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MEMBRANE-ASSOCIATED ORGANIZATIONAL PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human
membrane-associated
organizational proteins and to the use of these sequences in the diagnosis,
treatment, and prevention of
cell proliferative disorders, including cancer, and autoimmune/intlammatory,
neurological,
developmental, vesicle trafficking, reproductive, gastrointestinal, and renal
disorders.
BACKGROUND OF THE INVENTION
Cells are surrounded by plasma membranes which enclose the cell and maintain
an environment
inside the cell that is distinct from its surroundings. Eukaryotic organisms
are distinct from prokaryotes
in that they possess many intracellular organelle and vesicle structures
enclosed by membranes.
Membrane-associated organizational proteins are responsible for the
aggregation and assembly of
signaling and transport proteins at specialized regions of cellular,
organelle, and vesicular membranes.
For example, in postsynaptic signaling, membrane-associated organizational
proteins are responsible for
ion channel and receptor clustering. In Golgi-mediated transport and
secretion, membrane-associated
organizational proteins control cisternal stacking and vesicle docking.
Membrane-associated
organizational proteins also play a role in the formation of cell junctions,
regions of contact between
adjacent cells and between cells and the extracellular matrix. Cell junctions
influence cell shape, strength,
flexibility, motility, and adhesion.
PDZ Domains
A conserved protein domain called PDZ has been identified in various proteins
which act at the
cytosolic face of the plasma membrane. PDZ-containing proteins coordinate the
assembly of
multifunctional protein complexes involved in intercellular signaling events.
PDZ domains are
protein/protein interaction motifs involved, for example, in the localization
of channels, receptors,
signaling enzymes, and adhesion molecules to sites of cell-cell contact. PDZ
domains were named for
three proteins in which this domain was initially discovered: PSD-95
(postsynaptic density 95), Dlg
Droso hila lethal{ 1 )discs large-1 ), and ZO-1 (zonula occludens-1 ). These
proteins play important roles
in neuronal synaptic transmission, tumor suppression, and cell junction
formation, respectively. Since the
discovery of these proteins, over sixty additional PDZ-containing proteins
have been identified in diverse
prokaryotic and eukaryotic organisms. A large proportion of PDZ domains are
found in the eukaryotic
MAGUK (membrane-associated guanylate kinase) protein family, members of which
bind to the
intracellular domains of neuronal receptors and channels. However, PDZ domains
are also found in
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diverse membrane-localized proteins such as protein tyrosine phosphatases,
serine/threonine kinases, G-
protein cofactors, and synapse-associated proteins such as syntrophins and
neuronal nitric oxide synthase
(nNOS). Generally, about one to three PDZ domains are found in a given
protein, although up to nine
have been identified in a single protein. (See, e.g., Ponting, C.P. et al.
(1997) Bioessays 19:469-479;
Fanning, A.S. and J.M. Anderson (1999) J. Clin. Invest. 103:767-772.)
X-ray crystallography has shown that PDZ domains are generally compact
globular structures
containing about $0 to 100 amino acids which form six ~i-strands and two a-
helices. PDZ domains tend
to be rich in glycine residues which introduce turns in the poiypeptide chain
and promote compaction and
stability of the folded polypeptide. A GLGF (glycine-leucine-giycine-
phenylalanine) sequence motif is
conserved within some PDZ domains. This GLGF sequence is usually preceded by
an arginine found
about six residues upstream of GLGF. PDZ domains bind to a tripeptide motif
containing valine and
serine or threonine. Most tigands which bind PDZ domains contain this motif,
although some ligands
lack this motif or contain conservative substitutions therein.
PDZ-containing proteins are likely involved in disorders associated with
defective cell signaling
(Ponting, supra). For example, PDZ domains have been shown to play important
roles in development,
and the gene encoding the PDZ-containing protein LIM kinase 1 is deleted in
patients with Williams
syndrome, a complex developmental disorder.
PDZ-Mediated Neuronal Si naline
Cells communicate with and respond to their environment by receiving and
processing
extracellular signals. These signals take the form of growth factors,
hormones, cytokines, and peptides
which bind to activate specific plasma membrane receptors. The activated
receptors trigger intracellular
signal transduction pathways which culminate in a wide range of cellular
responses affecting gene
expression, protein secretion, cell cycle progression, and cell
differentiation. Initial events in signal
transduction require the proximity of intracellular signaling proteins to the
cytosolic domains of activated
plasma membrane receptors. These intracellular membrane-associated signaling
proteins couple the
activated receptor to downstream second messenger systems and play a key role
in the regulation and
coordination of complex, multiprotein signal transduction pathways.
PDZ proteins play an important role in the clustering of ion channels and
neurotransmitter
receptors at postsynaptic membranes. This organizational activity is essential
for neuronal development
and synaptic plasticity (Ponting, supra). Mutations that block clustering of
neuronal receptors and
channels cause perinatal lethality in mice. MAGUK proteins, in particular, are
important for clustering
neuronal receptors and ion channels responsive to glutamate, the predominant
excitatory neurotransmitter
in the mammalian hippocampus. Specifically, the PDZ domains of PSD-95, PSD-93,
SAP-97 (synapse-
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associated protein 97), SAP-102, and chapsyn 110 bind to the cytosolic C-
termini of N-methyl-D-
aspartate (NMDA) glutamate receptors and Shaker-type potassium channels,
causing them to cluster.
A novel synaptic PDZ protein, Homer, has recently been identified in rat
brain. Although Homer
may perform similar functions as MAGUK proteins, it is highly divergent from
MAGUK proteins and
may represent a novel and distinct PDZ protein family. Homer mRNA is 6.5 kb
long and encodes a
protein of 186 amino acids. Homer contains a single PDZ-like domain and binds
to the carboxy terminus
of phosphoinositide-linked metabotropic glutamate receptors. The PDZ-like
domain contains a GLGF
sequence and preceding arginine, as seen in the PDZ domains of proteins such
as PSD-95. Otherwise,
there is less than 10% amino acid sequence identity between Homer and the
reported members of the
PDZ family. Deletion constructs revealed that the amino-terminal 108 amino
acids of Homer, which
includes the GLGF sequence, is essential for the binding of Homer to glutamate
receptors. Expression of
Homer mRNA is strongly upregulated in the forebrain by seizure-and drug
induced neuronal activation
(Brakeman, P.R. et al. (1997) Nature 386:284-288).
Detailed immunohistochemical analysis revealed that Homer is enriched at
excitatory synapses.
Additionally, expression of the Homer gene is developmentally regulated by
synaptic activity, with peak
expression in the rat forebrain coinciding with increased synaptic activity
from the third to fifth postnatal
weeks. In the adult, Homer mRl~'A is rapidly induced in the hippocampus of
awakened rats. Homer may
also be linked to the regulation of dopamine receptors since it is rapidly
induced by cocaine in the
striatum (Brakeman, supra).
Taken together, these observations suggest that Homer may play a major role in
neuronal
function and development. Homer is likely to participate in signal
transduction and influence spatial
targeting of receptors. The selective expression of Homer at excitatory
synapses strongly supports a role
in synapse formation and in the regulation of glutamate mediated
neurotransmission.
Cisternal Stackin in Golgi
The Golgi apparatus (Golgi), an organelle composed of stacked disc-shaped
cisternal membranes,
is found adjacent to the nucleus during interphase in animal cells. The Golgi
contains enzymes that
modify secreted and membrane proteins posttranslationally as they traverse the
secretory pathway. Many
of the modifying enzymes function in an ordered sequence, and have unique
distributions within the
Golgi, suggesting that Golgi structure is important for their function.
Secreted and membrane proteins are
transported through the Golgi in specialized vesicles which bud from the donor
membrane and then dock
with and fuse to the target membrane. Cisternal stacking may be viewed as a
specialized form of docking
event, in which one cisterna docks with another without fusion. During
mitosis, the Golgi breaks down
into many small vesicles and tubules that are partitioned to the two daughter
cells. Vesicle fusion is
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inhibited during mitosis, possibly due to an inhibition of vesicle docking. As
the cell exits mitosis, the
Golgi cisternae reform and restack, and vesicle transport resumes.
Vesicles must be tethered to the target membrane prior to docking and fusion.
Giantin, a protein
on the vesicle surface, binds to pl 15, a cytosolic protein that is in turn
bound to its receptor GM130 on
the cytosolic surface of the Golgi. (iM130 is a tightly associated peripheral
membrane protein which has
a coiled-coil, elongated rod-like structure and appears to be a dimer. During
mitosis, GM130 is
phosphorylated by a cyclin-dependent kinase. This phosphorylation inhibits the
binding of pl 15 to
GM130, leading to a block in vesicle tethering and docking, and thus a block
in vesicle fusion.
Golgi ReAssembly Stacking Protein (GRASP65) was identified as a Golgi protein
which may
play roles in the initiation and/or maintenance of cisternal stacking and in
vesicle docking. Antibodies to
GRASP65 prevent cisternal stacking in a cell-free system. The rat GRASP65
gene, which is expressed in
all rat tissues tested, encodes a 65 kDa peripheral membrane protein which
appears to exist as a dimer.
Homologs of GRASP65 exist in the yeasts Saccharomyces cerevisiae and
Schizosaccharomyces nombe.
GRASP65 interacts with GM130 under both mitotic and nonmitotic conditions. The
GRASP65/GM 130 complex can interact with p 115, suggesting that all three
proteins function as a
complex with roles in vesicle docking and cisternal stacking. Like GM 130,
GRASP65 is heavily
phosphorylated during mitosis. GRASP65 is myristoylated at its N-terminus,
which may account for its
association with the Golgi membrane. The protein contains two imperfectly
repeated domains (from F16
through S108 and from Wl 12 through P202 in GI 4432587; SEQ ID NO:10) followed
by a serine-rich C-
terminal domain which may be involved in cell cycle regulation. 'The binding
site for GM 130 has been
mapped to amino acids 6194 through I201 within the second repeated domain. The
first repeated
domain, though similar to the second, does not bind GM130. The binding site
for GRASP65 on GM130
was mapped to the C-terminus of GM130, including the final four hydrophobic
amino acids. The
interaction of GRASP65 with GM 130 resembles the binding of PDZ domain-
containing proteins to their
ligands. The GYGY sequence within the GRASP65 binding site for GM 130 is
similar to the conserved
GLGF sequence of PDZ domains. In addition, PDZ proteins recognize the C-
terminal four amino acids
oftheir ligands. (See, e.g., Warren, Ci. and V. Malhotra (1998) Curr. Opin.
Cell Biol. 10:493-498; Lowe,
M. et al. ( 1998) Trends Cell Biol. 8:40-44; Barr, F.A. et al. ( 1997) Cell
91:253-262; Barr, F.A. et al.
(1998) EMBO J. 17:3258-3268.)
Defects in protein trafficking to organelles or the cell surface are involved
in numerous human
diseases and disorders. Defects in the trafficking of membrane-bound receptors
and ion channels are
associated with cystic fibrosis (cystic fibrosis transmembrane conductance
regulator), glucose-galactose
malabsorption syndrome (Na+/glucose cotransporter), hypercholesterolemia (low-
density lipoprotein
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receptor), and forms of diabetes mellitus (insulin receptor). Abnormal
hormonal secretion is linked to
disorders including diabetes insipidus (vasopressin), hyper- and hypoglycemia
(insulin, glucagon),
Grave's disease and goiter (thyroid hormone), and Cushing's and Addison's
diseases (adrenocorticotropic
hormone (ACTH) ).
Cancer cells secrete excessive amounts of hormones or other biologically
active peptides.
Disorders related to this excessive secretion include: fasting hypoglycemia
due to increased insulin
secretion from insulinoma-islet cell tumors; hypertension due to increased
epinephrine and .
norepinephrine secreted from pheochromocytomas of the adrenal medulla and
sympathetic paraganglia;
and carcinoid syndrome, which includes abdominal cramps, diarrhea, and
valvular heart disease, caused
by excessive amounts of vasoactive substances (serotonin, bradykinin,
histamine, prostaglandins, and
polypeptide hormones) secreted from intestinal tumors. Ectopic synthesis and
secretion of biologically
active peptides occurs including ACTH and vasopressin in lung and pancreatic
cancers, parathyroid
hormone in lung and bladder cancers, calcitonin in lung and breast cancers,
and thyroid-stimulating
hormone in medullary thyroid carcinoma.
Cell Junctions
Cell junctions are regions of contact between adjacent cells and between cells
and the
surrounding extracellular matrix. Cell junctions are comprised of both
intracellular and extracellular
protein complexes associated with the plasma membrane. In addition,
cytoskeletal filaments that traverse
the cytoplasm are anchored to the cell cortex by means of human cell junction
proteins. Cell junctions are
dynamic structures that are responsive to signals such as cytokines and growth
factors and are also
capable of signal transduction. The dynamic properties of cell junctions
influence cell shape, strength,
flexibility, motility, and adhesion. Cell-cell and cell-matrix contacts are
often disrupted in neoplastically
transformed cells, suggesting a mechanism for uncontrolled cell proliferation
and metastasis.
Tight junctions are present around the lateral circumference of epithelial or
endothelial cells.
(Reviewed in Balda, M.S. and K. Matter (1998) J. Cell Sci. 111:541-547;
Lampugnani, M.G. and E.
Dejana (1997) Curr. Opin. Cell Biol. 9:674-682.) Epithelia and endothelia are
monolayers of polarized
cells that separate a body compartment (the basolateral side) from the outside
environment or a
topologically equivalent space (the apical side). The apical and basolateral
domains are polarized,
containing different cell membrane components such as lipids and membrane-
associated proteins. Tight
junctions constitute a continuous, circumferential seal around cells, forming
a barrier to diffusion of
solutes across the cell sheet. Tight junctions also function as a boundary
between apical and basolateral
membrane domains, preventing lateral diffusion of membrane associated
proteins, such as receptors,
between compartments and thus maintaining cell polarity.
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Maintenance of epithelial cell polarity is essential for the proper function
of many epithelial
organs. In the kidney, for example, the functions of reabsorption and
secretion depend upon the polarized
insertion of specialized channels and transporters to apical membranes lining
the renal tubule lumen or
basolateral membranes adjacent to the insterstitium and blood space. Defective
polarization of membrane
proteins can lead to renal cystic diseases. (Wilson, P.D. (1997) Am. J.
Physiol. 272:F434-F442.) The
barrier function of tight junctions is also important, as disruptions to tight
junction permeability are
involved in a wide range of gastrointestinal pathologies. Agents such as
aspirin or ethanol which increase
gastric tight junction permeability initiate and amplify gastric mucosal
injury by allowing back-diffusion
of H+ ions into the mucosa. Abnormal tight junction permeability may also be
the cause of inflammatory
bowel disease such as Crohn's disease. Certain bacterial toxins cause
intestinal epithelial tight junction
abnormalities and contribute to diarrhea by dissipating electrochemical
gradients needed for proper
intestinal absorbtion and secretion. Structural and functional disruptions of
tight junctions are also
observed in inherited cholestatic liver disorders and cholestasis associated
with common bile duct
obstruction of the liver (Balda, M.S. et al. ( 1992) Yale J. Biol. Med. 65:725-
735).
The protein components of tight junctions include ZO-1 and ZO-2 (zona
occludens), cytoplasmic
proteins associated with the plasma membrane at tight junctions. ZO-l is a PDZ
domain-containing
protein which associates with spectrin and thus may link tight junctions to
the actin cytoskeleton. Other
cytoplasmic components oftight junctions include cingulin, 7H6 antigen,
symplekin, and small rab family
GTPases. 'The first identified component of the tight junction strands, which
form the actual junction
between cells, was the integral membrane protein occludin, a 65 kD protein
with four transmembrane
domains. ZO-1 binds to the carboxy-terminal region of occludin and may
localize occludin to the tight
junction. A recently identified family of proteins, the claudins, are also
components of tight junction
strands.
Claudins are 22-25 kD proteins which also contain four conserved transmembrane
domains, but
have no sequence homology to occludin (Furuse, M. et al. (1998) J. Cell Biol.
141:1539-1550). At least
eight members of the claudin family have been cloned from mice. Claudin-2, in
particular, is the most
ancestral member of the claudin family. The eight claudins studied so far all
have distinct tissue
distributions and claudin-6, in particular, appears to be developmentally
regulated (Morita, K. et al.
(1999) Proc. Natl. Acad. Sci. USA 96:511-516). Claudin-2 expression is
primarily restricted to the liver
and kidney, with low levels of expression in the brain (Furuse et al. supra).
Both claudin-1 and claudin-2
localize exclusively to the tight junction by immunofluorescence, and
introduction of cDNA for claudin-1
and claudin-2 into mouse fibroblasts lacking tight junctions could induce
tight junction formation (Furuse,
M. et al. (1998) J. Cell Biol. 143:391-401 ). As occludin expression induces
only a small number of short
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strands, the claudins appear to be the major structural components of tight
junction strands, with occludin
being an accessory protein.
Other classes of transmembrane proteins involved in different types of cell
junction formation
and cell adhesion are the integrins, cadherins, and selectins. Integrins are
transmembrane receptors at
focal adhesions, actin-based cell junctions that occur between cells and the
extracellular matrix. It now
appears that a second class of cell surface molecules modify the type of
adhesion mediated by primary
integrin receptors. In particular, the syndecans, a family of heparan sulfate
proteoglycans, act as co-
receptors in adhesion and modify the downstream organization of the
cytoskeleton. For example, .
although integrin is sufficient for attachment and spreading of primary
fibroblasts, a secondary signal
through interactions of matrix molecules with syndecans is needed for later
stages of focal adhesion and
stress fiber formation. The glycosaminoglycan chains of syndecans interact
with the heparan binding
domains of matrix proteins such as fibronectin, laminin, tenascin, and
collagens, as well as with growth
factors, proteases and protease inhibitors. Syndecans also interact with
components of downstream
signaling pathways, including protein kinase C and the src/cortactin pathway
(Woods, A. et al. (1998)
Matrix Biol. 17:477-483; Rapraeger, A.C. and V.L. Ott (1998) Curr. Opin. Cell
Biol. 10:620-628).
A recently discovered protein, syntenin, interacts with the cytoplasmic
domains of syndecans and
may form the link between syndecans and the cytoskeleton (Grootjans, J.J. et
al. (1997) Proc. Natl. Acad.
Sci. USA 94:13683-13688). Syntenin contains two tandem PDZ domains (from K110
through P193 and
from F 194 through I274 in GI 3342560; SEQ ID N0:12), both of which are
required to bind syndecan.
Syntenin coclusters with syndecans at the plasma membrane, and its
localization is affected by
overexpression of syndecans 1, 2, and 4. Overexpression of syntenin results in
cells that are larger,
flatter, and have many cell surface projections, demonstrating the effect of
syntenin on membrane
dynamics and microfilament organization (lirootjans et al. supra).
The discovery of new human membrane-associated organizational 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 disorders,
including cancer, and
autoimmune/inflammatory, neurological, developmental, vesicle trafficking,
reproductive,
gastrointestinal, and renal disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, human membrane-
associated
organizational proteins, referred to collectively as "HJNCT" and individually
as "HJNCT-1," "HJNCT-
2," "HJNCT-3," and "HJNCT-4." In one aspect, the invention provides a
substantially purified
polypeptide comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1-4,
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and fragments thereof.
The invention further provides a substantially purified variant having at
least 95% amino acid
identity to at least one of the amino acid sequences selected from the group
consisting of SEQ ID NO:1-4,
and fragments thereof. The invention also provides an isolated and purified
polynucleotide encoding the
polypeptide comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1-4,
and fragments thereof. The invention also includes an isolated and purified
polynucleotide variant having
at least 90% polynucleotide sequence identity to the polynucleotide encoding
the polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID NO:1-4,
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: l-4, and fragments
thereof. The invention
also provides an isolated and purified polynucleotide having a sequence which
is complementary to the
polynucleotide encoding the polypeptide comprising the amino acid sequence
selected from the group
consisting of SEQ ID NO: I-4, 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 NO:S-8, and fragments
thereof. The invention
further provides an isolated and purified polynucleotide variant having at
least 90% polynucleotide
sequence identity to the polynucleotide sequence selected from the group
consisting of SEQ ID NO:S-8,
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 NO:S-8, and fragments thereof.
The invention further provides an expression vector containing at least a
fragment of the
polynucleotide encoding the polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ ID NO:I-4, and fragments thereof. 1n another aspect, the
expression vector is
contained within a host cell.
The invention also provides a method for producing a polypeptide, the method
comprising the
steps of: (a) culturing the host cell containing an expression vector
containing at least a fragment of a
polynucleotide under conditions suitable for the expression of the
polypeptide; and (b) recovering the
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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:1-4, 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:1-4, 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 HJNCT, 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: I-4, 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 HJNCT, the method comprising administering
to a subject in need of
I S such treatment an effective amount of an antagonist of a polypeptide
having an amino acid sequence
selected from the group consisting of SEQ ID NO: I-4, and fragments thereof.
BRIEF DESCRIPTION OF THE FIGURES AND TABLES
Figures I A, 1 B, and I C show the amino acid sequence alignment between HJNCT-
1 (Incyte
Clone number 2687924; SEQ ID NO: I ) and rat Homer (GI 1913909; SEQ ID N0:9),
produced using the
multisequence alignment program of LASERGENE software (DNASTAR Inc, Madison
WI).
Figure 2 shows the amino acid sequence alignment between HJNCT-3 (Incyte Clone
number
2594049; SEQ ID N0:3) and mouse claudin-2 (GI 3335184; SEQ ID NO:1 I),
produced using the
multisequence alignment program of LASERGENE software.
Figures 3A and 3B show the amino acid sequence alignment between HJNCT-4
(Incyte Clone
number 5139028; SEQ ID N0:4) and mouse syntenin (GI 3342560; SEQ ID N0:12),
produced using the
multisequence alignment program of I,ASERGENE software.
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs), clone
identification numbers (clone IDs), cDNA libraries, and cDNA fragments used to
assemble full-length
sequences encoding HJNCT.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods and algorithms used for identification of HJNCT.
Table 3 shows useful fragments of each nucleic acid sequence; the tissue-
specific expression
patterns of each nucleic acid sequence as determined by northern analysis;
diseases, disorders, or
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conditions associated with these tissues; and the vector into which each cDNA
was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA clones
encoding HJNCT were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze HJNCT, along
with applicable
descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these may
vary. It is also to be understood that the terminology used herein is for the
purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will be
limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an," and
"the" include plural reference unless the context clearly dictates otherwise.
Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same meanings
as commonly understood by one of ordinary skill in the art to which this
invention belongs. Although any
machines, materials, and methods similar or equivalent to those described
herein can be used to practice
or test the present invention, the preferred machines, materials and methods
are now described. All
publications mentioned herein are cited for the purpose of describing and
disclosing the cell lines,
protocols, reagents and vectors which are reported in the publications and
which might be used in
connection with the invention. Nothing herein is to be construed as an
admission that the invention is not
entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"HJNCT" refers to the amino acid sequences of substantially purified HJNCT
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 HJNCT, increases
or prolongs the
duration of the effect of HJNCT. Agonists may include proteins, nucleic acids,
carbohydrates, or any
other molecules which bind to and modulate the effect of H.fNCT.
An "allelic variant" is an alternative form of the gene encoding HJNCT.
Allelic variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
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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 HJNCT include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polynucleotide the same as HJNCT or a
polypeptide with at least one functional characteristic of HJNCT. Included
within this definition are .
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
i0 the polynucleotide encoding HJNCT, and improper or unexpected hybridization
to allelic variants, with a
locus other than the normal chromosomal locus for the polynucleotide sequence
encoding HJNCT. 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 HJNCT. Deliberate
amino acid substitutions may be made on the basis of similarity in polarity,
charge, solubility,
IS hydrophobicity, hydrophilicity, and/or the amphipathic nature of the
residues, as long as the biological or
immunological activity of HJNCT 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; giycine and alanine; asparagine and glutamine; serine
and threonine; and
20 phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide, polypeptide,
or protein sequence, or a fragment of any of these, and to naturally occurring
or synthetic molecules. In
this context, "fragments," "immunogenic fragments," or "antigenic fragments"
refer to fragments of
I-IJNCT which are preferably at least 5 to about 15 amino acids in length,
most preferably at least 14
25 amino acids, and which retain some biological activity or immunological
activity of HJNCT. 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.
30 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 HJNCT,
decreases the amount
or the duration of the effect of the biological or immunological activity of
HJNCT. Antagonists may
include proteins, nucleic acids, carbohydrates, antibodies, or any other
molecules which decrease the
CA 02343696 2001-03-22
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effect of HJNCT.
The term "antibody" refers to intact molecules as well as to fragments
thereof, such as Fab,
F(ab')2, and Fv fragments, which are capable of binding the epitopic
determinant. Antibodies that bind
HJNCT polypeptides can be prepared using intact polypeptides or using
fragments containing small
peptides of interest as the immunizing antigen. The polypeptide ar
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 (ICL,H). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that fragment of a molecule (i.e.,
an epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to immunize
a host animal, numerous regions of the protein may induce the production of
antibodies which bind
specifically to antigenic determinants (given regions or three-dimensional
structures on the protein). An
antigenic determinant may compete with the intact antigen (i.e., the immunogen
used to elicit the immune
response) for binding to an antibody.
The term "antisense" refers to any composition containing a nucleic acid
sequence which is
complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules may be
produced by any method including synthesis or transcription. Once introduced
into a cell, the
complementary nucleotides combine with natural sequences produced by the cell
to form duplexes and to
block either transcription or translation. The designation "negative" can
refer to the antisense strand, and
the designation "positive" can refer to the sense strand.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" refers to the capability
of the natural, recombinant, or synthetic HJNCT, or of any oligopeptide
thereof, to induce a specific
immune response in appropriate animals or cells and to bind with specific
antibodies.
The terms "complementary" and "complementarity" refer to the natural binding
of
polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds
to the complementary
sequence "3' T-C-A 5'." Complementarity between two single-stranded molecules
may be "partial," such
that only some of the nucleic acids bind, or it may be "complete," such that
total complementarity exists
between the single stranded molecules. The degree of complementarity between
nucleic acid strands has
significant effects on the efficiency and strength of the hybridization
between the nucleic acid strands.
This is of particular importance in amplification reactions, which depend upon
binding between nucleic
acids strands, and in the design and use of peptide nucleic acid (PNA)
molecules.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising a
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given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding HJNCT or fragments
of HJNCT may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be associated
with a stabilizing agent such as a carbohydrate. In hybridizations, the probe
may be deployed in an
aqueous solution containing salts (e.g., NaCI), detergents (e.g., sodium
dodecyl sulfate; SDS), and other
components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
resequenced to resolve
uncalled bases, extended using the XL-PCR kit (Perkin-Elmer, Norwalk CT) in
the 5' and/or the 3'
direction, and resequenced, or which,has been assembled from the overlapping
sequences of more than
one Incyte Clone using a computer program for fragment assembly, such as the
GELVIEW fragment
assembly system (GCG, Madison WI). Some sequences have been both extended and
assembled to
produce the consensus sequence.
The term "correlates with expression of a polynucleotide" indicates that the
detection of the
presence of nucleic acids, the same or related to a nucleic acid sequence
encoding HJNCT, by northern
analysis is indicative of the presence of nucleic acids encoding HJNCT in a
sample, and thereby
correlates with expression of the transcript from the polynucleotide encoding
HJNCT.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to the chemical modification of a polypeptide
sequence, or a
polynucleotide sequence. Chemical modifications of a polynucleotide sequence
can include, for example,
replacement of hydrogen by an alkyl, acyl, or amino group. A derivative
polynucleotide encodes a
polypeptide which retains at least one biological or immunological function of
the natural molecule. A
derivative polypeptide is one modified by glycosylation, pegylation, or any
similar process that retains at
least one biological or immunological function of the polypeptide from which
it was derived.
The term "similarity" refers to a degree of complementarity. There may be
partial similarity or
complete similarity. The word "identity" may substitute for the word
"similarity." A partially
complementary sequence that at least partially inhibits an identical sequence
from hybridizing to a target
nucleic acid is referred to as "substantially similar." The inhibition of
hybridization of the completely
complementary sequence to the target sequence may be examined using a
hybridization assay (Southern
or northern blot, solution hybridization, and the like) under conditians 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
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reduced stringency conditions require that the binding of two sequences to one
another be a specific (i.e.,
a selective) interaction. The absence of non-specific binding may be tested by
the use of a second target
sequence which lacks even a partial degree of complementarity (e.g., less than
about 30% similarity or
identity). In the absence of non-specific binding, the substantially similar
sequence or probe will not
hybridize to the second non-complementary target sequence.
The phrases "percent identity" and "% identity" refer to the percentage of
sequence similarity
found in a comparison of two or more amino acid or nucleic acid sequences.
Percent identity can be
determined electronically, e.g., by using the MEGALIGN program (DNASTAR,
Madison WI) which.
creates alignments between two or more sequences according to methods selected
by the user, e.g., the
clustal method. (See, e.g., Higgins, D.G. and P.M. Sharp {1988) Gene 73:237-
244.) Parameters for each
method may be the default parameters provided by MEGALIGN or may be specified
by the user. The
clustal algorithm groups sequences into clusters by examining the distances
between all pairs. The
clusters are aligned pairwise and then in groups. The percentage similarity
between two amino acid
sequences, e.g., sequence A and sequence B, is calculated by dividing the
length of sequence A, minus
the number of gap residues in sequence A, minus the number of gap residues in
sequence B, into the sum
of the residue matches between sequence A and sequence B, times one hundred.
Gaps of low or of no
similarity between the two amino acid sequences are not included in
determining percentage similarity.
Percent identity between nucleic acid sequences can also be counted or
calculated by other methods
known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. ( 1990)
Methods Enzymol. 183:626-
645.) Identity between sequences can also be determined by other methods known
in the art, e.g., by
varying hybridization conditions.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain DNA
sequences of about 6 kb to 10 Mb in size, and which contain all of the
elements required for stable mitotic
chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules in which the amino
acid sequence
in the non-antigen binding regions has been altered so that the antibody more
closely resembles a human
antibody, and still retains its original binding ability.
"Hybridization" refers to any ;process by which a strand of nucleic acid binds
with a
complementary strand through base pairing.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A hybridization
complex may be formed in solution (e.g., C°t or R°t 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
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their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide sequence
resulting in the addition of one or more amino acid residues or nucleotides,
respectively, to the sequence
found in the naturally occurring molecule.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression of
various factors, e.g., cytokines, chemokines, and other signaling molecules,
which may affect cellular and
systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotides on
a substrate.
The terms "element" and "array element" in a microarray context, refer to
hybridizable
polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of HJNCT. For example,
modulation may
cause an increase or a decrease in protein activity, binding characteristics,
or any other biological,
functional, or immunological properties of HJNCT.
The phrases "nucleic acid" or "nucleic acid sequence," as used herein, refer
to a nucleotide,
oligonucleotide, polynucleotide, or any fragment thereof. These phrases also
refer to DNA or RNA of
genomic or synthetic origin which may be single-stranded or double-stranded
and may represent the sense
or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or
RNA-like material. In this
context, "fragments" refers to those nucleic acid sequences which comprise a
region of unique
polynucleotide sequence that specifically identifies SEQ ID NO:S-8, for
example, as distinct from any
other sequence in the same genome. For example, a fragment of SEQ ID NO:S-8 is
useful in
hybridization and amplification technologies and in analogous methods that
distinguish SEQ ID NO:S-8
from related polynucleotide sequences. A fragment of SEQ 1D NO:S-8 is at least
about 15-20 nucleotides
in length. The precise length of the fragment of SEQ ID NO:S-8 and the region
of SEQ ID NO:S-8 to
which the fragment corresponds are routinely determinable by one of ordinary
skill in the art based on the
intended purpose for the fragment. In some cases, a fragment, when translated,
would produce
polypeptides retaining some functional characteristic, e.g., antigenicity, or
structural domain
characteristic, e.g., ATP-binding site, of the full-length polypeptide.
The terms "operably associated" and "operably linked" refer to functionally
related nucleic acid
sequences. A promoter is operably associated or operably linked with a coding
sequence if the promoter
controls the translation of the encoded 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.
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The term "oiigonucleotide" refers to a nucleic acid sequence of at least about
6 nucleotides to 60
nucleotides, preferably about 15 to 30 nucleotides, and most preferably about
20 to 25 nucleotides, which
can be used in PCR amplification or in a hybridization assay or microarray.
"Oligonucleotide" is
substantially equivalent to the terms "amplimer," "primer," "oligomer," and
"probe," as these terms are
commonly defined in the art.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which comprises
an oligonucleotide of at least about S 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 HJNCT, or fragments thereof, or HJNCT itself, may comprise a bodily
fluid; an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or cDNA, in
solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a protein
or peptide and an agonist, an antibody, or an antagonist. The interaction is
dependent upon the presence
of a particular structure of the protein, e.g., the antigenic determinant or
epitope, recognized by the
binding molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A and
the antibody will reduce the amount of labeled A that binds to the antibody.
The term "stringent conditions" refers to conditions which permit
hybridization between
polynucleotides and the claimed polynucleotides. Stringent conditions can be
defined by salt
concentration, the concentration of organic solvent, e.g., formamide,
temperature, and other conditions
well known in the art. In particular, stringency can be increased by reducing
the concentration of salt,
increasing the concentration of formamide, or raising the hybridization
temperature.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are removed
from their natural environment and are isolated or separated, and are at least
about 60% free, preferably
about 75% free, and most preferably about 90% free from other components with
which they are naturally
associated.
A "substitution" refers to the replacement of one or more amino acids or
nucleotides by different
amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters, 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
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pores, to which polynucleotides or polypeptides are bound.
"Transformation" describes a process by which exogenous DNA enters and changes
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods well
known in the art, and may rely on any known method for the insertion of
foreign nucleic acid sequences
into a prokaryotic or eukaryotic host cell. The method for transformation is
selected based on the type of
host cell being transformed and may include, but is not limited to, viral
infection, electroporation, heat
shock, lipofection, and particle bombardment. The term "transformed" cells
includes stably transformed
cells in which the inserted DNA is capable of replication either as an
autonomously replicating plasmid or
as part of the host chromosome, as well as transiently transformed cells which
express the inserted DNA
or RNA for limited periods of time.
A "variant" of HJNCT 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 immunologicai 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 HJNCT. This definition may also include,
for example, "allelic" (as
defined above), "splice," "species," or "polymorphic" variants. A splice
variant may have significant
identity to a reference molecule, but will generally have a greater or lesser
number of polynucleotides due
to alternate splicing of exons during mRNA processing. The corresponding
polypeptide may possess
additional functional domains or an absence of domains. Species variants are
polynucleotide sequences
that vary from one species to another. The resulting polypeptides generally
will have significant amino
acid identity relative to each other. A polymorphic variant is a variation in
the polynucleotide sequence
of a particular gene between individuals of a given species. Polymorphic
variants also may encompass
"single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence
varies by one base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a propensity
for a disease state.
THE INVENTION
The invention is based on the discovery of new human membrane-associated
organizational
proteins (HJNCT), the polynucleotides encoding HJNCT, and the use of these
compositions for the
diagnosis, treatment, or prevention of cell proliferative disorders, including
cancer, and
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autoimmune/inflammatory, neurological, developmental, vesicle trafficking,
reproductive,
gastrointestinal, and renal disorders.
Table I lists the Incyte clones used to assemble full length nucleotide
sequences encoding
HJNCT. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs)
of the polypeptide
and nucleotide sequences, respectively. Column 3 shows the clone IDs of the
Incyte clones in which
nucleic acids encoding each HJNCT were identified, and column 4 shows the cDNA
libraries from which
these clones were isolated. Column 5 shows Incyte clones and their
corresponding cDNA libraries.
Clones for which cDNA libraries are not indicated were derived from pooled
cDNA libraries. The clones
in column 5 were used to assemble the consensus nucleotide sequence of each
HJNCT and are useful as
fragments in hybridization technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; column 6
shows homologous sequences as identified by BLAST analysis; and column 7 shows
analytical methods
used to characterize each polypeptide through sequence homology and protein
motifs.
As shown in Figures lA, 1B, and 1C, HJNCT-1 has chemical and structural
similarity with rat
Homer (GI 1913909; SEQ ID N0:9). In particular, HJNCT-1 and rat Homer share
72% identity.
Furthermore, HJNCT-1 and rat Homer share 100% identity within the region from
residue T65 to F93, as
shown in Figure lA. Within this region, HTNCT-1 contains a putative GLGF
segment from G90 to F93
and an arginine residue at R84. HJNCT-2 has chemical and structural similarity
with rat GRASP65 (GI
4432587; SEQ ID NO:10). In particular, HJNCT-2 and rat GRASP65 share 7I %
identity. HJNCT-2 and
rat GRASP65 are 100% identical from residue L50 to residue I62 of HJNCT-2, the
region shown to be
important for binding GM130 in GRASP65. Like rat GRASP65, HJNCT-2 contains
many possible
phosphorylation sites and has possible N-myristoylation sites. As shown in
Figure 2, HJNCT-3 has
chemical and structural homology with mouse claudin-2 (GI 3335184; SEQ ID
NO:11). In particular,
HJNCT-3 and mouse ciaudin-2 share 91% identity. As shown in Figures 3A and 3B,
HJNCT-4 has
chemical and structural homology with mouse syntenin (GI 3342560; SEQ ID
N0:12). In particular,
HJNCT-4 and mouse syntenin share 61% identity.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding HJNCT. The first column of Table
3 lists the nucleotide
SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1.
These fragments are
useful, for example, in hybridization or amplification technologies t:o
identify SEQ ID NO:S-8 and to
distinguish between SEQ ID NO:S-8 and related polynucleotide sequences. The
polypeptides encoded by
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these fragments are useful, for example, as immunogenic peptides. Column 3
lists tissue categories which
express NJNCT as a fraction of total tissues expressing HJNCT. Column 4 lists
diseases, disorders, or
conditions associated with those tissues expressing HJNCT as a fraction of
total tissues expressing
HJNCT. Of particular note is the expression of HJNCT-1 in reproductive tissue,
nervous tissue, and fetal
cell lines; the expression of HJNCT-2 in tissues associated with cancer and
cell proliferation disorders;
the expression of HJNCT-3 in cancerous, fetal, proliferating,
gastrointestinal, and urologic tissues; and
the expression of HJNCT-4 in cancerous, fetal, proliferating, and
gastrointestinal tissues. Column 5 lists
the vectors used to subclone each cDNA library.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
from which cDNA clones encoding HJNCT were isolated. Column 1 references the
nucleotide SEQ ID
NOs, column 2 shows the cDNA libraries from which these clones were isolated,
and column 3 shows the
tissue origins and other descriptive information relevant to the cDNA
libraries in column 2.
The invention also encompasses HJNCT variants. A preferred HJNCT variant is
one which has
at least about 85%, more preferably at least about 90%, and most preferably at
least about 95% amino
acid sequence identity to the HJNCT amino acid sequence, and which contains at
least one functional or
structural characteristic of HJNCT.
The invention also encompasses polynucleotides which encode HJNCT. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected from
the group consisting of SEQ ID NO:S-8, which encodes HJNCT.
The invention also encompasses a variant of a polynucleotide sequence encoding
HJNCT. In
particular, such a variant polynucleotide sequence will have at least about
85%, more preferably at least
about 90%, and most preferably at least about 95% polynucleotide sequence
identity to the polynucleotide
sequence encoding HJNCT. A particular aspect of the invention encompasses a
variant of a sequence
selected from the group consisting of SEQ ID NO:S-8 which has at least about
85%, more preferably at
least about 90%, and most preferably at least about 95% polynucleotide
sequence identity to a sequence
selected from the group consisting of SEQ ID NO:S-8. Any one of the
polynucleotide variants described
above can encode an amino acid sequence which contains at least one functional
or structural
characteristic of HJCNT.
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 HJNCT, some bearing
minimal similarity to the
polynucleotide sequences of any known and natural ly occurring gene, may be
produced. Thus, the
invention contemplates each and every possible variation of polynucleotide
sequence that could be made
by selecting combinations based on possible codon choices. These combinations
are made in accordance
with the standard triplet genetic code as applied to the polynucleotide
sequence of naturally occurring
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HJNCT, and all such variations are to be considered as being specifically
disclosed.
Although nucleotide sequences which encode HJNCT and its variants are
preferably capable of
hybridizing to the nucleotide sequence of the naturally occurring HJNCT under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding HJNCT 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 HJNCT
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 HJNCT
and
HJNCT 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 HJNCT 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 NO:S-8 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 Enzymoi. 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 dodecyi
sulfate (SDS), and the inclusion
or exclusion of carrier DNA, are well known to those skilled in the art.
Various levels of stringency are
accomplished by combining these various conditions as needed. In a preferred
embodiment, hybridization
will occur at 30°C in 750 mM NaCI, 75 mM trisodium citrate, and 1% SDS.
In a more preferred
embodiment, hybridization will occur at 37°C in 500 mM NaCI, 50 mM
trisodium citrate, 1 % SDS, 35%
formamide, and 100 ~sg/ml denatured salmon sperm DNA (ssDNA). In a most
preferred embodiment,
hybridization will occur at 42°C in 250 mM NaCI, 25 mM trisodium
citrate, 1 % SDS, 50 % fonmamide,
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and 200 ug/ml ssDNA. Useful variations on these conditions will be readily
apparent to those skilled in
the art.
The washing steps which follow hybridization can also vary in stringency. Wash
stringency
conditions can be defined by salt concentration and by temperature. As above,
wash stringency can be
increased by decreasing salt concentration or by increasing temperature. For
example, stringent salt
concentration for the wash steps will preferably be less than about 30 mM NaCI
and 3 mM trisodium
citrate, and most preferably less than about 15 mM NaCI and 1.5 mM trisodium
citrate. Stringent
temperature conditions for the wash steps will ordinarily include temperature
of at least about 25°C, more
preferably of at least about 42°C, and most preferably of at least
about 68°C. In a preferred embodiment,
wash steps will occur at 25°C in 30 mM NaCI, 3 mM trisodium citrate,
and 0.1 % SDS. In a more
preferred embodiment, wash steps will occur at 42°C in 1 S mM NaCI, 1.5
mM trisodium citrate, and
0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C
in IS mM NaCI, 1.5 mM
trisodium citrate, and 0.1% SDS. Additional variations on these conditions
will be readily apparent to
those skilled in the art.
Methods for DNA sequencing are well known in the art and may be used to
practice any of the
embodiments of the invention. The methods may employ such enzymes as the
Klenow 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 MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200
thermal cycler (MJ
Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Perkin-Elmer).
Sequencing is then
carried out using either the ABI 373 or 37 7 DNA sequencing system (Perkin-
Elmer), the MEGABACb
1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other
systems known in the art.
The resulting sequences are analyzed using a variety of algorithms which are
well known in the art. (See,
e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Bioloev, John Wiley &
Sons, New York NY,
unit 7.7; Meyers, R.A. { 1995) Molecular Biology and Biotechnolo~v, Wiley VCH,
New York NY, pp.
856-853.)
The nucleic acid sequences encoding HJNCT may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences, such
as promoters and regulatory elements. For example, one method which may be
employed, restriction-site
PCR, uses universal and nested primers to amplify unknown sequence from
genomic DNA within a
cloning vector. (See, e.g., Sarkar, G. ( 1993) PCR Methods Applic. 2:318-322.)
Another method, inverse
PCR, uses primers that extend in divergent directions to amplify unknown
sequence from a circularized
zl
CA 02343696 2001-03-22
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template. The template is derived from restriction fragments comprising a
known genomic locus and
surrounding sequences. (See, e.g., Triglia, T. et al. ( 1988) Nucleic Acids
Res. 16:8186.) A third method,
capture PCR, involves PCR amplification of DNA fragments adjacent to known
sequences in human and
yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR
Methods Applic.
1:111-119.) In this method, multiple restriction enzyme digestions and
ligations may be used to insert an
engineered double-stranded sequence into a region of unknown sequence before
performing PCR. Other
methods which may be used to retrieve unknown sequences are known in the art.
(See, e.g., Parker, J.D.
et al. ( 1991 ) Nucleic Acids Res. 19:3055-3060). Additionally, one may use
PCR, nested primers, and .
PROMOTERFINDER libraries (Ciontech, 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 HJNCT may be cloned in recombinant DNA molecules that direct expression
of HJNCT, 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 HJNCT.
The nucleotide sequences of the present invention can be engineered using
methods generally
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known in the art in order to alter HJNCT-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 glycosyIation
patterns, change codon preference, produce splice variants, and so forth.
In another embodiment, sequences encoding HJNCT may be synthesized, in whole
or in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. ( 1980) Nucleic Acids.
Symp. Ser. 7:215-223, and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively,
HJNCT 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 HJNCT, 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. I 82:392-421.) The
composition of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing. (See,
e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY.)
In order to express a biologically active HJNCT, the nucleotide sequences
encoding HJNCT 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. '1 hese 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
HJNCT. Such elements may vary in their strength and specificity. Specific
initiation signals may also be
used to achieve more e~cient translation of sequences encoding HJNCT. Such
signals include the ATG
initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases
where sequences encoding
HJNCT 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.)
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Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding HJNCT and appropriate transcriptional
and translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in vivo
genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning A Laboratory Manual,
S 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
encoding HJNCT. 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
1S the use intended for polynucleotide sequences encoding HJNCT. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding HJNCT can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1 plasmid
(Life Technologies). Ligation of sequences encoding HJNCT into the vector's
multiple cloning site
disrupts the IacZ gene, allowing a colorimetric screening procedure for
identification of transformed
bacteria containing recombinant molecules. In addition, these vectors may be
useful for in vitro
transcription, dideoxy sequencing, single strand rescue with helper phage, and
creation of nested deletions
in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster (1989) J.
Biol. Chem.
2ti4:SS03-5509.) When large quantities of HJNCT are needed, e.g. for the
production of antibodies,
vectors which direct high level expression of HJNCT may be used. For example,
vectors containing the
2S strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of HJNCT. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH promoters,
may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In
addition, such vectors direct
either the secretion or intracellular retention of expressed proteins and
enable integration of foreign
sequences into the host genome for stable propagation. (See, e.g., Ausubel,
1995, s. uara; Bitter, G.A. et
al. (1987) Methods Enzymol. 1S3:S16-544; and Scorer, C.A. et al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of HJNCT. 'Transcription of
sequences encoding
HJNCT may be driven viral promoters, e.g., the 3SS and 19S promoters of CaMV
used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO
J. 6:307-31 I).
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Alternatively, plant promoters such as the small subunit of RUBISCO or heat
shock promoters may be
used. (See, e.g., Coruzzi, G: et al. (1984) EMBO J. 3:1671-1680; Brogue, R. et
al. (1984) Science
224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-
105.) These constructs can be
introduced into plant cells by direct DNA transformation or pathogen-mediated
transfection. (See, e.g.,
The McGraw Hill Yearbook of Science and Technolo v (1992) McGraw Hill, New
York NY, pp.
191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases where
an adenovirus is used as an expression vector, sequences encoding HJNCT may be
ligated into an
adenovirus transcription/translation complex consisting of the late promoter
and tripartite leader
sequence. Insertion in a non-essential E 1 or E3 region of the viral genome
may be used to obtain
infective virus which expresses HJNCT in host cells. (See, e.g., Logan, J. and
T. Shenk ( I984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-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
HJNCT in cell lines is preferred. For example, sequences encoding HJNCT 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 and pat confer resistance to
chlorsulfuron and
phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al.
(1980) Proc. Natl. Acad. Sci.
CA 02343696 2001-03-22
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USA 77:3567-3570; Colbere-Garapin, F. et al. (1981} J. Mol. Biol. 150:1-14.)
Additional selectable
genes have been described, e.g., trpB and hisD, which alter cellular
requirements for metabolites. (See,
e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. USA
85:8047-8051.) Visible
markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech),13
glucuronidase and its substrate
13-glucuronide, or luciferase and its substrate luciferin may be used. These
markers can be used not only
to identify transformants, but also to quantify the amount of transient or
stable protein expression
attributable to a specific vector system. (See, e.g., Rhodes, C.A. (1995)
Methods Mol. Biol. 55:121-131.)
Although the presencelabsence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding HJNCT is inserted within a marker gene sequence, transformed
cells containing
sequences encoding HJNCT can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding HJNCT 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 HJNCT
and that express
HJNCT 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 HJNCT
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 HJNCT is preferred, but a
competitive binding assay may be
employed. These and other assays are well known in the art. (See, e.g.,
Hampton, R, et al. (1990)
Serological Methods a Laboratory Manual, APS Press, St. Paul MN, Sect. IV;
Coligan, J.E. et al. ( 1997)
Current Protocols in Immunolo v, 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 HJNCT
include oligolabeling,
nick translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the
sequences encoding HJNCT, 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
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CA 02343696 2001-03-22
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synthesize RNA probes in vitro by addition of an appropriate RNA poiymerase
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 HJNCT 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 HJNCT may be designed to contain signal sequences
which direct
secretion of HJNCT through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the inserted
sequences or to process the expressed protein in the desired fashion. Such
modifications of the
polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which cleaves a
"prepro" form of the protein may
also be used to specify protein targeting, folding, and/or activity. Different
host cells which have specific
cellular machinery and characteristic mechanisms for post-translational
activities (e.g., CHO, HeLa,
MDCK, HEK293, and WI38) are available from the American Type Culture
Collection (ATCC,
Manassas VA) and may be chosen to ensure the correct modification and
processing of the foreign
protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid sequences
encoding HJNCT 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 HJNCT protein
containing a
heterologous moiety that can be recognized by a commercially available
antibody may facilitate the
screening of peptide libraries for inhibitors of HJNCT 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 immunoaff7nity 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 HJNCT
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CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
encoding sequence and the heterologous protein sequence, so that HJNCT 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 HJNCT may
be achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract systems
(Promega). These systems
couple transcription and translation of protein-coding sequences operably
associated with the T7, T3, or
SP6 promoters. Translation takes place in the presence of a radioiabeled amino
acid precursor, preferably
'SS-methionine.
Fragments of HJNCT may be produced not only by recombinant production, but
also by direct
peptide synthesis using solid-phase techniques. (See, e.g., Creighton, supra,
pp. 55-60.) Protein synthesis
may be performed by manual techniques or by automation. Automated synthesis
may be achieved, for
example, using the ABI 431A peptide synthesizer (Perkin-Elmer). Various
fragments of HJNCT may be
synthesized separately and then combined to produce the full length molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists between
regions of HJNCT and human membrane-associated organizational proteins. In
addition, the expression
of HJNCT is closely associated with reproductive, neurological, developmental,
cancerous, fetal or
proliferating, gastrointestinal, and urologic tissues, and with inflammation
and the immune response.
Therefore, HJNCT appears to play a role in cell proliferative disorders,
including cancer, and
autoimmune/inflammatory, neurological, developmental, vesicle trafficking,
reproductive,
gastrointestinal, and renal disorders. In the treatment of disorders
associated with increased HJNCT
expression or activity, it is desirable to decrease the expression or activity
of HJNCT. In the treatment of
disorders associated with decreased H.fNCT expression or activity, it is
desirable to increase the
expression or activity of HJNCT.
Therefore, in one embodiment, HJNCT 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 HJNCT.
Examples of such disorders include, but are not limited to, a cell
proliferative disorder such as actinic
keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis,
mixed connective tissue disease
(MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,
psoriasis, primary
thrombocythemia, and cancer, 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
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autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS), Addison's
disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic
lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or .
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
t0 syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic lupus
erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial, fungal,
parasitic, protozoal, and helminthic infections, and trauma; a neurological
disorder such as epilepsy,
ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's
disease, Pick's disease,
Huntington's disease, dementia, Parkinson's disease and other extrapyramidal
disorders, amyotrophic
lateral sclerosis and other motor neuron disorders, progressive neural
muscular atrophy, retinitis
pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating
diseases, bacterial and viral
meningitis, brain abscess, subdural empyema, epidural abscess, suppurative
intracranial thrombophlebitis,
myelitis and radiculitis, viral central nervous system disease, prion diseases
including kuru,
Creutzfeldt-Jakob disease, and Gerstmann-Straussier-Scheinker syndrome, fatal
familial insomnia,
nutritional and metabolic diseases of the nervous system, neurofibromatosis,
tuberous sclerosis,
cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental
retardation and other
developmental disorders of the central nervous system, cerebral palsy,
neuroskeletal disorders, autonomic
nervous system disorders, cranial nerve disorders, spinal cord diseases,
muscular dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, akathesia,
amnesia, catatonia, diabetic
neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic
neuralgia, and Tourette's
disorder; a developmental disorder such as renal tubular acidosis, anemia,
Cushing's syndrome,
achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy,
gonadal dysgenesis,
WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation), Smith-
Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia, hereditary
keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and
neurofibromatosis,
hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea
and cerebral palsy, spina
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bifida, Williams syndrome, anencephaly, craniorachischisis, congenital
glaucoma, cataract, sensorineural
hearing loss, and any disorder associated with cell growth and
differentiation, embryogenesis, and
morphogenesis involving any tissue, organ, or system of a subject, e.g., the
brain, adrenal gland, kidney,
skeletal or reproductive system; 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, 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 a disorder of prolactin
production; infertility, including tubal
disease, ovulatory defects, and endometriosis; disruptions of the estrous
cycle, disruptions of the
menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome,
endometrial and
ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies,
and teratogenesis; cancer of
the breast, fibrocystic breast disease, and galactorrhea; disruptions of
spermatogenesis, abnormal sperm
physiology, cancer of the testis, cancer of the prostate, benign prostatic
hyperplasia, prostatitis, Peyronie's
disease, impotence, carcinoma of the male breast, and gynecomastia; a
gastrointestinal disorder such as
dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture,
esophageal carcinoma, dyspepsia,
indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis,
gastroparesis, antral or pyloric edema,
abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections
of the intestinal tract, peptic
ulcer, gastric ulcer, duodenal ulcer, cholelithiasis, cholecystitis,
cholestasis, pancreatitis, pancreatic
carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis,
passive congestion ofthe liver,
hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis,
Crohn's disease, Whipple's disease,
Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irritable
bowel syndrome, short bowel
syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired
immunodeficiency syndrome
(AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome,
hepatic steatosis,
hemochromatosis, Wilson's disease, alpha,-antitrypsin deficiency, Reye's
syndrome, primary sclerosing
cholangitis, liver infarction, portal vein obstruction and thrombosis,
centrilobular necrosis, peliosis
hepatis, hepatic vein thrombosis, veno-occlusive disease, preeclampsia,
eclampsia, acute fatty liver of
pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors including
nodular hyperplasias,
adenomas, and carcinomas; and a renal disorder such as renal amyloidosis,
hypertension, primary
aldosteronism, Addison's disease, renal failure, glomerulonephritis, chronic
glomerulonephritis,
tubulointerstitial nephritis, cystic disorders of the kidney and dysplastic
malformations such as polycystic
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disease, renal dysplasias, and cortical or medullary cysts, inherited
polycystic renal diseases (PRD) such
as recessive and autosomal dominant PRD, medullary cystic disease, meduliary
sponge kidney and
tubular dysplasia, Alport's syndrome, non-renal cancers which affect renal
physiology such as
bronchogenic tumors of the lungs or tumors of the basal region of the brain,
multiple myeloma,
adenocarcinomas of the kidney, metastatic renal carcinoma, and nephrotoxic
disorders including any
functional or morphologic change in the kidney produced by any pharmaceutical,
chemical, or biological
agent that is ingested, injected, inhaled, or absorbed. Some broad categories
of common nephrotoxic
agents are heavy metals, all classes of antibiotics, analgesics, solvents,
oxalosis-inducing agents, .
anticancer drugs, herbicides and pesticides, botanicals and biologicals, and
antiepileptics.
In another embodiment, a vector capable of expressing HJNCT 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 HJNCT including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
HJNCT 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 HJNCT
including, but not limited
to, those provided above.
In still another embodiment, an agonist which modulates the activity of HJNCT
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or activity of
HJNCT including, but not limited to, those listed above.
In a further embodiment, an antagonist of HJNCT may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of HJNCT.
Examples of such
disorders include, but are not limited to, those cell proliferative disorders,
including cancer, and
autoimmune/inflammatory, neurological, developmental, vesicle trafficking,
reproductive,
gastrointestinal, and renal disorders described above. In one aspect, an
antibody which specifically binds
HJNCT 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 HJNCT.
In an additional embodiment, a vector expressing the complement of the
polynucleotide encoding
HJNCT may be administered to a subject to treat or prevent a disorder
associated with increased
expression or activity of HJNCT 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
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WO 00/18915 PCT/US99/22082
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 HJNCT may be produced using methods which are generally known
in the art.
In particular, purified HJNCT may be used to produce antibodies or to screen
libraries of pharmaceutical
agents to identify those which specifically bind HJNCT. Antibodies to HJNCT
may also be generated
using methods that are well known in the art. Such antibodies may include, but
are not limited to,
polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments,
and fragments produced by
a Fab expression library. Neutralizing antibodies (i.e., those which inhibit
dimer formation) are especially
preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans, and
others may be immunized by injection with HJNCT or with any fragment or
oligopeptide thereof which
has immunogenic properties. Depending on the host species, various adjuvants
may be used to increase
immunological response. Such adjuvants include, but are not limited to,
Freund's, mineral gels such as
aluminum hydroxide, and surface active substances such as lysolecithin,
pluronic polyols, polyanions,
peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in
humans, BCG (bacilli
Calmette-Guerin) and Corvnebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to HJNCT
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 HJNCT 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 HJNCT 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.
(19$5) J. Immunol. Methods
81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030;
Cole, S.P. et al. (1984) Mol.
Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate antigen
specificity and biological activity, can be used. (See, e.g., Morrison, S.L.
et al. ( 1984) Proc. Natl. Acad.
Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and
Takeda, S. et 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 HJNCT-specific
single chain antibodies.
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Antibodies with related specificity, but of distinct idiotypic composition,
may be generated by chain
shuffling from random combinatorial immunoglobulin libraries. (See, e.g.,
Burton, D.R. (1991) Proc.
Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population or
by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the
literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA
86:3833-3837; Winter, G. et al.
(1991 ) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for HJNCT 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
HJNCT and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two
non-interfering HJNCT 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 HJNCT. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of HJNCT-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The ICe determined
for a preparation of polyclonal antibodies, which are heterogeneous in their
affinities for multiple HJNCT
epitopes, represents the average affinity, or avidity, of the antibodies for
HJNCT. The Ke determined for
a preparation of monoclonal antibodies, which are monospeciflc for a
particular HJNCT epitope,
represents a true measure of affinity. High-affinity antibody preparations
with ICa ranging from about 109
to 10'2 L/moie are preferred for use in immunoassays in which the HJNCT-
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 HJNCT, preferably in active form, from the antibody (Catty, D.
(1988) Antibodies.
Volume I: A Practical Approach, IRL Press, Washington, DC; Liddell, J.E. and
A. Cryer (1991) A
Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
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CA 02343696 2001-03-22
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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 HJNCT-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for antibody
quality and usage in various applications, are generally available. (See,
e.g., Catty, supra, and Coligan et
al. supra.)
In another embodiment of the invention, the polynucleotides encoding HJNCT, or
any fragment
or complement thereof, may be used for therapeutic purposes. In one aspect,
the complement of the
polynucleotide encoding HJNCT 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 HJNCT. Thus, complementary molecules or fragments may
be used to
modulate HJNCT 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 HJNCT.
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 ark
can be used to construct
vectors to express nucleic acid sequences complementary to the polynucleotides
encoding HJNCT. (See,
e.g., Sambrook, supra; Ausubel, 1995, supra.)
Genes encoding HJNCT can be turned off by transforming a cell or tissue with
expression vectors
which express high levels of a polynucleotide, or fragment thereof, encoding
HJNCT. Such constructs
may be used to introduce untranslatable sense or antisense sequences into a
cell. Even in the absence of
integration into the DNA, such vectors may continue to transcribe RNA
molecules until they are disabled
by endogenous nucleases. Transient expression may last for a month or more
with a non-replicating
vector, and may last even longer if appropriate replication elements are part
of the vector system.
As mentioned above, modifications of gene expression can be obtained by
designing
complementary sequences or antisense molecules (DNA, RNA, or PNA) to the
control, 5', or regulatory
regions of the gene encoding HJNCT. Oligonucleotides derived from the
transcription initiation site, e.g.,
between about positions -10 and +10 from the start site, are preferred.
Similarly, inhibition can be
achieved using triple helix base-pairing methodology. Triple helix pairing is
useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have been
described in the literature. (See, e.g., Gee, J.E. et al. ( 1994) in Huber,
B.E. and B.I. Carr, Molecular and
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CA 02343696 2001-03-22
WO 00/1$915 PCT/US99/220$2
Immunoloy'g c Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177.) A
complementary sequence
or antisense molecule may also be designed to block translation of mRNA by
preventing the transcript
from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example, engineered
hammerhead motif ribozyme molecules may specifically and efficiently catalyze
endonucleolytic
cleavage of sequences encoding HJNCT.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
IO scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA, GUU,
and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides, corresponding to
the region of the target gene containing the cleavage site, may be evaluated
for secondary structural
features which may render the oligonucleotide inoperable. The suitability of
candidate targets may also
be evaluated by testing accessibility to hybridization with complementary
oligonucleotides using
ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared by
any method known in the art for the synthesis of nucleic acid molecules. These
include techniques for
chemically synthesizing oligonucleotides such as solid phase phosphoramidite
chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in
vivo.transcription of DNA sequences
encoding HJNCT. 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 S' and/or 3' ends of
the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages within
the backbone of the molecule. This concept is inherent in the production of
PNAs and can be extended in
all of these molecules by the inclusion of nontraditional bases such as
inosine, queosine, and wybutosine,
as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine,
cytidine, guanine, thymine,
and uridine which are not as easily recognized by endogenous endonucleases.
Many methods for introducing vectors into cells or tissues are available and
equally suitable for
use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells taken
from the patient and clonally propagated for autologous transplant back into
that same patient. Delivery
by transfection, by liposome injections, or by polycationic amino palymers may
be achieved using
methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (
1997) Nat. Biotechnol.
CA 02343696 2001-03-22
WO 00/18915 PCT/US99122082
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
sterile composition, in conjunction with a pharmaceutically acceptable
carrier, for any of the therapeutic
effects discussed above. Such pharmaceutical compositions may consist of
HJNCT, antibodies to
HJNCT, and mimetics, agonists, antagonists, or inhibitors of HJNCT. 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, biocompatibie pharmaceutical carrier
including, but not limited to,
saline, buffered saline, dextrose, and water. The compositions may be
administered to a patient alone, or
in combination with other agents, drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered
by any number
of routes including, but not limited to, oral, intravenous, intramuscular,
intra-arterial, intramedullary,
intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain suitable
pharmaceutically-acceptable carriers comprising excipients and auxiliaries
which facilitate processing of
the active compounds into preparations which can be used pharmaceutically.
Further details on
techniques for formulation and administration may be found in the latest
edition of Remin ton's
Pharmaceutical Sciences (Maack Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using
pharmaceutically
acceptable carriers well known in the art in dosages suitable for oral
administration. Such carriers enable
the pharmaceutical compositions to be formulated as tablets, pills, dragees,
capsules, liquids, gels, syrups,
slurries, suspensions, and the like, for ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combining
active compounds
with solid excipient and processing the resultant mixture of granules
(optionally, after grinding) to obtain
tablets or dragee cores. Suitable auxiliaries can be added, if desired.
Suitable excipients include
carbohydrate or protein fillers, such as sugars, including lactose, sucrose,
mannitol, and sorbitol; starch
from corn, wheat, rice, potato, or other plants; cellulose, such as methyl
cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums,
including arabic and
tragacanth; and proteins, such as gelatin and collagen. If desired,
disintegrating or solubilizing agents
may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and
alginic acid or a salt thereof, such
as sodium alginate.
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Dragee cores may be used in conjunction with suitable coatings, such as
concentrated sugar
solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone,
carbopol gel, polyethylene
glycol, and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for
product identification or to
characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of gelatin,
as well as soft, sealed capsules made of gelatin and a coating, such as
glycerol or sorbitol. Push-fit
capsules can contain active ingredients mixed with fillers or binders, such as
lactose or starches,
lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
In soft capsules, the active
compounds may be dissolved or suspended in suitable liquids, such as fatty
oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be
formulated in aqueous
solutions, preferably in physiologically compatible buffers such as Hanks'
solution, Ringer's solution, or
physiologically buffered saline. Aqueous injection suspensions may contain
substances which increase
the viscosity of the suspension, such as sodium carboxymethyl cellulose,
sorbitol, or dextran.
Additionally, suspensions of the active compounds may be prepared as
appropriate oily injection
suspensions. Suitable lipophiiic 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 acids. Salts
tend to be more soluble in aqueous or other protonic solvents than are the
corresponding free base forms.
In other cases, the preferred preparation may be a lyophilized powder which
may contain any or all of the
following: 1 mM to 50 mM histidine, 0. I % 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 HJNCT, such
labeling would include amount, frequency, and method of administration.
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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 HJNCT or
fragments thereof, antibodies of HJNCT, and agonists, antagonists or
inhibitors of HJNCT, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by standard
pharmaceutical procedures in cell cultures or with experimental animals, such
as by calculating the EDSo
(the dose therapeutically effective in 50% of the population) or LDSO (the
dose lethal to 50% of the
population) statistics. The dose ratio of toxic to therapeutic effects is the
therapeutic index, which can be
expressed as the LDso/EDso ratio. Pharmaceutical compositions which exhibit
large therapeutic indices
are preferred. The data obtained from cell culture assays and animal studies
are used to formulate a range
of dosage for human use. The dosage contained in such compositions is
preferably within a range of
circulating concentrations that includes the EDso with little or no toxicity.
The dosage varies within this
range depending upon the dosage form employed, the sensitivity of the patient,
and the route of
administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the subject
requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the active
moiety or to maintain the desired effect. Factors which may be taken into
account include the severity of
the disease state, the general health of the subject, the age, weight, and
gender of the subject, time and
frequency of administration, drug combination(s), reaction sensitivities, and
response to therapy. Long
acting pharmaceutical compositions may be administered every 3 to 4 days,
every week, or biweekly
depending on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ~cg to 100,000 ,ug, up to a
total dose of about 1
gram, depending upon the route of administration. Guidance as to particular
dosages and methods of
delivery is provided in the literature and generally available to
practitioners in the art. Those skilled in the
art will employ different formulations for nucleotides than for proteins or
their inhibitors. Similarly,
delivery of polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
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DIAGNOSTICS
In another embodiment, antibodies which specifically bind HJNCT may be used
for the diagnosis
of disorders characterized by expression of HJNCT, or in assays to monitor
patients being treated with
HJNCT or agonists, antagonists, or inhibitors of HJNCT. Antibodies useful for
diagnostic purposes may
be prepared in the same manner as described above for therapeutics. Diagnostic
assays for HJNCT
include methods which utilize the antibody and a label to detect HJNCT 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 HJNCT, including ELISAs, RIAs, and FACS,
are known in
the art and provide a basis for diagnosing altered or abnormal levels of HJNCT
expression. Normal or
standard values for HJNCT expression are established by combining body fluids
or cell extracts taken
from normal mammalian subjects, preferably human, with antibody to HJNCT under
conditions suitable
for complex formation. The amount of standard complex formation may be
quantitated by various
methods, preferably by photometric means. Quantities of HJNCT 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 HJNCT may
be used for
diagnostic purposes. The polynucleotides which may be used include
oiigonucleotide 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 HJNCT
may be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
HJNCT, and to monitor regulation of HJNCT levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding HJNCT or closely related
molecules may be used to
identify nucleic acid sequences which encode HJNCT. The specificity of the
probe, whether it is made
from a highly specific region, e.g., the 5' regulatory region, or from a less
specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification
(maximal, high, intermediate, or
low), will determine whether the probe identifies only naturally occurring
sequences encoding HJNCT,
allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and should
preferably have at
least 50% sequence identity to any of the HJNCT encoding sequences, The
hybridization probes of the
subject invention may be DNA or RNA and may be derived from the sequence of
SEQ ID NO:S-8 or
from genomic sequences including promoters, enhancers, and intror~s ofthe
HJNCT gene.
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Means for producing specific hybridization probes for DNAs encoding HJNCT
include the
cloning of polynucleotide sequences encoding HJNCT or HJNCT derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may be
used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA polymerises and
the appropriate labeled nucleotides. Hybridization probes may be labeled by a
variety of reporter groups,
for example, by radionuclides such as 32P or'SS, or by enzymatic labels, such
as alkaline phosphatase
coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotide sequences encoding HJNCT may be used for the diagnosis of
disorders associated
with expression of HJNCT. Examples of such disorders include, but are not
limited to, a cell proliferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria, polycythemia
vera, psoriasis, primary thrombocythemia, and cancer, including
adenocarcinoma, leukemia, lymphoma,
melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of
the adrenal gland, bladder,
bone, bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver,
lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,
skin, spleen, testis, thymus,
thyroid, and uterus; an autoimmune/inflammatory disorder such as acquired
immunodeficiency syndrome
(AIDS), Addison's disease, adult respiratory distress syndrome, allergies,
ankylosing spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune thyroiditis,
autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED),
bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis,
dermatomyositis, diabetes mellitus,
emphysema, episodic iymphopenia with lymphocytotoxins, erythroblastosis
fetalis, erythema nodosum,
atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'
disease, Hashimoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis,
myasthenia gravis, myocardial
or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic lupus
erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial, fungal,
parasitic, protozoal, and helminthic infections, and trauma; a neurological
disorder such as epilepsy,
ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's
disease, Pick's disease,
Huntington's disease, dementia, Parkinson's disease and other extrapyramidal
disorders, amyotrophic
lateral sclerosis and other motor neuron disorders, progressive neural
muscular atrophy, retinitis
pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating
diseases, bacterial and viral
meningitis, brain abscess, subdural empyema, epidural abscess, suppurative
intracranial thrombophlebitis,
myelitis and radiculitis, viral central nervous system disease, prion diseases
including kuru,
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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
S nervous system disorders, cranial nerve disorders, spinal cord diseases,
muscular dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, akathesia,
amnesia, catatonia, diabetic
neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic
neuralgia, and Tourette's
disorder; a developmental disorder such as renal tubular acidosis, anemia,
Cushing's syndrome,
achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy,
gonadal dysgenesis,
WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation), Smith-
Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia, hereditary
keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and
neurofibromatosis,
hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea
and cerebral palsy, spina
bifida, Williams syndrome, anencephaly, craniorachischisis, congenital
glaucoma, cataract, sensorineural
hearing loss, and any disorder associated with cell growth and
differentiation, embryogenesis, and
morphogenesis involving any tissue, organ, or system of a subject, e.g., the
brain, adrenal gland, kidney,
skeletal or reproductive system; 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, 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 a disorder of prolactin
production; infertiiity, including tubal
disease, ovulatory defects, and endometriosis; disruptions of the estrous
cycle, disruptions of the
menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome,
endometrial and
ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies,
and teratogenesis; cancer of
the breast, fibrocystic breast disease, and galactorrhea; disruptions of
spermatogenesis, abnormal sperm
physiology, cancer of the testis, cancer of the prostate, benign prostatic
hyperplasia, prostatitis, Peyronie's
disease, impotence, carcinoma of the male breast, and gynecomastia; a
gastrointestinal disorder such as
dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture,
esophageal carcinoma, dyspepsia,
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indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis,
gastroparesis, antral or pyloric edema,
abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections
of the intestinal tract, peptic
ulcer, gastric ulcer, duodenal ulcer, cholelithiasis, cholecystitis,
cholestasis, pancreatitis, pancreatic
carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis,
passive congestion of the liver,
hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis,
Crohn's disease, Whipple's disease,
Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irritable
bowel syndrome, short bowel
syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired
immunodeficiency syndrome
(AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome,
hepatic steatosis,
hemochromatosis, Wilson's disease, alpha,-antitrypsin deficiency, Reye's
syndrome, primary sclerosing
I O cholangitis, liver infarction, portal vein obstruction and thrombosis,
centrilobular necrosis, peliosis
hepatis, hepatic vein thrombosis, veno-occlusive disease, preeclampsia,
eclampsia, acute fatty liver of
pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors including
nodular hyperplasias,
adenomas, and carcinomas; and a renal disorder such as renal amyloidosis,
hypertension, primary
aldosteronism, Addison's disease, renal failure, glomerulonephritis, chronic
glomerulonephritis,
tubulointerstitial nephritis,. cystic disorders of the kidney and dysplastic
malformations such as polycystic
disease, renal dysplasias, and cortical or medullary cysts, inherited
polycystic renal diseases (PRD) such
as recessive and autosomal dominant PRD, medullary cystic disease, medullary
sponge kidney and
tubular dysplasia, Alport's syndrome, non-renal cancers which affect renal
physiology such as
bronchogenic tumors of the lungs or tumors of the basal region of the brain,
multiple myeloma,
adenocarcinomas of the kidney, metastatic renal carcinoma, and nephrotoxic
disorders including any
functional or morphologic change in the kidney produced by any pharmaceutical,
chemical, or biological
agent that is ingested, injected, inhaled, or absorbed. Some broad categories
of common nephrotoxic
agents are heavy metals, all classes of antibiotics, analgesics, solvents,
oxalosis-inducing agents,
anticancer drugs, herbicides and pesticides; botanicals and biologicals, and
antiepileptics. The
polynucleotide sequences encoding HJNCT may be used in Southern or northern
analysis, dot blot, or
other membrane-based technologies; in PCR technologies; in dipstick, pin, and
multiformat ELISA-like
assays; and in microarrays utilizing fluids or tissues from patients to detect
altered HJNCT expression.
Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding HJNCT may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding HJNCT may be labeled by standard methods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a suitable
incubation period, the sample is washed and the signal is quantitated and
compared with a standard value.
If the amount of signal in the patient sample is significantly altered in
comparison to a control sample then
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the presence of altered levels of nucleotide sequences encoding HJNCT 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 iri 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 HJNCT, 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 HJNCT, under conditions suitable for hybridization or
amplification. Standard
hybridization may be quantified by comparing the values obtained from normal
subjects with values from
an experiment in which a known amount of a substantially purified
polynucleotide is used. Standard
values obtained in this manner may be compared with values obtained from
samples from patients who
are symptomatic for a disorder. Deviation from standard values is used to
establish the presence of a
disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated, hybridization
assays may be repeated on a regular basis to determine if the level of
expression in the patient begins to
approximate that which is observed in the normal subject. The results obtained
from successive assays
may be used to show the efficacy of treatment over a period ranging from
several days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development of
the disease, or may provide a means for detecting the disease prior to the
appearance of actual clinical
symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding HJNCT
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 HJNCT,
or a fragment of a polynucleotide complementary to the polynucleotide encoding
HJNCT, and will be
employed under optimized conditions for identification of a specific gene or
condition. Oligomers may
also be employed under less stringent conditions for detection or quantitation
of closely related DNA or
RNA sequences.
Methods which may also be used to quantify the expression of HJNCT include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from standard
curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244;
Duplaa, C. et al. (1993)
Anal. Biochem. 212:229-236.) The speed of quantitation of multiple samples may
be accelerated by
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running the assay in an ELISA format where the oligomer of interest is
presented in various dilutions and
a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as targets in a
microarray. The microarray can be
used to monitor the expression level of large numbers of genes simultaneously
and to identify genetic
variants, mutations, and polymorphisms. This information may be used to
determine gene function, to
understand the genetic basis of a disorder, to diagnose a disorder, and to
develop and monitor the
activities of therapeutic agents.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) Lf.S. Patent No. 5,605,662.)
In another embodiment of the invention, nucleic acid sequences encoding HJNCT
may be used to
generate hybridization probes useful in mapping the naturally occurring
genomic sequence. The
sequences may be mapped to a particular chromosome, to a specific region of a
chromosome, or to
artificial chromosome constructions, e.g., human artificial chromosomes
(HACs), yeast artificial
chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1
constructions, or single
chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price,
C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet. 7:149-
154.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome
mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al.
(1995) in Meyers, supra, pp.
965-968.) Examples of genetic map data can be found in various scientific
journals or at the Online
Mendelian Inheritance in Man (OMIM) site. Correlation between the location of
the gene encoding
HJNCT 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
4a
CA 02343696 2001-03-22
WO 00/1$915 PCT/US99/22082
techniques. Once the disease or syndrome has been crudely localized by genetic
linkage to a particular
genomic region, e.g., ataxia-telangiectasia to l 1q22-23, any sequences
mapping to that area may
represent associated or regulatory genes for further investigation. (See,
e.g., Gatti, R.A. et al. ( 1988)
Nature 336:577-580.) The nucleotide sequence of the subject invention may also
be used to detect
differences in the chromosomal location due to translocation, inversion, etc.,
among normal, carrier, or
affected individuals.
In another embodiment of the invention, HJNCT, 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 HJNCT and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. ( 1984) PCT application
W084/03564.) In this method, large numbers of different small test compounds
are synthesized on a
solid substrate. The test compounds are reacted with HJNCT, or fragments
thereof, and washed. Bound
HJNCT is then detected by methods well known in the art. Purified HTNCT 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 HJNCT specifically compete with a test compound
for binding HJNCT. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with HJNCT.
In additional embodiments, the nucleotide sequences which encode HJNCT 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. [Attorney Docket No. PF-0590 P, filed September 25,
1998], U.S. Ser No.
[Attorney Docket No. PF-0613 P, filed October 13, 1998], and U.S. Ser. No.
[Attorney Docket No. PF-
CA 02343696 2001-03-22
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0700 P, filed May 4, 1999], are hereby expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was isolated from tissues described in Table 4. For construction of the
LUNGNOT23 and
OVARTUT02 cDNA libraries, the frozen tissue was homogenized and lysed in
TRIZOL reagent (lgm
tissue/10 ml TRIZOL reagent; Life Technologies), a monophasic solution of
phenol and guanidine
isothiocyanate, using a Polytron PT-3000 homogenizes {Brinkman Instruments,
Westbury NY). After a
brief incubation on ice, chloroform was added (1:5 v/v), and the mixture was
centrifuged to separate the
phases. The upper aqueous phase was removed to a fresh tube, and isopropanol
was added to precipitate
RNA. The RNA was resuspended in RNase-free water and treated with DNase. The
RNA was re-
extracted as necessary with acid phenol-chloroform to increase purity, and the
RNA was reprecipitated
with sodium acetate and ethanol. For construction of the BLADNOT04 cDNA
library, the frozen tissue
was homogenized and lysed using a Polytron PT-3000 homogenizes (Brinkmann
Instruments) in
IS guanidinium isothiocyanate solution. The lysate was centrifuged over a 5.7
M CsCI cushion using an
SW28 rotor in an L8-70M ultracentrifuge (Beckman Instruments, Fullerton CA)
for 18 hours at 25,000
rpm at ambient temperature. RNA was extracted with acid phenol pH 4.7,
precipitated using 0.3 M
sodium acetate and 2.5 volumes of ethanol, resuspended in RNAse-free water,
and treated with DNase at
37°C. RNA extraction and precipitation steps were repeated, For
construction of the OVARDIT04
cDNA library, RNA was isolated directly from tissue lysates using other RNA
isolation kits, e.g., the
POLY(A)PURE mRNA purification kit (Ambion, Austin TX), or HPLC.
From each RNA preparation, poly(A+) RNA was isolated using the OLIGOTEX kit
(QIAGEN,
Chatsworth CA). Poly(A+) RNA was used for cDNA synthesis and construction of
each cDNA library
according to the recommended protocols in the SUPERSCRIPT plasmid system (Life
Technologies). The
cDNAs were fractionated on a SEPHAROSE CL4B column (Amersham Pharmacia
Biotech), and those
cDNAs exceeding 400 by were ligated into pINCY (Incyte Pharmaceuticals, Palo
Alto CA).
Recombinant plasmids were transformed into DHSa competent cells or
ElectroMAX~cells (Life
Technologies).
II. Isolation of cDNA Clones
Plasmid DNA was released from the cells and purified using the R.E.A.L. PREP
96 plasmid
purification kit (QIAGEN). The recommended protocol was employed except for
the following changes:
1) the bacteria were cultured in 1 ml of sterile Terrific Broth (Life
Technologies) with carbenicillin at 25
mg/1 and glycerol at 0.4%; 2) after the cultures were incubated for 19 hours,
the cells were lysed with 0.3
ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid
DNA pellets were each
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resuspended in 0.1 ml of distilled water. The DNA samples were stored at
4°C.
III. Sequencing and Analysis
cDNA sequencing reactions were processed using standard methods or high-
throughput
instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) thermal cycler or
the PTC-200 thermal
cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared using
reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing
kits such as the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
Electrophoretic
separation of cDNA sequencing reactions and detection of labeled
polynucleotides were carried out using
the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM
373 or 377
sequencing system (Perkin-Elmer) in conjunction with standard ABI protocols
and base calling software;
or other sequence analysis systems known in the art. Reading frames within the
cDNA sequences were
identified using standard methods (reviewed in Ausubel, 1997, supra, unit
7.7). Some of the cDNA
sequences were selected for extension using the techniques disclosed in
Example V.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilled in the art.
Table 5 summarizes the tools, programs, and algorithms used and provides
applicable descriptions,
references, and threshold parameters. The first column of Table S shows the
tools, programs, and
algorithms used, the second column provides brief descriptions thereof, the
third column presents
appropriate references, all of which are incorporated by reference herein in
their entirety, and the fourth
column presents, where applicable, the scores, probability values, and other
parameters used to evaluate
the strength of a match between two sequences (the higher the score, the
greater the homology between
two sequences). Sequences were analyzed using MACDNASIS PRO software (Hitachi
Software
Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and
polypeptide sequence alignments were generated using the default parameters
specified by the clustal
algorithm as incorporated into the MEGALIGN multisequence alignment program
(DNASTAR), which
also calculates the percent identity between aligned sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA sequences
and by masking ambiguous bases, using algorithms and programs based on BLAST,
dynamic
programing, and dinucleotide nearest neighbor analysis. The sequences were
then queried against a
selection of public databases such as the GenBank primate, rodent, mammalian,
vertebrate, and eukaryote
databases, and BLOCKS to acquire annotation using programs based on BLAST,
FASTA, and BLIMPS.
The sequences were assembled into full length polynucleotide sequences using
programs based on Phred,
Phrap, and Consed, and were screened for open reading frames using programs
based on GeneMark,
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BLAST, and FASTA. The full length polynucleotide sequences were translated to
derive the
corresponding full length amino acid sequences, and these full length
sequences were subsequently
analyzed by querying against databases such as the GenBank databases
(described above), SwissProt,
BLOCKS, PRINTS, Prosite, and Hidden Markov Model (HMM)-based protein family
databases such as
PFAM. HMM is a probabilistic approach which analyzes consensus primary
structures of gene families.
(See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide and
amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ ID NO:S-
8. Fragments from about 20 to about 4000 nucleotides which are useful in
hybridization and
amplification technologies were described in The Invention section above.
IV. Northern Analysis
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a gene
and involves the hybridization of a labeled nucleotide sequence to a membrane
on which RNAs from a
particular cell type or tissue have been bound. (See, e.g., Sambrook, supra,
ch. 7; Ausubel, 1995, supra,
ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in nucleotide databases such as GenBank or LIFESEQ (lncyte
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 BLAST score
100
The product score takes into account both the degree of similarity between two
sequences and the length
of the sequence match. For example, with a product score of 40, the match will
be exact within a 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 i 5 and 40,
although lower scores may
identify related molecules.
The results of northern analyses are reported as a percentage distribution of
libraries in which the
transcript encoding HJNCT occurred. Analysis involved the categorization of
cDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease/condition categories included cancer,
inflammation/trauma, cell
proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
sequence of interest was counted and divided by the total number of libraries
across all categories.
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Percentage values of tissue-specific and disease- or condition-specific
expression are reported in Table 3.
V. Extension of HJNCT Encoding Polynucleotides
The full length nucleic acid sequence of SEQ ID NO:S was 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 extension of an antisense
polynucleotide, and the other
was synthesized to initiate extension of a sense polynucleotide. Primers were
used to facilitate the
extension of the known sequence "outward" generating amplicons containing new
unknown nucleotide
sequence for the region of interest. The initial primers were designed from
the cDNA using OLIGO 4.06
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 target sequence at
temperatures of about 68°C to about 72°C. Any stretch of
nucleotides which would result in hairpin
structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries (Life Technologies) were used to extend the
sequence. If more
than one extension is necessary or desired, additional sets of primers are
designed to further extend the
IS. known region.
High fidelity amplification was obtained by following the instructions for the
XL-PCR kit
(Perkin-Elmer Corp., Norwalk, CT) and thoroughly mixing the enzyme and
reaction mix. PCR was
performed using the PTC-200 thermal cycler (MJ Research, Inc., Watertown, MA),
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 1 min (initial denaturation)
Step 2 65 C for 1 min
Step 3 68 C for 6 min
Step 4 94 C for 15 sec
Step 5 65 C for I min
Step 6 68 C for 7 min
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 min
Step 10 68 C for 7:15 min
Step 1 I Repeat steps 8 through 10 for an additional
12 cycles
Step 12 72 C for 8 min
Step 13 4 C (and holding)
A 5 ~cl to 10 ,ul aliquot of the reaction mixture was analyzed by
electrophoresis on a low
concentration (about 0.6% to 0.8%) agarose mini-gel to determine which
reactions were successful in
extending the sequence. Bands thought to contain the largest products were
excised from the gel, purified
using QIAQUICK DNA gel purification kit (Qiagen, Inc.), and trimmed of
overhangs using Klenow
49
CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
enzyme to facilitate religation and cloning.
After ethanol precipitation, the products were redissolved in 13 ,ul of
ligation buffer, l,ul T4-
DNA ligase (15 units) and 1~I T4 polynucleotide kinase were added, and the
mixture was incubated at
room temperature for 2 to 3 hours, or overnight at 16 ° C. Competent E.
coli cells (in 40 ,ul of appropriate
media) were transformed with 3 ,ul of ligation mixture and cultured in 80 ~l
of SOC medium. (See, e.g.,
Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at
37°C, the E. coli mixture was
plated on Luria Bertani (LB) agar (See, e.g., Sambrook, supra, Appendix A, p.
1 ) containing carbenicillin
(2x carb). The following day, several colonies were randomly picked from each
plate and cultured in 150
,ul of liquid LB/2x carb medium placed in an individual well of an appropriate
commercially-available
sterile 96-well microtiter plate. The following day, S gel of each overnight
culture was transferred into a
non-sterile 96-well plate and, after dilution 1:10 with water, S ~1 from each
sample was transferred into a
PCR array.
For PCR amplification, 18 ~1 of concentrated PCR reaction mix (3.3x)
containing 4 units of rTth
DNA polymerase, a vector primer, and one or both of the gene specific primers
used for the extension
reaction were added to each well. Amplification was performed using the
following conditions:
Step 1 94 C for 60 sec
Step 2 94 C for 20 sec
Step 3 55 C for 30 sec
Step 4 72 C for 90 sec
Step 5 Repeat steps 2 through 4 for an additional
29 cycles
Step 6 72 C for 180 sec
Step 7 4 C (and holding)
Aliquots of the PCR reactions were run on agarose gels together with molecular
weight markers.
The sizes of the PCR products were compared to the original partial eDNAs, and
appropriate clones were
selected, ligated into plasmid, and sequenced.
In like manner, the nucleotide sequence of SEQ ID NO:S is used to obtain 5'
regulatory
sequences using the procedure above, oligonucleotides designed for S'
extension, and an appropriate
genomic library.
The full length nucleic acid sequences of SEQ ID N0:6-8 were produced by
extension of
appropriate fragments of the full length molecules using oligonucleotide
primers designed from these
fragments. Primers were synthesized to initiate either 5' extension of the
known fragments or 3' extension
of the known fragments. 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 SO% 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
CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one extension
was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR was
performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction mix
contained DNA template, 200 nmol of each primer, reaction buffer containing
Mgz', (NH4)zSO4, and J3-
mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE
enzyme (Life
Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer pair PCI
A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 68°C, 2 min; Step
IO S: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, S min; Step 7:
storage at 4°C. In the alternative, the
parameters for primer pair T7 and SK+ were as follows: Step 1: 94°C, 3
min; Step 2: 94°C, 15 sec; Step
3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4
repeated 20 times; Step 6: 68°C, 5 min;
Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 Itl
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE and
0.5 ltl of undiluted PCR product into each well of an opaque fluorimeter plate
(Corning Costar, Acton
MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II (Labsystems
Oy, Helsinki, Finland) to measure the fluorescence of the sample and to
quantify the concentration of
DNA. A 5 ~1 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
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 iigase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham
Pharmacia
Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction
site overhangs, and
transfected into competent E. coli cells. Transformed cells were selected on
antibiotic-containing media,
individual colonies were picked and cultured overnight at 37°C in 384-
well plates in LB/2x carb liquid
media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham
Pharmacia Biotech) and Pfu DNA polyrnerase (Stratagene) with the following
parameters: Step 1: 94°C,
3 min; Step 2: 94°C, IS sec; Step 3: 60°C, 1 min; Step 4:
72°C, 2 min; Step 5: steps 2, 3, and 4 repeated
29 times; Step 6: 72°C, S min; Step 7: storage at 4°C. DNA was
quantified by PICOGREEN reagent
51
CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
(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 N0:6-8 are used to obtain
S' 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 NO:S-8 are employed to screen cDNAs,
genomic
DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about
20 base pairs, is
specifically described, essentially the same procedure is used with larger
nucleotide fragments.
Oligonucleotides are designed using state-of the-art software such as OLIGO
4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ,uCi of
['y-''-P) adenosine
triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase
(DuPont NEN, Boston MA).
The labeled oligonucleotides are substantially purified using a SEPHADEX G-25
supe~ne size exclusion
dextran bead column (Amersham Pharmacia Biotech). An aliquot containing I 0'
counts per minute of the
labeled probe is used in a typical membrane-based hybridization analysis of
human genomic DNA
digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst
1, Xba l, or Pvu II (DuPont
NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16 hours
at 40°C. To remove nonspecific signals, blots are sequentially washed
at room temperature under
increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography and compared.
VII. Microarrays
A chemical coupling procedure and an ink jet device can be used to synthesize
array elements on
the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An array
analogous to a dot or slot blot may
also be used to arrange and link elements to the surface of a substrate using
thermal, UV, chemical, or
mechanical bonding procedures. A typical array may be produced by hand or
using available methods
and machines and contain any appropriate number of elements. After
hybridization, nonhybridized
probes are removed and a scanner used to determine the levels and patterns of
fluorescence. The degree
of complementarity and the relative abundance of each probe which hybridizes
to an element on the
microarray may be assessed through analysis of the scanned images.
52
CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may
comprise the
elements of the microarray. Fragments suitable for hybridization can be
selected using software well
known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs,
ESTs, or fragments
thereof corresponding to one of the nucleotide sequences of the present
invention, or selected at random
from a cDNA library relevant to the present invention, are arranged on an
appropriate substrate, e.g., a
glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking
followed by thermal and
chemical treatments and subsequent drying. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470;
Shalom D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes are
prepared and used for .
hybridization to the elements on the substrate. The substrate is analyzed by
procedures described above.
VIII. Complementary Polynucleotides
Sequences complementary to the HJNCT-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring HJNC;T.
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 HJNCT. To inhibit
transcription, a
complementary oligonucleotide is designed from the most unique .5' sequence
and used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is
designed to prevent ribosomal binding to the HJNCT-encoding transcript.
1x. Expression of HJNCT
Expression and purification of HJNCT is achieved using bacterial or virus-
based expression
systems. For expression of HJNCT 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 HJNCT upon induction with isopropyl beta-D-thiogalactopyranoside
(IPTG). Expression of
HJNCT in eukaryotic cells is achieved by infecting insect or mammalian cell
lines with recombinant
Autosraphica californica nuclear polyhedrosis virus (AcMNPV), commonly known
as baculovirus. The
nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding
HJNCT by either
homologous recombination or bacterial-mediated transposition involving
transfer plasmid intermediates.
Viral infectivity is maintained and the strong polyhedrin promoter drives high
levels of cDNA
transcription. Recombinant baculovirus is used to infect Spodoptera fru~iperda
(Sf9) insect cells in most
cases, or human hepatocytes, in some cases. Infection of the latter requires
additional genetic
modifications to baculovirus. (See Engelhard, E.K. et al. ( 1994) Proc. Natl.
Acad. Sci. USA 91:3224-
53
CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.)
In most expression systems, HJNCT is synthesized as a fusion protein with,
e.g., glutathione S-
transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step, affinity-
based purification of recombinant fusion protein from crude cell lysates. GST,
a 26-kilodalton enzyme
from Schistosoma japonicum, enables the purification of fusion proteins on
immobilized glutathione
under conditions that maintain protein activity and antigenicity (Amersham
Pharmacia Biotech).
Following purification, the GST moiety can be proteolytically cleaved from
HJNCT 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 ( I
995, supra, ch 10 and 16).
Purified HJNCT obtained by these methods can be used directly in the following
activity assay.
X. Demonstration of HJNCT Activity
An assay for HJNCT-1 activity measures the PDZ-induced clustering of
transmembrane
1S receptors (Ponting, supra). Cultured cell lines are cotransfected with cDNA
encoding HJNCT-1 and
either EGF receptor or NMDA receptor. Control cell lines are transfected with
only one of the above
cDNAs. Clustering of EGF receptors or NMDA receptors in the cotransfected cell
lines is detected and
quantified using commercially available antibody specific to these receptors
in conjunction with indirect
immunofluorescence and image analysis systems. The amount of receptor
clustering is directly
proportional to the amount of HJNCT-1 activity.
HJNCT-2 activity is demonstrated by its ability to bind GM130 in an in vitro
assay (Barr (1998)
su ra . Coupled in vitro transcription-translation reactions are carried out
with plasmids encoding
HJNCT-2 and GM130 in the presence of radiolabeled ['SS]-methionine.
Immunoprecipitations are
performed on the in vitro transcription-translation reaction with antibodies
to HJNCT-2. The
2S immunoprecipitated material is analyzed by sodium dodecyl sulfate
polyacrylamide gel electrophoresis
and autoradiography to identify both HJNCT-2 and GM130 in the
immunoprecipitates. The GM130 spot
is cut out and counted in a radioisotope counter. The amount of radioactivity
recovered is proportional to
the activity of HJNCT-2 in the sample.
An assay for HJNCT-3 activity measures the ability of HJNCT-3 to induce the
formation of tight
junction strands when expressed in mouse fibroblasts (Furuse, M. et al. (1998)
J. Cell Biol. 143:391-401).
cDNA encoding HJNCT-3 is subcloned into a mammalian expression vector and
transfected into mouse L
fibroblasts. The transfected cells are analyzed by freeze-fracture electron
microscopy after
glutaraldehyde fixation and compared to untransfected cells. The presence of
networks of strands and
grooves at cell contact sites in transfected as compared to control cells is
indicative of HJNCT-3 activity.
S4
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An assay for HJNCT-4 activity measures the disruption of cytoskeletal filament
networks upon
overexpression of HJNCT-4 in cultured cell lines (Rezniczek, G.A. et al.
(1998) J. Cell Biol. 141:209-
225). cDNA encoding HJNCT-4 is subcloned into a mammalian expression vector
that drives high levels
of cDNA expression. This construct is transfected into cultured cells, such as
rat kangaroo PtIC2 or rat
bladder carcinoma 8046 cells. Actin filaments and intermediate filaments such
as keratin and vimentin
are visualized by immunofluorescence microscopy using antibodies and
techniques well known in the art.
The configuration and abundance of cyoskeletal filaments can be assessed and
quantified using confocal
imaging techniques. In particular, the appearance of cell surface projections
is indicative of HJNCT-4
activity.
XI. Functional Assays
HJNCT function is assessed by expressing the sequences encoding HJNCT at
physiologically
elevated levels in mammalian cell culture systems. eDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of eDNA
expression. Vectors of choice
include pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen, Carlsbad CA),
both of which
contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are
transiently transfected into a
human cell line, preferably of endothelial or hematopoietic origin, using
either liposome formulations or
electroporation. 1-2 beg of an additional plasmid containing sequences
encoding a marker protein are co-
transfected. Expression of a marker protein provides a means to distinguish
transfected cells from
nontransfected cells and is a reliable predictor of cDNA expression from the
recombinant vector. Marker
proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech),
CD64, or a CD64-GFP
fusion protein. Flow cytometry (FCM), an automated, laser optics-based
technique, is used to identify
transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic
state of the cells and other
cellular properties. F'CM 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 Cvtometry, Oxford,
New York NY.
The influence of HJNCT on gene expression can be assessed using highly
purified populations
of cells transfected with sequences encoding HJNCT and either CD64 or CDb4-
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
SS
CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success NY).
mRNA can be purified from the cells using methods well known by those of skill
in the art. Expression
of mRNA encoding HJNCT and other genes of interest can be analyzed by northern
analysis or
microarray techniques.
XII. Production of HJNCT Specific Antibodies
HJNCT 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 HJNCT amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides IS residues in length are synthesized using an ABI
431A peptide
synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH (Sigma-
Aldrich, St. Louis MO)
by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to
increase immunogenicity.
(See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with the oligopeptide-
ICLH complex in
complete Freund's adjuvant. Resulting antisera are tested for antipeptide
activity 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.
XI)CI. Purification of Naturally Occurring HJNCT Using Specific Antibodies
Naturally occurring or recombinant HJNCT is substantially purified by
immunoaffinity
chromatography using antibodies. specific for HJNCT. An immunaati3nity column
is constructed by
covalently coupling anti-HJNCT 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 HJNCT are passed over the immunoaffinity column, and the
column is washed
under conditions that allow the preferential absorbance of HJNCT (e.g., high
ionic strength buffers in the
presence of detergent). The column is eluted under conditions that disrupt
antibody/HJNCT 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
HJNCT is collected.
XIV. Identification of Molecules Which Interact with HJNCT
HJNCT, or biologically active fragments thereof, are labeled with'ZSI Bolton-
Hunter reagent.
(See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
56
CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
previously arrayed in the wells of a multi-well plate are incubated with the
labeled HJNCT, washed, and
any wells with labeled HTNCT complex are assayed. Data obtained using
different concentrations of
HJNCT are used to calculate values for the number, affinity, and association
of HJNCT with the
candidate molecules.
Various modifications and variations of the described methods and systems of
the invention will
be apparent to those skilled in the art without departing from the scope and
spirit of the invention.
Although the invention has been described in connection with specific
preferred embodiments, it should
be understood that the invention as claimed should not be unduly limited to
such specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which are obvious to
those skilled in molecular biology or related fields are intended to be within
the scope of the following
claims.
57
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CA 02343696 2001-03-22
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CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
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CA 02343696 2001-03-22
WO 00/18915 PCTNS99/22082
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CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
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CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
YUE, Henry
T,AT,, Preeti
CORLEY, Neil C.
GUEGLER, Karl J.
BAUGHN, Mariah R.
LU, Aina D.
TANG, Y. Tom
<120> MEMBRANE-ASSOCIATED ORGANIZATIONAL PROTEINS
<130> PF-0590 PCT
<140> To Be Assigned
<141> Herewith
<150> 09/161,043; unassigned; 09/172,189; unassigned; 09/305,329;
unassigned
<151> 1998-09-25; 1998-09-25; 1999-10-13; 1999-10-13; 1999-05-04;
1999-05-04
<160> 12
<170> PERL Program
<210> 1
<211> 361
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2687924CD1
<400> 1
Met Ser Thr Ala Arg Glu Gln Pro Ile Phe Ser Thr Arg Ala His
1 5 10 15
Val Phe Gln Ile Asp Pro Ala Thr Lys Arg Asn Trp Ile Pro Ala
20 25 30
Gly Lys His Ala Leu Thr Val Ser Tyr Phe Tyr Asp Ala Thr Arg
35 40 45
Asn Val Tyr Arg Ile Ile Ser Ile Gly Gly Ala Lys Ala Ile Ile
50 55 60
Asn Ser Thr Val Thr Pro Asn Met Thr Phe Thr Lys Thr Ser Gln
65 70 75
Lys Phe Gly Gln Trp Ala Asp Ser Arg Ala Asn Thr Val Tyr Gly
80 85 90
Leu Gly Phe Ala Ser Glu Gln His Leu Thr Gln Phe Ala Glu Lys
95 100 105
Phe Gln Glu Val Lys Glu Ala Ala Arg Leu Ala Arg Glu Lys Ser
110 115 120
Gln Asp Gly Gly Glu Leu Thr Ser Pro Ala Leu Gly Leu Ala Ser
1/11
CA 02343696 2001-03-22
WO 00/18915 PCTNS99/22082
125 130 135
His Gln Val Pro Pro Ser Pro Leu Val Ser Ala Asn Gly Pro Gly
140 145 150
Glu Glu Lys Leu Phe Arg Ser Gln Ser Ala Asp Ala Pro Gly Pro
155 160 165
Thr Glu Arg Glu Arg Leu Lys Lys Met Leu Ser Glu Gly Ser Val
170 175 180
Gly Glu Val Gln Trp Glu Ala Glu Phe Phe Ala Leu Gln Asp Ser
185 190 195
Asn Asn Lys Leu Ala Gly Ala Leu Arg Glu Ala Asn Ala Ala Ala
200 205 210
Ala Gln Trp Arg Gln Gln Leu Glu Ala Gln Arg Ala Glu Ala Glu
215 220 225
Arg Leu Arg Gln Arg Val Ala Glu Leu Glu Ala G1n Ala Ala Ser
230 235 240
Glu Val Thr Pro Thr Gly Glu Lys Glu Gly Leu Gly Gln Gly Gln
245 250 255
Ser Leu Glu Gln Leu Glu Ala Leu Val Gln Thr Lys Asp Gln Glu
260 265 270
Ile Gln Thr Leu Lys Ser Gln Thr Gly Gly Pro Arg Glu Ala Leu
275 280 285
Glu Ala Ala Glu Arg Glu Glu Thr Gln Gln Lys Val Gln Asp Leu
290 295 300
Glu Thr Arg Asn Ala Glu Leu Glu His Gln Leu Arg Ala Met Glu
305 310 315
Arg Ser Leu Glu Glu Ala Arg Ala Glu Arg Glu Arg Ala Arg Ala
320 325 330
Glu Val Gly Arg Ala Ala Gln Leu Leu Asp Val Arg Leu Phe Glu
335 340 345
Leu Ser Glu Leu Arg Glu Gly Leu Ala Arg Leu Ala Glu Ala Ala
350 355 360
Pro
<210> 2
<211> 300
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1320134CD1
<400> 2
Met Arg Phe Ser Gln Ser Glu Asp Phe Phe Thr Leu Ile Glu Ser
1 5 10 15
His Glu Gly Lys Pro Leu Lys Leu Met Val Tyr Asn Ser Lys Ser
20 25 30
Asp Ser Cys Arg Glu Val Thr Val Thr Pro Asn Ala Ala Trp Gly
35 40 45
Gly Glu Gly Ser Leu Gly Cys Gly Ile Gly Tyr Gly Tyr Leu His
50 55 60
Arg Ile Pro Thr Gln Pro Pro Ser Tyr His Lys Lys Pro Pro Gly
65 70 75
Thr Pro Pro Pro Ser Ala Leu Pro Leu Gly Ala Pro Pro Pro Asp
2/11
CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
80 85 90
Ala Leu Pro Pro Gly Pro Thr Pro Glu Asp Ser Pro Ser Leu Glu
95 100 105
Thr Gly Ser Arg Gln Ser Asp Tyr Met Glu Ala Leu Leu Gln Ala
110 115 120
Pro Gly Ser Ser Met Glu Asp Pro Leu Pro Gly Pro Gly Ser Pro
125 130 135
Ser His Ser Ala Pro Asp Pro Asp Gly Leu Pro His Phe Met Glu
140 145 150
Thr Pro Leu Gln Pro Pro Pro Pro Val Gln Arg Val Met Asp Pro
155 160 165
Gly Phe Leu Asp Val Ser Gly Ile Ser Leu Leu Asp Asn Ser Asn
170 175 180
Ala Ser Val Trp Pro Ser Leu Pro Ser Ser Thr Glu Leu Thr Thr
185 190 195
Thr Ala Val Ser Thr Ser Gly Pro Glu Asp Ile Cys Ser Ser Ser
200 205 210
Ser Ser His Glu Arg Gly Gly Glu Ala Thr Trp Ser Gly Ser Glu
215 220 225
Phe Glu Val Ser Phe Leu Asp Ser Pro Gly Ala Gln Ala Gln Ala
230 235 240
Asp His Leu Pro Gln Leu Thr Leu Pro Asp Ser Leu Thr Ser Ala
245 250 255
Ala Ser Pro Glu Asp Gly Leu Ser Ala Glu Leu Leu Glu Ala Gln
260 265 270
Ala Glu Glu Glu Pro Ala Ser Thr Glu Gly Leu Asp Thr Gly Thr
275 280 285
Glu Ala Glu Gly Leu Asp Ser Gln Ala Gln Ile Ser Thr Thr Glu
290 295 300
<210> 3
<211> 230
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2594049CD1
<400> 3
Met Ala Ser Leu Gly Leu Gln Leu Val Gly Tyr Ile Leu Gly Leu
1 5 10 15
Leu Gly Leu Leu Gly Thr Leu Val Ala Met Leu Leu Pro Ser Trp
20 25 30
Lys Thr Ser Ser Tyr Val Gly Ala Ser Ile Val Thr Ala Val Gly
35 40 45
Phe Ser Lys Gly Leu Trp Met Glu Cys Ala Thr His Ser Thr Gly
50 55 60
Ile Thr Gln Cys Asp Ile Tyr Ser Thr Leu Leu Gly Leu Pro Ala
65 70 75
Asp Ile Gln Ala Ala Gln Ala Met Met Val Thr Ser Ser Ala Ile
80 85 90
Ser Ser Leu Ala Cys Ile Ile Ser Val Val Gly Met Arg Cys Thr
95 100 105
3/11
CA 02343696 2001-03-22
WO 00/18915 PCTlUS99/22082
Val Phe Cys Gln Glu Ser Arg Ala Lys Asp Arg Val Ala Val Ala
110 115 120
Gly Gly Val Phe Phe Ile Leu Gly Gly Leu Leu Gly Phe Ile Pro
125 130 135
Val Ala Trp Asn Leu His Gly Ile Leu Arg Asp Phe Tyr Ser Pro
140 145 150
Leu Val Pro Asp Ser Met Lys Phe Glu Ile Gly Glu Ala Leu Tyr
155 160 165
Leu Gly Ile Ile Ser Ser Leu Phe Ser Leu Ile Ala Gly Ile Ile
170 175 180
Leu Cys Phe Ser Cys Ser Ser Gln Arg Asn Arg Ser Asn Tyr Tyr
185 190 195
Asp Ala Tyr Gln Ala Gln Pro Leu Ala Thr Arg Ser Ser Pro Arg
200 205 210
Pro Gly Gln Pro Pro Lys Val Lys Ser Glu Phe Asn Ser Tyr Ser
215 220 225
Leu Thr Gly Tyr Val
230
<210> 4
<211> 292
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5139028CD1
<400> 4
Met Ser Ser Leu Tyr Pro Ser Leu Glu Asp Leu Lys Val Asp Gln
1 5 10 15
Ala Ile Gln Ala Gln Val Arg Ala Ser Pro Lys Met Pro Ala Leu
20 25 30
Pro Val Gln Ala Thr Ala Ile Ser Pro Pro Pro Val Leu Tyr Pro
35 40 45
Asn Leu Ala Glu Leu Glu Asn Tyr Met Gly Leu Ser Leu Ser Ser
50 55 60
Gln Glu Val Gln Glu Ser Leu Leu Gln Ile Pro Glu Gly Asp Ser
65 70 75
Thr Ala Val Ser Gly Pro Gly Pro Gly Gln Met Val Ala Pro Val
80 85 90
Thr Gly Tyr Ser Leu Gly Val Arg Arg Ala Glu Ile Lys Pro Gly
95 100 105
Val Arg Glu Ile His Leu Cys Lys Asp Glu Arg Gly Lys Thr Gly
110 115 120
Leu Arg Leu Arg Lys Val Asp Gln Gly Leu Phe Val Gln Leu Val
125 130 135
Gln Ala Asn Thr Pro Ala Ser Leu Val Gly Leu Arg Phe Gly Asp
140 145 150
Gln Leu Leu Gln Ile Asp Gly Arg Asp Cys Ala Gly Trp Ser Ser
155 160 165
His Lys Ala His Gln Val Val Lys Lys Ala Ser Gly Asp Lys Ile
170 175 180
Val Met Val Val Arg Asp Arg Pro Phe Gln Arg Thr Val Thr Met
4/11
CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
185 190 195
His Lys Asp Ser Met Gly His Val Gly Phe Val Ile Lys Lys Gly
200 205 210
Lys Ile Val Ser Leu Val Lys Gly Ser Ser Ala Ala Cys Asn Gly
215 220 225
Leu Leu Thr Asn His Tyr Val Cys Glu Val Asp Gly Gln Asn Val
230 235 240
Ile Gly Leu Lys Asp Lys Lys Ile Met Glu Ile Leu Ala Thr Ala
245 250 255
Gly Asn Val Val Thr Leu Thr Ile Ile Pro Ser Val Ile Tyr Glu
260 265 270
His Met Val Lys Lys Leu Pro Pro Val Leu Leu His His Thr Met
275 280 285
Asp His Ser Ile Pro Asp Ala
290
<210> 5
<211> 1501
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2687924CB1
<400> 5
gcacgaaggg agtccccgga gttaccgacc tggggttgga ggaggcagaa gaggtgtccc 60
ccgaagttct ggacccagag acagggggcg tcgagggaat ctcggaagtt ccagggggcc 120
aaaccagtgc tcctgccacc tctctggctg ccccctagag cctgcccatc ccagcctgac 180
caatgtccac agccagggag cagccaatct tcagcacacg ggcgcacgtg ttccaaattg 240
acccagccac caagcgaaac tggatcccag cgggcaagca cgcactcact gtctcctatt 300
tctacgatgc cacccgcaat gtgtaccgca tcatcagcat cggaggcgcc aaggccatca 360
tcaacagcac tgtcactccc aacatgacct tcaccaaaac ttcccagaag ttcgggcagt 420
gggccgacag tcgcgccaac acagtctacg gcctgggctt tgcctctgaa cagcatctga 480
cacagtttgc cgagaagttc caggaagtga aggaagcagc caggctggcc agggagaaat 540
ctcaggatgg cggggagctc accagtccag ccctggggct cgcctcccac caggtgcccc 600
cgagccctct cgtcagtgcc aacggccccg gcgaggaaaa actgttccgc agccagagcg 660
ctgatgcccc cggccccaca gagcgcgagc ggctaaagaa gatgttgtct gagggctccg 720
tgggcgaggt acagtgggag gccgagtttt tcgcactgca ggacagcaac aacaagctgg 780
caggcgccct gcgagaggcc aacgccgccg cagcccagtg gaggcagcag ctggaggctc 840
agcgtgcaga ggccgagcgg ctgcggcagc gggtggctga gctggaggct caggcagctt 900
cagaggtgac ccccaccggt gagaaggagg ggctgggcca gggccagtcg ctggaacagc 960
tggaagctct ggtgcaaacc aaggaccagg agattcagac cctgaagagt cagactgggg 1020
ggccccgcga ggccctggag gctgccgagc gtgaggagac tcagcagaag gtgcaggacc 1080
tggagacccg caatgcggag ttggagcacc agctgcgggc gatggagcgc agcctggagg 1140
aggcacgggc agagcgggag cgggcgcggg ctgaggtggg ccgggcagcg cagctgctgg 1200
acgtcaggct gtttgagctg agtgagctgc gtgagggcct ggcccgcctg gctgaggctg 1260
cgccctgagc cggggctggt tttctatgaa cgattccggc ctgggatgcg ggccaggctg 1320
caggcggcat agttgggccc attcgtcctg gaaagggact ggggggtccc aacttagccc 1380
tgggtgggcc gggccgggct gggctggggt gggccccagt cggctctggt tgttggcagc 1440
tttggggctg tttttgagct tctcattgtg tagaatttct agatcccccg attacatttc 1500
g 1501
5/11
CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
<210> 6
<211> 1039
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1320134CB1
<400> 6
ctggatgtgg tgtgaagaga agggtgctgt ccatgagaag tgtctggttt tgttgaggct 60
ttagccctgg aagcaggcct caggtgctat gcgtttctca cagtccgagg acttctttac 120
gctcatcgag tctcatgagg ggaagccctt gaagctgatg gtgtataact ccaagtcaga 180
ctcctgccgg gaggtgactg taactcccaa cgcagcctgg ggtggagagg gcagtctggg 240
atgtggcatt ggctatgggt atctacaccg gatcccaact cagcccccca gctaccacaa 300
gaagccacct ggcaccccac caccttctgc tctaccactt ggtgccccac cacctgatgc 360
tctaccacct ggacccaccc ccgaggactc tccttccctg gagacaggtt ccaggcagag 420
tgactacatg gaggccctgc tgcaggcacc tggctcctcc atggaggatc cccttcctgg 480
gcctgggagt cccagccaca gtgctccaga ccctgatgga cttccccatt tcatggagac 540
tcctcttcag cccccacctc cagtgcagcg agttatggac ccaggcttcc tggacgtgtc 600
gggaatttct ctcttggaca acagcaatgc cagtgtgtgg cccagcctgc cctcttccac 660
agaactgacc accacagctg tctcaacctc agggccagag gacatctgct ccagcagcag 720
ttctcatgag cggggtggtg aggctacatg gtctgggtca gagtttgagg tctccttcct 780
ggacagccca ggtgcccaag cccaggcgga ccacctgcct cagctgactc ttcctgacag 840
tctcacctct gcagcctcac cagaagatgg gctgtccgcc gagctgcttg aagctcaggc 900
tgaggaggaa ccagcaagca cagagggcct agatactggg acggaggctg aggggctgga 960
cagccaggcc cagatctcta ccacagaata acaccctggg ctgtgacaag gcccatgatg 1020
acatttcatg aggcccaga 1039
<210> 7
<211> 2742
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2594049CB1
<400> 7
agaagtcagc ctggcagaga gactctgaaa tgagggatta gaggtgttca aggagcaaga 60
gcttcagcct gaagacaagg gagcagtccc tgaagacgct tctactgaga ggtctgccat 120
ggcctctctt ggcctccaac ttgtgggcta catcctaggc cttctggggc ttttgggcac 180
actggttgcc atgctgctcc ccagctggaa aacaagttct tatgtcggtg ccagcattgt 240
gacagcagtt ggcttctcca agggcctctg gatggaatgt gccacacaca gcacaggcat 300
cacccagtgt gacatctata gcacccttct gggcctgccc gctgacatcc aggctgccca 360
ggccatgatg gtgacatcca gtgcaatctc ctccctggcc tgcattatct ctgtggtggg 420
catgagatgc acagtcttct gccaggaatc ccgagccaaa gacagagtgg cggtagcagg 480
tggagtcttt ttcatccttg gaggcctcct gggattcatt cctgttgcct ggaatcttca 540
tgggatccta cgggacttct actcaccact ggtgcctgac agcatgaaat ttgagattgg 600
agaggctctt tacttgggca ttatttcttc cctgttctcc ctgatagctg gaatcatcct 660
ctgcttttcc tgctcatccc agagaaatcg ctccaactac tacgatgcct accaagccca 720
acctcttgcc acaaggagct ctccaaggcc tggtcaacct cccaaagtca agagtgagtt 780
caattcctac agcctgacag ggtatgtgtg aagaaccagg ggccagagct ggggggtggc 840
tgggtctgtg aaaaacagtg gacagcaccc cgagggccac aggtgaggga cactaccact 900
6/11
CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
ggatcgtgtc agaaggtgct gctgaggata gactgacttt ggccattgga ttgagcaaag 960
gcagaaatgg gggctagtgt aacagcatgc aggttgaatt gccaaggatg ctcgccatgc 1020
cagcctttct gttttcctca ccttgctgct cccctgccct aagtccccaa ccctcaactt 1080
gaaaccccat tcccttaagc caggactcag aggatccctt tgccctctgg tttacctggg 1140
actccatccc caaacccact aatcacatcc cactgactga ccctctgtga tcaaagaccc 1200
tctctctggc tgaggttggc tcttagctca ttgctgggga tgggaaggag aagcagtggc 1260
ttttgtgggc attgctctaa cctacttctc aagcttccct ccaaagaaac tgattggccc 1320
tggaacctcc atcccactct tgttatgact ccacagtgtc cagactaatt tgtgcatgaa 1380
ctgaaataaa accatcctac ggtatccagg gaacagaaag caggatgcag gatgggagga 1440
caggaaggca gcctgggaca tttaaaaaaa taaaaatgaa aaaaaaaccc agaacccatt 1500
tctcagggca ctttccagaa ttctctcata tttgtgggct gggatcaagc ctgcagcttg 1560
aggaaagcac aaggaaagga aagaagatct ggtggaaagc tcaggtggca gcggactctg 1620
actccactga ggaactgcct cagaagctgc gatcacaact ttggctgaag cccctgcctc 1680
actctagggc acctgacctg gcctcttgcc taaaccacaa ggctaagggc tatagacaat 1740
ggtttcctta ggaacagtaa accagttttt ctagggatgg cccttggctg ggggatgaca 1800
gtgtgggagc tgtggggtac tgaggaagac accattcctt gacggtgtct aagaagccag 1860
gtggatgtgt gtggtggctc cagtgggtgt ttctactctg ccagtgagag gcagccccct 1920
agaaactctt caggcgtaat ggaaaatcag ctcaaatgag atcaggcccc cccagggtcc 1980
acccacagag cactacagag cctctgaaag accatagcac caagcgagcc ccttcagatt 2040
cccccactgt ccatcggaag atgctccaga gtggctagag ggcatctaag ggctccagca 2100
tggcatatcc atgcccacgg tgctgtgtcc atgatctgag tgatagctgc actgctgcct 2160
gggattgcag ctgaggtggg agtggagaat ggttcccagg aagacagttc cacctctaag 2220
gtccgaaaat gttcccttta ccctggagtg ggagtgaggg gtcatacacc aaaggtattt 2280
tccctcacca gtctaggcat gactggcttc tgaaaaattc cagcacacct cctcgaacct 2340
cattgtcagc agagagggcc catctgttgt ctgtaacatg cctttcacat gtccaccttc 2400
ttgccatgtt ccagctgctc tcccaacctg gaaggccgtc tccccttagc caagtcctcc 2460
tcaggcttgg agaacttcct cagcgtcacc tccttcattg agccttctct gatcactcca 2520
tccctctcct acccctccct cccccaaccc tcaatgtata aattgcttct tgatgcttag 2580
cattcacaat ttttgattga tcgttatttg tgtgtgtgtg tccgatctca caagtatatt 2640
gtaaaccctt cggtgggtgg gggccatatc ctagacctct ctgtatcccc cagactatct 2700
gtaacagtgc caggcaccag aaggtgatca ataattcgta gc 2742
<210> 8
<211> 1473
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5139028CB1
<400> 8
ggaagccagc tgagagacag ggctacgttt cagggaggga aacagattca gcagcggcag 60
cagctggaga aggtcgtgga gcagcacctt gcctgcaggg tgttctgaga atcagccatg 120
tcatccctgt acccatctct agaggaccta aaagtggacc aagccattca ggcccaggtc 180
agagcctcac ccaagatgcc agccctgcca gtccaggcaa cagccatttc cccaccacca 240
gttttgtacc caaacttggc agaactggaa aattatatgg gtctttccct ctccagccaa 300
gaagtccagg agagcctgct tcagattcca gagggtgaca gtacagcggt ctcgggcccc 360
gggcccggcc agatggtggc accggtaacc gggtacagcc tgggcgtgcg gcgagctgag 420
atcaagcccg gggtgcgcga gatccacctg tgcaaggacg agcgcggcaa gaccgggctg 480
aggctgcgga aggtcgacca ggggctcttt gtgcagttgg tccaggccaa cacccctgca 540
tcccttgtgg ggctgcgctt tggggaccag ctcctgcaga ttgacgggcg tgactgtgct 600
gggtggagct cgcacaaagc ccatcaggtg gtgaagaagg catcaggcga taagattgtc 660
atggtggttc gggacaggcc gttccagcgg actgtcacca tgcacaagga cagcatgggc 720
7/11
CA 02343696 2001-03-22
WO 00/18915 PCTNS99/22082
rc-v,mv rv..i
cacgtcggct tcgtgatcaa gaaggggaag attgtctctc tggtcaaagg gagttctgcg 780
gcctgcaacg ggctcctcac caaccactac gtgtgtgagg tggacgggca gaatgttatc 840
gggctgaagg acaaaaagat catggagatt ctggccacgg ctgggaacgt tgtcaccctg 900
accatcatcc ccagtgtgat ctacgagcac atggtcaaaa agttgcctcc agtcctgctc 960
caccacacca tggaccactc catcccagat gcctgaagcc actgcagggc agggcaggca 1020
gggggggctt cccgccctcc tgcagcaaag ggcaaccacc ctcggatgat gggttgcagc 1080
cggcctgctg cttaaggtgg gggctgccat gaggggggcg tgtccaggag ggtgaccatg 1140
ggatggctta tacacacagg cctccttgga gcctcagact ccaagctagg ctgaggctca 1200
ggcagggccc acaggcagcc gattctcttg tgctgattta aatgctggac acggaggcag 1260
gctgtttaaa cgctgcttaa agtcgcaact gggccccttt caagaaattt tgctctacca 1320
ggaaaacagt tacacatttt aagagaacag agctacgttc tttgtgagag ctttttcctt 1380
gccttgactt gctctttgtc acagactgca taagttgtca gccttgacta tcttttgaat 1440
aaagatttga ttttaaacaa aaaaaaaaaa aaa 1473
<210> 9
<211> 189
<212> PRT
<213> Rattus norvegicus
<300>
<308> GenBank ID No: g1913909
<400> 9
Met Gly Lys Met Gly Glu Gln Pro Ile Phe Ser Thr Arg Ala His
1 5 10 15
Val Phe Gln Ile Asp Pro Asn Thr Lys Lys Asn Trp Val Pro Thr
20 25 30
Ser Lys His Ala Val Thr Val Ser Tyr Phe Tyr Asp Ser Thr Arg
35 40 45
Asn Val Tyr Arg Ile Ile Ser Leu Asp Gly Ser Lys Ala Ile Ile
50 55 60
Asn Ser Thr Ile Thr Pro Asn Met Thr Phe Thr Lys Thr Ser Gln
65 ~ 70 75
Lys Phe Gly Gln Trp Ala Asp Ser Arg Ala Asn Thr Val Tyr Gly
80 85 90
Leu Gly Phe Ser Ser Glu His His Leu Ser Lys Phe Ala Glu Lys
95 100 105
Phe Gln Glu Phe Lys Glu Ala Ala Arg Leu Ala Lys Glu Lys Ser
110 115 120
Gln Glu Lys Met Glu Leu Thr Ser Thr Pro Ser Gln Glu Ser Ala
125 130 135
Gly Gly Asp Leu Gln Ser Pra Leu Thr Pro Glu Ser Ile Asn Gly
140 145 150
Thr Asp Asp Glu Arg Thr Pro Asp Val Thr Gln Asn Ser Glu Pro
155 160 165
Arg Ala Glu Pro Ala Gln Asn Ala Leu Pro Phe Ser His Arg Tyr
170 175 180
Thr Phe Asn Ser Ala Ile Met Ile Lys
185
8/11
CA 02343696 2001-03-22
WO 00/18915 PCT/US99/2Z082
<210> 10
<211> 451
<212> PRT
<213> Rattus norvegicus
<300>
<308> GenBank ID No: 84432587
<400> 10
Met Gly Leu Gly Ala Ser Ser Glu Gln Pro Ala Gly Gly Glu Gly
1 5 10 15
Phe His Leu His Gly Val Gln Glu Asn Ser Pro Ala Gln Gln Ala
20 25 30
Gly Leu Glu Pro Tyr Phe Asp Phe Ile Ile Thr Ile Gly His Ser
35 40 45
Arg Leu Asn Lys Glu Asn Asp Thr Leu Lys Ala Leu Leu Lys Ala
50 55 60
Asn Val Glu Lys Pro Val Lys Leu Glu Val Phe Asn Met Lys Thr
65 70 75
Met Arg Val Arg Glu Val Glu Val Val Pro Ser Asn Met Trp Gly
80 85 90
Gly Gln Gly Leu Leu Gly Ala Ser Val Arg Phe Cys Ser Phe Arg
95 100 105
Arg Ala Ser Glu His Val Trp His Val Leu Asp Val Glu Pro Ser
110 115 120
Ser Pro Ala Ala Leu Ala Gly Leu Arg Pro Tyr Thr Asp Tyr Ile
125 130 135
Val Gly Ser Asp Gln Ile Leu Gln Glu Ser Glu Asp Phe Phe Thr
140 145 150
Leu Ile Glu Ser His Glu Gly Lys Pro Leu Lys Leu Met Val Tyr
155 160 165
Asn Ser Glu Ser Asp Ser Cys Arg Glu Val Thr Val Thr Pro Asn
170 175 180
Ala Ala Trp Gly Gly Glu Gly Ser Leu Gly Cys Gly Ile Gly Tyr
185 190 195
Gly Tyr Leu His Arg Ile Pro Thr Gln Pro Sex Ser Gln Tyr Lys
200 205 210
Lys Pro Pro Ser Ala Ser Ser Pro Gly Thr Pro Ala Lys Thr Pro
215 220 225
Gln Pro Asn Ala Phe Pro Leu Gly Ala Pro Pro Pro Trp Pro Ile
230 235 240
Pro Gln Asp Ser Ser Gly Pro Glu Leu Gly Ser Arg Gln Ser Asp
245 250 255
Tyr Met Glu Ala Leu Pro Gln Val Pro Gly Gly Phe Met Glu Glu
260 265 270
Gln Leu Pro Gly Pro Gly Ser Pro Gly His Gly Thr Ala Asp Tyr
275 280 285
Gly Gly Cys Leu His Ser Met Glu Ile Pro Leu Gln Pro Pro Pro
290 295 300
Pro Val Gln Arg Val Met Asp Pro Gly Phe Leu Asp Val Ser Gly
305 310 315
Met Ser Leu Leu Asp Ser Asn Asn Thr Ser Val Cys Pro Ser Leu
320 325 330
Ser Ser Ser Ser Leu Leu Thr Pro Thr Ala Val Ser Ala Leu Gly
335 340 345
9/11
CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
Pro Glu Asp Ile Gly Ser Ser Ser Ser Ser His Glu Arg Gly Gly
350 355 360
Glu Ala Thr Trp Ser Gly Ser Glu Phe Glu Ile Ser Phe Pro Asp
365 370 375
Ser Pro Gly Ser Gln Ala Gln Val Asp His Leu Pro Arg Leu Thr
380 385 390
Leu Pro Asp Gly Leu Thr Ser Ala Ala Ser Pro Glu Glu Gly Leu
395 400 405
Ser Ala Glu Leu Leu Glu Ala Gln Thr Glu Glu Pro Ala His Thr
410 415 420
Ala Ser Leu Asp Cys Met Ala Gln Thr Glu Gly Pro Ala Gly Gln
425 430 435
Val Gln Ala Ala Pro Asp Pro Glu Pro Gly Leu Cys Glu Gly Pro
440 445 450
Trp
<210> 11
<211> 230
<212> PRT
<213> Mus musculus
<300>
<308> GenBank ID No: 83335184
<400> 11
Met Ala Ser Leu Gly Val Gln Leu Val Gly Tyr Ile Leu Gly Leu
1 5 10 15
Leu Gly Leu Leu Gly Thr Ser Ile Ala Met Leu Leu Pro Asn Trp
20 25 30
Arg Thr Ser Ser Tyr Val Gly Ala Ser Ile Val Thr Ala Val Gly
35 40 45
Phe Ser Lys Gly Leu Trp Met Glu Cys Ala Thr His Ser Thr Gly
50 55 60
Ile Thr Gln Cys Asp Ile Tyr Ser Thr Leu Leu Gly Leu Pro Ala
65 70 75
Asp Ile Gln Ala Ala Gln Ala Met Met Val Thr Ser Ser Ala Met
80 85 90
Ser Ser Leu Ala Cys Ile Ile Ser Val Val Gly Met Arg Cys Thr
95 100 105
Val Phe Cys Gln Asp Ser Arg Ala Lys Asp Arg Val Ala Val Val
110 115 120
Gly Gly Val Phe Phe Ile Leu Gly Gly Ile Leu Gly Phe Ile Pro
125 130 135
Val Ala Trp Asn Leu His Gly Ile Leu Arg Asp Phe Tyr Ser Pro
140 145 150
Leu Val Pro Asp Ser Met Lys Phe Glu Ile Gly Glu Ala Leu Tyr
155 160 165
Leu Gly Ile Ile Ser Ala Leu Phe Ser Leu Val AIa Gly Val Ile
170 175 180
Leu Cys Phe Ser Cys Ser Pro Gln Gly Asn Arg Thr Asn Tyr Tyr
185 190 195
Asp Gly Tyr Gln Ala Gln Pro Leu Ala Thr Arg Ser Ser Pro Arg
200 205 210
10/11
CA 02343696 2001-03-22
WO 00/18915 PCT/US99/22082
Ser Ala Gln Gln Pro Lys Ala Lys Ser Glu Phe Asn Ser Tyr Ser
215 220 225
Leu Thr Gly Tyr Val
230
<210> 12
<211> 298
<212> PRT
<213> Mus musculus
<300>
<308> GenBank ID No: g3342560
<400> 12
Met Ser Leu Tyr Pro Ser Leu Glu Asp Leu Lys Val Asp Lys Val
1 5 10 15
Ile Gln Ala Gln Thr Ala Tyr Ser Ala Asn Pro Ala Ser Gln Ala
20 25 30
Phe Val Leu Val Asp Ala Ser Ala Ala Leu Pro Pro Asp Gly Asn
35 40 45
Leu Tyr Pro Lys Leu Tyr Pro Glu Leu Ser Gln Tyr Met Gly Leu
50 55 60
Ser Leu Asn Glu Ala Glu Ile Cys Glu Ser Met Pro Met Val Ser
65 70 75
Gly Ala Pro Ala Gln Gly Leu Val Ala Arg Pro Ser Ser Val Asn
80 85 90
Tyr Met Val Ala Pro Val Thr Gly Asn Asp Ala Gly Ile Arg Arg
95 100 105
Ala Glu Ile Lys Gln Gly Ile Arg Glu Val Ile Leu Cys Lys Asp
110 115 120
Gln Asp Gly Lys Ile Gly Leu Arg Leu Lys Ser Ile Asp Asn Gly
125 130 135
Ile Phe Val Gln Leu Val Gln Ala Asn Ser Pro Ala Ser Leu Val
140 145 150
Gly Leu Arg Phe Gly Asp Gln Val Leu Gln Ile Asn Gly Glu Asn
155 160 165
Cys Ala Gly Trp Ser Ser Asp Lys Ala His Lys Val Leu Lys Gln
170 175 180
Ala Phe Gly Glu Lys Ile Thr Met Thr Ile Arg Asp Arg Pro Phe
185 190 195
Glu Arg Thr Val Ile Met His Lys Asp Ser Ser Gly His Val Gly
200 205 210
Phe Ile Phe Lys Ser Gly Lys Ile Thr Ser Ile Val Lys Asp Ser
215 220 225
Ser Ala Ala Arg Asn Gly Leu Leu Thr Asp His His Ile Cys Glu
230 235 240
Ile Asn Gly Gln Asn Val Ile Gly Leu Lys Asp Ala Gln Ile Ala
245 250 255
Asp Ile Leu Ser Thr Ala Gly Thr Val Val Thr Ile Thr Ile Met
260 265 270
Pro Thr Phe Ile Phe Glu His Ile Ile Lys Arg Met Ala Pro Ser
275 280 285
Ile Met Lys Ser Leu Met Asp His Thr Ile Pro Glu Val
290 295
11/11
