Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02307709 2000-OS-OS
I0.
Tw0 HUMAN G-PROTEIN COUPLED RECEPTORS: EBV-INDUCED GPCR 2 (EBI-Z) AND EDG-1-
LIKE GPCR
This invention relates to newly identified polynucleotides, polypeptides
encoded by such
polynucleotides, the use of such polynucleotides and polypeptides, as well as
the production of
such polynucleotides and polypeptides. More particularly, the polypeptides of
the present
invention are a human EBV-induced G-protein coupled receptor (EBI-2) and a
human EDG-1-
like G-protein coupled receptor, sometimes hereinafter referred to singularly
as "GBR" or
"GPCR" and collectively as "GBRs." The invention also relates to inhibiting
the action of such
polypeptides.
At lease nine genes have beeri identified tfiat are apparently acitvated in
response to an
Epstein-Barr Virus (EBV} infection. One of two novel genes also identified in
such studies of
EBV infections was a novel GPCR-like cDNA molecule desingated EBV-induced G-
protein
coup.Ied receptor (EBI)-1.
Additionally, previously identified was an endothelium-differentiation gene
(EDG) that
was obtained from PMA-simulated-human endothelial cells. Rat and sheep
homologs of EDG-1
have been identified, which are also G-protein coupled receptors.
Tt is well established that many medically significant biological processes
are mediated by
proteins participating in signal transduction pathways that involve G-proteins
and/or second
messengers, e.g., cAMP (Lefkowitz, Nature, 351:353-354 (1991)). Herein these
proteins are
2 0 referred to as proteins participating in pathways with G-proteins or PPG
proteins. Some examples
of these proteins include the GPC receptors. such as those for adrenergic
agents and dopamine
(Kobilka, B.K., et al., PNAS, 84:46-50 (1987); Kobilka, B.K., et al., Science,
238:650-656
(1987); Bunzow, J.R_, et al., Nature, 336:783-787 (1988)), G-proteins
themselves, effector
proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and
actuator proteins, e.g.,
2 5 protein kinase A and protein kinase C (Simon, M.L, et al., Science,
252:802-8 (1991 )).
For example, in one form of signal transduction, the effect of hormone binding
is
activation of an enzyme. adenylate cyclase, inside the cell. Enzyme activation
by hormones is
dependent on the presence of the nucleotide GTP, and GTP also influences
hormone binding. A
G-protein connects the hormone receptors to adenylate cyclase. G-protein was
shown to
3 0 exchange GTP for bound GDP when activated by hormone receptors. The GTP-
carrying form
then binds Lo an activated adenylate cyclase. Hydrolysis of GTP to GDP,
catalyzed by the G-
protein itself, returns the G-protein to its basal, inactive form. Thus, the G-
protein serves a dual
role. as an intermediate that relays the signal from receptor to effector, and
as a clock that controls
CA 02307709 2000-OS-OS
2
the duration of the signal.
The membrane protein gene superfamily of G-protein coupled receptors has been
characterized as having seven putative transmembrane domains. The domains are
believed to
represent transmembrane a.-helices connected by extrace~lulai~ or cytoplasmic
loops, A function
G-protein is a trimer which consists of a variable alpha subunit coupled to a
much more tightly-
associated and constant beta and gamma subunits. A broad range of Iigands
(more than twenty)
have been identified which function through GPCRs. In general, bind of an
appropriate ligand to
a GPCR leads to the activation of the receptor. G-protein coupled receptors
include a wide range
of biologically active receptors: such as hormone, viral. growth factor and
neuroreceptors. Such
an activated receptor initiates the regulatory cycle of the G-protein. This
cycle consists of GTP
exchange for GDP, dissociation of the alpha and beta/gamma subunits,.
activation of the second
messenger pathway by a complex of GTP and th alpha subunit of the G-protein,
and return to the
resting state by GTP hydrolysis via the innate CrTP-ase activity of the G-
protein alpha subunit.A
G-protein coupled receptors have been characterized as including these seven
conserved
hydrophobic stretches of about_..20 to 30 amino acids, connecting at least
eight divergent
hydrophilic loops. The G-protein family of coupled receptors includes dopamine
receptors which
bind to neuroleptic drugs used for treating psychotic and neurological
disorders. Other examples
of members of this family include calcitonin, adrenergic, endothelin, cAMP,
adenosine.
muscarinicacetylcholine, serotonin, histamine, thrombin, kinin, follicle
stimulating hormone.
2 0 opsins and rhodopsins. odorant, cytomegalovirus receptors, etc.
Most GPRs have single conserved cysteine residues in each of the first nvo
extracellular
loops which form disulfide bonds that are believed to stabilize functional
protein structure. The 7
transmembrane regions are designated as TMI, TM2, TM3, TM4, TMS, TM6, and TM7.
TM3 is
also implicated in signal transduction.
2 5 Phosphorylation and lipidation (palmitylation or farnesylation) of
cysteine residues can
influence signal transduction of some GPRs. Most GPRs contain potential
phosphorylation sites
within the third cytoplasmic loop and/or the carboxy terminus. For several
GPRs. such as the (3-
adrenoreceptor, phosphoryiation by protein kinase A and/or specific receptor
kinases mediates '
receptor desensitization. .
3 0 The ligand binding sites of GPRs are believed to comprise a hydrophilic
socket formed by
several GPR transmembrane domains, which socket is surrounded by hydrophobic
residues of the
GPRs. The hydrophilic side of each GPR transmembrane helix is postulated to
face inward and
form the polar ligand binding site. TM3 has been implicated in several GPRs as
having a ligand
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3
binding site. such as including the 'fM3 aspartate residue. Additionally, TMS
serines, a TM6
asparagine and TM6 or TM7 phenylalanines or tyrosines are also implicated in
ligand binding.
GPRs can be intracellularly coupled by heterotrzmeric G- proteins to various
intracellular
enzymes, ion channels and transporters (see, Johnson et al., Endoc., Rev.,
10:312-331 (1989}).
Different G-protein a.-subunits preferentially stimulate particular effectors
to modulate various
biological functions in a cell. Phosphorylation of cytoplasmic residues of
GPRs has been
identified as an important mechanism for the regulation of G-protein coupling
of some GPRs.
G-protein coupled receptors are found in numerous sites within a mammalian
host, for
example, dopamine is a critical neurotransmitter in the central nervous system
and is a G-protein
coupled receptor ligand. ~ ~ '
In accordance with one aspect of the present invention, there are provided
novel
polypeptides. as well as antisense analogs thereof and biologically active and
diagnostically or
therapeutically useful fragments and derivatives thereof. The polypeptides of
the present
invention are of human origin.
7 5 In accordance with another aspect of the present invention, there are
provided isolated
nucleic acid molecules, including mRNAs, DNAs, CDNAS, genomic DNA as well as
antisense
analogs thereof and biologically active and diagnostically or therapeutically
useful fragments
thereof.
In accordance with a further aspect of the present invention, there is
provided a process for
2 0 producing such polypeptides by recombinant techniques which comprises
culturing recombinant
prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence
encoding a
polypeptide of the present invention, under conditions promoting expression of
said protein and
subsequent recovery of said protein.
In accordance with yet a further aspect of the present invention, there are
provided
2 5 antibodies against such polypeptides.
In accordance with another embodiment. there is provided a process for using
one or more
of the receptors according to the invention to screen for receptor antagonists
and/or agonists
' andlor receptor ligands.
In accordance with still another embodiment of the present invention there is
provided a
3 0 process of using such agonists to activate the polypeptide of the present
invention for the
treatment of conditions related to the underexpression of the polypeptide of
the present invention.
In accordance with another aspect of the present invention there is provided a
process of
using such antagonists for inhibiting the polypeptide of the present invention
for treating
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4
conditions associated with overexpression of the polypeptide of the present
invention.
In accordance with yet another aspect of the present invention there is
provided non-
naturally occurring synthetic, isolated and/or -recombinant polypeptides which
are fragments,
consensus fragments and/or sequences having conservative amino acid
substitutions, of at least
one transmembrane domain, such that the polypeptides of the present invention
may bind ligands.
or which rnay also modulate, quantitatively or qualitatively, Iigand binding
to the polypeptide of
the present invention.
In accordance with still another aspect of the present invention there are
provided
synthetic or recombinant polypeptides: conservative substitution derivatives
thereof, antibodies,
anti-idiotype antibodies, cormpositiorts and methods that can be useful as
potential modulators of
G-protein coupled receptor function, by binding to ligands .or modulating
ligand binding, due to
their expected biological properties, which may be used in diagnostic,
therapeutic andlor research
applications.
In accordance with another object of the present invention, there is provided
synthetic,
isolated or recombinant polypeptides which are designed to inhibit or mimic
various GPRs or
fragments thereof, as receptor types and subtypes.
In accordance with yet another object of the present invention, there is
provided a
diagnostic assay for detecting a disease or susceptibility to a disease
related to a mutation in a
nucleic acid sequence encoding a polypeptide of the present invention .
2 0 These and other aspects of the present invention should be apparent to
those skilled in the
art from the teachings herein.
The following drawings are illustrative of embodiments of the invention and
are not meant
to limit the scope of the invention as encompassed by the claims.
Figure I shows the cDNA sequence (SEQ ID NO:I ) and the corresponding deduced
2 5 amino acid sequence (SEQ ID N0:2) of the EBV-induced G-protein coupled
receptor of the
present invention. The polynucleotide sequence contains a 2249 nucleotide
sequence which
encodes a 342 amino acid ORF. In Figure 1, the standard one-Letter
abbreviation for amino acids
is used to illustrate the deduced amino acid sequence. Sequencing was
performed using a 373 -
Automated DNA sequencer (Applied Biosystems, Inc.). Sequencing accuracy is
predicted to be
3 0 greater than 97% accurate.
Figure 2 is an amino acid sequence comparison between the EBV-induced (EBI-2)
G-
Protein Coupled Receptor (upper line, see SEQ ID N0:2) and the human EBI-1 G-
Protein
Coupled Receptor (lower line. SEQ ID N0:17). The standard one-letter
abreviations are used to
CA 02307709 2000-OS-OS
represent the amino acid residues of the amino acid sequences illustrated. The
EBI-2 polypeptide
according to the invention shows approximately 25% identity and 49% similarity
to the amino
acid sequence of the EBI-1 gene over an approximately 350 amino acid stretch.
Figure 3 shows the eDNA sequence (SEQ ID-N0:3) and the corresponding deduced
5 amino acid sequence (SEQ ID N0:4) of the EDG-1-like G-protein coupled
receptor of the present
invention. The polynucleotide sequence contains a 1637 nucleotide sequence
which encodes a
260 amino acid ORF. In Figure 3, the standard one-letter abbreviation for
amino acids is used to
illustrate the deduced amino acid sequence. Sequencing was performed using a
373 Automated
DNA sequencer (Applied Biosystems, Inc.). Sequencing accuracy is predicted to
be greater than
97% accurate.
Figure 4 is an amino acid sequence comparison between the EDG-1-like G-Protein
Coupled Receptor (upper line, see SEQ ID N0:4) and the human EDG-1 orphan G-
Protein
Coupled Receptor (lower line, SEQ ID N0:18). The standard one-letter
abreviations are used to
represent the amino acid residues of the amino acid sequences illustrated. The
EDG-1-like
polypeptide according to the invention shows approximately 54% identity and
73% similarity to
the amino acid sequence of the human EDG-1 orphan G-protein Coupled Recptor
gene over two
regions totaling approximately 120 amino acids.
In accordance with an aspect of the present invention, there is provided an
isolated nucleic
acid (polynucleotide) which encode for the mature polypeptide having the
deduced amino acid
2 0 sequence of Figure 1 (SEQ ID N0:2) or for the mature polypeptide encoded
by the cDNA of the
clone deposited as ATCC Deposit No. 209003 on 4/28/97.
A polynucleatide encoding a EBI-2 polypeptide of the present invention may be
found in
a cDNA library from umbilical vein endothelial cells, neutrophil leukocyte
cells, and corpus
colosum cells. The polynucleotide of this invention was discovered in a cDNA
library derived
2 5 from umbilical vein endothelial cells. As described above, it is
structurally related to the G
protein-coupled receptor family. It contains an open reading frame encoding a
protein of 343
amino acid residues.
In accordance with an aspect of the present invention, there is provided an
isolated nucleic
acid {polynucleotide) which encode for the mature polypeptide having the
deduced amino acid
3 0 sequence of Figure 3 (SEQ ID N0:4) or for the mature polypeptide encoded
by the cDNA of the
clone deposited as ATCC Deposit No. 209004 on 4/28/97.
A polynucleotide encoding an EDG-1-like G-protein coupled receptor polypeptide
of the
present invention may be found in an activated neutrophil cDNA library,
cyciohexamine-treated
CA 02307709 2000-OS-OS
6
Raji cells, the RSR;I1 .bone marrow cell line, activated T-cells. tonsils, and
CD34-positive cord
blood cells. Northern blot anlyses indicate that the EDG-I-like receptor gene
is expressed
primarily in leukocytes, but expression may also be observed in placenta,
spleen, thymus, lung
and pancreas tissue. The polynucleotide of this invention Was discovered in a
cDNA library
S derived from activated neutrophils. As described above. it is structurally
related to the G protein-
coupled receptor family: It contains an open reading frame encoding a protein
of 261 amino acid
residues.
As noted above a great deal of the importance attributed to GPCR molecules
such as those
of the presently claimed. invention lies in the diversity of biological
functions in which they
.participate. For example, it is thought that, upon release form the alpha
subunit, the beta/gamma
subunit may also play a functional role in the regulation of signal
transduciton by activating the
arachidonic acid signal transduction pathway via the activation of
phospholipase A=. In addition.
GPCR molecules and their associated G-proteins have been implicated in the
coupling of vsisual
pigments to cGMP phosphodiesterase, phosphatidyl inositol (PI) turnover,
adenylyI cyclase signal
channels and other integral membrane enzymes to transporter proteins. As a
result, it is apparent
that novel GPCR molecules may prove useful in a wide variety of pharmaceutical
applications
including research and development. For example, target based screens for
small molecules and
other such pharmacologically valuable factors may be based on activating a
given GPCR. It has
also been observed that short peptides . may function by mimicking the GPCR
(temed
2 0 receptomimetics). Furthermore, monoclonal antibodies raised against such
factors may prove
useful as therapeutics in a number of capacities. Potential therapeutic,
and/or diagnositic
applications for such a factor may include such diverse clinical presentations
as heart disease,
mental illness, cancer, atherosclerosis, restenosis, Alzheimer s Disease,
Parkinson's Disease. and a
number of others.
2 5 Accordingly, the polynucleotides of the present invention may be in the
form of RNA or
in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
The DNA
may be double-stranded or single-stranded, and if single stranded may be the
coding strand or
non-coding (anti-sense) strand. The coding sequence which encodes the mature
EBI-2
polypeptide may be identical to the coding sequence shown in Figure I (SEQ ID
NO:l ) or that of
30 the deposited clone or may be a different coding sequence which coding
sequence, as a result of
the redundancy or degeneracy of the genetic code, encodes the same mature
polypeptide as the
DNA of Figure 1 .(SEQ ID NO:1) or the deposited cDNA. Similarly, the coding
sequence which
encodes the mature EDG-1-like G-protein coupled receptor polypeptide may be
identical to the
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7
coding sequence shown in Figure 3 (SEQ ID N0:3) or that of the deposited clone
or may be a
different coding sequence which coding sequence, as a result of the redundancy
or degeneracy of
the genetic code, encodes the same mature polypeptide as the DNA of Figure 3
(SEQ ID N0:3) or
the deposited cDNA.
The polynucleotides which encode either (a) the mature EBI-2 polypeptide of
Figure 1
{SEQ ID N0:2) or the mature EBI-2 polypeptide encoded by the deposited eDNA,
or (b) the
mature EDG-I-like G-protein coupled receptor polypeptide of Figure 3 (SEQ ID
N0:4) or the
mature EDG-1-like G-protein coupled receptor polypeptide encoded by the
deposited cDNA may
include: only the coding sequence for the mature polypeptide; the coding
sequence for the mature
polypeptide and additional coding sequence such ~s a leader or secretory
sequence or a proprotein
sequence; the coding sequence for the mature polypeptide (and optionally
additional coding
sequence) and non-coding sequence, such as introns or non-coding sequence 5'
and/or 3' of the
coding sequence for the mature polypeptide.
Thus, the term "polynucleotide encoding a polypeptide" encompasses a
polynucleotide
which includes only coding sequence for the polypeptide as well as a
polynucleotide which
includes additional coding and/or non-coding sequence.
The present invention further relates to variants of the hereinabove described
polynucleotides which encode for fragments. analogs and derivatives of (a) the
polypeptide
having the deduced amino acid sequence of Figure 1 (SEQ ID N0:2) or the
polypeptide encoded
2 0 by the cDNA of the deposited clone. or (2) the polypeptide having the
deduced amino acid
sequence of Figure 3 (SEQ ID N0:4) or the polypeptide encoded by the cDNA of
the deposited
clone, The variant of either of these two polynucleotides may be a naturally
occurring allelic
variant of the polynucleotide or a non-naturally occurring variant of the
polynucleotide.
Thus, the present invention includes polynucleotides encoding the same mature
2 S polypeptide as shown in Figure I (SEQ ID N0:2) or the same mature
polypeptide encoded by the
cDNA of the deposited clone as well as variants of such polynucleotides which
variants encode
for a fragment, derivative or analog of the polypeptide of Figure I (SEQ ID
N0:2) or the
polypeptide encoded by the cDNA of the deposited clone. Such nucleotide
variants include
deletion variants, substitution variants and addition or insertion variants.
3 0 Likewise, the present invention includes polynucleotides encoding the same
mature
polypeptide as shown in Figure 3 (SEQ ID N0:4) or the same mature polypeptide
encoded by the
cDNA of the deposited clone as well as variants of such polynucleotides which
variants encode
for a fragment. derivative or analog of the polypeptide of Figure 3 (SEQ ID
N0:4) or the
CA 02307709 2000-OS-OS
8
polypeptide encoded by the cDNA of the deposited clone. Such nucleotide
variants include
deletion variants, substitution variants and addition or insertion variants.
As hereinabove indicated, the polynucleotide may have a coding sequence which
is a '
naturally occurnng allelic variant of the coding sequence shown in Figure 1
{SEQ ID NO:1 ) or of
the coding sequence of the deposited clone. Also, as hereinabove indicated.
the polynucleotide
may have a coding sequence which is a naturally occurring allelic variant of
the coding sequence
shown in Figure 3 {SEQ ID N0:3) or of the coding sequence of the deposited
clone. As known in
the art. an allelic variant is an alternate form of a polynucleotide sequence
which may have a
substitution, deletion or addition of one or more nucleotides, which does not
substantially alter the
function of the encoded polypeptide. - ~ ~ .
The present invention also includes polynucleotides, wherein the. coding
sequence for the
mature polypeptide may be fused in the same reading frame to a polynucleotide
sequence which
aids in expression and secretion of a polypeptide from a host cell, for
example, a leader sequence
which functions as a secretory _sequence for controlling transport of a
polypeptide from the cell.
1 S The polypeptide having a leader, sequence is a preprotein and may have the
leader sequence
cleaved by the host cell to form the mature form of the polypeptide. The
polynucleotides may
also code for a proprotein which is the mature protein plus additional S'
amino acid residues. A
mature protein having a prosequence is a proprotein and is an inactive form of
the protein. Once
the prosequence is cleaved an active mature protein remains. Thus, for
example, the
2 0 polvnucleotide of the present invention may encode a mature protein, or a
protein having a
prosequence or for a protein having both a prosequence and a presequence
{leader sequence).
The polynucleotides of the present invention may also have the coding sequence
fused in
frame to a marker sequence which allows for purification of the polypeptide of
the present
invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9
vector to
2 S provide for purification of the mature polypeptide fused to the marker in
the case of a bacterial
host. or, for example, the marker sequence may be a hemagglutinin (HA) tag
when a mammalian
host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived
from the influenza
hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
The term "gene" means the segment of DNA involved in producing a polypeptide
chain; it
3 0 includes regions preceding and following the coding region (leader and
trailer) as well as
intervening sequences (introns) between individual coding segments (exons).
Fragments of the full length gene of the present invention may be used as
hybridization
probes for a cDNA or a genomic library to isolate the full length DNA and to
isolate other DNAs
CA 02307709 2000-OS-OS
9
which have a high sequence similarity to the gene or similar biological
activity. Probes of this
type preferably have at least I0, preferably at least I5, and even more
preferably at least 30 bases
and may contain, for example, at least 50 or more bases. In fact, probes of
this type having at
least up to I50 bases or greater may be preferably utilized: The probe may
also be used to
S identify a DNA clone corresponding to a full length transcript and a genomic
clone or clones that
contain the complete gene including regulatory and promotor regions, exons and
introns. An
example of a screen comprises isolating the coding region of the gene by using
the known DNA
sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides
having a sequence
complementary or identical to that of the gene or portion of the gene
sequences of the present
invention are used to screen a library of genom're DNA to determine which
members of the library
the probe hybridizes to.
It is also appreciated that such probes can be and are preferably labeled with
an
analytically detectable reagent to facilitate identification of the probe.
Useful reagents include but
are not limited to radioactivity, fluorescent dyes or enzymes capable of
catalyzing the formation
1 S of a detectable product. The probes are thus useful to isolate
complementary copies of DNA from
other sources or to screen such sources for related sequences.
The present invention further relates to polynucleotides which hybridize to
the
hereinabove-described sequences if there is at least 70%, preferably at least
90%, and more
preferably at least 95% identity between the sequences. (As indicated above,
70% identity would
2 0 include within such definition a 70 bps fragment taken from a 100 by
polynucleotide, for
example.) The present invention particularly relates to polynucleotides which
hybridize under
stringent conditions to the hereinabove-described polynucleotides. As herein
used, the term
"stringent conditions" means hybridization will occur only if there is at
least 95% and preferably
at least 97% identity between the sequences. The polynucleotides which
hybridize to the
2 5 hereinabove described polynucleotides in a preferred embodiment encode
enzymes which either
retain substantially the same biological. function or activity as the mature
polypeptide encoded by
the DNA of Figures l and 3 (SEQ ID NOS:2 and 4, respectively. In referring to
identity in the
case of hybridization, as known in the art, such identity refers to
complementarity of
polynucleotide segments.
3 0 Alternatively, the polynucleotide may have at least 15 bases, preferably
at least 30 bases,
and more preferably at least 50 bases which hybridize to any part of a
polynucleotide of the
i
present invention and which has an identity thereto, as hereinabove described,
and which may or
may not retain activity. For example, such polynucleotides may be employed as
probes for the
CA 02307709 2000-OS-OS
polynucleotides of SEQ ID NOS:I and 3, for example. for recovery of the
polynucleotide or as a
diagnostic probe or as a PCR primer.
Thus, the present invention is directed to polynucleotides having at least a
70% identity,
preferably at least 90% identity and more preferably at Zeasf a 95% identity
to a polynucleotide
5 which encodes either the polypeptide of SEQ ID N0:2, or the polypeptide of
SEQ ID N0:4, as
well as fragments thereof, which fragments have at least 15 bases, preferably
at least 30 bases,
more preferably at least 50 bases and most preferably fragments having up to
at least I50 bases or
greater, which fragments are at least 90% identical, preferably at least 95%
identical and most
preferably at least 97% identical to any portion of a polynucleotide of the
present invention.
10 The deposits) refeired to herein wilt-be,maintained under the terms of the
Budapest
Treaty on the International Recognition of the Deposit of Micro-organisms for
purposes of Patent
Procedure. These deposits are provided merely as convenience to those of skill
in the art and are
not an admission that a deposit is required under 35 U.S.C. 112. The sequence
of the
polynucleotides contained in the deposited materials, as well as the amino
acid sequence of the
polypeptides encoded thereby, are incorporated herein by reference and are
controlling in the
event of any conflict with any description of sequences herein. A license may
be required to
make, use or sell the deposited materials, and no such license is hereby
granted.
The present invention further relates to polypeptides which have the deduced
amino acid
sequences of Figures l and 3 (SEQ .ID NOS:2 and 4, respectively) as well as
fragments, analogs
2 0 and derivatives of such polypeptides.
The terms "fragment," "derivative" and "analog" when referring to (a) the
polypeptide of
Figure 1 (SEQ ID N0:2) or that encoded by the deposited cDNA, or (b) the
polypeptide of Figure
3 (SEQ ID N0:4), means a polypeptide which either retains substantially the
same biological
function or activity as such polypeptide, i.e. functions as a G-protein
coupled receptor, or retains
2 5 the ability to bind the ligand or the receptor even though the polypeptide
does not function as a G-
protein coupled receptor, for example, a soluble form of the receptor.
The polypeptide of the present invention may be a recombinant polypeptide, a
natural
polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
The fragment, derivative or analog of either (a) the golypeptide of Figure 1
(SEQ ID
3 0 N0:2) or that encoded by the deposited cDNA, (b) the polypeptide of Figure
3 (SEQ ID N0:4)
may be (i) one in which one or more of the amino acid residues are substituted
with a conserved
or non-conserved amino acid residue (preferably a conserved amino acid
residue) and such
substituted amino acid residue may or may not be one encoded by the genetic
code, or (ii) one in
CA 02307709 2000-OS-OS
which one or more of the amino acid residues includes a substituent group, or
(iii) one in which
the mature polypeptide is fused with another compound, such as a compound to
increase the half
life of the polypeptide (for example, polyethylene glycol), or (iv) one in
which the additional
amino acids are fused to the mature polypeptide, or (v) one iri~which a
fragment of the polypeptide
is soluble, i.e. not membrane bound, yet still binds ligands to the membrane
bound receptor. Such
fragments, derivatives and analogs are deemed to be within the scope of those
skilled in the art
from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably
provided in
an isolated form. and preferably are purified to homogeneity.
The term ''isolated" means that the material is removed from its original
environment
(e.g., the natural environment if it is naturally occurnng). For example, a
naturally-occurnng
polynucleotide or polypeptide present in a living animal is not isolated. but
the same
polynucleotide or polypeptide, separated from some or all of the coexisting
materials in the
natural system, is isolated. Such polynucleotides could be part of a vector
and/or_ such
polynucleotides or polypeptides could be part of a composition, and still be
isolated in that such
vector or composition is not part of its natural envirorunent.
The polypeptides of the present invention include the polypeptides of SEQ ID
NOS:2 and
4 (in particular the respective mature polypeptides) as well as polypeptides
which have of least
70% similarity (preferably at least a 70% identity) to either the polypeptide
of SEQ ID N0:2 or
2 0 the polypeptide of SEQ ID N0:4 and more preferably at least a 90%
similarity (more preferably
at least a 90% identity) to the polypeptide of SEQ ID N0:2 or of SEQ ID N0:4
and still more
preferably at least a 95% similarity (still more preferably a 90% identity) to
the polypeptide of
SEQ ID N0:2 or of SEQ ID N0:4 and also include portions of such polypeptides
with such
portion of the polypeptide generally containing at least 30 amino acids and
more preferably at
2 5 least ~0 amino acids.
As known in the art "similarity" between two polypeptides is determined by
comparing
the amino acid sequence and its conserved amino acid substitutes of one
polypeptide to the
sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present invention may be
employed far
3 0 producing the corresponding full-length polypeptide by peptide synthesis;
therefore, the
fragments may be employed as intermediates for producing the full-length
polypeptides.
Fragments or portions of the polynucleotides of the present invention may be
used to synthesize
full-length polynucleotides of the present invention.
CA 02307709 2000-OS-OS
12
The present invention also relates to a method for identifying andlor
isolating cells,
tissues, or classes of cells or tissues, by utilizing probes of the
polynucleotides that encode the
EBI-2 G-protein coupled receptor polypeptide or by utilizing an antibody
specific for the EBI-2 '
G-protein coupled receptor, fox example. Since the EBI-2 G-protein coupled
receptor
polypeptides according to the invention occur in vein endothelial cells,
neutrophil leukocyte cells
and corpus coiosum cells, the above probes or antibodies, for example, may be
utilized to identify
and/or isolate such cells. tissues or classes of cells or tissues.
The present invention further relates to a method for identifying and/or
isolating cells,
tissues, or classes of cells or tissues, by utilizing probes of the
polynucleotides that encode the
EDG-I-like G-protein coupled receptor polypeptide or by utilizing an antibody
specific for the
EDG-1-like G-protein coupled receptor polypeptide, for example. Since the EDG-
1-like G-
protein coupled receptor polypeptides according to the invention occur in
leukocyte, tonsil,
placenta, thymus, lung and pancreas tissue, the above probes or antibodies.
for example, may be
utilized to identify and/or isolate such cells, tissues or classes of cells or
tissues. -
The present invention also relates to vectors which include polynucleotides of
the present
invention. host cells which are genetically engineered with vectors of the
invention and the
production of polypeptides of the invention by recombinant techniques.
Host cells are genetically engineered (transduced or transformed or
transfected) with the
vectors of this invention which may be, far example, a cloning vector or an
expression vector.
2 0 The vector may be, for example, in the form of a plasmid, a viral
particle. a phage, etc. The
engineered host cells can be cultured in conventional nutrient media modified
as appropriate for
activating promoters, selecting transformants or amplifying the G-protein
coupled receptor genes.
The culture conditions, such as temperature, pH and the like, are those
previously used with the
host cell selected for expression, and will be apparent to the ordinarily
skilled artisan.
2 5 The polynueieotides of the present invention may be employed for producing
polypeptides
by recombinant techniques. Thus, for example, the polynucleotide may be
included in any one of
a variety of expression vectors for expressing a polypeptide. Such vectors
include chromosomal,
nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;
bacterial plasmids;
phage DNA;' baculovirus; yeast plasmids; vectors derived from combinations of
plasmids and
30 phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and
pseudorabies.
However, any other vector may be used as long as it is replicable and viable
in the host.
The appropriate DNA sequence may be inserted into the vector by a variety of
procedures.
In general, the DNA sequence is inserted into an appropriate restriction
endonuclease sites) by
CA 02307709 2000-OS-OS
l3
procedures known in the art. Such procedures and others are deemed to be
within the scope of
those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an
appropriate
expression control sequences) (promoter) to direct mRNA synthesis. As
representative examples
of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli.
lac or trp, the
phage lambda PL promoter and other promoters known to control expression of
genes in
prokaryotic or eukaryotic cells or their viruses. The expression vector also
contains a ribosome
binding site for translation initiation and a transcription terminator. The
vector may also include
appropriate sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more selectable
marker genes
to provide a phenotypic trait for selection of transformed host cells such as
dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin
resistance in E. coli.
The vector containing the appropriate DNA sequence as hereinabove described.
as well as
an appropriate promoter or control sequence, may be employed to transform an
appropriate host
to permit the host to express the protein.
As representative examples of appropriate hosts, there may be mentioned:
bacterial cells.
such as E. coli, Strentomvces, Salmonella iynhimurium; fungal cells, such as
yeast; insect cells
such as Drosophila S2 and S,~odoptera Sf9; animal cells such as CHO, COS or
Bowes melanoma;
2 0 adenoviruses; plant cells, etc. The selection of an appropriate host is
deemed to be within the
scope of those skilled in the art from the teachings herein.
More particularly, the present invention also includes recombinant constructs
comprising
one or more of the sequences as broadly described above. The constructs
comprise a vector, such
as a plasmid or viral vector, into which a sequence of the invention has been
inserted, in a forward
2 5 or reverse orientation. In a preferred aspect of this embodiment, the
construct further comprises
regulatory sequences, including, for example, a promoter, operabiy linked to
the sequence. Large
numbers of suitable vectors and promoters are known to those of skill in the
art. and are
commercially available. The following vectors are provided by way of example.
Bacterial:
pQE70, pQE60, pQE-9 (Qiagen), pbs, pDlO, phagescript, psiX174, pbluescript SK,
pbsks.
30 pNH8A, pNHl6a, pNHl8A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,
pDR540,
pRIT~ (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTI, pSG (Stratagene)
pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may
be used
as long as they are replicable and viable in the host.
' CA 02307709 2000-OS-OS
14
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol
transferase) vectors or other vectors with selectable markers. Two appropriate
vectors are
PKK232-8 and PCM7. Particular named bacterial promoters include lacl, lacZ,
T3, T7, gpt,
lambda PR, PL and trp. Eukaryotic promoters include CMV immediate early, HSV
thymidine
kinase, early and late SV40, LTIts from retrovirus, and mouse metallothionein-
I. Selection of the
appropriate vector and promoter is well within the level of ordinary skill in
the art.
In a further embodiment, the present invention relates to host cells
containing the above-
described constructs. The host cell can be a higher eukaryotic cell, such as a
mammalian cell, or a
lower eukaryotic cell, such as a yeast cell, or the host cell can be a
prokaryotic cell, such as a
bacterial cell. Introduction of the construct into~the host cell can be
effected by calcium phosphate
transfection. DEAF-Dextran mediated transfection, or electroporation. .
(Davis, L., Dibner, M.,
Batley. I., Basic Methods in Molecular Biology, ( 1986)).
The constructs in host cells can be used in a conventional manner to produce
the gene
product encoded by the recombinant sequence. Alternatively. the polypeptides
of the invention
can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in mammalian cells. yeast, bacteria, or other
cells under
the control of appropriate promoters. CeII-free translation systems can also
be employed to
produce such proteins using RNAs derived from the DNA constructs of the
present invention.
Appropriate cloning and expression vectors fox use with prokaryotic and
eukaryotic hosts are
described by Sambrook. et al.. Molecular Cloning: A Laboratory Manual. Second
Edition. Cold
Spring Harbor, N.Y., ( 1989), the disclosure of which is hereby incorporated
by reference.
Transcription of the DNA encoding the polypeptides of the present invention by
higher
eukaryotes is increased by inserting an enhancer sequence into the vector.
Enhancers are cis-
acting elements of DNA, usually about from 10 to 300 by that act on a promoter
to increase its
2 S transcription. Examples including the SV40 enhancer on the late side of
the replication origin by
100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of
the replication origin, and adenovirus enhancers.
Generally, recombinant expression vectors will include origins of replication
and
selectable markers permitting transformation of the host cell, e.g., the
ampicillin resistance gene
of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-
expressed gene to
direct transcription of a downstream structural sequence. Such promoters can
be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK),
a.-factor, acid
phosphatase. or heat shock proteins. among others. The heterologous structural
sequence is
CA 02307709 2000-OS-OS
assembled in appropriate phase with translation initiation and termination
sequences, and
preferably, a leader sequence capable of directing secretion of translated
protein into the
periplasmic space or extracellular medium. Optionally, the heterologous
sequence can encode a
fusion protein including .an N-terminal identification peptide imparting
desired .characteristics.
5 e.g., stabilization or simplified purification of expressed recombinant
product.
Useful expression vectors for bacterial use are constructed by inserting a
structural DNA
sequence encoding a desired protein together with suitable translation
initiation and termination
signals in operable reading phase with a functional promoter. The vector will
comprise one or
more phenotypic selectable markers and an origin of replication to ensure
maintenance of the
10 vector and to, if desirable, provide amplification -within the host.
Suitable prokaryotic hosts for
transformation include E. coli, Bacillus subtilis, Salmonella tvphimuri.um and
various species
within the genera Pseudomonas, Streptomyces. and Staphylococcus. although
others may also be
employed as a matter of choice.
As a representative but nonlimiting example, useful expression vectors for
bacterial use
15 can comprise a selectable marker and bacterial origin of replication
derived from commercially
available plasmids comprising genetic elements of the well known cloning
vector pBR322
(ATCC 37017). Such commercial vectors include, for example, pKK223-3
(Pharmacia Fine
Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These
pBR322
"backbone" sections are combined with an appropriate promoter and the
structural sequence to be
2 0 expressed.
Following transformation of a suitable host strain and growth of the host
strain to an
appropriate cell density, the selected promoter is induced by appropriate
means (e.g., temperature
shift or chemical induction) and cells are cultured for an additional period.
Cells are typically harvested by centrifugation. disrupted by physical or
chemical means,
2 5 and the resulting crude extract retained for further purification.
Microbial cells employed in expression of proteins can be disrupted by any
convenient
method. including freeze-thaw cycling, sonication. mechanical disruption, or
use of cell lysing
agents. such methods are well know to those skilled in the art.
Various mammalian cell culture systems can also be employed to express
recombinant
3 0 protein. Examples of mammalian expression systems include the COS-7 lines
of monkey kidney
fibroblasts. described by Gluzman, Cell, 23:175 ( 198 I ), and other cell
lines capable of expressing
a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
Mammalian
expression vectors will comprise an origin of replication, a suitable promoter
and enhancer, and
t
CA 02307709 2000-OS-OS
16
also any necessary ribosome binding sites, polyadenylation site, splice donor
and acceptor sites,
transcriptional termination sequences, and 5' flanking nontranscribed
sequences. DNA sequences
derived from the SV40 splice, and polyadenylation sites may be used to provide
the required
nontranscribed genetic elements.
The G-protein coupled receptor polypeptides can be recovered and purified from
recombinant cell cultures by methods including ammonium sulfate or ethanol
precipitation, acid
extraction, anion or cation exchange chromatography, phosphocellulose
chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite
chromatography and lectin chromatography. Protein refolding steps can be used.
as necessary, in
completing configuration of the mature protein: Rinally, high performance
liquid chromatography
(HPLC) can be employed for final purification steps.
The polypeptides of the present invention may be a naturally purified product.
or a product
of chemical synthetic procedures, or produced by recombinant techniques from a
prokaryotic or
eukaryotic host (for example, by bacterial, yeast, higher plant, insect and
mammalian cells in
culture). Depending upon the host employed in a recombinant production
procedure. the
polypeptides of the present invention may be glycosylated or may be non-
glycosylated.
Polypeptides of the invention may also include an initial methionine amino
acid residue.
The G-protein coupled receptor of the present invention may be employed in a
process for
screening for antagonists and/or agonists for the receptor.
2 0 In general, such screening procedures involve providing appropriate cells
which express
the receptor on the surface thereof. In particular, a polynucleotide encoding
the receptor of the
present invention is employed to transfect cells to thereby express the G-
protein coupled receptor.
Such transfection may be accomplished by procedures as hereinabove described.
One such screening procedure involves the use of the melanophores which are
transfected
2 5 - to express the G-protein coupled receptor of the present invention. Such
a screening technique is
described in PCT WO 92/01810 published February 6, 1992.
Thus, for example, such assay may be employed for screening for a receptor
antagonist by
contacting the melanophore cells which encode the G-protein coupled receptor
with both the
receptor ligand and a compound to be screened. Inhibition of the signal
generated by the Iigand
30 indicates that a compound is a potential antagonist for the receptor, i.e.,
inhibits activation of the
receptor.
The screen may be employed for determining an agonist by contacting such cells
with
compounds to be screened and determining whether such compound generates a
signal. i.e..
CA 02307709 2000-OS-OS
17
activates the receptor.
Other screening techniques include the use of cells which express the G-
protein coupled
receptor (for example, transfected CHO cells) in a system which measures
extracellular pH
changes caused by receptor activation, for example, as described in Science,
volume 246, pages
181-296 (October 1989). For example, potential agonists or antagonists may be
contacted with a
cell which expresses the G-protein coupled receptor and a second messenger
response, e.g. signal
transduction or pH changes, may be measured to determine whether the potential
agonist or
antagonist is effective.
Another such screening technique involves introducing RNA encoding the G-
protein
coupled receptor into xenopus. oocytes to transiently express the receptor.
The receptor oocytes
may then be contacted in the case of antagonist screening with the receptor
ligand and a
compound to be screened, followed by detection of inhibition of a calcium
signal.
Another screening technique involves expressing the G-protein coupled receptor
in which
the receptor is linked to a phospholipase C or D. As representative examples
of such cells, -there
may be mentioned endothelial cells, smooth muscle cells, embryonic kidney
cells, etc. The
screening for an antagonist or agonist may be accomplished as hereinabove
described by detecting
activation of the receptor or inhibition of activation of the receptor from
the phospholipase second
signal.
Another method involves screening for antagonists by determining inhibition of
binding
2 0 of labeled ligand to cells which have the receptor on the surface thereof.
Such a method involves
transfecting a eukaryotic cell with DNA encoding the G-protein coupled
receptor such that the
cell expresses the receptor on its surface and contacting the cell with a
potential antagonist in the
presence of a labeled form of a known ligand. The ligand can be labeled, e.g.,
by radioactivity.
The amount of labeled ligand bound to the receptors is measured, e.g., by
measuring radioactivity
2 5 of the receptors. If the potential antagonist binds to the receptor as
determined by a reduction of
labeled ligand which binds to the receptors, the binding of labeled ligand to
the receptor is
inhibited.
The present invention also provides a method for determining whether a ligand
not. known
to be capable of binding to a G-protein coupled receptor can bind to such
receptor which
3 0 comprises contacting a mammalian cell which expresses a G-protein coupled
receptor with the
ligand under conditions permitting binding of ligands to the G-protein coupled
receptor. detecting
the presence of a ligand which binds to the receptor and thereby determining
whether the ligand
binds to the G-protein coupled receptor. The systems hereinabove described for
determining
CA 02307709 2000-OS-OS
18
agonists and/or antagonists may also be employed for determining ligands which
bind to the
receptor.
In general, antagonists for G-protein coupled receptors which are determined
by screening
procedures may be employed for a variety of therapeutic purposes. For example,
such antagonists
have been employed for treatment of hypertension, angina pectoris. myocardial
infarction, ulcers.
asthma, allergies, psychoses, depression, migraine, vomiting, stroke, eating
disorders, migraine
headaches, cancer and benign prostatic hypertrophy.
Agonists for G-protein coupled receptors are also useful for therapeutic
purposes. such as
the treatment of asthzn.a; Parkinson's disease, acute heart failure,
hypotension. urinary retention,
and osteoporosis
Examples of G-protein coupled receptor antagonists include an antibody, or in
some cases
an oligonucleotide, which binds to the G-protein coupled receptor but does not
elicit a second
messenger response such that the activity of the G-protein coupled receptor is
prevented.
Antibodies include anti-idiotypic antibodies which recognize unique
determinants generally
1 S associated with the antigen-binding site of an antibody. Potential
antagonists also include
proteins which are closely related to the ligand of the G-protein coupled
receptor, i.e. a fragment
of the ligand, which have lost biological function and when binding to the G-
protein coupled
receptor, elicit no response.
A potential antagonist also includes an antisense construct prepared through
the use of
2.0 antisense technology. Antisense technology can be used to control gene
expression through
triple-helix formation or antisense DNA or RNA, both of which methods are
based on binding of
a polynucleotide to DNA or RNA. For example, the 5' coding portion of the
polynucleotide
sequence, which encodes for the mature polypeptides of the present invention,
is used to design an
antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A
DNA
2 S oligonucleotide is designed to be complementary to a region of the gene
involved in transcription
(triple helix -see Lee et al.; Nucl. Acids Res., 6:3073 (1979); Cooney et al,
Science, 241:456
(1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing
transcription and the
production of G-protein coupled receptor. The antisense RNA oligonucleotide
hybridizes to the '
mRNA in vivo and blocks translation of the mRNA molecule into the G-protein
coupled receptor
30 (antisense - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as
Antisense Inhibitors
of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides
described above
can also be delivered to cells such that the antisense RNA or DNA may be
expressed in vivo to
inhibit production of G-protein coupled receptor.
CA 02307709 2000-OS-OS
l9
Another potential antagonist is a small molecule which binds to the G-protein
coupled
receptor, making it inaccessible to ligands such that normal biological
activity is prevented.
Examples of small molecules include but are not limited to small peptides or
peptide-like
molecules.
Potential antagonists also include a soluble form of a G-protein coupled
receptor. e.g. a
fragment of the receptor, which binds to the ligand and prevents the Iigand
from interacting with
membrane bound G-protein coupled receptors.
The G-protein coupled receptor and antagonists or agonists may be employed in
combination with a suitable pharmaceutical earner. Such compositions comprise
a
therapeutically effective amount of the polypept'tde, and a pharmaceutically
acceptable carrier or
excipient. Such a carrier includes but is not limited to saline, buffered
saline, dextrose, water,
glycerol. ethanol, and combinations thereof. The formulation should suit the
mode of
administration.
The invention also provides a pharmaceutical pack or kit comprising one or _
more
containers filled with one or more of the ingredients of the pharmaceutical
compositions of the
invention. Associated with such containers) can be a notice in the form
prescribed by a
governmental agency regulating the manufacture, use or sale of pharmaceuticals
or biological
products, which notice reflects approval by the agency of manufacture, use or
sale for human
administration. In addition. the pharmaceutical compositions may be employed
in conjunction
2 0 with other therapeutic compounds.
The pharmaceutical compositions may be administered in a convenient manner
such as bs
the topical, intravenous, intraperitoneal, intramuscular, subcutaneous,
intranasal or intradermal
routes. The pharmaceutical compositions are administered in an amount which is
effective for
treating and/or prophylaxis of the specific indication. In general, the
pharmaceutical compositions
2 5 will be administered in an amount of at least about 10 g/kg body weight
and in most cases they
will be administered in an amount not in excess of about 8 mg/Kg body weight
per day. In most
cases, the dosage is from about 10 glkg to about 1 mg/kg body weight daily,
taking into account
' the routes of administration, symptoms. etc.
The G-protein coupled receptor polypeptides and antagonists or agonists which
are
30 polypeptides, may be employed in accordance with the present invention by
expression of such
polypeptides in vivo, which is often referred to as "gene therapy."
Thus, for example, cells from a patient may be engineered with a
polynucleotide (DNA or
RNA) encoding a polypeptide ex vivo, with the engineered cells then being
provided to a patient
CA 02307709 2000-OS-OS
to be treated with the polypeptide. Such methods are well-known in the art.
For example. cells
may be engineered by procedures known in the art by use of a retroviral
particle containing RNA
encoding a polypeptide of the-present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide in
vivo by, for
5 example. procedures known in the art. As known in the art, a producer cell
for producing a
retroviral particle containing RNA encoding the polypeptide of the present
invention may be
administered to a patient for engineering cells in vivo and expression of the
polypeptide in vivo.
These and other methods for administering a polypeptide of the present
invention by such method
should be apparent to those skilled in the art from the teachings of the
present invention. For
10 example, the expression vehicle for engineering cells may be other than a
retrovirus, for example,
an adenovirus which may be used to engineer cells in vivo after combination
with a suitable
delivery vehicle.
Retroviruses from which the retroviral plasmid vectors hereinabove mentioned
may be
derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen
necrosis virus,
15 retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian
leukosis virus, gibbon
ape leukemia virus, human immunodeficiency virus, adenovirus,
Myeloproliferative Sarcoma
Virus. and mammary tumor virus. In one embodiment, the retroviral plasmid
vector is derived
from Moloney Murine Leukemia Virus.
The vector includes one or more promoters. Suitable promoters which may be
employed
20 include. but are not limited to, the retroviral LTR; the SV40 promoter: and
the human
cytomegalovirus {CMV) promoter described in Miller. et al., Biotechnic~ues.
Vol. 7, No. 9. 980-
990 ( 1989), or any other promoter (e.g., cellular promoters such as
eukaryotic cellular promoters
including, but not limited to, the histone, pol III, and (3-actin promoters).
Other viral promoters
which may be employed include, but are not limited to, adenovirus promoters.
thymidine kinase
2 5 (TK) promoters, and B I 9 parvovirus promoters. The selection of a
suitable promoter will be
apparent to those skilled in the art from the teachings contained herein.
The nucleic acid sequence encoding the polypeptide of the present invention is
under the
control of a suitable promoter. Suitable promoters which may be employed
include. but are not
Iirnited to, adenoviral promoters, such as the adenoviral major late promoter:
or hetorologous
3 0 promoters, such as the cytomegalovirus (CMV) promoter; the respiratory
syncytial virus (RSV)
promoter; inducible promoters, such as the MMT promoter, the metallothionein
promoter; heat
shock promoters; the albumin promoter; the ApoAl promoter; human globin
promoters; viral
thymidine kinase promoters, such as the Herpes Simplex thymidine kinase
promoter: retroviral
CA 02307709 2000-OS-OS
21
LTRs (including the modified retroviral LTRs hereinabove described); the (3-
actin promoter; and
human growth hormone promoters. The promoter also may be the native promoter
which
controls the gene encoding the polypeptide.
The retroviral plasmid vector is employed to transduce packaging cell lines to
form
producer cell lines. Examples of packaging cells which may be transfected
include, but are not
limited to, the PESOl, PA317, ~r-2, y-AM, PAI2, T19-14X, VT-19-17-H2, yCRE,
SCRIP.
GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene
Therapy, Vol.
1, pgs. 5-14 (1990), which is incorporated herein by reference in its
entirety. The vector may
transduce the packaging cells through any means known in the art. Such means
include, but are
not limited to, electroporation..the use of liposomes, and CaP04
precipitation. In one alternative.
the retroviral plasmid vector may be encapsulated into a liposome, or coupled
to a lipid, and then
administered to a host.
The producer cell line generates infectious retroviral vector particles which
include the
nucleic acid sequences) encoding the polypeptides. Such retroviral vector
particles then may be .
employed, to transduce eukaryotic cells, either rn vitro or in vivo. The
transduced eukaryotic cells
will express the nucleic acid sequences) encoding the polypeptide. Eukaryotic
cells which may
be transduced include, but are not limited to, embryonic stem cells, embryonic
carcinoma cells, as
well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,
keratinocytes, endothelial
cells. and bronchial epithelial cells.
2 0 G-protein coupled receptors are ubiquitous in the mammalian host and are
responsible for
many biological functions, including many pathologies. Accordingly, it is
desirous to find
compounds which stimulate a G-protein coupled receptor and compounds which
antagonize a G-
protein coupled receptor.
This invention further provides a method of identifying compounds which
specifically
2 5 interact with, and bind to, the human G-protein coupled receptors on the
surface of a cell which
comprises contacting a mammalian cell comprising an isolated DNA molecule
encoding the G-
protein coupled receptor with a plurality of compounds, determining those
which bind to the
mammalian cell, and thereby identifying compounds which specifically interact
with and bind to a
human G-protein coupled receptor of the present invention.
3 0 This invention also provides a method of detecting expression of the G-
protein coupled
receptor on the surface of a cell by detecting the presence of mRNA coding for
a G-protein
coupled receptor which comprises obtaining total mRNA from the cell and
contacting the mRNA
so obtained with a nucleic acid probe comprising a nucleic acid molecule of at
least 1 ~
CA 02307709 2000-OS-OS
22
nucleotides capable of specifically hybridizing with a sequence included
within the sequence of a
nucleic acid molecule encoding a human G-protein coupled receptor under
hybridizing
conditions, detecting the presence of mRNA hybridized to the probe, and
thereby detecting the .
expression of the G-protein coupled receptor by the cell. '
This invention is also related to the use of the G-protein coupled receptor
gene as part of a
diagnostic assay for detecting diseases or susceptibility to diseases related
to the presence of
mutated G-protein coupled receptor genes. Such diseases are related to cell
transformation, such
as tumors and cancers.
Individuals carrying mutations in the human G-protein coupled receptor gene
may be
detected at the DNA level by a.variety of techniques. Nucleic acids for
diagnosis may be obtained
from a patient's cells, such as from blood, urine. saliva, tissue biopsy and
autopsy material. The
wgenomic DNA may be used directly for detection or may be amplified
enzymatically by using
PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA or cDNA
may also be
used for the same purpose. As an example, PCR primers complementary to the
nucleic acid
encoding the G-protein coupled .receptor protein can be used to identify and
analyze G-protein
coupled receptor mutations. For example, deletions and insertions can be
detected by a change in
size of the amplified product in comparison to the normal genotype. Point
mutations can be
identified by hybridizing amplified DNA to radiolabeled G-protein coupled
receptor RNA or
alternatively, radiolabeled G-protein coupled receptor antisense DNA
sequences. Perfectly
2 0 matched sequences can be distinguished from mismatched duplexes by RNase A
digestion or by
differences in melting temperatures.
Genetic testing based on DNA sequence differences may be achieved by detection
of
alteration in electrophoretic mobility of DNA fragments in gels with or
without denaturing agents.
Small sequence deletions and insertions can be visualized by high resolution
gel electrophoresis.
2 5 DNA fragments of different sequences may be distinguished on denaturing
formamide gradient
gels in which the mobilities of different DNA fragments are retarded in the
gel at different
positions according to their specific melting or partial melting temperatures
(see, e.g., Myers et
al., Science. 230:1242 (1985)).
Sequence changes at specific locations may also be revealed by nuclease
protection
3 0 assays, such as RNase and S 1 protection or the chemical cleavage method
(e.g., Cotton et al..
PNAS, USA, 85:4397-4401 (1985)).
Thus, the detection of a specific DNA sequence may be achieved by methods such
as
hybridization, RNase protection, chemical cleavage, direct DNA sequencing or
the use of
' CA 02307709 2000-OS-OS
23
restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP))
and Southern
blotting of genomic DNA. .
In addition to more conventional gel-electrophoresis and DNA sequencing,
mutations can
also be detected by in situ analysis.
The present invention also relates to a diagnostic assay for detecting altered
Levels of
soluble Forms of the receptor polypeptides of the present invention in various
tissues. Assays
used to detect levels of the soluble receptor polypeptides in a sample derived
from a host are well
known to those of skill in the art and include radioimmunoassays, competitive-
binding assays,
Western blot analysis and preferably as ELISA assay.
An ELISA'assay initially comprises preparing an antibody specific to antigens
of the G-
protein coupled receptor polypeptides, preferably a monoclonal antibody. In
addition a reporter
antibody is prepared against the monoclonal antibody. To the reporter antibody
is attached a
detectable reagent such as radioactivity, fluorescence or in this example a
horseradish peroxidase
enzyme. A sample is now removed from a host and incubated on a solid support,
e.g. a
polystyrene dish, that binds the proteins in the sample. Any free protein
binding sites on the dish
are then covered by incubating with a non-specific protein such as bovine
serum albumin. Next,
the monoclonal antibody is incubated in the dish during which time the
monoclonal antibodies
attach to any G-protein coupled receptor proteins attached to the polystyrene
dish. All unbound
monoclonal antibody is washed out with buffer. The reporter antibody linked to
horseradish
2 0 peroxidase is now placed in the dish resulting in binding of the reporter
antibody to any
monoclonal antibody bound to G-protein receptor proteins. Unattached reporter
antibody is then
washed out. Peroxidase substrates are then added to the dish and the amount of
color developed
in a given time period is a measurement of the amount of G-protein coupled
receptor proteins
present in a given volume of patient sample when compared against a standard
curve.
2 5 The sequences of the present invention are also valuable for chromosome
identification.
The sequence is specifically targeted to and can hybridize with a particular
location on an
individual human chromosome. Moreover, there is a current need for identifying
particular sites
on the chromosome. Few chromosome marking reagents based on actual sequence
data (repeat..
polymorphisms) are presently available for marking chromosomal location. The
mapping of
3 0 DNAs to chromosomes according to the present invention is an important
first step in correlating
those sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers
(preferably
1 ~-2~ bp) from the cDNA. Computer analysis of the 3' untranslated region is
used to rapidly
CA 02307709 2000-OS-OS
24
select primers that do not span more than one exon in the genomic DNA, thus
complicating the
amplification process. These primers are then used for PCR screening of
somatic cell hybrids
containing individual human. chromosomes. Only those hybrids containing the
human gene
corresponding to the primer will yield an amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a
particular DNA '
to a particular chromosome. Using the present invention with the same
oligonucleotide primers.
sublocalization can be achieved with panels of fragments from specific
chromosomes or pools of
large genomic clones in an analogous manner. Other mapping strategies that can
similarly be
used to map to its chromosome include in situ hybridization, prescreening with
labeled flow-
sorted chromosomes and preselection by hybridization to construct chromosome
specific-cDNA
libraries.
Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase
chromosomal
spread can be used to provide a precise chromosomal location in one step. This
technique can be
used with cDNA as short as SO or 60 bases. For a review of this technique, see
Verma et al.,
Hmnan Chromosomes: a Ivlanual of Basic Techniques, Pergamon Press, New York
(1988).
Once a sequence has been mapped to a precise chromosomal location, the
physical
position of the sequence on the chromosome can be correlated with genetic map
data. Such data
are found; for example, in V. McKusick, Mendelian Inheritance in Man
(available on line through
3ohns Hopkins University Welch Medical Library). The relationship between
genes and diseases
?_ 0 that have been mapped to the same chromosomal region are then identified
through linkage
analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic
sequence
between affected and unaffected individuals. If a mutation is observed in some
or all of the
affected individuals but not in any normal individuals, then the mutation is
likely to be the
2 S causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a
cDNA
precisely localized to a chromosomal region associated with the disease could
be one of between
SO and 500 potential causative genes. (This assumes 1 megabase mapping
resolution and one
gene per 20 kb).
3 0 The polypeptides, their fragments or other derivatives, or analogs
thereof, or cells
expressing them can be used as an immunogen to produce antibodies thereto.
These antibodies
can be, for example, polyclonal or monoclonal antibodies. The present
invention also includes
chimeric, single chain, and humanized antibodies, as well as Fab fragments, or
the product of an
CA 02307709 2000-OS-OS
Fab expression library. Various procedures known in the art may be used for
the production of
such antibodies and fragments. _ .
Antibodies generated against the polypeptides corresponding to a sequence of
the present
invention can be obtained by direct injection of the polypeptides into an
animal or by
S administering the polypeptides to an animal, preferably a nonhuman. The
antibody so obtained
will then bind the polypeptides itself. In this manner, even a sequence
encoding only a fragment
of the polypeptides can be used to generate antibodies binding the whale
native polypeptides.
Such antibodies can then be used to isolate the polypeptide from tissue
expressing that
polypeptide.
10 For preparation of monoclonal antibodies, any technique which provides
antibodies
produced by continuous cell line cultures can be used. Examples include the
hybridoma
technique (Kohler and Milstein. 1975. Nature. 256:495-497), the trioma
technique, the human B-
cell hvbridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the
EBV-
hybridoma technique to produce human monoclonal antibodies (Cole, et al.,
1985, in Monoclonal
15 Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain antibodies (U.S.
Patent
4,946,778) can be adapted to produce single chain antibodies to immunogenic
polypeptide
products of this invention. Also, transgenic mice may be used to express
humanized antibodies to
immunogenic polypeptide products of this invention.
2 0 The present invention will be further described with reference to the
following examples;
however. it is to be understood that the present invention is not limited to
such examples. All
parts or amounts, unless otherwise specified. are by weight.
In order to facilitate understanding of the following examples certain
frequently occurring
methods and/or terms will be described.
z S "Plasmids" are designated by a lower case p preceded and/or followed by
capital letters
and/or numbers. The starting plasmids herein are either commercially
available, publicly
available on an unrestricted basis, or can be constructed from available
plasmids in accord with
published procedures. In addition, equivalent plasmids to those described are
known in the art
and will be apparent to the ordinarily skilled artisan.
3 0 "Digestion" of DNA refers to catalytic cleavage of the DNA with a
restriction enzyme that
acts only at certain sequences in the DNA. The various restriction enzymes
used herein are
commercially available and their reaction conditions. cofactors and other
requirements were used
as would be known to the ordinarily skilled artisan. For analytical purposes,
typically 1 p.g of
' CA 02307709 2000-OS-OS
26
plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ~l of
buffer solution.
For the purpose of isolating DNA fragments for plasmid construction, typically
5 to 50 p.g of
DNA are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and
substrate amounts for particular restriction enzymes are specified by the
manufacturer. incubation
times of about I hour at 37°C are ordinarily used, but may vary in
accordance with the supplier's
instructions. After digestion the reaction is electrophoresed directly on a
polyacrylamide gel to
isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent
polyacrylamide gel
described by Goeddel. D. et al., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or
two
complementary polydeoxynucleotide strands which may be chemically synthesized.
Such
synthetic oligonucleotides have no 5' phosphate and thus will not ligate to
another oligonucleotide
without adding a phosphate with an ATP in the presence of a l:.inase. A
synthetic oligonucleotide
will ligate to a fragment that has not been dephosphorylated. -
"Ligation" refers to the- process of forming phosphodiester bonds between two
double
stranded nucleic acid fragments {Maniatis, T., et al., Id., p. 146). Unless
otherwise provided,
Iigation may be accomplished using known buffers and conditions with 10 units
to T4 DNA
ligase ("ligase") per O.S ug of approximately equimolar amounts of the DNA
fragments to be
Iigated.
2 0 Unless otherwise stated, transformation was performed as described in the
method of
Graham. F. and Van der Eb. A., Virology, 52:456-457 (1973).
Example 1
2 5 Bacterial Expression and Purification of EBT-2
The DNA sequence encoding EBI-2, ATCC # 209003, is initially amplified using
PCR
oligonucleotide primers corresponding to the S' sequences of the processed EBI-
2 protein (minus
the signal peptide sequence) and the vector sequences 3' to the EBI-2 gene
Additional
nucleotides corresponding to EBI-2 were added to the S' and 3' sequences
respectively. The ~' -
3 0 oligonucleotide primer has the sequence S' CCGAGGATCCATGCAAGCCGTCGACAAT 3'
(SEQ ID NO:S) contains a BamHI restriction enzyme site followed by 18
nucleotides of the EBI-
2 coding sequence starting from the presumed terminal amino acid of the
processed protein
codon. The 3' sequence S' CCGAGGATCCTTACATTGGAGTCTCTTC 3' (SEQ ID N0:6)
CA 02307709 2000-OS-OS
27
contains complementary sequences to BamHI site and is followed by 18
nucleotides of EBI-2.
The restriction enzyme sites correspond to the restriction enzyme sites on the
bacterial expression
vector pQE-60. (Qiagen, Inc., Chatsworth. CA, 91311 ). pQE-60 encodes
antibiotic resistance
(Amps, a bacterial origin of replication (ori), an IPTG-regulatable promoter
operator (P/O), a
ribosome binding site (RBS). a 6-His tag and restriction enzyme sites. pQE-60
was then digested
with Bamhl. The amplified sequences were ligated into pQE-60 and were inserted
in frame with
the sequence encoding for the histidine tag and the RBS. The ligation mixture
was then used to
transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described
in Sambrook, J. et
al.. Molecular Cloning: A Laboratory Manual. Cold Spring Laboratory Press,
(1989). MI5/rep4
contains multiple copies of the plasmid pREP4, which expresses the lacl
repressor and also
confers kanamycin resistance (Kan'~. Transformants are identified by their
ability to grow on LB
plates and ampicillin/kanamvcin resistant colonies were selected. Plasmid DNA
was isolated and
confi=.-med by restriction analysis. Clones containing the desired constructs
were grown overnight
(O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and
Kan (25
ug/ml). The O/N culture is used to inoculate a Large culture at a ratio of
1:100 to 1:250. The cells
were grown to an optical density 600 (O.D,6oo) of between 0.4 and 0.6. IPTG
("Isopropyl-B-D-
thioaalacto pyranoside") was then added to a final concentration of 1 mM. IPTG
induces by
inactivating the lacl repressor, clearing the P/O leading to increased gene
expression. Cells were
grown an extra 3 to 4 hours. Cells were then harvested by centrifugation. The
cell pellet was
2 0 solubilized in the chaotropic agent 6 Molar Guanidine HC1. After
clarification, solubilized hSca-2
was purified from this solution by chromatography on a Nickel-Chelate column
under conditions
that allow for tight binding by proteins containing the 6-His tag (Hochuli, E.
et al., J.
Chromatography 411:177-184 (1984)). hSca-2 (95 % pure was eluted from the
column in 6
molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3
molar guanidine
2 S HCI. 1 OOmM sodium phosphate. 10 mmolar glutathione (reduced) and 2 mmolar
glutathione
(oxidized). After incubation in this solution for 12 hours the protein was
dialyzed to 10 mmolar
sodium phosphate.
3 0 Example 2
Cloning and expression of EB1-2 using the baculovirus expression system
The DNA sequence encoding the full length EBI-2 protein, ATCC # 209003. was
arnpiified using PCR oiigonucleotide primers corresponding to the 5' and 3'
sequences of the
CA 02307709 2000-OS-OS
28
gene:
The 5' primer has the _ sequence 5' CCGAGGATCCGCCATCA-
TGCAAGCCGTCGACAAT 3' (SEQ ID N0:7) and contains a BamHI restriction enzyme
site (in
bold) followed by 6 nucleotides resembling an efficient signal for the
initiation of translation in
eukaryotic cells {Kozak, M., J. MoI. Biol., 196:947-950 (1987) which is just
behind the first 18
nucleotides of the EBI-2 gene (the initiation codon for translation "ATG" is
underlined).
The 3' primer has the sequence 5' CCGAGGATCCT-TACATTGGAGTCTCTTC 3' (SEQ
ID N0:8) and contains the cleavage site for the restriction endonuclease BamHI
and 18
nucleotides complementary to the 3' translated sequence of the extracellular
part of EBI-2. The
amplified sequences were isolated from a..lQ/o agarose gel using a
commercially available kit
("Geneclean," BIO 101 Inc., La Jolla. Ca.). The fragment was ~ then digested
with the
endonucleases BamHI. and purified again on a I% agarose gel. This fragment is
designated F?.
The vector pA2 (modification of pVL941 vector, discussed below) is used for
the
expression of the EBI-2 protein using the baculovirus expression system (for
review see:
Summers, M.D. and Smith, G.E: 1987, A manual of methods for bacuiovirus
vectors and insect
cell culture procedures, Texas Agricultural Experimental Station Bulletin No.
1555). This
expression vector contains the strong polyhedrin promoter of the Autographa
californica nuclear
polyhedrosis virus (AcMNPV) followed by the recognition sites for the
restriction endonucleases
Barnl-II. The polyadenylation site of the simian virus (SV)40 is used for
efficient
2 0 polvadenylation. For an easy selection of recombinant virus the beta-
galactosidase gene from
E.coli is inserted in the same orientation as the polyhedrin promoter followed
by the
polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are
flanked at both
sides by viral sequences for the cell-mediated homologous recombination of co-
transfected wild-
type viral DNA. Many other baculovirus vectors could be used in place of pA2
such as pRGI and
2 5 pA2-GP in which case the 5' primer are changed accordingly, and pAe373,
pVL941 and pAcIM 1
(Luckow, V.A. and Summers, M.D., Virology, 170:31-39).
The plasmid was digested with the restriction enzyme Baml-II and then
dephosphorylated
using calf intestinal phosphatase by procedures known in the art. The DNA was
then isolated
from a 1% .agarose gel using the commercially available kit ("Geneclean" BIO
101 Inc., La Jolla, .
3 0 Ca.). This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA
ligase.
E.coli HB 101 cells were then transformed and bacteria identified that
contained the plasmid
(pBacEBI-2) with the EBl-2 gene using the enzyme BaznHI. The sequence of the
cloned
CA 02307709 2000-OS-OS
29
fragment was confirmed by DNA sequencing.
p.g of the plasmid pBacEBI-2 was co-transfected with 1.0 ~g of a commercially
available Iinearized baculovinis ("BaculoGold baculovirus DNA", Pharmingen,
San Diego, CA.)
using the lipofection method (Felgner et al. Proc. Natl. Acad. Sci. USA,
84:7413-7417 (1987)).
5 l pg of BaculoGold virus DNA and 5 pg of the plasmid pBacEBI-2 were mixed in
a
sterile well Qf a microtiter plate containing 50 p.l of serum free , Grace's
medium (Life
Technologies Inc., Gaithersburg, MD). Afterwards 10 uI Lipofectin plus 90 p.l
Grace's medium
were added, mixed and incubated for I S minutes at room temperature. Then the
transfection
mixture was added drop-wise to the Sf9 insect cells (ATCC CRL 1711 ) seeded in
a 35 mm tissue
culture plate with 1 ml Grace's medium without serum. The plate was rocked
back and forth to
mix the newly added solution. The plate was then incubated for 5 hours at 27
C. After S hours
the
transfection solution was removed from the plate and 1 ml of Grace's insect
medium
supplemented with I O% fetal calf serum was added. The plate was put back into
an incubator and
1 S cultivation continued at 27 C for four days.
After four days the supernatant was collected and a plaque assay performed
similar as
described by Summers and Smith (supra). As a modification an agarose gel with
"Blue Gal" (Life
Technologies Ine., Gaithersburg) was used which allows an easy isolation of
blue stained plaques.
(A detailed description of a "plaque assay" can also be found in the user's
guide for insect cell
2 0 culture and baculovirology distributed by Life Technologies Inc.,
Gaithersburg, page 9-10).
Four days after the serial dilution, the virus was added to the cells and blue
stained plaques
were picked with the tip of an Eppendorf pipette. The agar containing the
recombinant viruses
was then resuspended in an Eppendoi~f tube containing 200 p.l of Grace's
medium. The agar was
removed by a brief centrifugation and the supernatant containing the
recombinant baculovirus was
2 S used to infect Sf~ cells seeded in 35 mm dishes. Four days later the
supernatants of these culture
dishes were harvested and then stored at 4 C.
Sf9 cells were grown in Grace's medium supplemented with 10% heat-inactivated
FBS.
The cells were infected with the recombinant baculovirus V-EBI-2 at a
multiplicity of infection
(MOI) of 2. Six hours later the medium was removed and replaced with SF900 II
medium minus
3 0 methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours
later 5 p.Ci of "5-
methionine and 5 pCi 35S cysteine (Amersham) were added. The cells were
further incubated for
16 hours before they were harvested by centrifugation and the labelled
proteins visualized by
SDS-PAGE and autoradiography.
CA 02307709 2000-OS-OS
- Example 3
Expression of Recombinant EBI-2 in COS cells
The expression of plasmid, EBI-2 HA is derived from a vector peDNAI/Amp
(Invitrogen)
containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3)
E.coli replication
origin. 4) .CMV promoter followed by a polylinker region, an SV40 intron and
polyadenylation
site. A DNA fragment encoding the entire EBI-2 precursor and a HA tag fused in
frame to its 3'
end was cloned into the polylinker region of the vector. therefore, the
recombinant protein
10 expression is directed under the CMV promoter. 'The HA tag corresponds to
an epitope derived
from the influenza hemagglutinin protein as previously described (I. ~ Wilson,
H. Niman, R.
Heighten. A Cherenson, M. Connolly. and R. Lemer, 19$4. CeII 37:767,. (1984)).
The infusion of
HA'tag to the target protein allows easy detection of the recombinant protein
with an antibody
that recognizes the HA epitope. -
15 _The plasmid construction strategy is described as follows:
The DNA sequence encoding EBI-2, ATCC # 209003, was constructed by PCR using
two
primers: the S' primer 5' CCGAGGATCCGCCATCATGCAAGCCGTCGACAAT 3' (SEQ ID
N0:9) contains a BamHI site followed by EBI-2 coding sequence starting from
the initiation
codon; the 3' sequence
20 5'CCGATCTAGATTAATCCCATACGACGTCCCAGACTACGCTCATTGGAGTCTCTTC3'
(SEQ ID NO:10) contains complementary sequences to XbaI site, translation stop
codon. HA tag
and EBI-2 coding sequence (not including the stop codon). Therefore, the PCR
product contains
a BamHI site, EBI-2 coding sequence followed by HA tag fused in frame, a
translation
termination stop codon next to the HA tag, and an Xbal site. The PCR amplified
DNA fragment
25 and the vector, pcDNAI/Amp, were digested with BamHI and XbaI restriction
enzyme and
ligated. The ligation mixture was transformed into E. coli strain SURE
(available from Stratagene
Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the
transformed culture
was plated on arnpicillin media plates and resistant colonies were selected.
Plasmid DNA was '
isolated from transformants and examined by restriction analysis for the
presence of the correct
3 0 fragment. For expression of the recombinant EBI-2 COS cells were
transfected with the
expression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T.
Maniatis, Molecular
Cloning: A Laboratory Manual, Cold Spring Laboratory Press, ( 1989)). The
expression. of the
EBI-2 HA protein was detected by radiolabelling and immunoprecipitation method
(E. Harlow.
CA 02307709 2000-OS-OS
31
D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
(1988)). Cells
were labelled for 8 hours with 35S-cysteine two days post transfection.
Culture media was then
collected and cells were lysed with detergent (RIPA buffer ( 150 mM NaCI, 1 %
NP-40, 0.1
SDS, 1% NP-40. 0.5% DOC, 50mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767
(1984)). Both cell
lysate and culture media were precipitated with an HA specific monoclonal
antibody. Proteins
precipitated were analyzed on 15% SDS-PAGE gels.
Example 4
_Fxnression via Gene Therapy
Fibroblasts are obtained from a subjeet~by skin biopsy. The resulting tissue
is placed in
tissue-culture medium and separated into small pieces. Small chunks of the
tissue are placed on a
wet surface of a tissue culture flask, approximately ten pieces are placed in
each flask. The flask
is turned upside down, closed tight and left at room temperature over night.
After 24 hours at
room temperature, the flask is inverted and the chunks of tissue remain fixed
to the bottom of the
flask and fresh media (e.g.; Ham's F12 media, with 10% FBS, penicillin and
streptomycin, is
added. This is then incubated at 37 C for approximately one week. At this
time, fresh media is
added and subsequently changed every several days. After an additional two
weeks in culture, a
monolaver of fibroblasts emerge. The monolayer is trypsinized and scaled into
larger flasks.
pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long
terminal
2 0 repeats of the Moloney marine sarcoma virus. is digested with EcoRI and
HindIII and
subsequently treated with calf intestinal phosphatase. The linear vector is
fractionated on agarose
gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention is amplified using
PCR
primers which correspond to the 5' and 3' end sequences respectively. The 5'
primer containing an
2 5 EcoRI site and the 3' primer further includes a HindIII site. Equal
quantities of the Moloney
marine sarcoma virus linear backbone and the amplified EcoRI and HindIII
fragment are added
together. in the presence of T4 DNA ligase. The resulting mixture is
maintained under conditions
appropriate for ligation of the two fragments. The ligation mixture is used to
transform bacteria
HB 101, which are then plated onto agar-containing kanamycin for the purpose
of confirming that
3 0 the vector had the gene of interest properly inserted.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture
to
confluent density in Dulbecco's Modified Eagles Medium (DMEM) with IO% calf
serum (CS),
penicillin and streptomycin. The MSV vector containing the gene is then added
to the media and
CA 02307709 2000-OS-OS
32
the packaging cells are transduced with the vector. The packaging cells now
produce infectious
viral particles containing the gene (the packaging cells are now referred to
as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the
media is
harvested from a 10 cm plate of confluent producer cells.. The spent media, -
containing the
infectious viral particles, is filtered through a millipore fitter to remove
detached producer cells
and this media is then used to infect fibroblast cells. Media is removed from
a sub-confluent plate
of fibroblasts and quickly replaced with the media from the producer cells.
This media is
removed and replaced with fresh media. If the titer of virus is high, then
virtually all fibroblasts
will be infected and no selection is required. If the titer is very low, then
it is necessary to use a
retroviral - -vector that has a selectable marker, such as neo or his.
The engineered fibroblasts are then injected into the host, either alone or
after having been
grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now
produce the protein
product.
Example 5 -
Bacterial Expression and Purification of EDG-1-Like Polvnentide
The DNA sequence encoding EDG-1-like polypeptide. ATCC # 209004, is initially
amplified using PCR oligonucleotide primers corresponding to the 5' sequences
of the processed
EDG-1-like polypeptide protein (minus the signal peptide sequence) and the
vector sequences 3'
to the EDG-1-like polypeptide gene. Additional nucleotides corresponding to
EBI-2 were added
2 0 to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has
the sequence
5' CCGAGGATC-CATGAACGCCACGGGGACC 3' (SEQ ID NO:I1) contains a BamHI
restriction enzyme site followed by 18 nucleotides of the EDG-1-like
polypeptide coding
sequence starting from the presumed terminal amino acid of the processed
protein codon. The 3'
sequence 5' CCGAGGATCCTCAGATGCTCCGCACGCT 3' (SEQ ID N0:12) contains
2 5 complementary sequences to BamHI site and is followed by 18 nucleotides of
EDG-1-like
polypeptide. The restriction enzyme sites correspond to the restriction enzyme
sites on the
bacterial expression vector pQE-60. (Qiagen, Inc., Chatsworth, CA, 91311 ).
pQE-60 encodes
antibiotic resistance (Amps, a bacterial origin of replication (ori), an IPTG-
regulatable promoter
operator (P/O), a ribosome binding site (RBS), a 6-His tag and restriction
enzyme sites. pQE-60 .
3 0 was then digested with BamhI. The amplified sequences were ligated into
pQE-60 and were
inserted in frame with the sequence encoding for the histidine tag and the
RBS. The ligation
mixture was then used to transform E. coli strain MIS/rep 4 (Qiagen, Inc.) by
the procedure
described in Sambrook. J. et al., Molecular Cloning: A Laboratory Manual, Cold
Spring
CA 02307709 2000-OS-OS
33
Laboratory Press. (1989). M15/rep4 contains multiple copies of the plasmid
pREP4, which
expresses the lacI repressor and also confers kanamycin resistance (Kan~.
Transformants are
identified by their ability to grow on LB plates and ampicillin/kanamycin
resistant colonies were
selected. Plasmid DNA was isolated and confimled by restriction analysis.
Clones containing the
desired constructs were grown overnight (O/N) in liquid culture in LB media
supplemented with
both Amp (100 ug/ml) and Kan (25 ug/ml). The 0/N culture is used to inoculate
a large culture at
a ratio of 1:100 to 1:250. The cells were grown to an optical density 600
(O.D.boo) of between 0.4
and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") was then added to a
final concentration
of I mM. IPTG induces by inactivating the lacl repressor. clearing the P/O
leading to increased
gene expression. Cells were , grown an extra 3~ to 4 hours. Cells were then
harvested by
centrifugation. The cell pellet was solubilized in the chaotropic agent
6.Molar Guanidine I-ICI.
After clarification. solubilized EBI-2 was purified from this solution by
chromatography on a -
Nickel-Chelate column under conditions that allow for tight binding by
proteins containing the 6-
His tag (Hochuli, E. et al., J. Chromatography 411:177-184 {1984)). EBI-2 (95
% pure was eluted
from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of
renaturation adjusted to
3 molar guanidine HC1, 100mM sodium phosphate, 10 mmolar glutathione (reduced)
and 2
mmolar glutathione (oxidized). After incubation in this solution for 12 hours
the protein was
dialyzed to 10 mmolar sodium phosphate.
Example 6
2 0 C_ lonin~ and expression of EDG-1-like nolvpeptide using the baculovirus
expression system
The DNA sequence encoding the full length EDG-1-like polypeptide protein. ATCC
#
209004, was amplified using PCR oligonucleotide primers corresponding to the
~' and 3'
sequences of the gene:
The 5' primer has the sequence ~' GCGAGGATCCGCCAT
2 5 CATGAACGCCACGGGGACC 3' (SEQ ID N0:13) and contains a BamHI restriction
enzyme
site (in bold) followed by 6 nucleotides resembling an efficient signal for
the initiation of
translation in eukaryotic cells (Kozak, M., J. Mol. BioL, 196:947-950 (I987)
which is just behind
the first 18 nucleotides of the EDG-1-like polypeptide gene (the initiation
codon for translation
"ATG" is underlined).
3 0 The 3' primer has the sequence 5' CCGAGGATCCTC-AGATGCTCCGCACGCT 3'
(SEQ ID N0:14) and contains the cleavage site for the restriction endonuclease
BamHI and 18
nucleotides complementary to the 3' translated sequence of the extracellular
part of EDG-l-like
polypeptide. The amplified sequences were isolated from a 1 % aearose gel
using a commercially
CA 02307709 2000-OS-O5
34
available kit {"Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment was
then digested with the
endonucleases BamHI, and purified again on a 1 % agarose gel. This fragment is
designated F2.
The vector pA2 (modification of pVL941 vector, discussed below) is used for
the -
expression of the EDG-I-like polypeptide protein using-the-~baculovirus
expression system (for
review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for
baculovirus vectors '
and insect cell culture procedures, Texas Agricultural Experimental Station
Bulletin No. 1555).
This expression vector contains the strong polyhedrin promoter of the
Autographa califomica
nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the
restriction
endonucleases BamHI. The polyadenylation site of the simian virus {SV)40 is
used for efficient
polyadenylation. For an easy selection of recombinant virus the beta-
galactosidase gene from
E.coli is inserted in the same orientation as the polyhedrin promoter followed
by the
polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are
flanked at both
sides by viral sequences for the cell-mediated homologous recombination of co-
transfected wild-
type viral DNA. Many other baculovirus vectors could be used in place of pA2
such as pRG l and
pA2=GP in which case the 5' primer are changed accordingly, and pAc373, pVL941
and pAcIMl
(Luckow, V.A. and Summers, M.D., Virology, 170:31-39).
The plasmid was digested with the restriction enzyme BamHI and then
dephosphorylated
using calf intestinal phosphatase by procedures known in the art. The DNA was
then iso~atea
from a 1% agarose gel using the commercially available kit ("Geneciean" BIO
101 Inc., La Jolla,
2 0 Ca.). This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 were Iigated with T4 DNA
ligase.
E.coli HB101 cells were then transformed and bacteria identified that-
contained the plasmid
(pBacEDG-1-like poiypeptide) with the EDG-1-like polypeptide gene using the
enzyme BamHI.
The sequence of the cloned fragment was confirmed by DNA sequencing.
5 p.g of the plasmid pBacEDG-1-like polypeptide was co-transfected with 1.0
~.g of a
commercially available linearized baculovirus ("BaculoGold baculovirus DNA",
Pharmingen,
San Diego, yCA.) using the lipofection method (Felgner et al. Proc. Natl.
Acad. Sci. USA.
84:7413-7417 ( 1987)).
l ~g of BaeuloGold virus DNA and 5 ~g of the plasmid pBacEDG-1-like
polypeptide
3 0 were mixed in a sterile well of a microtiter plate containing 50 ~l of
serum free Grace's medium
(Life Technologies Inc., Gaithersburg, MD). Afterwards 10 ~l Lipofectin plus
90 ul Grace's
medium were added', mixed and incubated for 15 minutes at room temperature.
Then the
transfection mixture was added drop-wise to the Sf~ insect cells (ATCC CRL 171
I ) seeded in a
CA 02307709 2000-OS-OS
35 mtn tissue culture plate with 1 ml Grace's medium without serum. The plate
was rocked back
and forth to mix the newly added solution. The plate was then incubated for 5
hours at 27 C.
After ~ hours the transfection solution was removed from the plate and 1 ml of
Grace's insect
medium supplemented with 10% fetal calf serum was added. The plate was put
back into an
5 incubator and cultivation continued at 27 C for four days.
After four days the supernatant was collected and a plaque assay performed
similar as
described by Summers and Smith (supra). As a modification an agarose gel with
"Blue Gal" (Life
Technologies Inc., Gaithersburg) was used which allows an easy isolation of
blue stained plaques.
(A detailed description of a "plaque assay" can also be found in the user's
guide for insect cell
10 culture and baculovirology distributed by Life Technologies Inc.,
Gaithersburg, page 9-10).
Four days after the serial dilution. the virus was added to the cells.and blue
stained plaques
were nicked with the tip of an Eppendorf pipette. The agar containing the
recombinant viruses
was then resuspended in an Eppendorf tube containing 200 ~l of Grace's medium.
The agar was
removed by a brief centrifugation and the supernatant containing the
recombinant baculovirus was
15 used to infect Sf9 cells seeded in 35 mm dishes. Four days later the
supernatants of these culture
dishes were harvested and then stored at 4 C.
SfT3 cells were grown in Grace's medium supplemented with 10% heat-inactivated
FBS.
The cells were infected with the recombinant baculovirus V-EDG-1-like
polypeptide at a
multiplicity of infection (MOI) of 2. Six hours later the medium was removed
and replaced. with
2 0 SF900 II medium minus methionine and cysteine (Life Technologies Inc.,
Gaithersburg). 4?
hours later 5 p.Ci of 'SS-methionine and 5 pCi'SS cysteine (Amersham) were
added. The cells
were further incubated for 16 hours before they were harvested by
centrifugation and the labelled
proteins visualized by SDS-PAGE and autoradiography.
2 5 Example 7
E~ression of Recombinant EDG-1-like Polyr~entide in COS cells
The expression of plasmid, EDG-1-like polypeptide HA is derived from a vector
peDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)
ampicillin resistance
gene. 3) E.coli replication origin, 4) CMV promoter followed by a polylinker
region, an SV40
30 intron and polyadenylation site. A DNA fragment encoding the entire EDG-1-
like polypeptide
precursor and a HA tag fused in frame to its 3' end was cloned into the
polylinker region of the
vector, therefore, the recombinant protein expression is directed under the
CMV promoter. The
HA tae corresponds to an epitope derived from the influenza hemagglutinin
protein as previously
CA 02307709 2000-OS-OS
36
described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R.
Lerner, 1984,
Cell 37:767, (1984)). The infusion of HA tag to the target protein allows easy
detection of the
i
recombinant protein with an antibody that recognizes the HA epitope. -
The plasmid construction strategy is described as ~ollovs:
The DNA sequence encoding EDG-1-like polypeptide. ATCC # 209004, was
constructed '
by PCR using two pnmers: the ~~ pnmer o
CCGAGGATCCGCCATCATGAACGCCACGGGGACC 3' (SEQ ID N0:15) contains a BamHI
site followed by EDG-1-like polypeptide coding sequence starting from the
initiation codon; the
;' sequence 5' CCGATCTAGATCAATCCCATACGACGTCCCAG-
ACTACGCTGATGCTCCGCACGCT 3' (SEQ~ ID, NO: I6) contains complementary sequences
to
XbaI site, translation stop codon, I-IA tag and EDG-I-like polypeptide coding
sequence (not
including the stop eodon). Therefore, the PCR product contains a BamHl site,
EDG-1-like
polypeptide coding sequence followed by I-IA tag fused in frame. a translation
termination stop
codon next to the HA tag, and an XbaI site. The PCR amplified DNA fragment and
the vector,
pcDNAI/Amp, were digested with BamHI and XbaI restriction enzyme and ligated.
The Iigation
mixture was transformed into E. coli strain SURE (available from Stratagene
Cloning Systems.
11099 Norih Torrey Pines Road, La Jolla, CA 92037) the transformed culture was
plated on
ampicillin media plates and resistant colonies were selected. Plasmid DNA was
isolated. from
transfonnants and examined by restriction analysis for the presence of the
correct fragment. For
expression of the recombinant EDG-1-like polypeptide COS cells were
transfected with the
expression vector by DEAE-DEXTRAN method (J. Sambrook. E. Fritsch. T.
Maniatis. Molecular
Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The
expression of the
EDG-1-like polypeptide HA protein was detected by radiolabelling and
immunoprecipitation
method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory
2 5 Press. ( 1988)). Cells were labelled for 8 hours with 'SS-cysteine two
days post transfection.
Culture media was then collected and cells were lysed with detergent (RIPA
buffer ( I 50 mM
NaCI, I% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5) (Wilson, I.
et al., Id.
37:767 (I9$4)). Both cell lysate and culture media were precipitated with an
HA specific
monoclonal antibody. Proteins precipitated were analyzed on I S% SDS-PAGE
gels.
Example 8
Expression via Gene TheranY
Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue
is placed in
CA 02307709 2000-OS-OS
37
tissue-culture medium and separated into small pieces. Small chunks of the
tissue are placed on a
wet surface of a tissue culture flask, approximately ten pieces are placed in
each flask. The flask
' is fumed upside down, closed tight and left at room temperature over night.
After 24 hours at
room temperature, the flask is inverted and the chunks of tissue remain fixed
to the bottom of the
S flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and
streptomycin, is
added. This is then incubated at 37 C for approximately one week. At this
time, fresh media is
added and subsequently changed every several days. After an additional two
weeks in culture, a
monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into
larger flasks.
pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long
terminal
repeats of the Moloney marine sarcoma virus, is digested with EcoRI and
HindIIl and
subsequently treated with calf intestinal phosphatase. The linear vector is
fractionated on agarose
gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention is amplified using
PCR
primers which correspond to the 5' and 3' end sequences respectively. The 5'
primer containing an
EcoF''I site and the 3' primer fizrther includes a HindIII site. Equal
quantities of the Moloney
marine sarcoma virus linear backbone and the amplified EcoRI and HindIII
fragment are added
together. in the presence of T4 DNA ligase: The resulting mixture is mamazmu
um~~ ..~i~u.~.".~~
appropriate for ligation of the two fragments. The Iigation mixture is used to
transform bacteria
HB 1 O 1, which are then plated onto agar-containing kanamycin for the purpose
of confirming that
2 0 the vector had the gene of interest properly inserted.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture
to
confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf
serum {CS),
penicillin and streptomycin. The MSV vector containing the gene is then added
to the media and
the packaging cells are transduced with the vector. The packaging cells now
produce infectious
2 5 viral particles containing the gene (the packaging cells are now referred
to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the
media is
harvested from a 10 cm plate of confluent producer cells. The spent media,
containing the
infectious viral particles, is filtered through a millipore filter to remove
detached producer cells
and this media is then used to infect fibroblast cells. Media is removed from
a sub-confluent plate
3 0 of fibroblasts and quickly replaced with the media from the producer
cells. This media is
removed and replaced with fresh media. If the titer of virus is high, then
virtually all fibroblasts
will be infected and no selection is required. If the titer is very tow, then
it is necessary to use a
retroviral - -vector that has a selectable marker, such as neo or his.
CA 02307709 2000-OS-OS
38
The engineered fibroblasts are then injected into the host, either alone or
after having
been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now
produce the
protein product. -
Numerous modifications and variations of the present iiwention are possible in
light of the
S .above teachings and, therefore, within the scope of the appended claims,
the invention may be
practiced otherwise than as particularly described.
CA 02307709 2000-OS-OS
39
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i)APPLICANT: Human Genome Sciences, Inc.
(ii) TITLE OF INVENTION: Two Human G-Protein Coupled Receptors:
EBV-Induced GPCR 2 (EBI-2) and EDG-1-Like GPCR
(iii) NUMBER OF SEQUENCES: 18
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: MBM & CO.
(B) STREET: P.O. BOX 809, STATION B
(C) CITY: OTTAWA
(D) PROVINCE: ONTARIO
(E) COUNTRY: CANADA
(F) POSTAL CODE: K1P 5P9
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE:
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: n/a
(B) FILING DATE: 05-MAY-2000
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SWAIN, Margaret
(B) REGISTRATION NUMBER: 10926
(C) REFERENCE/DOCKET NUMBER: 184-277a
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 613/567-0762
(B) TELEFAX: 613/563-7671
(2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2247 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
CA 02307709 2000-OS-OS
(A) CDS
NAME/KEY:
(B) 226...1 251
LOCATION:
(C)
IDENTIFICATION
METHOD:
(D)
OTHER
INFORMATION:
(xi ) ON: SEQ ID
SEQUENCE N0:1:
DESCRIPTI
GCACGAGGAA CAGAACACTT C GGTCAGAT TAC AAGAGCACTCAAGACTT
60
TCTCATGTC AG
TACTGACGAA AACTCAGGAA CTATC AAAGAGGT TTG GCAACTAAACTAAGACA
120
ATCCT AC
TTAAAAG GAA AATACCAGAT CCACTCTGC GCTGCAAT AAC TACTACTTACTGGATAC180
G AG
ATTCAAACCC TCCAGAATCA CAGTTATCA TAACCAACAAG AA TG CC 237
A GG A CAA GTC
G
M et ln la
G A Val
1
GACAAT CTC ACC GCG CCTGGG AAC ACCAGT CTG TGC ACCAGA GAC 285
TCT
AspAsn Leu Thr Ala ProGly Asn ThrSer Leu Cys ThrArg Asp
Ser
5 10 15 20
TACAAA ATC ACC GTC CTCTTC CCA CTGCTC TAC ACT GTCCTG TTT 333
CAG
TyrLys Ile Thr Val LeuPhe Pro LeuLeu Tyr Thr ValLeu Phe
Gln
25 30 35
TTTGTT GGA CTT ACA AATGGC CTG GCGATG AGG ATT TTCTTT CAA 381
ATC
PheVal Gly Leu Thr AsnGly Leu AlaMet Arg Ile PhePhe Gln
Ile
40 45 50
ATCCGG AGT AAA AAC TTTATT ATT TTTCTT AAG AAC ACAGTC ATT 429
TCA
IleArg Ser Lys Asn PheIle Ile PheLeu Lys Asn ThrVal Ile
Ser
55 60 65
TCTGAT CTT CTC ATT CTGACT TTT CCATTC AAA ATT CTTAGT GAT 477
ATG
SerAsp Leu Leu Ile LeuThr Phe ProPhe Lys Ile LeuSer Asp
Met
70 75 80
GCCAAA CTG GGA GGA CCACTG AGA ACTTTT GTG TGT CAAGTT ACC 525
ACA
AlaLys Leu Gly Gly ProLeu Arg ThrPhe Val Cys GlnVal Thr
Thr
85 90 95 100
TCCGTC ATA TTT TTC ACAATG TAT ATCAGT ATT TCA TTCCTG GGA 573
TAT
SerVal Ile Phe Phe ThrMet Tyr IleSer Ile Ser PheLeu Gly
Tyr
105 110 115
CTGATA ACT ATC CGC TACCAG AAG ACCACC AGG CCA TTTAAA ACA 621
GAT
LeuIle Thr Ile Arg TyrGln Lys ThrThr Arg Pro PheLys Thr
Asp
120 125 130
TCCAAC CCC AAA CTC TTGGGG GCT AAGATT CTC TCT GTTGTC ATC 669
AAT
SerAsn Pro Lys Leu LeuGly Ala LysIle Leu Ser ValVal Ile
Asn
135 140 145
TGGGCA TTC ATG TTA CTCTCT TTG CCTAAC ATG ATT CTGACC AAC 717
TTC
TrpAla Phe Met Leu LeuSer Leu ProAsn Met Ile LeuThr Asn
Phe
150 155 160
CA 02307709 2000-OS-OS
41
AGGCAG CCG AGAGAC AAG AATGTG AAG AAA TGCTCT TTCCTT AAA TCA 765
ArgGln Pro ArgAsp Lys AsnVal Lys Lys CysSer PheLeu Lys Ser
165 170 175 180
GAGTTC GGT CTAGTC TGG CATGAA ATA GTA AATTAC ATCTGT CAA GTC 813
GluPhe Gly LeuVal Trp HisGlu Ile Val AsnTyr IleCys Gln Val
185 190 195
ATTTTC TGG ATTAAT TTC TTAATT GTT ATT GTATGT TATACA CTC ATT 861
IlePhe Trp IleAsn Phe LeuIle Val Ile ValCys TyrThr Leu Ile
200 205 210
ACAAAA GAA CTGTAC CGG TCATAC GTA AGA ACGAGG GGTGTA GGT AAA 909
ThrLys Glu LeuTyr Arg SerTyr Val Arg ThrArg GlyVal Gly Lys
215 220 225
GTCCCC AGG AAAAAG GTG AACGTC AAA GTT TTCATT ATCATT GCT GTA 957
ValPro Arg LysLys Val AsnVal Lys Val PheIle IleIle Ala Val
230 235 240
TTCTTT ATT TGTTTT GTT CCTTTC CAT TTT GCCCGA ATTCCT TAC ACC 1005
PhePhe Ile CysPhe Val ProPhe His Phe AlaArg IlePro Tyr Thr
245 250 255 260
CTGAGC CAA ACCCGG GAT GTCTTT GAC TGC ACTGCT GAAAAT ACT CTG 1053
LeuSer Gln ThrArg Asp ValPhe Asp Cys ThrAla GluAsn Thr Leu
265 270 275
TTCTAT GTG AAAGAG AGC ACTCTG TGG TTA ACTTCC TTAAAT GCA TGC 1101
PheTyr Val LysGlu Ser ThrLeu Trp Leu ThrSer LeuAsn Ala Cys
280 285 290
CTGGAT CCG TTCATC TAT TTTTTC CTT TGC AAGTCC TTCAGA AAT TCC 1149
LeuAsp Pro PheIle Tyr PhePhe Leu Cys LysSer PheArg Asn Ser
295 300 305
TTGATA AGT ATGCTG AAG TGCCCC AAT TCT GCAACA TCTCTG TCC CAG 1197
LeuIle Ser MetLeu Lys CysPro Asn Ser AlaThr SerLeu Ser Gln
310 315 320
GACAAT AGG AAAAAA GAA CAGGAT GGT GGT GACCCA AATGAA GAG ACT 1245
AspAsn Arg LysLys Glu GlnAsp Gly Gly AspPro AsnGlu Glu Thr
325 330 335 340
CCAATG TAAACAAATT CAATCTCTTTGTGT TCAGAACTCG 1301
AACTAAGGAA
ATATTT
ProMet
TTAAAGCAAA GCGCTAAGTA AAAATATTAA CTGACGAAGA AGCAACTAAG TTAATAATAA 1361
TGACTCTAAA GAAACAGAAG ATTACAAAAG CAATTTTCAT TTACCTTTCC AGTATGAAAA 1421
GCTATCTTAA AATATAGAAA ACTAATCTAA ACTGTAGCTG TATTAGCAGC AAAACAAACG 1481
ACATCCAATT GTCATGCTGC ATGCAAAACT ACACAGAATT CATGTTTTGG CAGAGTTTTG 1541
GCAAAATGAG TAATCATATA ATATTTACTG TAATTTTTAA AATACATTAT CGTTCACAAT 1601
CA 02307709 2000-OS-OS
42
TTTATTTTTT CATAATCAAC TAAGGAAGAA CGATCAATTG GATATAATCT TCTTACCAAA 1661
AATGATAGTT AAAATGTATA TATATCCTAG TCCCCTAACC AAATCCTGAC CTATTGGGAT 1721
ACTTATAAAA ATTTAAGTAA GTGGGATACA CAAAGAATAA TAACTATTAA CTTTTCATTA 1781
TTAGCCAAAA ACCTAAGGGA TTTAAACTAA TTGAAACTGT ATTTGATTGG ACTTAATTTT 1841
TTATGTTTAT TTAGAAGATA AAGATTTAAG AAGACCTTTA CAATAAAGAG AAGAAATATC 1901
GAAGTCATTA AAATAAGGAG ACTTACTTTT ATGACATTCT AATACTAAAA AATATAGAAA 1961
TATTTCCTTA ATTCTAGAGA AACTAGTTTT ACTAATTTTT TACAACTTCA ATAATACCAT 2021
CACTGACACT TACCTTTATT AATTAGCTTC TAGAAAATAG CTGCTAATTA GGTTAATGAA 2081
CATTTTACCT TAGTGAAAAA AAATTAATTA AATATGATTA CAAAGTTGCA CAGCATAACT 2141
ACTGAGAGGA AAGTGATTGA TCTGTTTGTA ATTACTTGTT TGTATTGGTG TGTATAAAAT 2201
ACAAATTTAC ATTAAACTCT AAATCATTAA AAAAAA 2247
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 342 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Gln Ala Val Asp Asn Leu Thr Ser Ala Pro Gly Asn Thr Ser Leu
1 5 10 15
Cys Thr Arg Asp Tyr Lys Ile Thr Gln Val Leu Phe Pro Leu Leu Tyr
20 25 30
Thr Val Leu Phe Phe Val Gly Leu Ile Thr Asn Gly Leu Ala Met Arg
35 40 45
Ile Phe Phe Gln Ile Arg Ser Lys Ser Asn Phe Ile Ile Phe Leu Lys
50 55 60
Asn Thr Val Ile Ser Asp Leu Leu Met Ile Leu Thr Phe Pro Phe Lys
65 70 75
Ile Leu Ser Asp Ala Lys Leu Gly Thr Gly Pro Leu Arg Thr Phe Val
90 95
Cys Gln Val Thr Ser Val Ile Phe Tyr Phe Thr Met Tyr Ile Ser Ile
100 105 110
CA 02307709 2000-OS-OS
43
Ser Phe Leu Gly Leu Ile Thr Ile Asp Arg Tyr Gln Lys Thr Thr Arg
115 120 125
Pro Phe Lys Thr Ser Asn Pro Lys Asn Leu Leu Gly Ala Lys Ile Leu
130 135 140
Ser Val Val Ile Trp Ala Phe Met Phe Leu Leu Ser Leu Pro Asn Met
145 150 155 160
Ile Leu Thr Asn Arg Gln Pro Arg Asp Lys Asn Val Lys Lys Cys Ser
165 170 175
Phe Leu Lys Ser Glu Phe Gly Leu Val Trp His Glu Ile Val Asn Tyr
180 185 190
Ile Cys Gln Val Ile Phe Trp Ile Asn Phe Leu Ile Val Ile Val Cys
195 200 205
Tyr Thr Leu Ile Thr Lys Glu Leu Tyr Arg Ser Tyr Val Arg Thr Arg
210 215 220
Gly Val Gly Lys Val Pro Arg Lys Lys Val Asn Val Lys Val Phe Ile
225 230 235 240
Ile Ile Ala Val Phe Phe Ile Cys Phe Val Pro Phe His Phe Ala Arg
245 250 255
Ile Pro Tyr Thr Leu Ser Gln Thr Arg Asp Val Phe Asp Cys Thr Ala
260 265 270
Glu Asn Thr Leu Phe Tyr Val Lys Glu Ser Thr Leu Trp Leu Thr Ser
275 280 285
Leu Asn Ala Cys Leu Asp Pro Phe Ile Tyr Phe Phe Leu Cys Lys Ser
290 295 300
Phe Arg Asn Ser Leu Ile Ser Met Leu Lys Cys Pro Asn Ser Ala Thr
305 310 315 320
Ser Leu Ser Gln Asp Asn Arg Lys Lys Glu Gln Asp Gly Gly Asp Pro
325 330 335
Asn Glu Glu Thr Pro Met
340
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1637 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
CA 02307709 2000-OS-OS
44
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 50...1201
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GGCACGAGCC CACCCTGCGT CGGGCCTCAG TCAGCCCCCG GGGGAGGCC ATG AAC GCC 58
Met Asn Ala
1
ACG GGG ACC CCG GTG GCC CCC GAG TCC TGC CAA CAG CTG GCG GCC GGC 106
Thr Gly Thr Pro Val Ala Pro Glu Ser Cys Gln Gln Leu Ala Ala Gly
10 15
GGG CAC AGC CGG CTC ATT GTT CTG CAC TAC AAC CAC TCG GGC CGG CTG 154
Gly His Ser Arg Leu Ile Val Leu His Tyr Asn His Ser Gly Arg Leu
20 25 30 35
GCC GGG CGC GGG GGG CCG GAG GAT GGC GGC CTG GGG GCC CTG CGG GGG 202
Ala Gly Arg Gly Gly Pro Glu Asp Gly Gly Leu Gly Ala Leu Arg Gly
40 45 50
CTG TCG GTG GCC GCC AGC TGC CTG GTG GTG CTG GAG AAC TTG CTG GTG 250
Leu Ser Val Ala Ala Ser Cys Leu Val Val Leu Glu Asn Leu Leu Val
55 60 65
CTG GCG GCC ATC ACC AGC CAC ATG CGG TCG CAA CGC TGG GTC TAC TAT 298
Leu Ala Ala Ile Thr Ser His Met Arg Ser Gln Arg Trp Val Tyr Tyr
70 75 80
TGC CTG GTG AAC ATT ACG ATG AGT GAC CTG CTC ACG GGC GCG GCC TAC 346
Cys Leu Val Asn Ile Thr Met Ser Asp Leu Leu Thr Gly Ala Ala Tyr
85 90 95
CTG GCC AAC GTG CTG CTG TCG GGG GCC CGC ACC TTC CGT CTG GCG CCC 394
Leu Ala Asn Val Leu Leu Ser Gly Ala Arg Thr Phe Arg Leu Ala Pro
100 105 110 115
GCC CAG TGG TTC CTA CGG AAG GGC CTG CTC TTC ACC GCC CTG GCC GCC 442
Ala Gln Trp Phe Leu Arg Lys Gly Leu Leu Phe Thr Ala Leu Ala Ala
120 125 130
TCC ACC TTC AGC CTG CTC TTC ACT GCA GGG TTG CGC TTT GCC ACC ATG 490
Ser Thr Phe Ser Leu Leu Phe Thr Ala Gly Leu Arg Phe Ala Thr Met
135 140 145
GTG CGG CCG GTG GCC GAG AGC GGG GCC ACC AAG ACC AGC CGC GTC TAC 538
Val Arg Pro Val Ala Glu Ser Gly Ala Thr Lys Thr Ser Arg Val Tyr
150 155 160
GGC TTC ATC GGC CTC TGC TGG CTG CTG GCC GCG CTG CTG GGG ATG CTG 586
Gly Phe Ile Gly Leu Cys Trp Leu Leu Ala Ala Leu Leu Gly Met Leu
165 170 175
CA 02307709 2000-OS-OS
CCT TTG CTG GGC TGG AAC TGC CTG TGC GCC TTT GAC CGC TGC TCC AGC 634
Pro Leu Leu Gly Trp Asn Cys Leu Cys Ala Phe Asp Arg Cys Ser Ser
180 185 190 195
CTT CTG CCC CTC TAC TCC AAG CGC TAC ATC CTC TTC TGC CTG GTG ATC 682
Leu Leu Pro Leu Tyr Ser Lys Arg Tyr Ile Leu Phe Cys Leu Val Ile
200 205 210
TTC GCC GGC GTC CTG GCC ACC ATC ATG GGC CTC TAT GGG GCC ATC TTC 730
Phe Ala Gly Val Leu Ala Thr Ile Met Gly Leu Tyr Gly Ala Ile Phe
215 220 225
CGC CTG GTG CAG GCC AGC GGG CAG AAG GCC CCA CGC CCA GCG GCC CGC 778
Arg Leu Val Gln Ala Ser Gly Gln Lys Ala Pro Arg Pro Ala Ala Arg
230 235 240
CGC AAG GCC CGC CGC CTG CTG AAG ACG GTG CTG ATG ATC CTG CTG GCC 826
Arg Lys Ala Arg Arg Leu Leu Lys Thr Val Leu Met Ile Leu Leu Ala
245 250 255
TTC TTG GTG TGC TGG GGA CCA CTC TTC GGG CTG CTG CTG GCC GAC GTC 874
Phe Leu Val Cys Trp Gly Pro Leu Phe Gly Leu Leu Leu Ala Asp Val
260 265 270 275
TTT GGC TCC AAC CTC TGG GCC CAG GAG TAC CTG CGG GGC ATG GAC TGG 922
Phe Gly Ser Asn Leu Trp Ala Gln Glu Tyr Leu Arg Gly Met Asp Trp
280 285 290
ATC CTG GCC CTG GCC GTC CTC AAC TCG GCG GTC AAC CCC ATC ATC TAC 970
Ile Leu Ala Leu Ala Val Leu Asn Ser Ala Val Asn Pro Ile Ile Tyr
295 300 305
TCC TTC CGC AGC AGG GAG GTG TGC AGA GCC GTG CTC AGC TTC CTC TGC 1018
Ser Phe Arg Ser Arg Glu Val Cys Arg Ala Val Leu Ser Phe Leu Cys
310 315 320
TGC GGG TGT CTC CGG CTG GGC ATG CGA GGG CCC GGG GAC TGC CTG GCC 1066
Cys Gly Cys Leu Arg Leu Gly Met Arg Gly Pro Gly Asp Cys Leu Ala
325 330 335
CGG GCC GTC GAG GCT CAC TCC GGA GCT TCC ACC ACC GAC AGC TCT CTG 1114
Arg Ala Val Glu Ala His Ser Gly Ala Ser Thr Thr Asp Ser Ser Leu
340 345 350 355
AGG CCA AGG GAC AGC TTT CGC GGC TCC CGC TCG CTC AGC TTT CGG ATG 1162
Arg Pro Arg Asp Ser Phe Arg Gly Ser Arg Ser Leu Ser Phe Arg Met
360 365 370
CGG GAG CCC CTG TCC AGC ATC TCC AGC GTG CGG AGC ATC TGAAGTTGCA 1211
Arg Glu Pro Leu Ser Ser Ile Ser Ser Val Arg Ser Ile
375 380
GTCTTGCGTG TGGATGGTGC AACCACCGGG TGCGTGCCAG GCAGGCCCTC CTGGGGTACA 1271
GGAAGCTGTG TGCACGCAAC CTCGCCCTGT ATGGGGAGCA GGGAACGGGA CAGGCCCCCA 1331
CA 02307709 2000-OS-OS
46
TGGACTTGCC CGGTGGCCTC TCGGGGCTTC TGACGCCATA TGGACTTGCC CATTGCCTAT 1391
GGCTCACCCT GGACAAGGAG GCAACCACCC CACCTCCCCG TAGGAGCAGA GAGCACCCTG 1451
GTGTGGGGGC GAGTGGGTTC CCCACAACCC CGCTTCTGTG TGATTCTGGG GAAGTCCCGG 1511
CCCCTCTCTG GGCCTCAGTA GGGCTCCCAG GCTGCAAGGG GTGGACTGTG GGATGCATGC 1571
CCTGGCAACA TTGAAGTTCG ATCATGGTAA 1631
1637
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 384 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Asn Ala Thr Gly Thr Pro Val Ala Pro Glu Ser Cys Gln Gln Leu
1 5 10 15
Ala Ala Gly Gly His Ser Arg Leu Ile Val Leu His Tyr Asn His Ser
20 25 30
Gly Arg Leu Ala Gly Arg Gly Gly Pro Glu Asp Gly Gly Leu Gly Ala
35 40 45
Leu Arg Gly Leu Ser Val Ala Ala Ser Cys Leu Val Val Leu Glu Asn
50 55 60
Leu Leu Val Leu Ala Ala Ile Thr Ser His Met Arg Ser Gln Arg Trp
65 70 75 80
Val Tyr Tyr Cys Leu Val Asn Ile Thr Met Ser Asp Leu Leu Thr Gly
85 90 95
Ala Ala Tyr Leu Ala Asn Val Leu Leu Ser Gly Ala Arg Thr Phe Arg
100 105 110
Leu Ala Pro Ala Gln Trp Phe Leu Arg Lys Gly Leu Leu Phe Thr Ala
115 120 125
Leu Ala Ala Ser Thr Phe Ser Leu Leu Phe Thr Ala Gly Leu Arg Phe
130 135 140
Ala Thr Met Val Arg Pro Val Ala Glu Ser Gly Ala Thr Lys Thr Ser
145 150 155 160
Arg Val Tyr Gly Phe Ile Gly Leu Cys Trp Leu Leu Ala Ala Leu Leu
CA 02307709 2000-OS-OS
47
165 170 175
Gly Met Leu Pro Leu Leu Gly Trp Asn Cys Leu Cys Ala Phe Asp Arg
180 185 190
Cys Ser Ser Leu Leu Pro Leu Tyr Ser Lys Arg Tyr Ile Leu Phe Cys
195 200 205
Leu Val Ile Phe Ala Gly Val Leu Ala Thr Ile Met Gly Leu Tyr Gly
210 215 220
Ala Ile Phe Arg Leu Val Gln Ala Ser Gly Gln Lys Ala Pro Arg Pro
225 230 235 240
Ala Ala Arg Arg Lys Ala Arg Arg Leu Leu Lys Thr Val Leu Met Ile
245 250 255
Leu Leu Ala Phe Leu Val Cys Trp Gly Pro Leu Phe Gly Leu Leu Leu
260 265 270
Ala Asp Val Phe Gly Ser Asn Leu Trp Ala Gln Glu Tyr Leu Arg Gly
275 280 285
Met Asp Trp Ile Leu Ala Leu Ala Val Leu Asn Ser Ala Val Asn Pro
290 295 300
Ile Ile Tyr Ser Phe Arg Ser Arg Glu Val Cys Arg Ala Val Leu Ser
305 310 315 320
Phe Leu Cys Cys Gly Cys Leu Arg Leu Gly Met Arg Gly Pro Gly Asp
325 330 335
Cys Leu Ala Arg Ala Val Glu Ala His Ser Gly Ala Ser Thr Thr Asp
340 345 350
Ser Ser Leu Arg Pro Arg Asp Ser Phe Arg Gly Ser Arg Ser Leu Ser
355 360 365
Phe Arg Met Arg Glu Pro Leu Ser Ser Ile Ser Ser Val Arg Ser Ile
370 375 380
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
CCGAGGATCC ATGCAAGCCG TCGACAAT 2g
CA 02307709 2000-OS-OS
48
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CCGAGGATCC TTACATTGGA GTCTCTTC 28
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
CCGAGGATCC GCCATCATGC AAGCCGTCGA CART 34
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
CCGAGGATCC TTACATTGGA GTCTCTTC 28
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
CA 02307709 2000-OS-OS
49
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
CCGAGGATCC GCCATCATGC AAGCCGTCGA CAAT 34
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CCGATCTAGA TTAATCCCAT ACGACGTCCC AGACTACGCT CATTGGAGTC TCTTC 55
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
CCGAGGATCC ATGAACGCCA CGGGGACC 28
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
CCGAGGATCC TCAGATGCTC CGCACGCT 28
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02307709 2000-OS-OS
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
GCGAGGATCC GCCATCATGA ACGCCACGGG GACC 34
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
CCGAGGATCC TCAGATGCTC CGCACGCT 28
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
CCGAGGATCC GCCATCATGA ACGCCACGGG GACC 34
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
CCGATCTAGA TCAATCCCAT ACGACGTCCC AGACTACGCT GATGCTCCGC ACGCT 55
(2) INFORMATION FOR SEQ ID N0:17:
CA 02307709 2000-OS-OS
51
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 348 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Ile Gln Met Ala Asn Asn Phe Thr Pro Pro Ser Ala Thr Pro Gln Asn
1 5 10 15
Asp Cys Asp Leu Tyr Ala His His Ser Thr Ala Arg Ile Val Met Pro
20 25 30
Leu His Tyr Ser Leu Val Phe Ile Ile Gly Leu Val Gly Asn Leu Leu
35 40 45
Ala Leu Val Val Ile Val Gln Asn Arg Lys Lys Ile Asn Ser Thr Thr
50 55 60
Leu Tyr Ser Thr Asn Leu Val Ile Ser Asp Ile Leu Phe Thr Thr Ala
65 70 75 80
Leu Pro Thr Arg Ile Ala Tyr Tyr Ala Met Gly Phe Asp Trp Arg Ile
85 90 95
Gly Asp Ala Leu Cys Arg Ile Thr Ala Leu Val Phe Tyr Ile Asn Thr
100 105 110
Tyr Ala Gly Val Asn Phe Met Thr Cys Leu Ser Ile Asp Arg Phe Ile
115 120 125
Ala Val Val His Pro Leu Arg Tyr Asn Lys Ile Lys Arg Ile Glu His
130 135 140
Ala Lys Gly Val Cys Ile Phe Val Trp Ile Leu Val Phe Ala Gln Thr
145 150 155 160
Leu Pro Leu Leu Ile Asn Pro Met Ser Lys Gln Glu Ala Glu Arg Ile
165 170 175
Thr Cys Met Glu Tyr Pro Asn Phe Glu Glu Thr Lys Ser Leu Pro Trp
180 185 190
Ile Leu Leu Gly Ala Cys Phe Ile Gly Tyr Val Leu Pro Leu Ile Ile
195 200 205
Ile Lys Ile Cys Tyr Ser Gln Ile Cys Cys Lys Leu Phe Arg Thr Ala
210 215 220
Lys Gln Asn Pro Leu Thr Glu Lys Ser Gly Val Asn Lys Lys Ala Leu
225 230 235 240
Asn Thr Ile Ile Leu Ile Ile Val Val Phe Val Leu Cys Phe Thr Pro
245 250 255
Tyr His Val Ala Ile Ile Gln His Met Ile Lys Lys Leu Arg Phe Ser
260 265 270
Asn Phe Leu Glu Cys Ser Gln Arg His Ser Phe Gln Ile Ser Leu His
275 280 285
Phe Thr Val Cys Leu Met Asn Phe Asn Cys Cys Met Asp Pro Phe Ile
290 295 300
Tyr Phe Phe Ala Cys Lys Gly Tyr Lys Arg Lys Val Met Arg Met Leu
305 310 315 320
Lys Arg Gln Val Ser Val Ser Ile Ser Ser Ala Val Lys Ser Ala Pro
325 330 335
Glu Glu Asn Ser Arg Glu Met Thr Glu Thr Gln Met
340 345
CA 02307709 2000-OS-OS
51.1
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 381 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Met Gly Pro Thr Ser Val Pro Leu Val Lys Ala His Arg Ser Ser Val
1 5 10 15
Ser Asp Tyr Val Asn Tyr Asp Ile Ile Val Arg His Tyr Asn Tyr Thr
20 25 30
Gly Lys Leu Asn Ile Ser Ala Asp Lys Glu Asn Ser Ile Lys Leu Thr
35 40 45
Ser Val Val Phe Ile Leu Ile Cys Cys Phe Ile Ile Leu Glu Asn Ile
50 55 60
Phe Val Leu Leu Thr Ile Trp Lys Thr Lys Lys Phe His Arg Pro Met
65 70 75 80
Tyr Tyr Phe Ile Gly Asn Leu Ala Leu Ser Asp Leu Leu Ala Gly Val
85 90 95
Ala Tyr Thr Ala Asn Leu Leu Leu Ser Gly Ala Thr Thr Tyr Lys Leu
100 105 110
Thr Pro Ala Gln Trp Phe Leu Arg Glu Gly Ser Met Phe Val Ala Leu
115 120 125
Ser Ala Ser Val Phe Ser Leu Leu Ala Ile Ala Ile Glu Arg Tyr Ile
130 135 140
Thr Met Leu Lys Met Lys Leu His Asn Gly Ser Asn Asn Phe Arg Leu
145 150 155 160
Phe Leu Leu Ile Ser Ala Cys Trp Val Ile Ser Leu Ile Leu Gly Gly
165 170 175
Leu Pro Ile Met Gly Trp Asn Cys Ile Ser Ala Leu Ser Ser Cys Ser
180 185 190
Thr Val Leu Pro Leu Tyr His Lys His Tyr Ile Leu Phe Cys Thr Thr
195 200 205
Val Phe Thr Leu Leu Leu Leu Ser Ile Val Ile Leu Tyr Cys Arg Ile
210 215 220
Tyr Ser Leu Val Arg Thr Arg Ser Arg Arg Leu Thr Phe Arg Lys Asn
225 230 235 240
Ile Ser Lys Ala Ser Arg Ser Ser Glu Asn Val Ala Leu Leu Lys Thr
245 250 255
Val Ile Ile Val Leu Ser Val Phe Ile Ala Cys Trp Ala Pro Leu Phe
260 265 270
Ile Leu Leu Leu Leu Asp Val Gly Cys Lys Val Lys Thr Cys Asp Ile
275 280 285
Leu Phe Arg Ala Glu Tyr Phe Leu Val Leu Ala Val Leu Asn Ser Gly
290 295 300
Thr Asn Pro Ile Ile Tyr Thr Leu Thr Asn Lys Glu Met Arg Arg Ala
305 310 315 320
Phe Ile Arg Ile Met Ser Cys Cys Lys Cys Pro Ser Gly Asp Ser Ala
325 330 335
Gly Lys Phe Lys Arg Pro Ile Ile Ala Gly Met Glu Phe Ser Arg Ser
CA 02307709 2000-OS-OS
51.2
340 345 350
Lys Ser Asp Asn Ser Ser His Pro Gln Lys Asp Glu Gly Asp Asn Pro
355 360 365
Glu Thr Ile Met Ser Ser Gly Asn Val Asn Ser Ser Ser
370 375 380
