Oligomers may
also be employed under less stringent conditions for detection or quantitation
of closely related DNA or
RNA sequences.
Methods which may also be used to quantitate the expression of HCYT include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from standard
curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244;
Duplaa, C. et al. (1993)
Anal. Biochem. 229-236.) The speed of quantitation of multiple samples may be
accelerated by running
the assay in an ELISA format where the oligomer of interest is presented in
various dilutions and a
37
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
spectrophotometric or coiorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as targets in a
microarray. The microarray can be
used to monitor the expression level of large numbers of genes simultaneously
and to identify genetic
variants, mutations, and polymorphisms. This information may be used to
determine gene function, to
understand the genetic basis of a disorder, to diagnose a disorder, and to
develop and monitor the
activities of therapeutic agents.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(I996) Proc. Natl. Acad. Sci.
93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalon, D. et al. (i995)
PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci.
94:2150-2155; and Heller,
M.J. et al. ( 1997) U.S. Patent No. 5,605,662.)
In another embodiment of the invention, nucleic acid sequences encoding HCYT
may be used to
generate hybridization probes useful in mapping the naturally occurring
genomic sequence. The
IS sequences may be mapped to a particular chromosome, to a specific region of
a chromosome, or to
artificial chromosome constructions, e.g., human artificial chromosomes
(HACs), yeast artificial
chromosomes (PACs), bacterial artificial chromosomes (BACs), bacterial P1
constructions, or single
chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat
Genet. I5:345-355; Price, C.M.
(1993) Blood Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet. 7:149-154.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome
mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al.
(1995) in Meyers, supra, pp.
965-968.) Examples of genetic map data can be found in various scientific
journals or at the Online
Mendefian Inheritance in Man (OMIM) site. Correlation between the location of
the gene encoding
HCYT on a physical chromosomal map and a specific disorder, or a
predisposition to a specific disorder,
may help define the region of DNA associated with that disorder. The
nucleotide sequences of the
invention may be used to detect differences in gene sequences among normal,
carrier, and affected
individuals.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps. Often
the placement of a gene on the chromosome of another mammalian species, such
as mouse, may reveal
associated markers even if the number or arm of a particular human chromosome
is not known. New
sequences can be assigned to chromosomal arms by physical mapping. This
provides valuable
information to investigators searching for disease genes using positional
cloning or other gene discovery
techniques. Once the disease or syndrome has been crudely localized by genetic
linkage to a particular
genomic region, e.g., ataxia-telangiectasia to l 1q22-23, any sequences
mapping to that area may represent
associated or regulatory genes for further investigation. (See, e.g., Gatti,
R.A. et al. ( 1988) Nature
38
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WO 00/06730 PCT/US99/17167
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, HCYT, 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 HCYT arid 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 HCYT, or fragments
thereof, and washed. Bound
HCYT is then detected by methods well known in the art. Purified HCYT 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 HCYT specifically compete with a test compound
for binding HCYT. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with HCYT.
In additional embodiments, the nucleotide sequences which encode HCYT 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. [Atty Docket No. PF-0566 P, filed August 4, 1998],
U.S. Ser. No. [Atty Docket
No. PF-0568 P, filed July 31, 1998] and U.S. Ser. No.: [Atty Docket No. PF-
0578 P, filed August 19,
1998], are hereby expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some tissues
were homogenized and lysed in guanidinium isothiocyanate, while others were
homogenized and lysed in
39
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WO 00/06730 PCT/US99/17167
phenol or in a suitable mixture of denaturants, such as TRIZOL (Life
Technologies), a monophasic
solution of phenol and guanidine isothiocyanate. The resulting lysates were
centrifuged over CsCI
cushions or extracted with chloroform. RNA was precipitated from the lysates
with either isopropanol or
sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA purity.
In some cases, RNA was treated with DNase. For most libraries, poly(A+) RNA
was isolated using oligo
d(T~coupled paramagnetic particles (Promega), OLIGOTEX latex particles
(QIAGEN, Valencia CA), or
an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated
directly from tissue
lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA
purification kit (Ambion, Austin
TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997,x, units
5.1-6.6). Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-1000
bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography
(Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs
were ligated into
compatible restriction enzyme sites of the polylinker of a suitable plasmid,
e.g., PBLUESCRIPT plasmid
(Stratagene), pSPORTI plasmid (Life Technologies), or pINCY (Incyte
Pharmaceuticals, Palo Alto CA).
Recombinant plasmids were transformed into competent E.E. coli cells including
XL1-Blue, XL1-
BIueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life
Technologies.
II. Isolation of cDNA Clones
Plasmids were recovered from host cells by inin vivo excision, using the
UNIZAP vector system
(Stratagene) or cell Iysis. Plasmids were purified using at least one of the
following: a Magic or
WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep
purification kit (Edge
Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,
QIAWELL 8
Ultra Plasmid purification systems or the REAL Prep 96 plasmid kit from
QIAGEN. Following
precipitation, plasmids were resuspended in 0.1 ml of distilled water and
stored, with or without
lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell Iysates using direct
link PCR in a high-
throughput format (Rao, V.B. ( 1994) Anal. Biochem. 216:1-14). Host cell lysis
and thermal cycling steps
were carried out in a single reaction mixture. Samples were processed and
stored in 384-welt plates, and
the concentration of amplified plasmid DNA was quantified fluorometrically
using PICOGREEN reagent
(Molecular Probes, Eugene OR) and a Fluoroskan II fluorescence scanner
(Labsystems Oy, Helsinki,
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
Finland).
III. Sequencing and Analysis
The cDNAs were prepared for sequencing using the ABI CATALYST 800 (Perkin-
Elmer) or the
HYDRA microdispenser (Bobbins Scientific) or MICROLAB 2200 (Hamilton) systems
in combination
with the PTC-200 thermal cyclers (MJ Research). The cDNAs were sequenced using
the ABI PRISM
373 or 377 sequencing systems (Perkin-Elmer) and standard ABI protocols, base
calling software, and
kits. 1n one alternative, cDNAs were sequenced using the MEGABACE 1000 DNA
sequencing system
(Molecular Dynamics). In another alternative, the cDNAs were amplified and
sequenced using the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer). In
yet another
alternative, cDNAs were sequenced using solutions and dyes from Amersham
Pharmacia Biotech.
Reading frames for the ESTs were determined using standard methods as reviewed
in Ausubel (1997,
unit 7.7). Some of the cDNA sequences were selected for extension using the
techniques disclosed
in Example V.
The polynucleotide sequences derived from cDNA, extension, and shotgun
sequencing were
assembled and analyzed using a combination of software programs which utilize
algorithms well known
to those skilled in the art. Table 5 summarizes the software programs,
descriptions, references, and
threshold parameters used. The first column of Table 5 shows the tools,
programs, and algorithms used,
the second column provides a brief description thereof, the third column
presents the references which are
incorporated by reference herein, and the fourth column presents, where
applicable, the scores,
probability values, and other parameters used to evaluate the strength of a
match between two sequences
(the higher the probability the greater the homology). Sequences were analyzed
using MACDNASIS
PRO software (Hitachi Software Engineering, S. San Francisco CA) and LASERGENE
software
(DNASTAR).
The polynucleotide sequences were validated by removing vector, linker, and
polyA sequences
and by masking ambiguous bases, using algorithms and programs based on BLAST,
dynamic
programing, and dinucleotide nearest neighbor analysis. The sequences were
then queried against a
selection of public databases such as GenBank primate, rodent, mammalian,
vertebrate, and eukaryote
databases, and BLOCKS to acquire annotation, using programs based on BLAST,
FASTA, and BLIMPS.
The sequences were assembled into full length polynucleotide sequences using
programs based on Phred,
Phrap, and Consed, and were screened for open reading frames using programs
based on GeneMark,
BLAST, and FASTA. The full length polynucleotide sequences were translated to
derive the
corresponding full length amino acid sequences, and these full length
sequences were subsequently
analyzed by querying against databases such as the GenBank databases
(described above), SwissProt,
BLOCKS, PRINTS, PFAM, and Prosite.
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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, sub, ch.
7; Ausubel, 1995, supra,
ch. 4 and i6.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in nucleotide databases such as GenBank or LIFESEQ database (Incyte
Pharmaceuticals). This
analysis is much faster than multiple membrane-based hybridizations. In
addition, the sensitivity of the
computer search can be modified to determine whether any particular match is
categorized as exact or
similar. The basis of the search is the product score, which is defined as:
seguence identity x % maximum B A$~' score
100
The product score takes into account both the degree of similarity between two
sequences and the length
of the sequence match. For example, with a product score of 40, the match will
be exact within a 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 tower scores may
identify related molecules.
The results of northern analyses are reported a percentage distribution of
libraries in which the
transcript encoding HCYT 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 categories included cancer,
inflammation/trauma, fetal,
neurological, and pooled. For each category, the number of libraries
expressing the sequence of interest
was counted and divided by the total number of libraries across all
categories. Percentage values of
tissue-specific and disease expression are reported in Table 3.
V. Extension of HCYT Encoding Polynucleotides
Full length nucleic acid sequences (SEQ ID N0:9-14) were produced by extension
of the
component fragments described in Table I, Column 5, using oligonucleotide
primers based on those
fragments. Primers were used to facilitate the extension of the known sequence
"outward" generating
amplicons containing new unknown nucleotide sequence for the region of
interest. The initial primers
were designed from the cDNA using OLIGO 4.06 (National Biosciences, Inc.), 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 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 (The
42
CA 02335656 2001-O1-22
_ WO 00/06730 PCT/US99/17167
Perkin-Elmer Corp.) and thoroughly mixing the enzyme and reaction mix. PCR was
performed using the
PTC-200 thermal cycler (MJ Research, Inc.), beginning with 40 pmol of each
primer and the
recommended concentrations of all other components of the kit, with the
following parameters:
Step 1 94 C for I min (initial denaturation)
Step 2 65 C for 1 min
Step 3 68 C for 6 min
Step 4 94 C for 15 sec
Step 5 65 C for 1 min
Step 6 68 C for 7 min
i0 Step 7 Repeat steps 4 through 6 for an additional
15 cycles
Step 8 94 C for 15 sec
Step 9 65 C for 1 m in
Step 10 68 C for 7:15 min
Step 11 Repeat steps 8 through 10 for an
additional 12 cycles
15 Step 12 72 C for 8 min
Step 13 4 C (and holding)
A 5 ul to 10 ~d 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
20 extending the sequence. Bands thought to contain the largest products were
excised from the gel, purified
using QIAQUICKTM (QIAGEN Inc.), and trimmed of overhangs using Klenow enryme
to facilitate
reiigation and cloning.
After ethanol precipitation, the products were redissolved in 13 ~.d of
ligation buffer, l~.d T4-DNA
ligase ( I S units) and l,ul T4 polynucleotide kinase were added, and the
mixture was incubated at room
25 temperature for 2 to 3 hours, or overnight at 16° C. Competent E.E.
coli cells (in 40 ~d of appropriate
media) were transformed with 3 ~l of ligation mixture and cultured in 80 ~.el
of SOC medium. (See, e.g.,
Sambrook, su~a, Appendix A, p. 2.) After incubation for one hour at
37°C, the E.E. coli mixture was
plated on Luria Bertani (LB) agar (See, e.g., Sambrook,,, Appendix A, p. 1)
containing carbenicillin
(2x carb). The following day, several colonies were randomly picked from each
plate and cultured in 150
30 ~1 of liquid LB/2x carb medium placed in an individual well of an
appropriate commercially-available
sterile 96-weal microtiter plate. The following day, 5 E.d of each overnight
culture was transferred into a
non-sterile 96-well plate and, after dilution 1:10 with water, S E.d from each
sample was transferred into a
PCR array..
For PCR amplification, 18 /d of concentrated PCR reaction mix (3.3x)
containing 4 units of rTth
35 DNA polymerise, a vector primer, and one or both of the gene specific
primers used for the extension
reaction were added to each well. Amplification was performed using the
following conditions:
Step 1 94 C for 60 sec
Step 2 94 C for 20 sec
Step 3 55 C for 30 sec
40 Step 4 72 C for 90 sec
Step 5 Repeat steps 2 through 4 for an additional
29 cycles
Step 6 72 C for 180 sec
Step 7 4 C (and holding)
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WO 00/06730 PCT/US99/17167
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 N0:9-14 are used to obtain
5' regulatory
sequences using the procedure above, oligonucieotides designed for 5'
extension, and an appropriate
genomic library.
The full length nucleic acid sequences of SEQ ID NO:15-16 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
Mgr+, (NH4)ZS04, and ~i-
mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech), ELONGASE
enryme (Life
Technologies), and Pfu DNA polymerise (Stratagene), with the following
parameters for primer pair PCI
A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, IS sec; Step 3:
60°C, I 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, IS sec; Step
3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4
repeated 20 times; Step 6: 68°C, 5 min;
Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 pl
PICOGREEN
qusntitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes) 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 S ul to 10 ul aliquot of the reaction mixture was analyzed by
electrophoresis on a 1 % agarose mini-gel
to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC I 8 vector (Amersham
Pharmacia Biotech). For shotgun
44
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WO 00/06730 PCT/ITS99/17167
sequencing, the digested nucleotides were separated on low concentration (0.6
to 0.8%) agarose gels,
fragments were excised, and agar digested with Agar ACE (Promega). Extended
clones were religated
using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham
Pharmacia
Biotech), treated with Pfu DNA polymerise (Stratagene) to fill-in restriction
site overhangs, and
transfected into competent E. coli cells. Transformed cells were selected on
antibiotic-containing media,
individual colonies were picked and cultured overnight at 37°C in 384-
well plates in LB/2x carb liquid
media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise
(Amersham
Pharmacia Biotech) and Pfu DNA polymerise (Stratagene) with the following
parameters: Step 1: 94~,
3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4:
72°C, 2 min; Step 5: steps 2, 3, and 4 repeated
29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C. DNA was
quantified by PICOGREEN reagent
(Molecular Probes) as described above. Samples with low DNA recoveries were
reamplified using the
same conditions as described above. Samples were diluted with 20%
dimethysulphoxide (1:2, v/v), and
sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC
DIRECT kit
(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle
sequencing ready reaction
kit (Perkin-Elmer).
In Like manner, the nucleotide sequences of SEQ ID NO:1 S-16 are used to
obtain 5' regulatory
sequences using the procedure above,.oligonucleotides designed for such
extension, and an appropriate
genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:9-16 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, Inc.) and labeled by combining 50 pmol of each oligomer, 250
E.aCi of [y 'zP] adenosine
triphosphate (Amersham Pharmacia Biotech, Ltd.), and T4 polynucleotide kinase
(DuPont NEN, Boston,
MA). The labeled oligonucleotides are substantially purified using a SEPHADEX
G-25 superfine size
exclusion dextrin bead column (Amersham Pharmacia Biotech, Ltd.). An aliquot
containing I(Ycounts
per minute of the labeled probe is used in a typical membrane-based
hybridization analysis of human
genomic DNA digested with one of the following endonucleases: Ase I, Bgl II,
Eco RI, Pst I, Xba I, or
Pvu II (DuPont NEN, Boston, MA).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (NYTRAN Plus, Schleicher & Schuell, Durham, NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature under
increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate. After
XOMAT AR film (Eastman Kodak, Rochester, NY) is exposed to the blots,
hybridization patterns are
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
compared visually.
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, sub.) 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, LIV, 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 (SSTs), 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., W cross-linking
followed by thermal and
chemical treatments and subsequent drying. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470;
Shalon, D. et 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 HCYT-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring HCYT. Although
use of oligonucleotides
comprising from about 15 to 30 base pairs is described, essentially the same
procedure is used with
smaller or with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO 4.06
software (National Biosciences) and the coding sequence of HCYT. To inhibit
transcription, a
complementary oligonucleotide is designed from the most unique 5' sequence and
usedto prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is
designed to prevent ribosomal binding to the HCYT-encoding transcript.
IX. Expression of HCYT
Expression and purification of HCYT is achieved using bacterial or virus-based
expression
systems. For expression of HCYT 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 HCYT upon induction with isopropyl beta-D-thiogalactopyranoside
(IPTG). Expression of
46
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
HCYT in eukaryotic cells is achieved by infecting insect or mammalian cell
lines with recombinant
Autoeraohica californica nuclear polyhedrosis virus (AcMNPV), commonly known
as baculovirus. The
nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding
HCYT 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 Spoaontera
fr,~eioerda (Sf~7) insect cells in most
cases, or human hepatocytes, in some cases. Infection of the latter requires
additional genetic
modifications to baculovirus. (See Engelhard, E. K. et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3224-
3227; Sandig, V, et al. (1996) Hum. Gene Ther. 7:1937-1945.)
In most expression systems, HCYT is synthesized as a fusion protein with,
e.g., glutathione S-
transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step, affinity-
based purification of recombinant fusion protein from crude cell lysates. GST,
a 26-kilodalton enzyme
from Schistosoma ja oni ~m, enables the purification of fusion proteins on
immobilized glutathione
under conditions that maintain protein activity and antigenicity (Amersham
Pharmacia Biotech).
I S Following purification, the GST moiety can be proteolytically cleaved from
HCYT at specifically
engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity
purification using
commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman
Kodak). 6-His, a
stretch of six consecutive histidine residues, enables purification on metal-
chelate resins (QIAGEN).
Methods for protein expression and purification are discussed in Ausubel
(1995, supra, ch 10 and 16).
Purified HCYT obtained by these methods can be used directly in the following
activity assay.
X. Demonstration of HCYT Activity
HCYT activity may be measured by effects of the proteins on cellular
locomotion. In vitro cell
motility (locomotion) assays are performed as follows. Myosin is diluted to
200 ltg/ml in buffer C (25
mM imidazole, pH 7.4, 25 mM KCI, 4 mM MgClz, 1 mM EGTA, 10 mM dithiothreitol),
applied to a flow
cell coated with nitrocellulose, and blocked with buffer C containing 0.5
mg/ml BSA (CBSA). A
solution of phalloidin-labeled actin is perfused followed by 1 mM ATP in CBSA
to remove myosin
heads that bind actin in a rigor fashion. After washing with CBSA to remove
the excess nonfluorescent
actin, a solution of rhodamine-phalloidin-labeled actin and HCYT in CBSA is
introduced. Active
movement is initiated at room temperature by introducing CBSA containing 1 mM
ATP and oxygen
scavenger enzymes. Images (recorded using a Zeiss standard microscope (Zeiss,
New York NY)
equipped with a Hamamatsu SIT camera) of moving myotubes are tracked for up to
30 sec, and
translocation velocities calculated using the myotube centroids to establish
initial and final positions for 2
sec or 4 sec samples during the continuous movement.
Alternatively, an assay for HCYT activity measures the binding affinity of
HCYT for actin as
described by Hammell, R.L. and Hitchcock-DeGregori, S.E. (1997, J. Biol. Chem.
272:22409-22416).
HCYT and actin are prepared from In vitro recombinant cDNA expression systems
and the N-terminus of
47
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
HCYT is acetylated using methods well known in the art. Binding of N-terminal
acetyl-HCYT to actin is
measured by cosedimentation at 25°C in a Beckman model TL-100
centrifuge as described. The bound
and free HCYT are determined by quantitative densitometry of SDS-
polyacrylamide gels stained with
Coomassie Blue. Apparent binding constants (IC,PP) and Hill coefficients (H)
are determined by using
methods well known in the art to fit the data to the equation as described by
Hammell and Hitchcock-
DeGregori ( 1997, supra). The HCYT:actin ratio determined using densitometry
is normalized. Hammell
and Hitchcock-DeGregori ( 1997, supra) have shown that saturation of binding
corresponds to a
HCYT:actin molar ratio of 0.14, a stoichiometry of 1 HCYT:7 actin.
XI. Functional Assays
HCYT function is assessed by expressing the sequences encoding HCYT 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
IS contain the cytomegalovirus promoter. 5-10 E.cg of recombinant vector are
transiently transfected into a
human cell line, preferably of endothelial or hematopoietic origin, using
either liposome formulations or
electroporation. 1-2 ~cg of an additional plasmid containing sequences
encoding a marker protein are co-
transfected. Expression of a marker protein provides a means to distinguish
transfected cells from
nontransfected cells and is a reliable predictor of cDNA expression from the
recombinant vector. Marker
proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech),
CD64, or a CD64-GFP
fusion protein. Flow cytometry (FCM), an automated, laser optics-based
technique, is used to identify
transfected cells expressing GFP or CD64-GFP, and to evaluate properties, for
example, their apoptotic
state. FCM detects and quantifies the uptake of fluorescent molecules that
diagnose events preceding or
coincident with cell death. These events include changes in nuclear DNA
content as measured by staining
of DNA with propidium iodide; changes in cell size and granularity as measured
by forward light scatter
and 90 degree side light scatter; down-regulation of DNA synthesis as measured
by decrease in
bromodeoxyuridine uptake; alterations in expression of cell surface and
intracellular proteins as measured
by reactivity with specific antibodies; and alterations in plasma membrane
composition as measured by
the binding of fluorescein-conjugated Annexin V protein to the cell surface.
Methods in flow cytometry
are discussed in Ormerod, M. G. ( 1994) Flow ~rtom _er,.", Oxford, New York
NY.
The influence of HCYT on gene expression can be assessed using highly purified
populations of
cells transfected with sequences encoding HCYT 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
48
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
encoding HCYT and other genes of interest can be analyzed by northern analysis
or microarray
techniques.
XII. Production of HCYT Specific Antibodies
HCYT substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the HCYT amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, , ch. 11.)
Typically, oligopeptides 15 residues in length are synthesized using an ABI
431A Peptide
Synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to ICLH (Sigma-
Aldrich, St. Louis MO)
by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to
increase immunogenicity.
(See, e.g., Ausubel, 1995, sub.) Rabbits are immunized with the oligopeptide-
KLH complex in
complete Freund's adjuvant. Resulting antisera are tested for antipeptide
activity by, for example, binding
the peptide to plastic, blocking with 1 % BSA, reacting with rabbit antisera,
washing, and reacting with
radio-iodinated goat anti-rabbit IgG.
XIII. Purification of Naturally Occurring HCYT Using Specific Antibodies
Naturally occurring or recombinant HCYT is substantially purified by
immunoaffinity
chromatography using antibodies specific for HCYT. An immunoaffinity column is
constructed by
covalently coupling anti-HCYT 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 HCYT are passed over the immunoaffinity column, and the
coiumn is washed
under conditions that allow the~preferential absorbance of HCYT (e.g., high
ionic strength buffers in the
presence of detergent). The column is eluted under conditions that disrupt
antibody/HCYT 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
HCYT is collected.
XIV. Identification of Molecules Which Interact with HCYT
HCYT, or biologically active fragments thereof, are labeled with'~'I Bolton-
Hunter reagent.
(See, e.g., Bolton et al. ( 1973) Biochem. J. 133:529.) Candidate molecules
previously arrayed in the
wells of a multi-well plate are incubated with the labeled HCYT, washed, and
any wells with labeled
HCYT complex are assayed. Data obtained using different concentrations of HCYT
are used to calculate
values for the number, affinity, and association of HCYT with the candidate
molecules.
Various modifications and variations of the described methods and systems of
the invention will
49
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
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 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
BANDMAN, Olga
TANG, Y. Tom
YUE, Henry
CORLEY, Neil C.
GUEGLER, Karl J.
AZIMZAI, Yalda
PATTERSON, Chandra
LAL, Preeti
BAUGHN, Mariah R.
<120> HUMAN CYTOSKELETAL PROTEINS
<130> PF-0568 PCT
<140> To Be Assigned
<191> Herewith
<150> 09/127,665
<151> 1998-07-31
<160> 17
<170> PERL Program
<210> 1
<211> 284
<212> PRT
<213> Homo sapiens
<z2o>
<221> misc feature
<223> Incyte Clone No: 1274060
<400> 1
Met Glu Ala Ile Lys Lys Lys Met Gln Met Leu Lys Leu Asp Lys
1 5 10 15
Glu Asn Ala Ile Asp Arg Ala Glu Gln Ala Glu Ala Asp Lys Lys
20 25 30
Ala Ala Glu Asp Lys Cys Lys Gln Val Glu Glu Glu Leu Thr His
35 40 45
Leu Gln Lys Lys Leu Lys Gly Thr Glu Asp Glu Leu Asp Lys Tyr
50 55 60
Ser Glu Asp Leu Lys Asp Ala Gln Glu Lys Leu Glu Leu Thr Glu
65 70 75
Lys Lys Ala Ser Asp Ala Glu Gly Asp Val Ala Ala Leu Asn Arg
80 85 90
Arg Ile Gln Leu Val Glu Glu Glu Leu Asp Arg Ala Gln Glu Arg
95 100 105
Leu Ala Thr Ala Leu Gln Lys Leu Glu Glu Ala Glu Lys Ala Ala
110 115 120
Asp Glu Ser Glu Arg Gly Met Lys Val Ile Glu Asn Arg Ala Met
125 130 135
Lys Asp Glu Glu Lys Met Glu Ile Gln Glu Met Gln Leu Lys Glu
140 145 150
Ala Lys His Ile Ala Glu Glu Ala Asp Arg Lys Tyr Glu Glu Val
155 160 165
Ala Arg Lys Leu Val Ile Leu Glu Gly Glu Leu Glu Arg Ala Glu
170 175 180
Glu Arg Ala Glu Val Ser Glu Leu Lys Cys Gly Asp Leu Glu Glu
185 190 195
1/21
CA 02335656 2001-O1-22
WO 00/06730 PCT1US99/17167
Glu Leu Lys Asn Val Thr Asn Asn Leu Lys Sex Leu Glu Ala Ala
200 205 210
Ser Glu Lys Tyr Ser Glu Lys Glu Asp Lys Tyr Glu Glu Glu Ile
215 220 225
Lys Leu Leu Ser Asp Lys Leu Lys Glu Ala Glu Thr Arg Ala Glu
230 235 240
Phe Ala Glu Arg Thr Val Ala Lys Leu Glu Lys Thr Ile Asp Asp
245 250 255
Leu Glu Glu Lys Leu Ala Gln Ala Lys Glu Glu Asn Val Gly Leu
260 265 270
His Gln Thr Leu Asp Gln Thr Leu Asn Glu Leu Asn Cys Ile
275 280
<210> 2
<211> 158
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1577078
<400> 2
Met Lys Val Ile Glu Asn Arg Ala Met Lys Asp Glu Glu Lys Met
1 S 10 15
Glu Ile Gln Glu Met Gln Leu Lys Glu Ala Lys His Ile Ala Glu
20 25 30
Glu Ala Asp Arg Lys Tyr Glu Glu Val Ala Arg Lys Leu Val Ile
35 40 45
Leu Glu Gly Glu Leu Glu Arg Ala Glu Glu Arg Ala Glu Val Ser
50 55 60
Glu Leu Lys Cys Gly Asp Leu Glu Glu Glu Leu Lys Asn Val Thr
65 70 75
Asn Asn Leu Lys Ser Leu Glu Ala Ala Ser Glu Lys Tyr Ser Glu
80 85 90
Lys Glu Asp Lys Tyr Glu Glu Glu Ile Lys Leu Leu Ser Asp Lys
95 100 105
Leu Lys Glu Ala Glu Thr Arg Ala Glu Phe Ala Glu Arg Thr Val
110 115 120
Ala Lys Leu Glu Lys Thr Ile Asp Asp Leu Glu Glu Lys Leu Ala
125 130 135
Gln Ala Lys Glu Glu Asn Val Gly Leu His Gln Thr Leu Asp Gln
140 145 150
Thr Leu Asn Glu Leu Asn Cys Ile
155
<210> 3
<211> 208
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte Clone No: 1426711
<400> 3
2/21
CA 02335656 2001-O1-22
WO 00/Ob730 PCT/US99/171b7
Met Asp Ala Ile Lys Lys Lys Met Gln Met Leu Lys Leu Asp Lys
1 5 10 15
Glu Asn Ala Leu Asp Arg Ala Glu Gln Ala Glu Ala Asp Lys Lys
20 25 30
Ala Ala Glu Asp Arg Ser Lys Gln Leu Glu Glu Asp Ile Ala Ala
35 40 45
Lys Glu Lys Leu Leu Arg Val Ser Glu Asp Glu Arg Asp Arg Val
50 55 60
Leu Glu Glu Leu His Lys Ala Glu Asp Ser Leu Leu Ala Ala Glu
65 70 75
Glu Ala Ala Ala Lys Ala Glu Ala Asp Val Ala Ser Leu Asn Arg
80 B5 90
Arg Ile Gln Leu Val Glu Glu Glu Leu Asp Arg Ala Gln Glu Arg
95 100 105
Leu Ala Thr Ala Leu Gln Lys Leu Glu Glu Ala Glu Lys Ala Ala
110 115 120
Asp Glu Ser Glu Arg Gly Met Lys Val Ile Glu Ser Arg Ala Gln
125 130 135
Lys Asp Glu Glu Lys Met Glu Ile Gln Glu Ile Gln Leu Lys Glu
140 145 150
Ala Lys His Ile Ala Glu Asp Ala Asp Arg Lys Tyr Glu Glu Val
155 160 165
Ala Arg Lys Leu Val Ile Ile Glu Ser Asp Leu Glu Arg Ala Glu
170 175 180
Glu Arg Ala Gly Glu Gly Leu Asp Lys Asp Arg Arg Ala Ala Thr
185 190 195
His His Pro Ser Pro His Pro His Pro Leu Leu Glu Phe
200 205
<210> 4
<211> 156
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte Clone No: 1676756
<900> 4
Met Ile Lys Arg Val Leu Leu Glu Arg Leu Glu Asn Thr Arg Lys
1 5 10 15
Leu Arg Glu Leu Thr Glu Gly Arg Thr Leu Asp Trp Pro Gln Asn
20 25 30
Arg Ile Thr Glu Val Ser Ala Lys Arg Gln Ile Val Thr Glu Tyr
35 40 45
Arg Glu Lys Gly Lys Arg Asn Tyr Glu Glu Lys Lys Arg Asp Leu
50 55 60
Glu Gly Arg Ser Arg Arg Tyr Asn Leu Cys Ile Ile Gly Ile Pro
65 70 75
Glu Thr Glu Asp Arg Ala Ser Gly Ala Glu Thr Ile Lys Asp Leu
80 85 90
Leu Glu Lys Asn Phe Pro Glu Leu Lys Asn Glu Leu Asp Leu Gln
95 100 105
Met Glu Lys Ala His Arg Ile Pro Leu Lys Phe Asn Glu Lys Lys
110 115 120
Ala Ala Ser Arg His Ile Arg Val Thr Phe Leu Asn Phe Lys Asp
125 130 135
Glu Thr Phe Tyr Lys His Pro Val Arg Glu Ser Arg Leu Leu Thr
190 145 150
Lys Gly Gln Lys Ser Gly
155
3/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/IJS99/17167
<210> 5
<211> 876
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte Clone No: 1843770
<400> 5
Met Ala Ser Asp Ala Ser His Ala Leu Glu Ala Ala Leu Glu Gln
1 5 10 15
Met Asp Gly Ile Ile Ala Gly Thr Lys Thr Gly Ala Asp Leu Ser
20 25 30
Asp Gly Thr Cys Glu Pro Gly Leu Ala Ser Pro Ala Ser Tyr Met
35 40 45
Asn Pro Phe Pro Val Leu His Leu Ile Glu Asp Leu Arg Leu Ala
50 55 60
Leu Glu Met Leu Glu Leu Pro Gln Glu Arg Ala Ala Leu Leu Ser
65 70 75
Gln Ile Pro Gly Pro Thr Ala Ala Tyr Ile Lys Glu Trp Phe Glu
80 85 90
Glu Ser Leu Ser Gln Val Asn His His Ser Ala Ala Ser Asn Glu
95 100 105
Thr Tyr Gln Glu Arg Leu Ala Arg Leu Glu Gly Asp Lys Glu Ser
110 115 120
Leu Ile Leu Gln Val Ser Val Leu Thr Asp Gln Val Glu Ala Gln
125 130 135
Gly Glu Lys Ile Arg Asp Leu Glu Val Cys Leu Glu Gly His Gln
140 145 150
Val Lys Leu Asn Ala Ala Glu Glu Met Leu Gln Gln Glu Leu Leu
155 160 165
Ser Arg Thr Ser Leu Glu Thr Gln Lys Leu Asp Leu Met Thr Glu
170 175 180
Val Ser Glu Leu Lys Leu Lys Leu Val Gly Met Glu Lys Glu Gln
185 190 195
Arg Glu Gln Glu Glu Lys Gln Arg Lys Ala Glu Glu Leu Leu Gln
200 205 210
Glu Leu Arg His Leu Lys Ile Lys Val Glu Glu Leu Glu Asn Glu
215 220 225
Arg Asn Gln Tyr Glu Trp Lys Leu Lys Ala Thr Lys Ala Glu Val
230 235 290
Ala Gln Leu Gln Glu Gln Val Ala Leu Lys Asp Ala Glu Ile Glu
245 250 255
Arg Leu His Ser Gln Leu Ser Arg Thr Ala Ala Leu His Ser Glu
260 265 270
Ser His Thr Glu Arg Asp Gln Glu Ile Gln Arg Leu Lys Met Gly
275 280 285
Met Glu Thr Leu Leu Leu Ala Asn Glu Asp Lys Asp Arg Arg Ile
290 295 300
Glu Glu Leu Thr Gly Leu Leu Asn Gln Tyr Arg Lys Val Lys Glu
305 310 315
Ile Val Met Val Thr Gln Gly Pro Ser Glu Arg Thr Leu Ser Ile
320 325 330
Asn Glu Glu Glu Pro Glu Gly Gly Phe Ser Lys Trp Asn Ala Thr
335 340 345
Asn Lys Asp Pro Glu Glu Leu Phe Lys Gln Glu Met Pro Pro Arg
350 355 360
Cys Ser Ser Pro Thr Val Gly Pro Pro Pro Leu Pro Gln Lys Ser
365 370 375
Leu Glu Thr Arg Ala Gln Lys Lys Leu Ser Cys Ser Leu Glu Asp
380 385 390
Leu Arg Ser Glu Ser Val Asp Lys Cys Met Asp Gly Asn Gln Pro
395 400 405
4/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
Phe Pro Val Leu Glu Pro Lys Asp Ser Pro Phe Leu Ala Glu His
410 415 420
Lys Tyr Pro Thr Leu Pro Gly Lys Leu Ser Gly Ala Thr Pro Asn
425 430 435
Gly Glu Ala Ala Lys Ser Pro Pro Thr Ile Cys Gln Pro Asp Ala
990 445 450
Thr Gly Ser Ser Leu Leu Arg Leu Arg Asp Thr Glu Ser Gly Trp
455 460 465
Asp Asp Thr Ala Val Val Asn Asp Leu Ser Ser Thr Ser Ser Gly
470 475 480
Thr Glu Ser Gly Pro Gln Ser Pro Leu Thr Pro Asp Gly Lys Arg
485 490 495
Asn Pro Lys Gly Ile Lys Lys Phe Trp Gly Lys Ile Arg Arg Thr
500 505 510
Gln Ser Gly Asn Phe Tyr Thr Asp Thr Leu Gly Met Ala Glu Phe
515 520 525
Arg Arg Gly Gly Leu Arg Ala Thr Ala Gly Pro Arg Leu Ser Arg
530 535 540
Thr Arg Asp Ser Lys Gly Gln Lys Ser Asp Ala Asn Ala Pro Phe
545 550 555
Ala Gln Trp Ser Thr Glu Arg Val Cys Ala Trp Leu Glu Asp Phe
560 565 570
Gly Leu Ala Gln Tyr Val Ile Phe Ala Arg Gln Trp Val Ser Ser
575 580 585
Gly His Thr Leu Leu Thr Ala Thr Pro Gln Asp Met Glu Lys Glu
590 595 600
Leu Gly Ile Lys His Pro Leu His Arg Lys Lys Leu Val Leu Ala
605 610 615
Val Lys Ala Ile Asn Thr Lys Gln Glu Glu Lys Ser Ala Leu Leu
620 625 630
Asp His Ile Trp Val Thr Arg Trp Leu Asp Asp Ile Gly Leu Pro
635 640 645
Gln Tyr Lys Asp Gln Phe His Glu Ser Arg Val Asp Gly Arg Met
650 655 660
Leu Gln Tyr Leu Thr Val Asn Asp Leu Leu Phe Leu Lys Val Thr
665 670 675
Ser Gln Leu His His Leu Ser Ile Lys Cys Ala Ile His Val Leu
680 685 690
His Val Asn Lys Phe Asn Pro His Cys Leu His Arg Arg Pro Ala
695 700 705
Asp Glu Ser Asn Leu Ser Pro Ser Glu Val Val Gln Trp Ser Asn
710 715 720
His Arg Val Met Glu Trp Leu Arg Ser Val Asp Leu Ala Glu Tyr
725 730 735
Ala Pro Asn Leu Arg Gly Ser Gly Val His Gly Gly Leu Ile Ile
740 795 750
Leu Glu Pro Arg Phe Thr Gly Asp Thr Leu Ala Met Leu Leu Asn
755 760 765
Ile Pro Pro Gln Lys Thr Leu Leu Arg Arg His Leu Thr Thr Lys
770 775 780
Phe Asn Ala Leu Ile Gly Pro Glu Ala Glu Gln Glu Lys Arg Glu
785 790 795
Lys Met Ala Ser Pro Ala Tyr Thr Pro Leu Thr Thr Thr Ala Lys
800 805 810
Val Arg Pro Arg Lys Leu Gly Phe Ser His Phe Gly Asn Ile Arg
815 820 825
Lys Lys Lys Phe Asp Glu Ser Thr Asp Tyr Ile Cys Pro Met Glu
830 835 840
Pro Ser Asp Gly Val Ser Asp Ser His Arg Val Tyr Ser Gly Tyr
845 850 855
Arg Gly Leu Ser Pro Leu Asp Ala Pro Glu Leu Asp Gly Leu Asp
860 865 870
Gln Val Gly Gln Ile Ser
875
5/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
<210> 6
<211> 806
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte Clone No: 3768043
<400> 6
Met Glu Glu Pro Gly Ala Thr Pro Gln Pro Tyr Leu Gly Leu Val
1 5 10 15
Leu Glu Glu Leu Arg Arg Val Val Ala Ala Leu Pro Glu Ser Met
20 25 30
Arg Pro Asp Glu Asn Pro Tyr Gly Phe Pro Ser Glu Leu Val Val
35 40 45
Cys Ala Ala Val Ile Gly Phe Phe Val Val Leu Leu Phe Leu Trp
50 55 60
Arg Ser Phe Arg Ser Val Arg Ser Arg Leu Tyr Val Gly Arg Glu
65 70 75
Gln Lys Leu Gly Ala Thr Leu Ser Gly Leu Ile Glu Glu Lys Cys
80 85 90
Lys Leu Leu Glu Lys Phe Ser Leu Ile Gln Lys Glu Tyr Glu Gly
95 100 105
Tyr Glu Val Glu Ser Ser Leu Glu Asp Ala Ser Phe Glu Lys Glu
110 115 120
Ala Ala Glu Glu Ala Arg Ser Leu Glu Ala Thr Cys Glu Lys Leu
125 130 135
Asn Arg Ser Asn Ser Glu Leu Glu Asp Glu Ile Leu Cys Leu Glu
190 145 150
Lys Asp Leu Lys Gln Glu Lys Ser Lys His Ser Gln Gln Asp Glu
155 160 165
Leu Met Ala Asp Ile Ser Lys Ser Ile Gln Ser Leu Glu Asp Glu
170 175 180
Ser Lys Ser Leu Lys Ser Gln Ile Ala Glu Ala Lys Ile Ile Cys
185 190 195
Lys Thr Phe Lys Met Ser Glu Glu Arg Arg Ala Ile Ala Ile Lys
200 205 210
Asp Ala Leu Asn Glu Asn Ser Gln Leu Gln Thr Ser His Lys Gln
215 220 225
Leu Phe Gln Gln Glu Ala Glu Val Trp Lys Gly Glu Val Ser Glu
230 235 240
Leu Asn Lys Gln Lys Ile Thr Phe Glu Asp Ser Lys Val His Ala
245 250 255
Glu Gln Val Leu Asn Asp Lys Glu Asn His Ile Lys Th'r Leu Thr
260 265 270
Gly His Leu Pro Met Met Lys Asp Gln Ala Ala Val Leu Glu Glu
275 280 285
Asp Thr Thr Asp Asp Asp Asn Leu Glu Leu Glu Val Asn Ser Glu
290 295 300
Ser Glu Asn Gly Ala Tyr Leu Asp Asn Pro Pro Lys Gly Ala Leu
305 310 315
Lys Lys Leu Ile His Ala Ala Lys Leu Asn Ala Ser Leu Lys Thr
320 325 330
Leu Glu Gly Glu Arg Asn Gln Ile Tyr Ile Gln Leu Ser Glu Val
335 340 395
Asp Lys Thr Lys Glu Glu Leu Thr Glu His Ile Lys Asn Leu Gln
350 355 360
Thr Gln Gln Ala Ser Leu Gln Ser Glu Asn Thr His Phe Glu Asn
365 370 375
Glu Asn Gln Lys Leu Gln Gln Lys Leu Lys Val Met Thr Glu Leu
380 385 390
6/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99117167
Tyr Gln Glu Asn Glu Met Lys Leu His Arg Lys Leu Thr Val Glu
395 400 905
Glu Asn Tyr Arg Leu Glu Lys Glu Glu Lys Leu Ser Lys Val Asp
410 415 420
Glu Lys Ile Ser His Ala Thr Glu Glu Leu Glu Thr Tyr Arg Lys
425 930 435
Arg Ala Lys Asp Leu Glu Glu Glu Leu Glu Arg Thr Ile His Ser
440 445 450
Tyr Gln Gly Gln Ile Ile Ser His Glu Lys Lys Ala His Asp Asn
455 460 465
Trp Leu Ala Ala Arg Asn Ala Glu Arg Asn Leu Asn Asp Leu Arg
470 475 980
Lys Glu Asn Ala His Asn Arg Gln Lys Leu Thr Glu Thr Glu Leu
985 490 495
Lys Phe Glu Leu Leu Glu Lys Asp Pro Tyr Ala Leu Asp Val Pro
500 505 510
Asn Thr Ala Phe Gly Arg Glu His Ser Pro Tyr Gly Pro Ser Pro
515 520 525
Leu Gly Trp Pro Ser Ser Glu Thr Arg Ala Phe Leu Ser Pro Pro
530 535 540
Thr Leu Leu Glu Gly Pro Leu Arg Leu Ser Pro Leu Leu Pro Gly
595 550 555
Gly Gly Gly Arg Gly Ser Arg Gly Pro Gly Asn Pro Leu Asp His
560 565 570
Gln Ile Thr Asn Glu Arg Gly Glu Ser Ser Cys Asp Arg Leu Thr
575 580 585
Asp Pro His Arg Ala Pro Ser Asp Thr Gly Ser Leu Ser Pro Pro
590 595 600
Trp Asp Gln Asp Arg Arg Met Met Phe Pro Pro Pro Gly Gln Ser
605 610 615
Tyr Pro Asp Ser Ala Leu Pro Pro Gln Arg Gln Asp Arg Phe Cys
620 625 630
Ser Asn Ser Gly Arg Leu Ser Gly Pro Ala Glu Leu Arg Ser Phe
635 690 695
Asn Met Pro Ser Leu Asp Lys Met Asp Gly Ser Met Pro Ser Glu
650 655 660
Met Glu Ser Ser Arg Asn Asp Thr Lys Asp Asp Leu Gly Asn Leu
665 670 6?5
Asn Val Pro Asp Ser Ser Leu Pro Ala Glu Asn Glu Ala Thr Gly
680 685 690
Pro Gly Phe Val Pro Pro Pro Leu Ala Pro Ile Arg Gly Pro Leu
695 700 705
Phe Pro Val Asp Ala Arg Gly Pro Phe Leu Arg Arg Gly Pro Pro
710 715 720
Phe Pro Pro Pro Pro Pro Gly Ala Met Phe Gly Ala Ser Arg Asp
725 730 735
Tyr Phe Pro Pro Arg Asp Phe Pro Gly Pro Pro Pro Ala Pro Phe
740 745 750
Ala Met Arg Asn Val Tyr Pro Pro Arg Gly Phe Pro Pro Tyr Leu
755 760 765
Pro Pro Arg Pro Gly Phe Phe Pro Pro Pro Pro His Ser Glu Gly
770 775 780
Arg Ser Glu Phe Pro Ser Gly Leu Ile Pro Pro Ser Asn Glu Pro
785 790 795
Ala Thr Glu His Pro Glu Pro Gln Gln Glu Thr
800 805
<210> 7
<211> 2992
<212> PRT
<213> Homo sapiens
7/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
<220>
<221> misc feature
<223> Incyte Clone No: 1655208
<400> 7
Met Glu Thr Arg Ser Pro Gly Leu Asn Asn Met Lys Pro Gln Ser
1 5 10 15
Leu Gln Leu Val Leu Glu Glu Gln Val Leu Ala Leu Gln Gln Gln
20 25 30
Met Ala Glu Asn Gln Ala Ala Ser Trp Arg Lys Leu Lys Asn Ser
35 40 45
Gln Glu Ala Gln Gln Arg Gln Ala Thr Leu Val Arg Lys Leu Gln
50 55 60
Ala Lys Val Leu Gln Tyr Arg Ser Trp Cys Gln Glu Leu Glu Lys
65 70 75
Arg Leu Glu Ala Thr Gly Gly Pro Ile Pro Gln Arg Trp Glu Asn
80 85 90
Val Glu Glu Pro Asn Leu Asp Glu Leu Leu Val Arg Leu Glu Glu
95 100 105
Glu Gln Gln Arg Cys Glu Ser Leu Ala Glu Val Asn Thr Gln Ile
110 115 120
Arg Leu His Met Glu Lys Ala Asp Val Val Asn Lys Ala Leu Arg
125 130 135
Ala Asp Val Glu Lys Leu Thr Val Asp Trp Ser Arg Ala Arg Asp
140 195 150
Glu Leu Met Arg Lys Glu Ser Gln Trp Gln Met Glu Gln Glu Phe
155 160 165
Phe Lys Gly Tyr Leu Lys Gly Glu His Gly Arg Leu Leu Ser Leu
170 175 180
Trp Arg Glu Val Val Thr Phe Arg Arg His Phe Leu Glu Met Lys
185 190 195
Ser Ala Thr Asp Arg Asp Leu Met Glu Leu Lys Ala Glu His Val
200 205 210
Arg Leu Ser Gly Ser Leu Leu Thr Cys Cys Leu Arg Leu Thr Val
215 220 225
Gly Ala Gln Ser Arg Glu Pro Asn Gly Ser Gly Arg Met Asp Gly
230 235 240
Arg Glu Pro Ala Gln Leu Leu Leu Leu Leu Ala Lys Thr Gln Glu
245 250 255
Leu Glu Lys Glu Ala His Glu Arg Ser Gln Glu Leu Ile Gln Leu
260 265 270
Lys Ser Gln Gly Asp Leu Glu Lys Ala Glu Leu Gln Asp Arg Val
275 280 285
Thr Glu Leu Ser Ala Leu Leu Thr Gln Ser Gln Lys Gln Asn Glu
290 295 300
Asp Tyr Glu Lys Met Ile Lys Ala Leu Arg Glu Thr Val Glu Ile
305 310 315
Leu Glu Thr Asn His Thr Glu Leu Met Glu His Glu Ala Ser Leu
320 325 330
Ser Arg Asn Ala Gln Glu Glu Lys Leu Ser Leu Gln Gln Val Ile
335 340 345
Lys Asp Ile Thr Gln Val Met Val Glu Glu Gly Asp Asn Ile Ala
350 355 360
Gln Gly Ser Gly Leu Glu Asn Ser Leu Glu Leu Glu Ser Ser Ile
365 370 375
Phe Ser Gln Phe Asp Tyr Gln Asp Ala Asp Lys Ala Leu Thr Leu
380 385 390
Val Arg Ser Val Leu Thr Arg Arg Arg Gln Ala Val Gln Asp Leu
395 400 405
Arg Gln Gln Leu Ala Gly Cys Gln Glu Ala Val Asn Leu Leu Gln
410 415 420
Gln Gln His Asp Gln Trp Glu Glu Glu Gly Lys Ala Leu Arg Gln
925 430 435
Arg Leu Gln Lys Leu Thr Gly Glu Arg Asp Thr Leu Ala Gly Gln
440 445 450
8/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
Thr Val Asp Leu Gln Gly Glu Val Asp Ser Leu Ser Lys Glu Arg
455 460 465
Glu Leu Leu Gln Lys Ala Arg Glu Glu Leu Arg Gln Gln Leu Glu
470 475 480
Val Leu GIu Gln Glu Ala Trp Arg Leu Arg Arg Val Asn Val Glu
485 490 495
Leu Gln Leu Gln Gly Asp Ser Ala Gln Gly Gln Lys Glu Glu Gln
500 505 510
Gln Glu Glu Leu His Leu Ala Val Arg Glu Arg Glu Arg Leu Gln
515 520 525
Glu Met Leu Met Gly Leu Glu Ala Lys Gln Ser Glu Ser Leu Ser
530 535 540
Glu Leu Ile Thr Leu Arg Glu Ala Leu Glu Ser Ile His Leu Glu
545 550 555
Gly Glu Leu Leu Arg Gln Glu Gln Thr Glu Val Thr Ala Ala Leu
560 565 570
Ala Arg Ala Glu Gln Ser Ile Ala Glu Leu Ser Ser Ser Glu Asn
575 580 585
Thr Leu Lys Thr Glu Val Ala Asp Leu Arg Ala Ala Ala Val Lys
590 595 600
Leu Ser Ala Leu Asn Glu Ala Leu Ala Leu Asp Lys Val Gly Leu
605 610 615
Asn Gln Gln Leu Leu Gln Leu Glu Glu Glu Asn Gln Ser Val Cys
620 625 630
Ser Arg Met Glu Ala Ala Glu Gln Ala Arg Asn Ala Leu Gln Val
635 690 695
Asp Leu Ala Glu Ala Glu Lys Arg Arg Glu Ala Leu Trp Glu Lys
650 655 660
Asn Thr His Leu Glu Ala Gln Leu Gln Lys Ala Glu Glu Ala Gly
665 670 675
Ala Glu Leu Gln Ala Asp Leu Arg Asp Ile Gln Glu Glu Lys Glu
680 685 690
Glu Ile Gln Lys Lys Leu Ser Glu Ser Arg His Gln Gln Glu Ala
695 700 705
Ala Thr Thr Gln Leu Glu Gln Leu His Gln Glu Ala Lys Arg Gln
7I0 715 720
Glu Glu Val Leu Ala Arg Ala Val Gln Glu Lys Glu Ala Leu Val
725 730 735
Arg Glu Lys Ala Ala Leu Glu Val Arg Leu Gln Ala Val Glu Arg
790 745 750
Asp Arg Gln Asp Leu Ala Ala Gln Leu Gln Gly Leu Ser Ser Ala
755 760 765
Lys Glu Leu Leu Glu Ser Ser Leu Phe Glu Ala Gln Gln Gln Asn
770 775 780
Ser Val Ile Asp Glu Pro Gln Gly Gln Leu Glu Val Gln Ile Gln
785 790 795
Thr Val Thr Gln Ala Lys Glu Val Ile Gln Gly Glu Val Arg Cys
800 805 810
Leu Lys Leu Glu Leu Asp Thr Glu Arg Ser Gln Ala Glu Gln Glu
815 820 825
Arg Asp Ala Ala Ala Arg Gln Leu Ala Gln Ala Glu Gln Glu Gly
830 835 840
Lys Thr Ala Leu Glu Gln Gln Lys Ala Ala His Glu Lys Glu Val
895 850 855
Asn Gln Leu Arg Glu Lys Trp Glu Lys Glu Arg Ser Trp His Gln
860 865 870
Gln Glu Leu Ala Lys Ala Leu Glu Ser Leu Glu Arg Glu Lys Met
875 880 885
Glu Leu Glu Met Arg Leu Lys Glu Gln Gln Thr Glu Met Glu Ala
890 895 900
Ile Gln Ala Gln Arg Glu Glu Glu Arg Thr Gln Ala Glu Ser Ala
905 910 915
Leu Cys Gln Met Gln Leu Glu Thr Glu Lys Glu Arg Val Ser Leu
920 925 930
Leu Glu Thr Leu Leu Gln Thr Gln Lys Glu Leu Ala Asp Ala Ser
9/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
935 940 945
Gln Gln Leu-Glu Arg Leu Arg Gln Asp Met Lys Val Gln Lys Leu
950 955 960
Lys Glu Gln Glu Thr Thr Gly Ile Leu Gln Thr Gln Leu Gln Glu
965 970 975
Ala Gln Arg Glu Leu Lys Glu Ala Ala Arg Gln His Arg Asp Asp
980 985 990
Leu Ala Ala Leu Gln Glu Glu Ser Ser Ser Leu Leu Gln Asp Lys
995 1000 1005
Met Asp Leu Gln Lys Gln Val Glu Asp Leu Lys Ser Gln Leu Val
1010 1015 1020
Ala Gln Asp Asp Ser Gln Arg Leu Val Glu Gln Glu Val Gln Glu
1025 1030 1035
Lys Leu Arg Glu Thr Gln Glu Tyr Asn Arg Ile Gln Lys Glu Leu
1040 1045 1050
Glu Arg Glu Lys Ala Ser Leu Thr Leu Ser Leu Met Glu Lys Glu
1055 1060 1065
Gln Arg Leu Leu Val Leu Gln Glu Ala Asp Ser Ile Arg Gln Gln
1070 1075 1080
Glu Leu Ser Ala Leu Arg Gln Asp Met Gln Glu Ala Gln Gly Glu
1085 1090 1095
Gln Lys Glu Leu Ser Ala Gln Met Glu Leu Leu Arg Gln Glu Val
1100 1105 1110
Lys Glu Lys Glu Ala Asp Phe Leu Ala Gln Glu Ala Gln Leu Leu
1115 1120 1125
Glu Glu Leu Glu Ala Ser His Ile Thr Glu Gln Gln Leu Arg Ala
1130 1135 1190
Ser Leu Trp Ala Gln Glu Ala Lys Ala Ala Gln Leu His Leu Arg
1145 1150 1155
Leu Arg Ser Thr Glu Ser Gln Leu Glu Ala Leu Ala Ala Glu Gln
1160 1165 1170
Gln Pro Gly Asn Gln Ala Gln Ala Gln Ala Gln Leu Ala Ser Leu
1175 1180 1185
Tyr Ser Ala Leu Gln Gln Ala Leu Gly Ser Val Cys Glu Ser Arg
1190 1195 1200
Pro Glu Leu Ser Gly Gly Gly Asp Ser Ala Pro Ser Val Trp Gly
1205 1210 1215
Leu Glu Pro Asp Gln Asn Gly Ala Arg Ser Leu Phe Lys Arg Gly
1220 1225 1230
Pro Leu Leu Thr Ala Leu Ser Ala Glu Ala Val Ala Ser Ala Leu
1235 1240 1245
Leu Lys Leu His Gln Asp Leu Trp Lys Thr Gln Gln Thr Arg Asp
1250 1255 1260
Val Leu Arg Asp Gln Val G1n Lys Leu Glu Glu Arg Leu Thr Asp
1265 1270 1275
Thr Glu Ala Glu Lys Ser Gln Val His Thr Glu Leu Gln Asp Leu
1280 1285 1290
Gln Arg Gln Leu Ser Gln Asn Gln Glu Glu Lys Ser Lys Trp Glu
1295 1300 1305
Gly Lys Gln Asn Ser Leu Glu Ser Glu Leu Met Glu Leu His Glu
1310 1315 1320
Thr Met Ala Ser Leu Gln Ser Arg Leu Arg Arg Ala Glu Leu Gln
1325 1330 1335
Arg Met Glu Ala Gln Gly Glu Arg Glu Leu Leu Gln Ala Ala Lys
1340 1345 1350
Glu Asn Leu Thr Ala Gln Val Glu His Leu Gln Ala Ala Val Val
1355 1360 1365
Glu Ala Arg Ala Gln Ala Ser Ala Ala Gly Ile Leu Glu Glu Asp
1370 1375 1380
Leu Arg Thr Ala Arg Ser Ala Leu Lys Leu Lys Asn Glu Glu Val
1385 1390 1395
Glu Ser Glu Arg Glu Arg Ala Gln Ala Leu Gln Glu Gln Gly Glu
1400 1405 1410
Leu Lys Val Ala Gln Gly Lys Ala Leu Gln Glu Asn Leu Ala Leu
1415 1920 1425
10/21
CA 02335656 2001-O1-22
WO 00/06730 PCTNS99/17167
Leu Thr Gln Thr Leu Ala Glu Arg Glu Glu Glu Val Glu Thr Leu
1430 1435 1440.
Arg Gly Gln Ile Gln Glu Leu Glu Lys Gln Arg Glu Met Gln Lys
1445 1950 1455
Ala Ala Leu Glu Leu Leu Ser Leu Asp Leu Lys Lys Arg Asn Gln
1460 1465 1470
Glu Val Asp Leu Gln Gln Glu Gln Ile Gln Glu Leu Glu Lys Cys
1475 1480 1485
Arg Ser Val Leu Glu His Leu Pro Met Ala Val Gln Glu Arg Glu
1990 1495 1500
Gln Lys Leu Thr Val Gln Arg Glu Gln Ile Arg Glu Leu Glu Lys
1505 1510 1515
Asp Arg Glu Thr Gln Arg Asn Val Leu Glu His Gln Leu Leu Glu
1520 1525 1530
Leu Glu Lys Lys Asp Gln Met Ile Glu Ser Gln Arg Gly Gln Val
1535 1590 1545
Gln Asp Leu Lys Lys Gln Leu Val Thr Leu Glu Cys Leu Ala Leu
1550 1555 1560
Glu Leu Glu Glu Asn His His Lys Met Glu Cys Gln Gln Lys Leu
1565 1570 1575
Ile Lys Glu Leu Glu Gly Gln Arg Glu Thr Gln Arg Val Ala Leu
1580 1585 1590
Thr His Leu Thr Leu Asp Leu Glu Glu Arg Ser Gln Glu Leu Gln
1595 1600 1605
Ala Gln Ser Ser Gln Ile His Asp Leu Glu Ser His Ser Thr Val
1610 1615 1620
Leu Ala Arg Glu Leu Gln Glu Arg Asp Gln Glu Val Lys Ser Gln
1625 1630 1635
Arg Glu Gln Ile Glu Glu Leu Gln Arg Gln Lys Glu His Leu Thr
1640 1645 1650
Gln Asp Leu Glu Arg Arg Asp Gln Glu Leu Met Leu Gln Lys Glu
1655 1660 1665
Arg Ile Gln Val Leu Glu Asp Gln Arg Thr Arg Gln Thr Lys Ile
1670 1675 1680
Leu Glu Glu Asp Leu Glu Gln Ile Lys Leu Ser Leu Arg Glu Arg
1685 1690 1695
Gly Arg Glu Leu Thr Thr Gln Arg Gln Leu Met Gln Glu Arg Ala
1700 1705 1710
Glu Glu Gly Lys Gly Pro Ser Lys Ala Gln Arg Gly Ser Leu Glu
1715 1720 1725
His Met Lys Leu Ile Leu Arg Asp Lys Glu Lys Glu Val Glu Cys
1730 1735 1740
Gln Gln Glu His Ile His Glu Leu Gln Glu Leu Lys Asp Gln Leu
1745 1750 1755
Glu Gln Gln Leu Gln Gly Leu His Arg Lys Val Gly Glu Thr Ser
1760 1765. 1770
Leu Leu Leu Ser Gln Arg Glu Gln Glu Ile Val Val Leu Gln Gln
1775 1780 1785
Gln Leu Gln Glu Ala Arg Glu Gln Gly Glu Leu Lys Glu Gln Ser
1790 1795 1800
Leu Gln Ser Gln Leu Asp Glu Ala Gln Arg Ala Leu Ala Gln Arg
lao5 lslo 1815
Asp Gln Glu Leu Glu Ala Leu Gln Gln Glu Gln Gln Gln Ala Gln
1820 1825 1830
Gly Gln Glu Glu Arg Val Lys Glu Lys Ala Asp Ala Leu Gln Gly
1835 1840 1845
Ala Leu Glu Gln Ala His Met Thr Leu Lys Glu Arg His Gly Glu
1850 1855 1860
Leu Gln Asp His Lys Glu Gln Ala Arg Arg Leu Glu Glu Glu Leu
1865 1870 1875
Ala Val Glu Gly Arg Arg Val Gln Ala Leu Glu Glu Val Leu Gly
1880 1885 1890
Asp Leu Arg Ala Glu Ser Arg Glu Gin Glu Lys Ala Leu Leu Ala
1895 1900 1905
Leu Gln Gln Gln Cys Ala Glu Gln Ala Gln Glu His Glu Val Glu
11/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
1910 1915 1920
Thr Arg Ala Leu Gln Asp Ser Trp Leu Gln Ala Gln Ala Val Leu
1925 1930 1935
Lys Glu Arg Asp Gln Glu Leu Glu Ala Leu Arg Ala Glu Ser Gln
1940 1995 1950
Ser Ser Arg His Gln Glu Glu Ala Ala Arg Ala Arg Ala Glu Ala
1955 1960 1965
Leu Gln Glu Ala Leu Gly Lys Ala His Ala Ala Leu Gln Gly Lys
1970 1975 1980
Glu Gln His Leu Leu Glu Gln Ala Glu Leu Ser Arg Ser Leu Glu
1985 1990 1995
Ala Ser Thr Ala Thr Leu Gln Ala Ser Leu Asp Ala Cys Gln Ala
2000 2005 2010
His Ser Arg Gln Leu Glu Glu Ala Leu Arg Ile Gln Glu Gly Glu
2015 2020 2025
Ile Gln Asp Gln Asp Leu Arg Tyr Gln Glu Asp Val Gln Gln Leu
2030 2035 2040
Gln Gln Ala Leu Ala Gln Arg Asp Glu Glu Leu Arg His Gln Gln
2045 2050 2055
Glu Arg Glu Gln Leu Leu Glu Lys Ser Leu Ala Gln Arg Val Gln
2060 2065 2070
Glu Asn Met Ile Gln Glu Lys Gln Asn Leu Gly Gln Glu Arg Glu
2075 2080 2085
Glu Glu Glu Ile Arg Gly Leu His Gln Ser Val Arg Glu Leu Gln
2090 2095 2100
Leu Thr Leu Ala Gln Lys Glu Gln Glu Ile Leu Glu Leu Arg Glu
2105 2110 2115
Thr Gln Gln Arg Asn Asn Leu Glu Ala Leu Pro His Ser His Lys
2120 2125 2130
Thr Ser Pro Met Glu Glu Gln Ser Leu Lys Leu Asp Ser Leu Glu
2135 2140 2145
Pro Arg Leu Gln Arg Glu Leu Glu Arg Leu Gln Ala Ala Leu Arg
2150 2155 2160
Gln Thr Glu Ala Arg Glu Ile Glu Trp Arg Glu Lys Ala Gln Asp
2165 2170 2175
Leu Ala Leu Ser Leu Ala Gln Thr Lys Ala Ser Val Ser Ser Leu
2180 2185 2190
Gln Glu Val Ala Met Phe Leu Gln Ala Ser Val Leu Glu Arg Asp
2195 2200 2205
Ser Glu Gln Gln Arg Leu Gln Asp Glu Leu Glu Leu Thr Arg Arg
2210 2215 2220
Ala Leu Glu Lys Glu Arg Leu His Ser Pro Gly Ala Thr Ser Thr
2225 2230 2235
Ala Glu Leu Gly Ser Arg Gly Glu Gln Gly Val Gln Leu Gly Glu
2290 2245 2250
Val Ser Gly Val Glu Ala Glu Pro Ser Pro Asp Gly Met Glu Lys
2255 2260 2265
Gln Ser Trp Arg Gln Arg Leu Glu His Leu Gln Gln Ala Val Ala
2270 2275 2280
Arg Leu Glu Ile Asp Arg Ser Arg Leu Gln Arg His Asn Val Gln
2285 2290 2295
Leu Arg Ser Thr Leu Glu Gln Val Glu Arg Glu Arg Arg Lys Leu
2300 2305 2310
Lys Arg Glu Ala Met Arg Ala Ala Gln Ala Gly Ser Leu Glu Ile
2315 2320 2325
Ser Lys Ala Thr Ala Ser Ser Pro Thr Gln Gln Asp Gly Arg Gly
2330 2335 2340
Gln Lys Asn Ser Asp Ala Lys Cys Val Ala Glu Leu Gln Lys Glu
2345 2350 2355
Val Val Leu Leu Gln Ala Gln Leu Thr Leu Glu Arg Lys Gln Lys
2360 2365 2370
Gln Asp Tyr Ile Thr Arg Ser Ala Gln Thr Ser Arg Glu Leu Ala
2375 2380 2385
Gly Leu His His Ser Leu Ser His Ser Leu Leu Ala Val Ala Gln
2390 2395 2400
12/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
Ala Pro Glu Ala Leu Glu Ala Thr Arg Leu
Thr Val Glu Arg Asp
2405 2410 241
Glu Ser Leu Thr Leu Thr Ser Gly Pro Leu
Gln Ser Pro Val Leu
2420 2425 2430
His Pro Ser Pro Thr Gln Ala Ser Arg
Ser Thr Ala
2435 2940
<210> 8
<211> 153
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte Clone No: 2195418
<400> B
Met Ala Arg Asn Thr Leu Ser Ser Arg Phe Arg Arg Val Asp Ile
1 5 10 15
Asp Glu Phe Asp Glu Asn Lys Phe Val Asp Glu Gln Glu Glu Ala
20 25 30
Ala Ala Ala Ala Ala Glu Pro Gly Pro Asp Pro Ser Glu Val Asp
35 90 45
Gly Leu Leu Arg Gln Gly Asp Met Leu Arg Ala Phe His Ala Ala
50 55 60
Leu Arg Asn Sex Pro Val Asn Thr Lys Asn Gln Ala Val Lys Glu
65 70 75
Arg Ala Gln Gly Val Val Leu Lys Val Leu Thr Asn Phe Lys Ser
80 85 90
Ser Glu Ile Glu Gln Ala Val Gln Ser Leu Asp Arg Asn Gly Val
95 100 105
Asp Leu Leu Met Lys Tyr Ile Tyr Lys Gly Phe Glu Lys Pro Thr
110 115 120
Glu Asn Ser Ser Ala Val Leu Leu Gln Trp His Glu Lys Ala Leu
125 130 135
Ala Val Gly Gly Leu Gly Ser Ile Ile Arg Val Leu Thr Ala Arg
140 145 150
Lys Thr Val
<210> 9
<211> 1185
<212> DNA
<2I3> Homo Sapiens
<220>
<221> misc feature
<223> Incyte Clone No: 1274060
<400> 9
cacagccaca gcccctgact gccgcagccc ccacagagcc cgccgcgcac cccacgtccc 60
ccacgccagc gcccagccat ggaggccatc aagaagaaaa tgcagatgct gaagttggac 120
aaggagaatg ccatcgaccg cgcggagcag gcggaggcgg ataagaaagc cgctgaggac 180
aagtgcaagc aggtggagga ggagctgacg cacctccaga agaaactaaa agggacagag 290
gacgagctgg ataaatattc cgaggacctg aaggacgcgc aggagaagct ggagctcacg 300
gagaagaagg cctccgacgc tgaaggtgat gtggccgccc tcaaccgacg catccagctc 360
gttgaggagg agttggacag ggctcaggaa cgactggcca cggccctgca gaagctggag 420
gaggcagaaa aagctgcaga tgagagtgag agaggaatga aggtgataga aaaccgggcc 480
atgaaggatg aggagaagat ggagattcag gagatgcagc tcaaagaggc caagcacatt 590
gcggaagagg ctgaccgcaa atacgaggag gtagctcgta agctggtcat cctggagggt 600
13/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
gagctggaga gggcagagga gcgtgcggag gtgtctgaac taaaatgtgg tgacctggaa 660
gaagaactca agaatgttac taacaatctg aaatctctgg aggctgcatc tgaaaagtat 720
tctgaaaagg aggacaaata tgaagaagaa attaaacttc tgtctgacaa actgaaagag 780
gctgagaccc gtgctgaatt tgcagagaga acggttgcaa aactggaaaa gacaattgat 890
gacctggaag agaaacttgc ccaggccaaa gaagagaacg tgggcttaca tcagacactg 900
gatcagacac taaacgaact taactgtata taagcaaaac agaagagtct tgttccaaca 960
gaaactctgg agctccgtgg gtctttctct tctcttgtaa gaagttcctt ttgttattgc 1020
catcttcgct ttgctggaaa tgtcaagcaa attatgaata catgaccaaa tattttgtat 1080
cggagaagct ttgagcacca tgttaaatct cattccttcc cttttttttt caaaaaaaaa 1140
aagagatagg attgagtgga agggtagggg aggaaaaaaa aaaaa 1185
<210> 10
<211> 1050
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1577078
<400> 10
gtttttccca gacattttcc gggaagatca tactgaaatt ttggtatgag tgtaaattcc 60
ctatggcctg gactcctggg tgggctttga cagggaagat caggttaaca gagggcagga 120
catgggggag gctcccactg gtggctggcc tgatacttct taacatggct gcacctccga 180
cctccccagg ctgaaggtga tgtggccgcc ctcaaccgac gcatccagct cgttgaggag 240
gagttggaca gggctcagga acgactggcc acggccctgc agaagctgga ggaggcagaa 300
aaagctgcag atgagagtga gagaggaatg aaggtgatag aaaaccgggc catgaaggat 360
gaggagaaga tggagattca ggagatgcag ctcaaagagg ccaagcacat tgcggaagag 420
gctgaccgca aatacgagga ggtagctcgt aagctggtca tcctggaggg tgagctggag 480
agggcagagg agcgtgcgga ggtgtctgaa ctaaaatgtg gtgacctgga agaagaactc 540
aagaatgtta ctaacaatct gaaatctctg gaggctgcat ctgaaaagta ttctgaaaag 600
gaggacaaat atgaagaaga aattaaactt ctgtctgaca aactgaaaga ggctgagacc 660
cgtgctgaat ttgcagagag aacggttgca aaactggaaa agacaattga tgacctggaa 720
gagaaacttg cccaggccaa agaagagaac gtgggcttac atcagacact ggatcagaca 780
ctaaacgaac ttaactgtat ataagcaaaa cagaagagtc ttgttccaac agaaactctg 840
gagctccgtg ggtctttctc ttctcttgta agaagttcct tttgttattg ccatcttcgc 900
tttgctggaa atgtcaagca aattatgaat acatgaccaa atattttgta tcggagaagc 960
tttgagcacc atgttaaatc tcattccttc cctttttttt tcaaaaaaaa aaagagatag 1020
gattgagtgg aagggtaggg gaggaaaaaa
1050
<210> 11
<211> 729
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte Clone No: 1426711
<400> 11
cgctcctccg cccgaccgcg cgctcgcccc gccgctcctg ctgcagcccc agggcccctc 60
gccgccgcca ccatggacgc catcaagaag aagatgcaga tgctgaagct cgacaaggag 120
aacgccttgg atcgagctga gcaggcggag gccgacaaga aggcggcgga agacaggagc 180
aagcagctcg aggaggacat cgcggccaag gagaagttgc tgcgggtgtc ggaggacgag 240
cgggaccggg tgctggagga gctgcacaag gcggaggaca gcctcctggc cgccgaagag 300
gccgccgcca aggctgaagc cgacgtagct tctctgaaca gacgcatcca gctggttgag 360
gaagagttgg atcgtgccca ggagcgtctg gcaacagctt tgcagaagct ggaggaagct 420
gagaaggcag cagatgagag tgagagaggc atgaaagtca ttgagagtcg agcccaaaaa 480
gatgaagaaa aaatggaaat tcaggagatc caactgaaag aggcaaagca cattgctgaa 540
gatgccgacc gcaaatatga agaggtggcc cgtaagctgg tcatcattga gagcgacctg 600
gaacgtgcag aggagcgggc tggagaaggc ctggataaag acagaagggc ggcaacacac 660
14/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
caccccagcc cccaccccca ccctctcctt gagtttctgt gaattaaaat atttgcaaat 720
ccaaaaaaa 729
<210> 12
<211> 2068
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte Clone No: 1676756
<400> 12
gcagccagaa ccgagtcagc cataaagcta cgcgctaggc tcttggccct gacgtgaggc 60
gcgcagagat ggcgtaacgg gaatagtttt caacgtctat ttcattccct gcttcagagg 120
acctctttaa tctttgattt tggtccctgt ttctaagaaa agcaactgaa aaggtcgtaa 180
taccgcccct gagaaaaaag gagcagcgct aaataatcga gaaaatgcct cctcttgaaa 290
cggatataga gatggaaaca agatataaga aggattgaga atcatataat acaggagctt 300
aaacacctat gcgcgatgat aaagagggta ctattagagc gcttggaaaa taccaggaag 360
ttgagagagt taacagaagg gcgcacgctg gattggccac aaaatcgaat tactgaagta 420
agtgcaaaac gacaaattgt cacagaatac agagaaaagg ggaaaagaaa ttacgaggag 480
aaaaagagag atctagaggg ccggtccagg agatacaatc tatgcataat aggaatacct 540
gaaactgagg acagagcaag tggagctgaa acaataaagg atctacttga aaaaaatttt 600
ccagaattga agaacgaact agatctacaa atggaaaagg ctcataggat acctttaaag 660
tttaatgaaa agaaagcagc atctagacat atccgggtga cgtttttgaa tttcaaagac 720
gaaacatttt acaagcatcc agtcagagaa agcaggttac ttacaaaggg gcaaaagtca 780
ggctgacctc agatttttct cctgcaattc taaatgccag aagacagtgg aacaatatct 840
agagtgttaa gggaaaataa ttttgagcca agaattatat actctgccaa gttatcattt 900
ctttacaaag gaaactggaa gacattctta gatatacagg ggttaggaaa gtatatcaac 960
caagaacttt ccctgaaaat tttgctgaag gatttactgc agctaacaga gaacctgaat 1020
taaaataaga atagggaggc aattgtatga aagaactgat ggtatgcatt aaaactagtt 1080
aaggagcatt aagtttaaat tgttatgtat agggcattaa aactaaatac aaaaatagaa 1140
acataagctg ggtgtaaaat ttcagtttac gtttaaactg gaagggcata ccacatgttc 1200
aatatattgg acaaatactg ttttattttt agcatgtagt attgtagata caaatacgca 1260
tgttcttaag ctaaacctaa gtaaaagggc tttgtcactg tagatttgac aaataacatt 1320
ctaaatgtca aatctgacaa ttggaaaaca gtacagtttt atttccctta tttttaagta 1380
tagcaggaaa aaaataacaa gtttgtccat gttactactg atactgtaaa gaaacttcca 1490
aatcactggt ttgggagggg gtggatacat cctcattttc tgtacatgat gcatggggaa 1500
aaacagtagt tactgcatca tatagttcca agtaaatcat atagttccaa gtaaatttga 1560
catggaaata agtgttaact acaaaaatag aatatataca ttccaatgtt tggaaaagaa 1620
gtggtaaaaa gaaaaatagc aaaaagacac taaaattagc aaaaagtaga ttgacagtac 1680
agatgaccta taaactggtt aaatagccct attacaattg aaattgggtc aaaaatcaat 1790
ctctaacctg agatacattt gtataaaaca aatgatcaaa gagtttaaag ataatggaaa 1800
ctataaatca tagaaatagc aacatttttc ttaaaatcta aacataagac attttatgta 1860
aaacatacaa tataggatgt aacatttaac aaactatatg gtaaataata tgacattaat 1920
atataaaata tatgttccag atcttgatgt gggtgatagt tttacttatg tacatcgata 1980
tacatctatt taaaaacatg tacatttaaa ttgttgcatt ttatgtattt aaaatttatt 2090
gagtaatcct tctgtatgta ttattaat 2068
<210> 13
<211> 3191
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1843770
<900> 13
aggcaggcaa aaggagtcat ggcttctgat gctagtcatg cgctggaagc tgccctggag 60
caaatggacg ggatcattgc aggcactaaa acaggtgcag atcttagtga tggtacttgt 120
gagcctggac tggcttcccc ggcctcctac atgaacccct tcccggtgct ccatctcatc 180
15/21
aaagctgcag atgagagtga gagaggaatg aaggtgatag aa
CA 02335656 2001-O1-22
WO 00/06730 PC'T/US99/17167
gaggacttga ggctggcctt ggagatgctg gagcttcctc aggagagagc agccctcctg 290
agccagatcc ctggcccaac agctgcctac ataaaggaat ggtttgaaga gagc.ttgtcc 300
caggtaaacc accacagtgc tgctagtaat gaaacctacc aggaacgctt ggcacgtcta 360
gaaggggata aggagtccct catattgcag gtgagtgtcc tcacagacca agtagaagcc 420
cagggagaaa agattcgaga cctggaagtg tgtctggaag gacaccaggt gaaactcaat 480
gctgctgaag agatgcttca acaggagctg ctaagccgca catctcttga gacccagaag 540
ctcgatctga tgactgaagt gtctgagctg aagctcaagc tggttggcat ggagaaggag 600
cagagagagc aggaggagaa gcagagaaaa gcagaggagt tactgcaaga gctcaggcac 660
ctcaaaatca aagtggaaga gttggaaaat gaaaggaatc agtatgaatg gaagctaaag 720
gccactaagg ctgaagtcgc ccagctgcaa gaacaggtgg ccctgaaaga tgcagaaatt 780
gagcgtctgc acagccagct ctcccggaca gcagctctcc acagtgagag tcacacagag 840
agagaccaag aaattcaacg tctgaaaatg gggatggaaa ctttgctgct tgccaatgaa 900
gataaggacc gtcggataga ggagcttacg gggctgttaa accagtaccg gaaggtaaag 960
gagattgtga tggtcactca agggccttcg gagagaactc tctcaatcaa tgaagaagaa 1020
ccggagggag gtttcagcaa gtggaacgct acaaataagg accctgaaga attatttaaa 1080
caagagatgc ctccaagatg tagctctcct acagtggggc cacctccatt gccacagaaa 1140
tcactggaaa ccagggctca gaaaaagctc tcttgtagtc tagaagactt gagaagtgaa 1200
tctgtggata agtgtatgga tgggaaccag cccttcccgg tgttagaacc caaggacagc 1260
cctttcttgg cggagcacaa atatcccact ttacctggga agctttcagg agccacgccc 1320
aatggagagg ctgccaaatc tcctcccacc atctgccagc ctgacgccac ggggagcagc 1380
ctgctgaggc tgagagacac agaaagtggc tgggatgaca ctgctgtggt caatgacctc 1940
tcatccacat catcgggcac tgaatcaggt cctcagtctc ctctgacacc agatggtaaa 1500
cggaatccca aaggcattaa gaagttctgg ggaaaaatcc gaagaactca gtcaggaaat 1560
ttctacactg acacgctggg gatggcagag tttcgacgag gtgggctccg ggcaaccgca 1620
gggccaagac tctctaggac cagggactcc aagggacaga aaagtgacgc caatgccccc 1680
tttgcccagt ggagcacaga gcgtgtgtgt gcatggctgg aggactttgg cctggctcag 1740
tatgtgatct ttgccaggca gtgggtatct tctggccaca ccttattgac agccacccct 1800
caggacatgg aaaaggagct aggaattaag cacccactcc acaggaagaa gcttgtttta 1860
gcagtgaaag ccatcaacac caaacaggag gagaagtctg cactgctaga ccacatttgg 1920
gtgacaaggt ggcttgatga tattggctta ccccagtaca aagaccagtt tcatgaatct 1980
agagttgacg gacgaatgct gcaataccta actgtgaacg atttactctt cttaaaagtc 2040
accagccaac tacatcatct cagcatcaaa tgtgccattc acgtgctgca tgtcaacaag 2100
ttcaaccccc actgcctgca ccggcggcca gctgatgaga gtaacctttc tccttcagaa 2160
gttgtacagt ggtccaacca cagggtgatg gagtggttac gatctgtgga cctggcagag 2220
tatgcaccca atcttcgagg gagtggagtc catggaggcc tcattatcct ggagccacgc 2280
ttcactgggg acaccctggc tatgcttctc aacatccccc cacaaaagac gctcctcagg 2340
cgccacctga ccaccaagtt caatgccttg attggtccgg aggctgaaca ggaaaagcga 2400
gagaaaatgg cctcaccagc ttacacacca ctgaccacca cagccaaagt ccggccaagg 2460
aaactaggat tttcacactt cggaaacata agaaaaaaga agttcgatga atcgacggac 2520
tacatttgcc caatggagcc cagtgacggt gtcagtgata gtcacagggt ctacagtggc 2580
taccggggcc tcagccccct tgatgcccct gaactggatg ggctggacca ggtgggacag 2640
attagctgat gcccttgtca cctgccctct gtgcaccctg agagctcaca gtaacactgt 2700
gtgtgtcacc atataactgc acctcacccc cgcacgtgtg catgactcgc agagaatatt 2760
ccagcaattg tgtacccctg ggccagtctc tttgaaccct gagggtggcc aggatctgga 2820
gctgcatctc taaggggcca ggctttgggg accattgcca aaggtggact caggaggaaa 2880
gacacttaaa gacactttta catgtctagt aattcttgat gttcatcttc agcaccagtg 2990
gaaacacatg aacttcgatg caggtccaga gaccatggac actcccacga ggctcagctc 3000
tcaggcaccc cctacacttc agttgaggga aaagctcaag tgccttaggc ccgtggacca 3060
cagtcttggc tgagatcaaa gggatgagca acagggactt ctgccacagt gacaatggaa 3120
ttgtgttgtg ccttacttca gaggtggtct cttctttctt gtaataaaag caatatttat 3180
gcggaaagca t 3191
<210> 14
<211> 3164
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte Clone No: 3768043
<900> 14
aggtttaatc catgaagaag acagcaattt taaaagtgta ttcaccaaaa aataaagctt 60
caaaatatgt gatgtgaaaa ctgccagaac taaggcgggc cgggctcaga ccagcgctgc 120
16/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
ctcaggatgt aaagtgtaac aagagggcca ggggaggtgg tgggggacaa catgggcctg 180
tgaggcctgt gggtgcccgc gttccccagc tccccccgca gcccgctcca cagtggtccg 240
ctccggttgg ttgtcacgtg cgcattcggg ttccagaccc aaggctgcgt gttctccacc 300
gcttgttgtg gccagtgtta ctgcggtgac cgccagagca gcctcgacgc tatggaggag 360
cctggtgcta cccctcagcc ctacctgggg ctggtcctgg aggagctacg cagagttgtg 920
gcagcactac ctgagagtat gagaccagat gagaatcctt atggttttcc atcggaactg 980
gtggtatgtg cagctgttat tggatttttt gttgttctcc tttttttgtg gagaagtttt 540
agatcggtta ggagtcggct ttacgtggga agagagcaaa aacttggtgc aacgctttct 600
ggactaattg aagaaaaatg taaactactt gaaaaattta gccttattca aaaagagtat 660
gaaggctatg aagtagagtc atctttagag gatgccagct ttgagaagga ggcagcagaa 720
gaagcacgaa gtttggaggc aacctgtgaa aagctgaaca ggtccaattc tgaacttgag 780
gatgaaatcc tctgtctaga aaaagactta aaacaagaga aatctaaaca ttctcaacaa 840
gatgaattga tggcggatat ttcaaaaagt atacagtctc tagaagatga gtcaaaatcc 900
ctcaaatcac aaatagctga agccaaaatc atctgcaaga catttaaaat gagtgaagaa 960
cgacgggcta tagcaataaa agatgctttg aatgaaaatt ctcaacttca gacaagccat 1020
aaacagcttt ttcagcaaga agctgaagta tggaaaggag aagtgagtga acttaataaa 1080
cagaaaataa catttgaaga ctccaaagta cacgcagaac aagttctgaa tgataaagaa 1140
aatcacatca agaccctgac tggacacttg ccaatgatga aagatcaggc tgctgtgctt 1200
gaagaagaca caacggatga tgataacctg gaattagaag tgaacagtga atcggaaaat 1260
ggtgcttact tagataatcc tccaaaagga gctttgaaga aactgattca tgctgctaag 1320
ttaaatgctt ctttaaaaac cttagaagga gaaagaaacc aaatttatat tcagttgtct 1380
gaagttgata aaacaaagga agagcttaca gagcatatta aaaatcttca gactcaacaa 1440
gcatctttgc agtcagaaaa cacacatttt gaaaatgaga atcagaagct tcaacagaaa 1500
cttaaagtaa tgactgaatt atatcaagaa aatgaaatga aactccacag gaaattaaca 1560
gtagaggaaa attatcggtt agagaaagaa gagaaacttt ctaaagtaga tgaaaagatc 1620
agccatgcca ctgaagagct ggagacctat agaaagcgag ccaaagatct tgaagaagaa 1680
ttggagagaa ctattcattc ttatcaaggg cagattattt cccatgagaa aaaagcacat 1740
gataattggt tggcagctcg gaatgctgaa agaaacctca atgatttaag gaaagaaaat 1800
gctcacaaca gacaaaaatt aactgaaaca gagcttaaat ttgaactttt agaaaaagat 1860
ccttatgcac tcgatgttcc aaatacagca tttggcagag agcattcccc atatggtccc 1920
tcaccattgg gttggccttc atctgaaaca agagcttttc tctctcctcc aactttgttg 1980
gagggtccac tcagactctc acctttgctt ccagggggag gaggaagagg ctcacgaggc 2040
ccagggaatc ctctggacca tcagattacc aatgaaagag gagaatcaag ctgtgatagg 2100
ttaaccgatc ctcatagggc tccctctgac actgggtctc tgtcacctcc atgggaccag 2160
gaccgtagga tgatgtttcc tccgccagga caatcatatc ctgattcagc ccttcctcca 2220
caaaggcaag acagattttg ttctaattct ggtagactgt ctggaccagc agaactcaga 2280
agttttaata tgccttcttt ggataaaatg gatgggtcaa tgccttcaga aatggaatcc 2340
agtagaaatg ataccaaaga tgatcttggt aatttaaatg tgcctgattc atctctccct 2400
gctgaaaatg aagccactgg ccctggcttt gttcctccac ctcttgctcc aatcagaggt 2460
ccattgtttc cagtggatgc aagaggccca ttcttgagaa gaggacctcc tttcccccca 2520
cctcctccag gagccatgtt tggagcttct cgagattatt ttccaccaag ggatttccca 2580
ggtccaccac ctgctccatt tgcaatgaga aatgtctatc caccgagggg ttttcctcct 2640
taccttcccc caagacctgg atttttcccc ccacccccac attctgaagg tagaagtgag 2700
ttcccctcag gtttgattcc accttcaaat gagcctgcta ctgaacatcc agaaccacag 2760
caagaaacct gacaatattt ttgctctctt caaaagtaat tttgactgat ctcattttca 2820
gtttaagtaa ctgctgttac ttaagtgatt acacttttgc tcaaattgaa gcttaatgga 2880
attataattc tcaggatagt attttgtaaa taaagatgat ttaaatatga atcttatgag 2940
taaattattt caattttatt ttagacggta taactatttc aatttgatta atccactatt 3000
atataaacaa tagtgggagt tttatatatg taatctttca ggtggggagg ctttaaattc 3060
tgaagtctgt gtctttatgc caagaactgt atttactgtg gttgtggaca aatgtgaaag 3120
taactttatg cttaaataaa ttatagttga tttaaaaaaa aaaa 3169
<210> 15
<211> 7962
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte Clone No: 1655208
<400> 15
aaaagtagga aggtagagtt gttggcagaa atcctgggat aagagaatag tttcctggaa 60
gatctgtgcc tccaaccagc agagagggat tgagcttcat tgaactcaac agagccaaca 120
17/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
tttcatagca ccatgttcaa gaggaggttg aagtggcatg gcaatggtta gagaccctgc 180
tgggcgtgaa caccctctgg ctacctaggg acctgtgggc ctaccacctg gtgccctcat 240
ggagacaaga agccctgggt tgaacaacat gaagccccag tcactgcagc tggtactgga 300
agagcaggtg ctggcactac agcagcagat ggcagagaat caggcagcct cctggcggaa 360
gctgaagaac tcccaggagg cccagcagag acaagcaacc cttgtgagga agctgcaggc 420
caaggtgctg cagtaccgaa gctggtgcca agagctggag aagcggctag aagccactgg 480
aggaccaatc ccccagaggt gggaaaatgt ggaggagcca aacctggatg agctgctggt 540
ccgattggag gaggagcaac agaggtgtga gagtctagca gaggtgaaca cccagattcg 600
actgcacatg gaaaaagctg acgtggtgaa taaagccctt agggcagatg tggaaaaact 660
gacagtggac tggagccggg cccgggatga gctaatgagg aaggagagcc agtggcagat 720
ggagcaggag ttcttcaagg gctacctgaa aggggagcac ggtcgccttc tcagtctatg 780
gcgggaggtt gtgacattcc gacgccactt cctggaaatg aagtcagcta ctgacagaga 840
tctgatggag ctaaaagctg agcatgtgag gctttcaggg tctctgttga cctgttgtct 900
gcgcttgact gtgggagcac agtctcggga acccaacgga tctggaagaa tggatgggcg 960
ggagccggcc cagctgctgc tgctactagc caagacccag gagctggaga aggaagccca 1020
tgaaaggagc caggagttaa tacagctgaa gagtcaaggg gatctggaga aggctgaact 1080
tcaggaccgg gtgaccgagc tctctgctct gttgacccag tctcagaagc aaaatgaaga 1140
ttatgaaaag atgataaagg ctctgagaga gacagtggag atcctggaga caaatcacac 1200
agaattaatg gaacatgaag catctcttag taggaatgcg caagaggaga agttgtcttt 1260
acagcaggtg atcaaggata taacccaggt catggtggaa gaaggggaca atatagccca 1320
aggctctggt cttgagaact ctttggaatt ggagtctagt atcttctccc agtttgatta 1380
ccaagatgca gacaaggctc ttactctggt gcgttcagtg ctgactcgga gacgccaggc 1440
tgtgcaggac ctaaggcagc agcttgcagg ctgtcaagag gctgtgaact tgttgcaaca 1500
gcagcatgat cagtgggagg aagagggcaa agccttgaga cagcggctgc agaagctcac 1560
tggggagcgg gacactctgg cagggcagac tgtggacctc cagggagagg tggactctct 1620
cagcaaggag cgagagctgc tgcagaaggc cagggaagag ctgcggcagc agctggaggt 1680
gctagagcag gaggcatggc gcctgcgaag ggtaaatgtg gagcttcagc tgcaggggga 1740
ctctgcccag ggccagaagg aggaacagca ggaggagctg cacctggctg tccgggagag 1800
ggagcgtctt caggagatgc tgatgggcct ggaagccaaa cagtcagaat cactcagtga 1860
actgatcact cttcgggaag ccctggagtc aattcacctg gaaggggagt tactgaggca 1920
agagcaaacg gaagtgaccg cagcgctggc tagggcagag cagtcaattg cagagctgtc'1980
gagttctgaa aacaccctga agacagaagt agctgatctt cgggctgcag ctgtcaagct 2040
cagtgcctta aatgaggctt tggcgttaga taaagttggg ctgaaccagc agcttctcca 2100
gttagaggag gagaaccagt ctgtgtgcag cagaatggag gccgcagagc aggcgagaaa 2160
tgctttgcag gtcgacctgg cggaggcaga gaagaggagg gaagccctgt gggaaaagaa 2220
cactcacctg gaggctcagc tgcagaaagc tgaggaggct ggggctgagc tgcaggcaga 2280
tctcagggac atccaagaag agaaggaaga aattcaaaag aaactaagtg agtcacgtca 2340
ccagcaggag gcagccacga ctcagctgga gcagctacat caggaggcaa agcgacagga 2900
agaagtgctt gccagggcag tccaggagaa ggaggcccta gtacgagaga aagcggctct 2460
agaggtgcgg ctgcaggccg tggagcgtga ccggcaggac ctcgctgcac aactacaggg 2520
gctcagctca gccaaggagc tactggagag cagtctgttt gaagcccaac aacaaaattc 2580
tgtgatagac gagccgcagg ggcagctgga ggtccagatt caaactgtca ctcaagccaa 2690
ggaagtaatc caaggggaag tgaggtgcct gaagctggaa ctggacactg aacggagtca 2700
ggcagagcag gagcgggatg ctgcagccag acagctggcc caggctgagc aagaagggaa 2760
gactgccttg gagcagcaga aggcagccca tgagaaagag gtgaaccagc tccgggagaa 2820
atgggagaag gagcgctcct ggcaccagca ggagctggca aaggctctgg agagcttaga 2880
aagggaaaaa atggagctgg aaatgaggct aaaggagcag cagacagaaa tggaggccat 2940
ccaggcccag agggaagaag aacggaccca ggcagagagt gccctatgcc agatgcagct 3000
ggaaacagag aaggagagag tatccctcct ggagacactg ctgcagacgc agaaggagct 3060
agcagatgcc agccaacaac tggaacgact gaggcaggac atgaaagtcc agaaattaaa 3120
ggagcaggag accactggga tactacagac ccagctccag gaggctcaac gggagctgaa 3180
ggaggcagcc cggcagcaca gagatgacct tgctgccctc caagaagaga gcagctccct 3240
gctgcaggat aagatggacc tgcagaagca ggtggaggac ttgaagtctc agctggtggc 3300
ccaggatgac tcccagaggc tggtggagca ggaggttcag gagaagctga gagagaccca 3360
ggagtataac cgaattcaga aggagctgga gagagagaaa gccagcctga ctctgtcact 3420
gatggaaaag gaacagagac tccttgtttt acaagaagct gactctattc gacaacaaga 3980
gctgagtgcc ctgcgccagg acatgcagga ggcccaggga gaacagaaag agctcagtgc 3590
tcagatggaa ttactaaggc aagaggtgaa ggaaaaggag gctgactttc tggcccagga 3600
agcacagctg ctggaggagc tggaggcgtc tcatatcacg gagcagcagc tgcgagcctc 3660
cttgtgggcc caggaagcca aggcagccca actacacctg cgactgcgca gcacagagag 3720
ccagctagaa gcgctggccg cagagcagca gcccgggaac caggcccagg cccaggccca 3780
gctggccagc ctctactctg ccctgcagca ggccctgggg tctgtttgtg agagcaggcc 3890
tgagctgagt ggtgggggag actctgctcc ttccgtctgg ggccttgagc cagaccagaa 3900
tggagctagg agcctcttta agagagggcc cctgctgact gctctctccg ctgaggcagt 3960
agcatctgcc ctcctcaagc ttcatcaaga cctgtggaag actcaacaga cccgggatgt 4020
18/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
tctgagggat caggtccaga aactggaaga gcgtctaact gatactgagg ctgagaagag 4080
ccaggtccac acagagttgc aggatctgca gagacagctc tcccagaatc aggaagagaa 4140
atccaagtgg gaaggaaagc agaactccct agaatctgag ctgatggaac tacatgaaac 4200
tatggcatcc ttacagagtc gcctgcggag agcagagcta cagcgaatgg aagcccaggg 9260
tgagcgagag ttacttcagg cagccaagga gaacctgaca gcccaggtgg aacacctgca 9320
agcagctgtc gtagaagcca gggctcaggc aagtgctgct ggcatcctgg aagaagacct 4380
gagaacggct cgctcagcac tgaagctgaa aaatgaggaa gtagagagtg agcgtgagag 4440
agcccaggct ctgcaagagc agggcgaact gaaggtggcc caagggaagg ctctgcaaga 9500
gaatttggcc ctcctgaccc agaccctagc tgaaagagaa gaggaggtgg agactctgcg 4560
gggacaaatc caggaactgg agaagcaacg ggaaatgcag aaggctgctt tggaattgct 9620
gtctctggac ctgaagaaga ggaaccaaga ggtagatctg cagcaagaac agattcagga 4680
gctagagaag tgtaggtctg ttttagagca tctgcccatg gccgtccagg agcgagagca 9740
gaagctgact gtgcagaggg agcagatcag agagctcgag aaggatcggg agactcagag 4800
gaacgtcttg gagcatcagc ttctagaact tgagaagaaa gaccaaatga ttgagtccca 9860
gagaggacag gttcaggacc tgaaaaagca gttggttact ctggaatgcc tggccctgga 9920
actggaggaa aaccatcaca agatggagtg ccagcaaaaa ctgatcaagg agctggaggg 4980
ccagagggaa acccagagag tggctttgac ccaccttacg ctggacctag aagaaaggag 5090
ccaggagctg caggcacaaa gcagccagat ccatgacctg gagagccaca gcaccgttct 5100
ggcaagagag ctgcaggaga gggaccagga ggtgaagtct cagcgagaac agatcgagga 5160
gctgcagagg cagaaagagc atctgactca ggatctcgag aggagagacc aggagctgat 5220
gctgcagaag gagaggattc aggttctcga ggatcagagg acccggcaga ccaagatcct 5280
ggaggaggac ctggaacaga tcaagctgtc cttgagagag cgaggccggg agctgaccac 5390
tcagaggcag ctgatgcagg aacgggcaga ggaagggaag ggcccaagta aagcacagcg 5900
cgggagccta gagcacatga agctgatcct gcgtgataag gagaaggagg tggaatgtca 5960
gcaggagcat atccatgaac tccaggagct caaagaccag ctggagcagc agctccaggg 5520
cctgcacagg aaggtaggtg agaccagcct cctcctgtcc cagcgagagc aggaaatagt 5580
ggtcctgcag cagcaactgc aggaagccag ggaacaaggg gagctgaagg agcagtcact 5640
tcagagtcaa ctggatgagg cccagagagc cctagcccag agggaccagg aactggaggc 5700
tctgcagcaa gaacagcagc aggcccaggg acaggaggag agggtgaagg aaaaggcaga 5760
cgccctccag ggagctctgg agcaagccca tatgacactg aaggagcgtc atggagagct 5820
tcaggaccac aaggaacagg cacgaaggct ggaggaagag ctggcagtgg agggacggcg 5880
ggtccaggcc ctggaggagg tgctgggaga cctaagggct gagtctcggg aacaggagaa 5990
agctctgttg gccctccagc agcagtgtgc tgagcaggca caggagcatg aggtggagac 6000
cagggccctg caggacagct ggctgcaggc ccaggcagtg ctcaaggaac gggaccagga 6060
gctggaagct ctgcgggcag aaagtcagtc ctcccggcat caggaggagg ctgcccgggc 6120
ccgggctgag gctctgcagg aggcccttgg caaggctcat gctgccctgc aggggaaaga 6180
gcagcatctc ctcgagcagg cagaattgag ccgcagtctg gaggccagca ctgcaaccct 6240
gcaagcctcc ctggatgcct gccaggcaca cagtcggcag ctggaggagg ctctgaggat 6300
acaagaaggt gagatccagg accaggatct ccgataccag gaggatgtgc agcagctgca 6360
gcaggcactt gcccagaggg atgaagagct gagacatcag caggaacggg agcagctgct 6420
ggagaagtct ctggcccaga gggtccaaga gaatatgatc caagagaagc agaatctggg 6980
gcaagagaga gaagaggagg agataagggg ccttcatcag agtgtaaggg agctacagct 6540
gactctagcc caaaaggaac aggagattct ggagctgagg gagacccagc aaaggaacaa 6600
cctggaagcc ttaccccaca gccacaaaac ctccccaatg gaggaacaat ctctaaaact 6660
tgattcttta gagcccaggc tgcagcggga gctggagcgg ctacaggcag ccctgagaca 6720
gacagaagcc agggagattg agtggaggga gaaggcccag gacttggcac tctccctagc 6780
gcagaccaag gccagtgtca gcagtctgca ggaggtagcc atgttcctac aagcctctgt 6840
cctggagcgg gactcagaac agcaaaggct gcaggatgaa ctggagctca ccagacgggc 6900
tctggagaag gagcggctac acagcccagg tgcaaccagc acagcagaac tggggtccag 6960
aggggagcag ggtgtgcagc tgggagaggt ctcaggagtg gaggctgagc ctagtcctga 7020
tggaatggag aagcagtcat ggagacaaag gcttgaacac ctgcagcaag cagtggcccg 7080
gctggagatt gacaggagca ggctgcagcg ccacaatgtc cagctgcgga gtaccttgga ?190
gcaggtggag cgagaacgga ggaagctgaa gagggaggcc atgcgtgcgg cccaggcagg 7200
gtccctagag atcagcaagg ccacggcttc ttcacccaca cagcaggatg ggagaggaca 7260
gaagaactca gatgccaagt gtgtggctga actgcagaaa gaggtggtcc tgctgcaagc 7320
tcagctgact ttggagcgga agcagaagca ggactacatc acccgctcag cacagaccag 7380
ccgtgagcta gcaggcctgc accacagcct.ctcacactca cttcttgccg tggcccaggc 7440
ccctgaggcc actgtcctgg aggcagagac ccgcaggctg gatgagtccc tgactcaaag 7500
tctgacatcc ccagggccag tcctgctaca ccccagcccc agcactaccc aagccgcctc 7560
caggtagcag ccacagccag gagcacacag acagaagact gtgtcatggg tcatggcccc 7620
tccgcacacc tacaggtttg ccaaaggaaa agcctggctc tgttaggcac ccaggagccc 7680
caggtcggcg ggtgttccca ggaagaggaa gtaaatctgc aaccctgggg aggaccccaa 7740
ctcacctggg aatgaggcaa attgcatttg cttgctccct atggaatcac ccagaggggt 7800
gccttgccct ggctgaggga catgtactgc ctctcatcta gaatttattt tcctagcact 7860
tcaccacctc cttcatcttc tccttcaaca ataaaccctg accaaatgat tcaaaaaaaa 7920
19/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
tataaaaaag accagtccaa gcttattccc tttagtgagg tt 7962
<210> 16
<211> 1373
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte Clone No: 2195418
<900> 16
cagccgggca gccgcttccc gcccccgagc aggagccggt gcgagcggag cagagccgag 60
gtcgggccgc gagcggagcc ggctgagcgg gcgccgagct cccgccatgg cccggaacac 120
gctgtcctcg cgcttccgcc gggtggacat cgacgaattt gacgagaaca aatttgtgga 180
cgagcaggag gaggcggcgg cggcggcggc ggagccaggc ccggacccga gcgaggtgga 240
cgggctcctg cggcaagggg acatgcttcg ggcattccat gcagccttgc ggaactctcc 300
cgtcaacacc aagaatcaag ctgtgaagga gcgagcccag ggcgtggtgc tgaaagtgct 360
cacaaacttc aagagcagtg agattgagca ggctgtgcag tcactggaca gaaacggcgt 420
tgacttgtta atgaagtaca tttataaagg ctttgagaag cccacagaaa atagcagcgc 480
agtgttactc cagtggcacg aaaaggcctt agcagtagga ggactaggct ccattataag 540
agttcttaca gcaagaaaga ctgtttaaaa aaaataaaaa gactcatgtt accttgagaa 600
gaattctgga tgcccaggct ggtgaagaag ggattgacaa tggaccatct tcctaggaac 660
tcccaagtaa actatttcag gacatgtatc tgctgaaatg tattttattt tcaaggtgga 720
ggggaaaatc gtctgtttcc taaatcctgt ttaggatctg atagtctatg cctttgtctc 780
cgagtactgc agaactgaca ttttgacggt ctaccagcgt ggcggctggt gttggtcaga 890
tgcacctgtg tgcactgggg gagggatggt ttgggcaggt gcagatccaa gggctgtggt 900
aaacgggaga gcttgtgttt ttgaagtgga aaaaaaccca agagtttgta cagacatcct 960
gtcttcccag agaaggtgga cactcttggg ctcattgtaa agtgcctgct gcatcaataa 1020
agctcttggc ttattagtct atacattgcg gtgtgtttcg tgtatgtaaa aaaaaatggt 1080
aatgaatggg atggtaatga atgagagttc agttgttgtt ccggaaaccc gatgtggaag 1140
gagtagacct gtgtccctgt tgagccaccc ctgggagcga gcatggcaat ccacaggccc 1200
tctgccacag gacgccagcc tcggcctcag agctgccggc tgctgcagag aggtgtttgc 1260
tgaataaact atttattgtt tcttattcct ttgatttgta tgtaattaat tttggagctt 1320
atttaattaa tttaataaag tgccaaacat ttaataatta aaaaaaaaaa aaa 1373
<210> 17
<211> 151
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> GenBank ID No: g2282042
<400> 17
Met Ser Lys Asn Thr Val Ser Ser Ala Arg Phe Arg Lys Val Asp
1 5 10 15
Val Asp Glu Tyr Asp Glu Asn Lys Phe Val Asp Glu Glu Asp Gly
20 25 30
Gly Asp Gly Gln Ala Gly Pro Asp Glu G40y Glu Val Asp Ser Cys
35 45
Leu Arg Gln Gly Asn Met Thr Ala Ala Leu Gln Ala Ala Leu Lys
50 55 60
Asn Pro Pro Ile Asn Thr Lys Ser Gln Ala Val Lys Asp Arg Ala
65 70 75
Gly Ser Ile Val Leu Lys Val Leu Ile Ser Phe Lys Ala Asn Asp
80 85 90
Ile Glu Lys Ala Val Gln Ser Leu Asp Lys Asn Gly Val Asp Leu
95 100 105
Leu Met Lys Tyr Ile Tyr Lys Gly Phe Glu Ser Pro Ser Asp Asn
20/21
CA 02335656 2001-O1-22
WO 00/06730 PCT/US99/17167
110 115 120
Ser Ser Ala Met Leu Leu Gln Trp His Glu Lys Ala Leu Ala Ala
125 130 135
Gly Gly Val Gly Ser Ile Val Arg Val Leu Thr Ala Arg Lys Thr
140 195 150
Val
21/21
