Note: Descriptions are shown in the official language in which they were submitted.
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~WAN ~:u~Or~sr~-lv K~ .O~
T]his invention relates to newly identified
polynucleotides, polypeptides encoded by such
polynucleotides, the use of such polynucleotides an,d
polype~ptides, as well as the production of such
polynucleotides and polypeptides. The polypeptides o~ the
present invention are human 7-tr~n~m~mhralle G-protein coupled
receptors. More particularly, the polypeptides of the
present invention are neuropeptide receptor polypeptides,
sometimes hereinafter referred to as neuropeptide receptor
polypeptides. The invention also relates to inhibiting the
action of such polypeptides.
Obesity i8 the comm--n~st nutritional disorder in Western
societies. More than three in ten adult Americans weigh at
least 2096 in excess o~ their ideal body weight (Burroa, M.,
The New York Times, 17 July 1994). Increased body weight is
an important public health problem because it is associated
with '~ype II diabetes, hypertension, hyperlipidemia and
certain cancers (Grundy, S.M., and Barnett, J.P., Disease-a-
Month, 36:645-696 (1990)).
~ ive single-gene mutations in the mouse obesity gene
(ob) which result in an obese phenotype have been described
(Friedman, J.M. & Leibel, R. L., Cell, 66:217-220 (1990)).
The cloning and sequencing of the mouse ob gene and its human
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homologue have been reported ~Zhany, Y., et al., Nature,
372:425-431 (1994)). The ob gene encodes a 4.5-kb adipose
ti~sue mRNA with a highly conserved 167-amino-acid open
reading frame. The predicted amino-acid sequence is 84~
identical between human and mouse and has features of a
secreted protein. The ob gene product may function as part
of a signalling pathway from adipose tissue that acts to
regulate the size of the body fat depot ( d. 425).
Of the brain regions implicated in the regulation of
feeding behavior, the ventromedial nucleus of the
hypothAlAmll~ (VMH) i8 considered to be the most important
satiety center in the central nervous system (CNS). The
energy balance in mAmmAls is therefore postulated to be
controlled by a feedhack loop in which the amount of stored
energy is sensed by the hypothAlAmll~, which adjusts food
intake and energy expenditure to mA;ntAin a constant body
weight (Ombeck, J.R., Yale J. Biol. Med., 20:545-552 (1948)
and Kennedy, G.C., Proc. R. Soc.148:578-592 (1953)). In the
lipostasis theory, the size of the body fat depot is
regulated by the CNS, with a product of body fat metabolism
affecting energy hAlAnce by interacting with the hypothalamus
(Kennedy, G.C., Proc. R. Soc.148:578-592 (1953)).
The ;nAh;lity to identify the putative signal from fat
has h;n-lered the validation of the lipostasis theory. The
possibility that at least one component of the signalling
system circulates in the bloodstream was first suggested by
Hervey (Dietrich, W., et al., Genetics, 131:423-447 (1992)),
who showed that the transfer of blood from an An;mAl with a
VMH lesion across a vascular graft to an untreated An;~Al (a
parabiosis experiment) resulted in a reduction of food intake
in the intact An;mAl It is now significant that there is
evidence that the ob gene product is secreted, suggesting
that ob may encode this circulating factor.
The ob signal may act directly or indirectly on the CNS
to inhibit food intake and/or regulate energy expenditure as
CA 02220036 1997-10-31
W O 96~34877 PCT/US95/05616
part oi- a homeostatic mechanism to m~n~in constancy of the
adipose mass (Zhang, Y., et al., Nature, 372:425-431, 431
~1994)'l. The ob gene apparently encodes a protein secreted
by fat., and mutations apparently prevent translation or
expression of the gene (Rink, T., Nature, 372:406-407
(1994)l.
Parabiosis experiments suggest thaLt the ob receptor i8
encoded by the mouse db (diabetes) gene (Coleman, D.L.,
Diabetologia, 14:141-148 (1978)). Mice having a mutation in
the db gene are al~o obese, with the de~ect possibly beiny a
receptor defect. (Id. at 406).
Neuropeptide Y is similar to the ob gene product in that
it mediates the feeding response. Neuropeptide Y acts on at
least ~our types of neuropeptide Y receptors called Y" Y2, Y3
and an atypical Y~ receptor, which mediates the feeding
response stimulated by neuropeptide Y.
Neuropeptide Y has a wide range of biological functions.
Neuropeptide Y is found to be widely distributed in th.e
central nervous system (CNS) and the peripheral nervou~
system. (PNS). In the PNS, neuropeptide Y is found in thLe
noradrenergic sympathetic innervation o:E blood vessels and
other smooth muscle tissues and in neurons within the enteric
nervous system. Neuropeptide Y ;mmllnoreactive fibers al~o
occur in the non-vascular smooth muscle, surrounding exocrinLe
glands and surface epithelia. Neuropeptide Y also occurs in
subpopulations of neurons and is generally co-localized with
other neurotransmitters, particular nora.drenaline.
]:n the CNS, neuropeptide Y is cont~ n~ in GABAergi.c
interneurons in higher centers and in pre~om~nAntly
catechol~m~nergic cells that project further caudally. For
examp]e, neuropeptide Y is contained in interneurons in the
cortex, hippocampus, amygdala, basal forebrain and striatum,
whereas in the brain stem, neuropeptide Y is ~contained in
~ noradrenergic neurons of the A, and A2 groups in the medulla,
and l_he locus coeruleus (LC). In the hypoth~1~mll~,
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neuropeptide Y is found pre~min~ntly in the arcuate nucleus
and lateral hypoth~
Within the peripheral nervous system, neuropeptide Y is
present in postganglionic sympathetic nerves, and is co-
localized as stated above with other neurotransmitters,
including catechol ~mi nPfi, When used pharmacologically,
neuropeptide Y has been shown to have a potent
vasoconstrictor activity as well as dramatically potentiating
the vasoconstriction caused by many other pressor agents.
Particularly high concentrations of neuropeptide Y are found
in the sympathetic nerves supplying the coronary, cerebral
and renal vasculature and when infused into these vascular
beds, neuropeptide Y causes prolonged vasoconstriction that
is not reversed by adrenergic blocking agents. These
observations have lead to the proposal that neuropeptide Y is
the candidate transmitter for pathological vasospasm, a major
cause o~ morbidity and mortality when involving the coronary
and cerebral vessels.
Neuropeptide Y also appears to be involved in
interaction with the renin angiotensin system. Neuropeptide
Y contA;n;ng sympathetic nerve terminals are found on the
juxta-glomerular apparatus of the renal cortex and
neuropeptide Y influences renin release. These data,
together with the ~Pmon~tration of all durations in
neuropeptide Y concentrations in hypertensive ~n;m~l models
and the pressor response to infusion of the peptide, have
resulted in implications of this peptide in hypertension.
Within the central nervous system neuropeptide Y is
located pre~om;n~ntly within interneurons where it appears to
have a regulatory role. It therefore has widespread and
diverse effects including effects on memory and a possible
role in Al7he;mPr~s disease. Neuropeptide Y is the most
potent known substance to cause an increase in feeding and
may play a role in the genetic basis of Type II Diabetes
Mellitus. Neuropeptide Y may also play a role as a
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regulat.ory agent and pituitary function as well as potential
neuromodulatory function in stress responses and in
reprod~lctive function.
In accordance with one aspect of the present invention~
~ there aLre provided novel mature receptor polypeptides as well
as bio]ogically active and diagnostically or therapeutically
~ u8eful fragments, analogs and derivatives thereo~. The
receptor polypeptides of the present inv~ntion are of human
origin.
~ n accordance with another aspect of the presen~
invent:ion, there are provided isolated nucleic acid molecules
encoding the receptor polypeptides of the present invention,
includ:ing mRNAs, DNAs, cDNAs, genomic DNA as well as
antisense analogs thereof and biologically active and
diagno,stically or therapeutically useful fragments thereof..
I:n accordance with a further aspect of the present
invention, there are provided processes for producing such
receptor polypeptides by recomhin~nt techniq~ues comprising
culturing rec~mbin~nt prokaryotic and/or eukaryotic host
cells, cont~;n;ng nucleic acid seq[uences encoding the
receptor polypeptides of the present invention, under
conditions promoting expression of sai.d polypeptides an.d
subsequent recovery of said polypeptides.
I.n accordance with yet a further aspect of the presen.t
invent.ion, there are provided antibodies against such
receptor polypeptides.
In accordance with another aspect of the present
invent:ion there are provided methods of screening for
compo~mds which bind to and activate or inhibit activation of
the receptor polypeptides of the present invention.
:[n accordance with still another embo~-im~nt of the
present invention there are provi.ded processes of
~m; nistering compounds to a host which bind to and activat:e
the receptor polypeptide of the present invention which axe
usefu:L in the prevention and/or treatment of obesity,
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hyperlipidemia, certain cancers, to stimulate neuronal
growth, to regulate neurotransmission, to ~nh~nce activity
levels and utilization of ingested foods.
In accordance with another aspect ~of the present
invention there is provided a method of ~m;n; stering the
receptor polypeptides of the present invention via gene
therapy to treat conditions related to underexpression of the
polypeptides or underexpression of a ligand to the receptor
polypeptide.
In accordance with still another embodiment of the
present invention there are provided processes of
administering compounds to a host which bind to and ; nh; h~ t
activation of the receptor polypeptides of the present
invention which are useful in the prevention and/or treatment
of Al~he;mer's disease, Type II Diabetes Mellitus, epilepsy,
stress, anxiety, hypertension, cardiovascular disea~e,
psychotic conditions and obesity caused by neuropeptide Y.
In accordance with yet another aspect of the present
invention, there are provided nucleic acid probes comprising
nucleic acid molecules of sufficient length to specifically
hybridize to the polynucleotide sequences of the present
invention.
In accordance with still another aspect of the present
invention, there are provided diagnostic assays for detecting
diseases related to mutations in the nucleic acid sequences
encoding such polypeptides and for detecting an altered level
of the soluble form of the receptor polypeptides.
In accordance with yet a further aspect of the present
invention, there are provided processes for utilizing such
receptor polypeptides, or polynucleotides encoding such
polypeptides, for in vitro purposes related to scientific
research, synthesis of DNA and manufacture of DNA vectors.
These and other aspects of the present invention should
be apparent to those skilled in the art from the teachings
herein.
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The following drawings are illustrative o~ embodiments
of the invention and are not meant to limit the scope of the
invention as encomr~sed by the cl ~ms .
F:igure 1 shows the cDNA sequence and the corresponding
deduced amino acid sequence of the neuropeptide receptor
polypeptide of the present invention. The st~n~rd one-
~ letter abbreviation for amino acids is u~ed. Se~l~nc~ng was
performed using a 373 Automated DNA sequencer ~Applied
Biosystems, Inc.).
Figure 2 shows the cDNA sequence and the correspondingdeduced amino acid sequence of the neuropeptide receptor
splice variant 1 polypeptide o~ the pre~ent invention. The
st~n~rd one-letter abbreviation for amino acids is used.
P~igure 3 shows the cDNA sequence and the corresponding
deduced amino acid sequence o~ the neuropeptide receptor
splice! variant 2 polypeptide of the pre~ent invention. The
st~n~l~d one-letter abbreviation for amino acids is used.
Figure 4 illustrates the amino acid sequence and seven
tr~nsmemhrane regions of the neuropeptide receptor. The
tr~nfim~mh~ane regions are underlined and denoted with a TM.
~ igure 5 illustrates the amino aci~ sequence and seven
tr~n~m~mhrane regions of the neuropeptide receptor splice
variant 1. The tr~nsm~mhrane regions are underlined and
denoted with a TM.
]Figure 6 illustrates the amino acid sequence and seven
transmembrane regions of the neuropeptide receptor splice
variant 2. The transmemhrane regions are underlined and
denoted with a TM.
Figure 7 shows the amino acid homology between the human
neuropeptide receptor polypeptide of the present invention
(and the human neuropeptide Y~ receptor).
The receptor polypeptides of the present invention are
recepltors for ligands, both known and unl~nown, which modulate
the activity of cells in both the central nervous system and
peripheral tissues regulated by the central nervous system.
,
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~ CA 02220036 1997-10-31 5 ~ 16
IPEA/US 3 1 MAR 1997
Exam~les of such ligands are neuropeptide Y, substance P, the
human ob gene product and neurokinin B. Accordingly,
modulation of the activity of receptor polypeptides of the
pre~ent invention will have a broad range of therapeutic and
diagnostic applications, particularly with respect to the
treatment of obesity.
The present inventors have isolated a full-length cDNA
clone encoding a human neuropeptide receptor polypeptide.
The present full-length cDNA has been mapped to a location on
human chromosome 1 position p31-34 which corresponds to a
location on the mouse chromosome 4 where the db gene is
found. The mouse db gene is thought to encode the receptor
~or the obesity gene product
In accordance with an aspect of the present invention,
there are provided isolated nucleic acids (polynucleotides)
which encode for the mature polypeptide having the deduced
amino acid sequence of Figures 2 (S~Q ID NO:2) or ~or the
mature polypeptide encoded by the cDNA of the clone~s)
depasited as ATCC Deposit No. 97128 on April 28, 1995.
The polynucleotide of this invention was discovered in
a cDNA library derived from human adult hypoth~l~m~ It is
structurally related to the G protein-coupled receptor
family. The neuropeptide receptor polypeptide cont~in~ an
open reading frame encoding a protein of 402 amino acid
residues. The neuropeptide receptor protein exhibits the
highe~t degree of homology to human neuropeptide Yl receptor
protein with 52 % ~imilarity and 26 % identity over the
entire amino acid sequence.
The polynucleotides of the present invention may be in
the form of RNA or in the form of DNA, which DNA incl~des
cDN~, 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 sequences which encode the mature polypeptide may be
identical to the coding sequence shown in Figure 1 (SFQ ID
,~lOED S~
CA 02220036 1997-10-31
W 096/34877 PCTrUS95/05616
NO:1) or that of the deposited clone(s) or may be a di~feren~
coding se~uence which coding sequence, aLs a result of the
re~lln~ncy or degeneracy of the genetic code, encodes the
same n~lture polypeptide as the DNA of Figure 1 (SEQ ID NO:1)
or the deposited cDNA(s).
The polynLucleotides which encode for the mature
~ polypeotide of Figure 2 (SEQ ID NO:2) or for the mature
polypeptide encoded by the deposited c~NA(s) mi3Ly include:
only the coding sequence for the mature polypeptide; the
coding se~uence 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 mi3Lture polypeptide.
T]lus, 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 n.on-coding sequence.
T]he present invention further relates to variants of the
herPin.~bove described polynucleotides which encode for
fragme:nts, analogs and derivatives of the polypeptides having
the delduced amino acid sequence of Figure 2 (SEQ ID NO:2) or
the polypeptide encoded by the cDNA of the deposited
clone(s). The variants of the polynucleotide may be
naturally occurring allelic variants of the polynucleotides
or non-naturally occurring variants of the polynucleotides.
Thus, the present invention includes polynucleotides
encoding the same mature polypeptide as shown in Figure 2
(SEQ ID NO:2) or the same maLture polypeptide encoded by the
cDNA of the deposited clone(s) as well as variants of such
polYnucleotide which variants encode ~or a fragment,
deriva.tive or analog of the polypeptide of Figure 2 (SEQ ID
NO:2) or the polypeptide encoded by the cDNA of the deposited
clone~s). Such nucleotide variants inclu,de deletion
variants, substi~ution variants and addition or insertion
variants. Specific examples of such variants include the
_g_
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polynucleotide sequences as set forth in SEQ ID NOS:3 and 5
which encode for ~plice variant 1 and 2, respectively, of the
polypeptide of the present invention.
As hereinabove indicated, the polynucleotides may have
a coding sequence which is a naturally occurring allelic
variant of the coding sequence shown in Figure 1 (SEQ ID
NO:1) or of the coding sequence of the deposited clone(s).
As known in the art, an allelic variant is an alternate form
of polynucleotide sequences which may have a substitution,
deletion or addition of one or more nucleotides, which does
not sub~tantially alter the function of the encoded
polypeptides.
The polynucleotides may also encode for a soluble form
of the neuropeptide receptor polypeptide which is the
extracellular portion of the polypeptide which has been
cleaved from the TM and intracellular ~m~i n of the full-
length polypeptide of the present invention.
The polynucleotides of the present invention may also
have the coding sequence fused in frame to a m~rker 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 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 m~mm~l ian
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 present invention further relates to
polynucleotides which hybridize to the her~;nAhove-described
sequences if there is at least 70~, preferably at least 90%,
and more preferably at least 95% identity between the
sequences. The present invention particularly relates to
polynucleotides which hybridize under stringent conditions to
the hereinabove-described polynucleotides. As herein used,
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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 hereinabove described polynucleotides
in a preferred ~mho~m~nt encode polypeptides which either
retain substantially the same biological $unction or activity
as the mature polypeptide encoded by the cDNAs of Figure 1
(SBQ I]D NO:1) or the deposited cDNA(s), i.e. function as a
soluble neuropeptide receptor by retA~n~ng the ability t~
bind the ligands for the receptor even though the polypeptide
does not function as a membrane bound neuropeptide receptor,
for example, by eliciting a second messenger response.
Alternatively, the polynucleotides may be
polynurleotides which have at least 20 bases, preferably 30
bases ,and more preferably at least 50 bases which hybridize
to a polynucleotide of the present invention and which have
an identity thereto, as her~nAhove described, and which does
not rel_ain activity. Such polynucleotides may be employed as
probes for the polynucleotide of SEQ ID NO: 1, or for
variants thereof, for example, for recovery of the
polynucleotide or as a diagnostic probe or as a PCR primer
The deposit~s) referred to herein will be ~AtntA~n~
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.
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The present invention ~urther relates to a polypeptide
which has the deduced amino acid sequence of Figure 2 (SEQ ID
NO:2) or which has the amino acid sequence encoded by the
deposited cDNA(s), as well as fragments, analogs and
derivatives of such polypeptide.
The terms ~fragment," "derivative'l and "analog" when
referring to the polypeptide of Figure 2 (SEQ ID NO:2) or
that encoded by the deposited cDNA(s), means polypeptides
which either retain substantially the same biological
function or activity as such polypeptides, i.e., function as
a soluble neuropeptide receptor by retA;n~ng the ability to
bind the ligands of the receptors even though the
polypeptides do not function as membrane bound neuropeptide
receptors. An analog includes a proprotein which can be
activated by cleavage of the proprotein portion to produce an
active mature polypeptide. Specific examples are splice
variant 1 and 2 of Figures 2 and 3 (SEQ ID NO:4 and 6),
respectively.
The polypeptides of the present invention may be
reComh;n~nt polypeptides, natural polypeptides or synthetic
polypeptides, preferably recombinant polypeptides.
A fragment, derivative or analog of the polypeptide of
Figure 2 (SEQ ID NO:2) or that encoded by the deposited
cDNA(s) 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, (ii) one in which one
or more of the amino acid residues includes a substituent
group, (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), (iv) one in which the additional amino acids are
fused to the mature polypeptide, such as sequence which is
employed for purification of the mature polypeptide sequence
CA 02220036 1997-10-31
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or (iV'I splice variants of the mature polypeptide whieh may
have one or more amino acids deleted from the mature
polypeptide yet still retain activity c~rresponding to the
mature polypeptide. Such fragments, derivatives and analogs
are dee~med to be within the scope of those skilled in the art
from the t~h~ngs herein.
The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
pre~erably are purified to homogeneity.
The tenm "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes reyions preceding
and following the coding region "leader and trailer" as well
as intervening sequences (introns) between individual coding
segments (exons).
I~he term "isolated" means that the material is removed
from its original environment (e.g., the natural envi~unllle~t
if it is naturally occurring). For example, a naturally-
occurring polynucleotide or polypeptide present in a living
~ni~l iS not isolated, but the same polynucleotide or
polypeptide, separated from some or all of the coexisting
materi.als in the natural system, is isolated. Such
polymlcleotides could be part of a vector and/or such
polymlcleotides or polypeptides could be part of a
compol,ition, and still be isolated in that such vector or
composition is not part of its natural environm~nt.
The present invention also relates to vectors which
inclu~e 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, for example, a cloning vector or an
expression vector. The vector m~y be, for example, in the
form of a plasmid, a viral particle, a phage, etc. The
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engineered host cells can be cultured in conventional
nutrient media modified as appropriate for activating
promoters, selecting transformants or amplifying the human
neuropeptide 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.
The polynucleotides of the present invention may be
employed for producing polypeptides by recom.binant
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
comh;nAtions of plasmids and 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
~n~onllclease site(s) by 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 sequence(s)
(promoter) to direct mRNA synthesis. As representative
examples of such promoters, there may be mentioned: hTR or
SV40 promoter, the E. coli. lac or trP, the phage 1 Amh~lA 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.
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The v~ector may also include appropriate sequences for
ampli~ying expression.
In addition, the expre8sion vectors pre~erably 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 ~or eukaryoti.c
cell culture, or such as tetracycline or ampicilli.n
resistance in E. coli.
I'he vector cont~ ni n~ the appropriate DNA sequence as
hereinabove described, as well as an appropriate promoter or
contrcll sequence, may be employed to transform an appropriate
host to permit the host to express the protein.
~ -s representative examples of a~.o~,iate hosts, there
may be mentioned: bacterial cells, such as E. coli,
strePt:omyces~ S~lm~nella tYphimurium; fungal cells, such as
yeast; insect cells such as Droso~hila S2 and S~odo~tera Sf9;
~n~l cells such as CHO, COS or Bowes m~l ~no~ i
adenoviruses; plant cells, etc. The selection of an
appropriate host is deemed to be within the scope of tho~e
skilled in the art from the teachings herein.
r~Ore particularly, the present invention also includes
reco~inant constructs comprising one or more of the
sequences as broadly described above. The construc~s
compr:ise a vector, such as a plasmid or viral vector, in1o
which a sequence of the invention has been inserted, in a
forward or reverse orientation. In a preferred aspect of this
embodiment, the construct further comprises regulatory
sequences, including, $or example, a promoter, operably
linked to the sequence. Large numbers o~ suitable vectors
and promoters are known to those of skill in the art, and are
co~m~rcially available. The following vectors are provid,ed
by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),
pbs, pDlO, phagescript, psiXl74, pbluescript~ SK, pbsks,
pNH8A, p~l6a, pNHl8A, pNH46A (Stratagene!; pTRC99a, pKK223-
3, p~K233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO,
.
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pSV2CAT, pO&44, pXT1, 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.
Promoter regions can be selected from any desired gene
using CAT (chlor~mph~n;col transferase) vectors or other
vectors with selectable markers. Two appropriate vectors are
PKK232-8 and PCM7. Particular named bacterial promoters
include lacI, lacZ, T3, T7, gpt, lambda PR~ PL and trp.
Eukaryotic promoters include CMV ~mm~ te early, HSV
thymidine kinase, early and late SV40, LTRs 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 cont~;n;ng the above-described constructs. The
host cell can be a higher eukaryotic cell, such as a
m~mm~lian 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, DEAE-
Dextran mediated transfection, or electroporation (Davis, L.,
Dibner, M., Battey, I., Basic Methods in Molecular Biology,
~1986)).
The constructs in host cells can be used in a
conventional m~nner to produce the gene product encoded by
the recombinant sequence. Alternatively, the polypeptides of
the invention can be synthetically produced by conventional
peptide synthesizers.
Fragments of the polypeptides of the present invention
may be employed for producing the corresponding full-length
polypeptide by peptide synthesis, therefore, the fragments
may be employed as intermediates ~or producing the full-
length polypeptides. Fragments of the polynucleotides of the
present invention may be used in a similar m~nner to
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synthe~size the full-length polynucleotides of the present
invention.
Mature proteins can be expressed in mAmm~1~An cells,
yeast, bacteria, or other cells under the control of
a~l~Liate promoters. Cell-free translation systems can
also b~e employed to produce such protein~ using RNAs derived
from the DNA constructs of the present invention.
Appropriate cloning and expression vectors for use with
prokaryotic and eukaryotic hosts are de~cribed by Sambrook,
et al., Molecular Cloning: A Laboratory ~nll~l, Second
~dition, 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 ~nh~ncer sequence into the vector. Rnh~ncers
are cis-acting el~m~nts of DNA, usually about from 10 to 3~0
bp that act on a promoter to increase its transcription.
Examples including the SV40 ~nh~ncer on the late side of the
replication origin bp 100 to 270, a cytomegalovirus early
promoter ~nh~ncer, the polyoma ~nh~ncer on the late side of
the replication origin, and adenovirus enhancers.
Generally, reco~h; n~nt expression vectors will include
origins of replication and selectable markers permitting
transf-ormation of the host cell, e.g., the aml?icillin
resist:ance 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
promol-ers can be derived from operons PnCo~ng glycolytic
enzymes such as 3-phosphoglycerate kinase (PGK), ~-factor,
acid ]?hosphatase, or heat shock proteins, among others. The
heterologous structural sequence is assembled in a~o~riate
phase with translation initiation and termination sequences,
and preferably, a leader sequence capable of directing
secretion o~ translated protein into the periplasmic space or
extracellular medium. Optionally, the heterologous sequence
~ , .
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can encode a fusion protein including an N-terminal
identification peptide imparting desired characteristics,
e.g., stabilization or simplified purification of expressed
recomh~n~nt 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 vector and to, if
desirable, provide amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli,
Bacillus subtilis, Salmonella tY~himurium and various species
within the genera Psell~omon~s~ 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 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'l
sections are combined with an appropriate promoter and the
structural se~uence to be 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, and the resulting
crude extract retained for further purification.
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Microbial cells employed in expression o~ proteins cc~n
be disrupted by any convenient method, including freeze-thi~w
cycling, sonication, mechanical disruption, or use o~ ce].l
lysing agents, such methods are well know to those skilled i.n
the art.
~ arious mAmm~lian cell culture systems can also be
employed to express recom-h~nAnt protein. ~xamples of
m~ l ian expression Cystems include ~he COS-7 lines of
monkey kidney fibroblasts, described by ~luzman, Cell, 23:175
(1981), and other cell lines cApAbl~ of expressing a
comlpat.ible vector, for example, the C127, 3T3, CHO, HeLa and
BHK cell lines. ~A~'l ian eXprecsion vectors will comprise
an origin of replication, a suitable promoter and PnhAncer,
and also any necessary ribosome binding sites,
polyaclenylation site, splice donor and acceptor sites,
transc:riptional termination sequences) and 5' flankillg
nontricmscribed sequences. DNA sequences derived from the
SV40 splice, and polyadenylation sites m~y be used to provide
the required nontranscribed genetic ele~ents.
The neuropeptide receptor polypeptide of the present
invenl_ion can be recovered and purified from recombinant cell
cultu:res by methods including Ammon~um sulfate or ethanol
precipitation, acid extraction, anion or cation ~xrh~nge
chrom~tography, phosphocellulose chromatography, hydrophobic
interiaction chromatography, affinil:y chromatography,
hydroxylapatite chromatography and lectin chromatography.
Protein refolding steps can be used, as necessary, in
completing configuration of the mature protein. Finally,
high performance liquid chromatography (HPLC) can be employed
for final purification steps.
The neuropeptide receptor polypeptide of the present
invention may be a naturally purified product, or a product
of chemical synthetic procedures, or produced by recomh~nAnt
techniques from a prokaryotic or eukaryotic host (for
exam.F~le, by bacterial, yeast, higher plant, insect and
CA 02220036 1997-10-31
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m~mmAl~An cells in culture). Depending upon the host
employed in a reco~h~ n~nt 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 polynucleotides and polypeptides of the present
invention may be employed as research reagents and materials
for discovery of treatments and diagnostics to human disease.
The human neuropeptide receptor polypeptides of the
present invention may be employed in a process for screening
compounds which bind to and activate the receptor polypeptide
and for compounds which bind to and inhibit activation of the
receptor polypeptides of the present invention.
In general, the neuropeptide receptor in isolated,
immobilized or cell bound form is contacted with a plurality
of compounds and those compounds are selected which bind to
and interact with the receptor. The h; nA; ng or interaction
can be measured directly by using radioactively labeled
compounds of interest or by the second messenger effect
resulting from the interaction or binding of the candidate
compound. Alternatively, the candidate compounds can be
subjected to competition screening assays, in which a known
ligand, preferably labeled with an analytically detectable
reagent, most preferably radioactivity, is introduced with
the compound to be tested and the compound~s capacity to
inhibit or ~nhAnce the binding of the labeled ligand is
measured. Compounds are screened for their increased
afffinity and selectivity to the receptor polypeptide of the
present invention.
One such screening procedure involves the use of
melanophores which are transfected to express the
neuropeptide receptor of the present invention. Such a
screening technique is described in PCT WO 92/01810 published
February 6, 1992.
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For example, to screen for compounds which i nhi bit
activation of the receptor polypeptide of the present
invention, the compound and a ligand ~lown to bind to the
receptor are both contacted with the melanophore cell~.
Tnh; hi tion of the signal generated by the ligand indicates
that t:he compound inhibits activation of the receptor.
~ he screen rnay be employed for determining a compo~ld
which binds to and activate~ the receptor polypeptide of the
present invention by contacting such cells with com~ounds ~o
be screened and deter~ining whether such compound generates
a signal, i.e., activates the receptor.
Other examples include the use of cells which express a
neuropeptide receptor of the present invention (for example,
transiected CHO cells) in a system which nnea~ures extra-
cellu:Lar pH changes caused by receptor activation, for
exarnp:Le, as described in Science, volume 246, pages 181-296
(October 1989). ~or example, compounds may be contacted with
a celL which expresses an neuropeptide receptor polypepti~e
of the present invention and a second messenger response,
e.g. signal transduction or pH changes, may be measured to
deten~ine whether the potential compound is effective as ~n
activator or inhibitor.
~ nother example involves introducing RNA encoding a
neuropeptide receptor of the present invention into Xenopus
oocytes to transiently express the receptor. The oocytes may
then be contacted with the receptor ligand and a compound to
be screened, followed by detection of ~ nh- hi tion of or an
increase in intracellular calcium.
Another exan~ple involves expres~3ing a neuropeptide
receptor polypeptide of the present invention on the surface
of a cell wherein the receptor i~ linked to a phospholipase
C or D. As representative examples of such cells there n~y
be rnentioned endothelial cells, smooth mu~scle cells,
embr~onic kidney cells, etc. The screening may be
accomplished as hereinabove described by detecting activation
CA 02220036 1997-10-31
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of the receptor or ~nh~ h; tion of activation of the receptor
from the phospholipase second signal.
Another method involves determining ;nh; h; tion of
h;n~ing of labeled ligand to cells which have a neuropeptide
receptor on the surface thereof. Such a method involves
transfecting a eukaryotic cell with DNA encoding an
neuropeptide receptor polypeptide of the present invention
such that the cell expresses the receptor on its surface and
contacting the cell with a compound 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 of the receptors. If the compound 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.
Another screening technique involves expre~sing a
neuropeptide receptor polypeptide on the surface of a cell
wherein the receptor is linked to a second messenger to
increase cytosolic calcium levels in transfected CHO cells.
An example of such a method comprises transfecting CHO cells
with a nucleic acid sequence encoding a receptor of the
present invention such that the receptor is expressed on the
surface thereof. The transfected cell is then incubated in
a reaction mixture with labeled calcium in the presence of a
compound to be screened. The ability of the compound to
increase calcium up-take or ;nh;h;t calcium up~take can then
be determined by measuring the amount of labeled calcium
transported into the cells by taking advantage of the label,
e.g., radioactivity.
Compounds may also be identified by the above methods
which bind to specific subregions within the CNS that are
important for specific behaviors through indirect
interactions with a neuropeptide receptor polypeptide of the
present invention.
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To mea~ure intracellular cyclic AMP levels, cyclic A~P
is assayed in whole cells treated for 15 minutes at 37~C wi~.h
100 micromolar isobutylmethyl~Anth~ne (IBMX; Sigma).
TransfeCted cells (1 x 106 / 0.5 ml reaction) are incubated
with ~0 micromolar forskolin and various concentrations of
known or unknown ligands to the receptor. Reactions are
terminated with the addition of HCl to O.lM, incubation at
room t:emperature for 15 minutes, neutralization and sample
dilution in 50 mM sodium acetate, pH 6.2. Cyclic AMP i.s
quantified by using a radioimmlln~as8ay (Dupont/NEN~.
~ o measure levels of intracellular calcium, transfected
cells are suspended in loading medium (modified RPMI 1640
medium/10 mM Hepes/1~ newborn calf serum) and incubated in a
spinner ~lask at 37~C ~or 2.5 hour at 1 x 106 cells per ml.
Cells are then treated with 1 micromolar Fura-2 acetoxymethyl
ester (fura-2 AM; Molecular Probes) for 30 minutes at 37~C,
washed twice with loading medium, and resuspended at 5 x 106
cells~ml. Tmm~iAtely be~ore fluorescence ~;pectroscop~,r,
cells are recovered by centrifugatioIl at 1000 rpm aIld
resuspended at 1 x 10 cells/ml in a modified Krebs buffer
(135 mM NaCl/4.7 mM KCl/1.2 mM MgSO4/1.2 mM KH2PO4/5 mM
NaHCO3/l mM CaCl2/2.8 mM glucose/10 mM hepes, pH 7.~)
cont~tn;ng sulfinpyrazone. Bombesin is purchased ~rom Si~na
and Auspep. Fluorescence recordings are made on a Hitachi
fluorescence spectrometer (F4010) at 340 nm (excitation) and
505 m~ (emission) over 10 minutes with slit widths of 5 ~m
and response time of 2 seconds. Intracellular calcium is
quantified by using equations described by Grynkiewicz, ,et
al., ,J. Bio. Chem. 260:3440-3450, 1985.
The invention also provides a method of treating and/or
preventing obesity ~y A~m; ni stering to a host a compound
which binds to and activates the receptor polypeptides of the
present invention. Such a compound is o~her than the ob gene
product disclosed in Zhang, et al., Nature, 372:425-431
(1994). The receptor polypeptide of the present invention
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maps to a human chromosome which corresponds to the position
of the mouse chromosome which encodes for the receptor of the
ob gene product. The human ob gene encodes a "satiety"
factor which binds to and activates the receptor polypeptide
of the present invention. Accordingly, a compound which
activates the receptor of the present invention will decrease
appetite and prevent obesity.
The compounds described above may also be employed to
enhance activity level, modify eating behavior, PnhAnce
utilization of ingested foods and regulate deposition of fat
stores. Conditions related to obesity may also be treated by
the compounds which bind to and activate the receptor
polypeptides of the present invention including
hyperli~;m~, type II diabetes and certain cancers.
These compounds may also be employed to treat and/or
prevent other conditions related to an underexpression of the
receptor polypeptide of the present invention or ligands
which bind thereto, for example, to stimulate neuronal
growth.
Specific examples of compounds which inhibit activation
of the receptor polypeptides of the present invention include
an antibody, or in some cases an oligonucleotide, which binds
to the receptor but does not elicit a second messenger
response such that the activity of the receptor i8 prevented.
Another example is proteins which are closely related to
the ligands of the receptor, i.e. a fragment of the ligand,
which have lost biological function and when binding to the
receptor, elicit no response.
Another example includes an antisense construct prepared
through the u~e of 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 se~uence, which encodes for the mature
,
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W O 96/34877 PCT/US95/05616
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 oligonucleotide is designed to be
complementary to a region of the gene involved in
transcription (triple helix -see hee et al., Nucl. Aci~ds
Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988);
and r)ervan et al., Science, 251: 1360 (1991)), thereby
preventing transcription and the production of a neuropeptide
recepltor polypeptide of the present invention. The antisense
RNA o:Ligonucleotide hybridizes to the m~NA in vivo and blocks
translation of the mRNA molecule into the receptor (antisen~e
- Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides
as Antisense Inhibitors of Gene Bxpression, CRC Pres~, Boca
Raton, FL (1988)). The oligonucleotides de8cribed above can
al80 ]~e delivered to cells such that the antisense RNA or DNA
m~Ly be expressed in vivo to t nh~ h~ t production of the
receptors.
~ nother example is a small molecule which binds to a
neuropeptide receptor polypeptide of the present invention,
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 and neuropeptide Y fragments and/or derivatives.
Soluble forms of a neuropeptide receptor polypeptide of
the present invention, e.g., a fragment of the receptor,
which binds to the ligand and prevents the ligand from
interacting with membrane bound receptors may also t nh; h; t
activation of the receptor polypeptides of the present
invention.
This invention additionally provides a method of
utilizing such compounds which inhibit activation for
treat:ing abnorm~Ll conditions related to an excess of activity
of ~ neuropeptide receptor polypeptide of the present
invention for treating obesity since the neuropeptide
receptor polypeptides of the present invention may bind
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neuropeptide Y which is the most potent known substance to
cause an increase in feeding behavior and type II Diabetes
Mellitus since neuropeptide Y may play a role in the genetic
basis of this disease.
The compounds which inhibit activation of the receptor
polypeptides of the present invention may be employed to
treat and/or prevent hypertension since neuropeptide Y
stimulates renin release and neuropeptide Y is known to have
potent vasoconstrictor activity when involving the coronary
and cerebral vessels.
The compounds may also be employed to treat Al~h~im~r~s
disease since neuropeptide Y receptors are prevalent in the
central nervous system and are localized pre~ominAntly within
interneurons where they appear to have regulatory roles in
memory and Al~heim~rs disease.
The compounds may also be employed to suppress
excitatory transmission by neuropeptide Y in the hippocampus
and therefore may be employed to treat epileptic seizure,
stress and anxiety.
The prevalence of neuropeptide Y receptors in the
central nervous system indicates that the compounds which
inhibit the neuropeptide receptor polypeptides of the present
invention may be used as an antipsychotic drug by regulating
neurotransmission.
The compounds which inhibit the receptor polypeptides of
the present invention may also be employed to treat
pathological vasospasm involving coronary and cerebral
vessels.
This invention also provides a method for determining
whether a ligand not known to be capable of binding to a
neuropeptide receptor of the present invention can bind
thereto which comprises contacting the ligand to be
identified with a cell comprising the coding se~uence of a
neuropeptide receptor and expressing same on its ~urface
under conditions sufficient for binding of ligands previously
CA 02220036 1997-10-31
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identified as htn~tng to such a receptor. In other
emboAtm~nts cell membrane fractions comprising the receptor
or isolated receptors free or tmmnh;lized on solid suppor~s
may be used to measure binding of the ligand to be tested
When r~ecombinant cells are used for purposes of expression of
the receptor it is preferred to use cells with little or no
endogenous receptor activity so that htnAtn~, if any, is d~e
to the presence of the expressed receptor of interest.
Preferred cells include human embryonic kidney cells, monkey
kidney (HEK-293 cells), fibroblast (COS) cells, ~htne~e
hamster ovary (CH0) cells, Droso~hila or murine L-cells. It
is al!~o preferred to employ as a host cell, one in which a
recep1:0r responsive second messenger system exists. Well
known second messenger systems include increases or decreas~s
in phosphoinositide hydrolysis, adenylate cyclase, guanyla~e
cyclase, or ion ch~nnel activity in response to ligand
binding to extracellular receptor A~; n~ . In a further
embodiment a specifically designed indicator of receptor
hi nAi1ng can be constructed. For example, a fusion protein
can b~e made by fusing the receptor of this invention with a
protein Anm~tn which is sensitive to receptor ligand h;nAtng
Such a ~om~in referred to here as an indicator ~om~in is
capable, itself, or in association with accessory molecules,
of generating an analytically detectable signal which is
indicative or receptor ligand binding.
This invention also provides a method of detecting
expression of a neuropeptide receptor polypeptide of the
present invention on the surface o~ a cell by detecting the
presence of mRNA coding for the receptor which comprises
obt~intng total mRNA from the cell and contacting the mRNA so
obtained with a nucleic acid probe comprising a nucleic acid
molecule of at least lO nucleotides capable of specifically
hybridizing with a sequence included within th~e sequence of
a nucleic acid molecule encoding the receptor under
hybridizing conditions, detecting the presence of mRNA
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W 096/34877 PCTIUS95/05616
hybridized to the probe, and thereby detecting the expression
of the receptor by the cell.
The present invention also provides a method for
identifying receptors related to the receptor polypeptides of
the present invention. These related receptors may be
identified by homology to a neuropeptide receptor polypeptide
of the present invention, by low stringency cross
hybridization, or by identifying receptors that interact with
related natural or synthetic ligands and or elicit similar
behaviors after genetic or pharmacological blockade of the
neuropeptide receptor polypeptides of the present invention.
Fragments of the genes may be used as a hybridization
probe for a cDNA library to isolate other genes which have a
high sequence similarity to the genes of the present
invention, or which have similar biological activity. Probes
of this type preferably have 50 bases or more. The probe may
also be used to identify a cDNA clone corresponding to a full
length transcript and a genomic clone or clones that contain
the complete gene of the present invention including
regulatory and promoter regions, exons and introns. An
example of a screen of this type 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 to that of
the genes of the present invention are used to ~creen a
library of human cDNA, genomic DNA or mRNA to determine which
members of the library the probe hybridizes to.
The neuropeptide receptor polypeptides and compounds
identified above which are polypeptide~, 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
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W 096/.34877 PCTrUS9510~616
provicled to a patient 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 contA~nin~ RNA encoding a polypeptide o$
the present invention.
C;~m; l Arly~ cells may be engineered in vivo for
expression of a polypeptide in vivo by, for example,
proceclures known in the art. As known in the art, a producer
cell ~or producing a retroviral particle cont~ntng RNA
encodiLng the polypeptide of the present invention may ~e
n-l stered to a patient for engineering cells in vivo alld
expression of the polypeptide in vivo. These and other
metho~s for ~min; stering a polypeptide of the prese~t
inven1ion by such method should be apparent to those skilled
in the art from the teachings of the present invention. For
examp:Le, the expression vehicle for engineering cells may ~e
other than a retrovirus, for example, an adenovirus which may
be used to engineer cells in vivo after co~h~n~tion with a
suita]ble 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, retroviruses such as Rous Sarcoma Virus, Harvey
Sarcoma Virus, avian leukosis virus, gibbon ape leukemia
virus, human ~mml~nodeficiency virus, adenovirus,
Myeloproliferative Sarcoma Virus, and ~mm~ry 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 include, but are not limited
to, 1_he retroviral LTR; the SV40 promoter; and the hum~n
cytomegalovirus (CMV) promoter described in Miller, et al.,
~iotechnic~ues, Vol. 7, No. 9, 980-990 (1989), or any other
~ promc>ter (e.g., cellular promoters such as eukaryotic
cellular promoters including, but n~t limited to, the
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histone, pol III, and ~-actin promoters). Other viral
promoters which may be employed include, but are not limited
to, adenovirus promoters, thymidine kinase (TK) promoters,
and B19 parvovirus promoters. The selection of a suitable
promoter will be apparent to those skilled in the art from
the teachings cont~;ned herein.
The nucleic acid sequence encoding the polypeptide o~
the present invention is under the control of a suitable
promoter. Suitable promoters which may be employed include,
but are not limited to, adenoviral promoters, such a the
adenoviral major late promoter; or hetorologous 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 ApoAI promoter; human
globin promoters; viral thymidine kinase promoters, such as
the Herpes Simplex thymidine kinase promoter; retroviral LTRs
(including the modified retroviral LTRs here;n~hove
described); the ~-actin promoter; and human growth hormone
promoters. The promoter also may be the native promoter
which controls the genes encoding the polypeptides.
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 PE501, PA317, ~-2, ~-AM, PA12, T19-14X,
VT-19-17-H2, ~CRE, ~CRIP, 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 re~erence
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 CaPO4 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.
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The producer cell line generates infectious retroviral
vector particles which include the nucleic acid sequence (8)
encod:ing the polypeptides. Such retroviral vector particles
then may be employed, to transduce eukaryotic cells, either
in vitro or in vivo. The transduced eukaryotic cells will
express the nucleic acid sequence~s) encoding the
polypeptide. Eukaryotic cells which may be transduced
inclu~e, but are not limited to, em~ryonic stem cells,
embry~nic carC~n~m~ cells, as well as hematopoietic stem
cells, hepatocytes, fibroblasts, myoblasts, keratinocytes,
endothelial cell~, and bro~ch;~l epithelial cells.
'rhe soluble neuropeptide receptor polypeptides and
com~pounds which bind to and activate or inhibit activation of
a receptor of the present invention may also be employed in
combination with a suitable pharmaceutical carrier. Such
compositions comprise a therapeutically effective amount of
the soluble neuropeptide receptor polypeptide or compounds,
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
com~prising one or more cont~;ners filled with one or more of
the iLngredients of the pharmaceutical compositions of the
inven,tion. Associated with such cont~;ner(s) can be a notice
in t~le 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 ~m; n; stration. In
addit:ion, the soluble neuropeptide receptor polypeptides or
compounds of the present invention may be employed in
conjtmction with other therapeutic compounds.
The pharmaceutical compositions may be administered in
a convenient m~nner such as by the topical, intravenous,
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intraperitoneal, intramuscular, subcutaneous, intranasal or
intradermal routes. The pharmaceutical compositions are
~m; n~ stered in an amount which is effective for treating
and/or prophylaxis of the speCific indication. In general,
the pharmaceutical compositions will be ~m; n; stered in an
amount of at least about 10 ~g/kg body weight and in most
cases they will be ~m; n; stered in an amount not in excess of
about 8 mg/Kg body weight per day. In most cases, the dosage
is from about 10 ~g/kg to about 1 mg/kg body weight daily,
taking into account the routes of ~m~ n; stration, s~mptoms,
etc.
The present invention also contemplates the use of the
genes of the present invention as a diagnostic, for example,
some diseases result from inherited defective genes. These
genes can be detected by comparing the sequences of the
defective gene with that of a normal one. Subsequently, one
can verify that a "mutant" gene is associated with abnormal
receptor activity. In addition, one can insert mutant
receptor genes into a suitable vector for expression in a
functional assay sy6tem (e.g., colorimetric assay, expression
on MacConkey plates, complementation experiments, in a
receptor deficient strain of HEK293 cells) as yet another
means to verify or identify mutations. Once "mutant" genes
have been identified, one can then screen population for
carriers of the "mutant" receptor gene.
Individuals carrying mutations in the gene of the
present invention may be detected at the DNA level by a
variety of techniques. Nucleic acids used for diagnosis may
be obtained from a patient's cells, including but not limited
to such as from blood, urine, saliva, tissue biopsy and
autopsy material. The genomic 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 complimentary to the nucleic acid of the
CA 02220036 1997-10-31
W 096/.34877 PCTrUS95/0~616
instant invention can be used to identi~y and analyze
mutat:ions in the gene o~ the present invention. For example,
delet:ions and insertions can be detected by a change in size
of the amplified product in co~r~ri.son to the normal
genotype. Point mutations can be identified by hybridizing
ampli:Eied DNA to radio labeled RNA of the invention or
alternatively, radio labeled antisense DNA sequences of the
invenltion. Perfectly matched sequences can be distingll;sh~A
from mismatched duplexes by RNase ~ digestion or by
differences in melting temperatu~es. Su~h a diagno8tic would
be pa~ticularly useful for prenatal or even neonatal testing.
Sequence differences between the re~erence gene and
~mutants" may be revealed by the direct DNA sequencing
methold. In addition, cloned DNA segments may be used as
probes to detect specific DNA segments. The sensitivity of
this method is greatly ~nh~nced when combined with PCR. For
example, a secluence primer is used with double stranded PCR
product or a single stranded template molecule generated by
a modified PCR. The secluence determination is performed by
conventional procedures with radio labeled nucleotide or by
an automatic sequencing procedure with fluorescent-tags.
Genetic testing based on DNA secluence differences may be
achieved by detection o~ alterations in the electrophoretic
mobility of DNA fragments in gels with or without denaturing
agents. Secluences changes at specific locations may also be
revealed by nucleus protection assays, such RNase and S1
protection or the chemical cleavage method (e.g. Cotton, et
al., PNAS, USA, 85:4397-4401 1985).
In addition, some diseases are a result of, or are
characterized by changes in gene expression which can be
detec:ted by changes in the mRNA. Alternatively, the genes of
the present invention can be used as a reference to identify
individuals expressing a decrease of functio~s associated
with receptors of this type.
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The present invention al~o relates to a diagnostic assay
for detecting altered levels of soluble forms of the
neuropeptide 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 ho~t
are well known to those of skill in the art and include
radioi ~ ~noA~says, competitive-h;n~ing assays, Western blot
analysis and preferably as 8LISA assay.
An BLISA assay initially comprises preparing an antibody
~pecific to antigens of the neuropeptide receptor
polypeptides, preferably a monoclonal antibody. In addition
a ~e~o-Ler antibody is prepared against the monoclonal
Ant;hoAy To the reporter antibody is attAch~ 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 su~oLL, e.g. a
polystyrene dish, that binds the proteins in the sample. Any
free protein h;n~ing sites on the dish are then covered by
incubating with a non-specific protein such as bovine serum
A lhl-m; n Next, the monoclonal antibody is incubated in the
dish during which time the monoclonal antiho~es attach to
any neuropeptide receptor proteins attAche~ to the
poly~tyrene dish. All unbound monoclonal antibody is ~ ~e~
out with buffer. The ~~-Ler antibody linked to horseradish
peroxidase is now placed in the dish resulting in binding of
the reporter antibody to any monoclonal antibody bound to
neuropeptide receptor proteins. Unattached reporter antibody
is then washed out. Peroxida~e substrates are then added to
the dish and the amount of color developed in a given time
period is a mea~urement of the ~-.~,~ of neuropeptide
receptor proteins present in a given volume of patient sample
~ when compared against a stAn~Ard curve.
The se~lences of the present invention are also valuable
for chromosome identification. The sequence is specifically
targeted to and can hybridize with a particular location on
--34--
SI~BSTlllrrE SHEET (RULE 2~)
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an individual human chromosome. Moreover, there is a current
need ior identifying particular sites on the chromosome. Few
chromosome marking reagents based on actual sequence data
(repeat polymorphisms) are presently available ~or marking
chrom~somal location. The mapping of DNAs to chromosomes
according to the present invention is an important first step
in correlating those seq[uences with genes associated with
disea~3e.
]3riefly, seq~uences can be mapped to chromosomes by
prepa:ring PCR primers (pre~erably 15-25 bp) ~rom the cDN~.
Compu~er analysis o~ the 3' untranslated region is used to
rapidly select primers that do not span more than one exon in
the genomic DNA, thus complicating the ampli$ication process.
These primers are then used for PCR screening of somatic cell
hybril~s cont~n~ng individual human chromosomes. Only those
hybri~s cont~n;ng the human gene corresponding to the primer
will yield an amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure
~or 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 m~nner. 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) o~ a cDNA
clone to a met~ph~e chromosomal spread can be used to
provide a precise chromosomal location in one step. This
techniq[ue can be used with cDNA as short as 50 or 60 bases.
For a review o~ this technique, ee ~erma et al., Human
Chromosomes: a ~nll~l o~ Basic Techniq[ues, Pely~oll Press,
New York (1988).
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The above techniques were utilized to map the gene
corresponding to the neuropeptide receptor of the present
invention to chromosome 1 position 31-34.
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, MPn~plian
Inheritance in Man (available on line through Johns Hopkins
University Welch Medical Library). The relationship between
genes and diseases 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 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 50 and 500 potential causative genes. (This
assumes 1 megabase mapping resolution and one gene per 20
kb).
The polypeptides, their fragments or other derivatives,
or analogs thereof, or cells expressing them can be used as
an immllnogen to produce antibodies thereto. These antibodies
can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes chim~ric, single chain,
and hllm~nized antibodies, as well as Fab fragments, or the
product of an 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
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obtained by direct injection of the polypeptides into an
~ntm;~ l or by ~r~m~n~fitering the polypeptides to an ~n~m~l,
prefe:rably a nonhllm~n. The antibody so obt~; n~rl will then
bind the polypeptides itself. In this m~nnPr, even a
sequence encoding only a fragment of the polypeptides can be
used to generate antibodies binding the whole native
polypleptides. Such antibodies can then be used to isolate
the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any techniclue
which provides antibodies produced by continuous cell line
cultures can be used. Examples include the hybridoma
techniclue (Kohler and Milstein, 1975, Nature, 256:495-497),
the trioma techniclue, the human B-cell hybridoma technique
(Kozbor et al., 1983, Tmmllnology Today 4:72), and the EBV-
hybridoma technique to produce human monoclonal antibodies
(Cole, et al., 1985, in Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniclues described for the production of single chain
antibodies (U.S. Patent 4,946,778) can be adapted to produce
single chain antibodies to ;mmllnogenic polypeptide products
of this invention. Also, transgenic mice may be used to
express hllm~n~zed antibodies to ~nnogenic polypeptide
products of this invention.
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 suLch
examples. All parts or amounts, unless otherwise specified,
are by weight.
In order to facilitate underst~n-~ng of the ~ollowing
exam;c)les certain frecluently occurring methods and/or terms
will be described.
"Plasmids" are designated by a lower case p preceded
and/or followed by capital letters and/or numbers. The
start:ing plasmids herein are either co~rcially available,
publicly available on an unrestricted basis, or can be
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W 096/34877 PCT/US95/05616
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.
"Digestion" of DNA refers to catalytic cleavage of the
DNA with a restriction enzyme that acts only at certain
se~uences in the DNA. The various restriction enzymes used
herein are c~m~rcially 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 ~g of 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 ~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 1 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 kinase. 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
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1PEA~VS 3 ~ MAR ~97
frag~ents (Maniatis, T , et al., Id., p. 146). Unl~ess
otherwise provided, ligation may be accomplished using known
buffers and conditions with 10 units to T4 DNA liga8e
("ligase") per 0.5 ~g of approximately equimolar amounts of
the DNA fragments to be ligated.
Unless otherwise stated, transform~tion was performed as
described in the method o~ Graham, F. and van der Eb, A.,
Virology, 52:456-457 (1973).
Example 1
Bac~rial Expression and Purification of the Neuropeptide
Recel~tor
The DNA sequence encoding for neuropeptide receptor,
ATCC # 97128 is initially amplified using PCR oligonucleotide
primers corresponding to the 5' and 3' end sequences of the
proc,-ssed neuropeptide receptor gen~ (minus the si~lal
peptide sequence) and the vector sequences 3~ to the gene.
Addiltional nucleotides corresponding to neuropeptide recep~or
nucleotide sequence are added to the 5~ and 3' sequences
respectively. The 5' oligonucleotide primer has the sequence
5' C~CTAAAGG-l-lAATGGAGCCCTCAGCCACC 3' (SEQ ID NO:7) cont~;n~
a Hind III restriction enzyme site followed by 18 nucleotides
of neuropeptide receptor coding sequence starting from the
preslumed terminal amino acid of the processed protein codon.
The :3~ sequence 5' ACAAGTCCTTGTC~-l-L~-lAGAGGGC 3' (SEQ ID N0:8)
and contains an Xbal site. The restriction enzyme sites
corrlespond to the restriction enzyme sites on the bacterial
exprlession vector pQE-9 (Qiagen, Inc. Chatsworth, CA). pQE-9
enco,des antibiotic resistance (Ampr), 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-9 is then digested with Hind
III ~d Xbal. The amplified sequences are ligated into pQ~-9
and are inserted in ~rame with the sequence encoding for the
histidine tag and the RBS. The ligation mixture is then used
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to trans~onm E. coli strain M15/rep 4 (Qiagen, Inc.) by the
procedure described in Sambrook, J. et al., Molecular
Cloning: A Laboratory ~n~ , Cold Spring Laboratory Press,
(1989). M15/rep4 cnnt~'n~ multiple copies of the plasmid
pREP4, which expresses the lacI repressor and also confers
kanamycin resistance (Kanr). Trans~ormants are identi~ied by
their ability to grow on LB plates and ampicillin/kanamycin
resistant colonies are selected. Plasmid DNA is isolated and
confirmed by restriction analysis. Clones cont~;n~ng the
desired constructs are grown overnight (0/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 are
grown to an optical density 600 (O.D.~) of between 0.4 an.d
0.6. IPTG (~IIsopropyl-B-D-thiogalacto pyranosidell) is then
added to a ~inal concentration of 1 mM. IPTG induces by
inactivating the lacI repressor, clearing the P/O leading to
increased gene expression. Cells are grown an extra 3 to 4
hours. Cells are then harvested by centrifugation. The cell
pellet: is solubilized in the chaotropic agent 6 Molar
Guanicline HCl. After clarification, solubilized neuropeptide
recept:or is purified from this solution ~y chromatography on
a NicX:el-Chelate column under conditions that allow for tight
binding by proteins cont~n~ng the 6-His tag (Hochuli, E. et
al., J. Chromatography 411:177-184 (1984). The protein is
elutec~ from the column in 6 molar guanidine HCl pH 5.0 and
for the purpose of renaturation adjusted to 3 molar guanidine
HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced)
and 2 mmolar glutathione (oxidized). After incu~ation i.n
this solution for 12 hours the protein is dialyzed to 10
mmola:r sodium phosphate.
ExamPle 2
Expression of Recombinant Neuro~e~tide Rece~tor in COS cells
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IPEA/US 3 ~ 1997
The expression of plasmid, neuropeptide receptor HA is
derived from a vector pcDNA3/Amp (Invitrogen) cont~;n;ng~
SV40 origin of replication, 2) ampicillin resistance gene, 3)
E.coli replication origin, 4) CMV promoter followed by a
polylinker region, a Sv40 intron and polyadenylation site.
A DNA fragment encoding the entire neuropeptide receptor
precursor and a HA tag fused in frame to its 3' end is cloned
into the polylinker region of the vector, therefore, the
recombinant protein expression is directed under the CMV
promoter. The HA tag corresponds to an epitope derived from
the influenza hemagglutinin protein as pre~iously described
(I. Wilson, H. ~iman, R. Heighten, A Cherenson, M. Connolly,
and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag
to the target protein allows ea~y detection of the
recombinant protein with an antibody that recognizes the HA
epitope.
The plasmid construction strategy is described as
follows:
The DNA sequence encoding for neuropeptide receptor,
ATCC # 97128, is constructed by PCR using two primers: the
5' primer 5' CCTAGGATGCCCCTCTGCTGCAGCGG 3' (S~Q ID NO:9)
contains a BamHI site; the 3' sequence 5~ ACAA~l~-l-l~l
CCTTCTAGAGGGC 3' (SEQ ID NO:10) contains co~plementary
sequences to an XbaI site, translation stop codon, and the
last 17 nucleotides of the neuropeptide receptor coding
sequence (not including the stop codon). Therefore, the PCR
prod.uct contains a BamHI site, coding sequence, a translation
term~ination stop codon and an XbaI site. The PCR amplified
DNA fragment and the vector, pcDNA3/Amp, are digested with
BamH:I and XbaI restriction enzymes and ligated. The ligation
mixture is transformed into E. coli strain SUR~ (Stratagene
Cloning Systems, La Jolla, CA) the transformed culture is
plated on ampicillin media plates and re~istant colonies are
selected. Plasmid DNA is isolated from transformants and
exam.ined by restriction analysis for the presence of the
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CA 02220036 1997-10-31 PCT/lJS9 5 / O 5 6 t6
1P~IS 3 1 MAR 1997
correct fragment. For expression of the recombinant
neuropeptide receptor, COS cells are transfected with the
expression vector by DEAE-DEXTRAN me~hod (J. Sambrook, E.
Frit;sch, T. Maniatis, Molecular Cloning: A Laboratory Manual,
Cold Spring Laboratory Press, (1989)). The expression of the
neuropeptide receptor HA protein is detected by radio-
labelling and immllnoprecipitation method (E. Harlow, D. Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, (1988)). Cells are labelled for 8 hours
with 35S-cysteine two days post transf~ction. Culture media
are then collected and cells are lysed with detergent (RIPA
buffer (150 mM NaCl, 1% NP-40, 0.1~ SDS, 1% NP-40, 0.5% DOC,
50m~[ Tris, pH 7.5). (Wilson, I. et al., Id. 37:767 (1984)).
sotklcell lysate and culture media are precipitated with a HA
specific monoclonal antibody. Proteins precipitated are
analyzed on 15% SDS-PAGE gels.
Example 3
Clor.Linq and exPression of Neuropeptide Rece~tor usinq the
baculovirus expression s~stem
The DNA sequence encoding the fu]l length neuropeptide
receptor protein, ATCC # 97128, is amplified using PCR
olicronucleotide primers corresponding to the 5' and 3'
seqllences of the gene:
The 5~ primer has the sequence 5~ CGGGATCCGCCATCATGGAG
CCCTCAGCCACC 3~ (SEQ ID NO:11) and contains a BamHI
rest:riction enzyme site (in bold) followed by 6 nucleotides
resembling an efficient signal for the initiation of
translation in eukaryotic cells (J. Mol. Biol. 1987, 196,
947-950, Xozak, M.). The initiation codon for translation
"ATG" is underlined).
The 3~ primer has the sequence 5~ ACAAGT~ ~lCCTTCT
AGA~GGC 3' (SEQ ID NO:12) and contains the cleavage site for
the restriction endonuclease XbaI and 5 nucleotides
comE~lementary to the 3~ non-translated sequence of the
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AMENDE~
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W 096i34877 PCTrUS95/05616
neuropeptide receptor gene. The amplified sequences are
isolated from a 1% agarose gel using a romm~cially available
kit ("Geneclean," BI0 101 Inc., La Jolla, Ca.). The fragment
is then digested with the en~onllcleases BamHI and XbaI and
then purified as described in ~xample 1. This fragment is
designated F2.
The vector pA2 (modification of pVL941 vector, discussed
below) is used for the expression of the neuropeptide
receptor protein using the baculovirus expression system (for
review see: Summers, M.D. and Smith, G.E. 1987, A m~nll~l of
methods for baculovirus vectors and insect cell culture
procedures, Texas Agricultural Experim~ntAl Station Bulletin
N0:1, 3 and 5555). This e~?ression vector contains the
strong polyhedrin promoter of the Autographa californica
nuclear polyhidrosis virus (AcMNPV) followed by the
recognition sites for the restriction ~n~nllcleases BamHI and
XbaI. The polyadenylation site of the simian virus (SV)40 is
used for efficient polyadenylation. For an easy selection of
reco~lbinant viruses 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
homo]ogous recQm~in~tion of co-transfected wild-type viral
DNA. Many other baculovirus vectors could be used in place
of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and
Summers, M.D., Virology, 170:31-39).
The plasmid is digested with the restriction enzymes
BamH:[ and XbaI and then dephosphorylated using calf
intestinal phosphatase by procedures ~own in the art. The
DNA is then isolated from a 1% agarose gel as described in
Exam]?le 1. This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 are
liga~ed with T4 DNA ligase. DH5~ are then transformed and
bactleria identified that cont~in~ the plasmid (pBac
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W 096/34877 PCTrUS95/05616
neuropeptide receptor) with the neuropeptide receptor gene
using the enzyme~; BaTnHI and XbaI. The sequence o~ the cloned
fragment is confirmed by DNA sequencing.
5 llg of the plasmid pBac neuropeptide receptor are co-
transfected with 1.0 ~g of a commercially available
linearized baculovirus ("BaculoGoldn' baculovirus DNA",
Pharmingen, San Diego, CA.) using the lipo~ection method
(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417
(1987)).
l~g of BaculoGold~ virus DNA and 5 ~g of the plasmid
pBac neuropeptide receptor are mixed in a sterile well o~ a
microtiter plate cont~;n;ng 50 ~1 of serum free Grace's
medium (Life Technologies Inc., Gaithersburg, ~).
Afterwards 10 ~Ll Lipofectin plus 90 ~l Grace's medium are
added, mixed and incubated for 15 minutes at room
temperature. Then the transfection mixture is added drop
wise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35
mm tissue culture plate with 1 ml Grace' medium without
serum. The plate is rocked back and ~orth to mix the newly
added solution. The plate is then incubated ~or 5 hours at
27~C. After 5 hours the transfection solution is removed
from the plate and 1 ml of Grace's insect medium supplemented
with 10% fetal calf serum is added. The plate is put back
into an incubator and cultivation continued at 27~C i~or four
days.
A~ter four days the supernatant is 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) is used which
allows an easy isolation of blue stained plaques. (A
detailed description of a "plaque assay" can also be ~ound in
the user's guide for insect cell culture and baculovirology
distributed by Life Technologies Inc., Gaithersburg, page 9-
10) .
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W 096/34877 PCTrUS95/05616
Four days after the serial dilution the virus is added
to the cells and blue sti~n~ plac~ues are picked with the tip
of an Eppendorf pipette. The agar conti~;ntng the reComhtni~nt
viruses is then resuspended in an Eppendorf tube contAtntng
200 ~1 of Grace~s medium. The agar is removed by a brief
centrifugation and the supernatant cont~tntng the recnmhtni~nt
baculoviruses is used to infect Sf9 cells seeded in 35 mm
~t hPs Four days later the supernatants of these culture
dishes are harvested and then stored at 4~C.
Sf9 cells are grown in Grace's medium supplemented with
10% ~eat-inactivated FBS. The cells are infected with the
recombinant baculovirus V-neuropeptide receptor at a
multiplicity of infection (MOI) of 2. Six hours later the
medium is removed and replaced with SF900 II medium minus
methionine and cysteine (Life Technologies Inc.,
Gait~ersburg). 42 hours later 5 ~Ci of 35S-methionine and 5
~Ci 3-'S cysteine (Amersham) are added. The cells are $urther
incubated for 16 hours before they are harvested by
centrifugation and the labelled proteins visualized by SDS-
PAGE and autoradiography.
Exam~le 4
Expression via Gene TherapY
Fibroblasts are obt~nP~ from a subject 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 t:urned upside down, closed tight; and left at room
temperature over night. After 24 hours at room temperature,
the ~Elask is inverted and the chunks oE tissue remain fixed
to the bottom of the flask and fresh media (e.g., Ham's F12
medii~, with 10~ FBS, penicillin and streptomyc~n, is added.
This is then incubated at 37~C for approximately one week.
At this time, fresh media is added and subsequently changed
,
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W 096/34877 PCT/US9S/05616
every ~everal 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 murine
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
EcoRI site and the 3' primer having contains a HindIII site.
Equal quantities of the Moloney murine sarcoma virus linear
backbone and the EcoRI and HindIII fragment are added
together, in the presence of T4 DNA ligase. The resulting
mixture is maintAin~ under conditions appropriate for
ligation of the two fragments. The ligation mixture is used
to transform bacteria HB101, which are then plated onto agar-
containing kanamycin for the purpose of confirming that 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 viral particles cont~intng 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, contA;n~ng 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
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a sub-confluent plate of fibroblasts and ~uickly 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
marke~r, 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 ~rotein product.
Numerous modifications and variations o~ the present
inven.tion are possible in light of the above teachings and,
there!fore, within the scope of the appended claims, the
inven.tion may be practiced otherwise than as particularly
described.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: LI, ET AL.
(ii) TITLE OF lNv~NllON: Hum~n Neuropeptide Receptor
(iii) NUMBER OF SEQUENCES: 12
(iv) rORR~:~P~)NL~N~:~; ADDRESS:
(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,
CECCHI, STEWART & OLSTEIN
(B) STREET: 6 BECKER FARM ROAD
(C) CITY: ROSELAND
(D) STATE: NEW JERSEY
(E) ~:OIJN1~Y: USA
(F) ZIP: 07068
(v) COM~ul~ READABLE FORM:
(A) MEDIUM TYPE: 3.5 INCH DISKETTE
(B) COM~ul~: IBM PS/2
(C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WORD PERFECT 5.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: concurrently
(C) CLASSIFICATION:
(vii) ATTORNEY/AGENT INFORMATION:
(A) NAME: FERRARO, GREGORY D.
(B) REGISTRATION NUMBER: 36,134
(C) REFERENCE/DOCKET NUMBER: 325800-268
(viii) TELECOMMnNICATION INFORMATION:
(A) TELEPHONE: 201-994-1700
(B) TELEFAX: 201-994-1744
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUEN OE CHARACTERISTICS
(A) LENGTH: 1209 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRA-NL~ Nlt:SS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ATGGAGCCCT CAGCCACCCC AGGGGCCCAG AlGGGG~lCC CC'C-~-lGGCAG CAGAGAGCCG 60
CCL-lL~lGC CTCCAGACTA TGAAGATGAG ~ L'lCLGCT Al~-l~l~GCG TGATTATCTG 120
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TACCCA;~AAC AGTATGAGTG G~1-~-1'~ATC CCAGCCTATG ~LGG~ C~lC~LGGCC 180
L-1~L~a~GCA ACACGCTGGT CTGCCTGGCC GTGTGGCGGA ACCACCACAT GAGGACAGTC 240
ACCAAC~rACT TCATTGTCAA C~1'~L~C-1~ GCTGACGTTC TGGTGACTGC TATCTGCCTG 300
CCGGCC~GCC TG~1~aLW A CATCACTGAG TCCTGGCTGT TCGGCCATGC C~-l.-lG~AAG 360
GTCA'LCI_C~1' ATCTACAGGC '1'~'1~a'LCC~L~ TCAGTGGCAG TGCTAACTCT CAGCTTCATC 420
GCCCTG~;ACC GCTGGTATGC CATCTGCCAC CCACTATTGT TCAAGAGCAC AGCCCGGCGG 480
GCC~Ia'L~aGCT CCA'1'~-L~GG CATCTGGGCT GTGTCGCTGG CCATCATGGT GCCCCAGGCT 540
GCAGTC~TGG AATGCAGCAG TGTGCTGCCT GAGCTAGCCA ACCGCACACG G~-1~-11'~-1'~A 600
~1~-L~L~ATG AACGCTGGGC AGATGACCTC TATCCCAAGA TCTACCACAG TTG~-l-L~-l-l-L 660
ATTGTC~CCT ACCTGGCCCC ACT WW CCTC ATGGCCATGG CCTATTTCCA GATATTCCGC 720
AAC~1~L~GG GCCGCCAGAT CCCCGGCACC ACCTCAGCAC TGGTGCW AA CTGGAAGCGC 780
CCCTCAraACC AG~-1GGGG~A CCTGGAGCAG GGCCTGAGTG GAGAGCCCCA GCCCCGGGGC 840
CGCGCCrTCC TGGCTGAAGT GAAGCAGATG CGTGCACGGA GGAAGACAGC CAAGATGCTG 900
ATGW TGaTGC TG-LW 1~11 CGCC~L~L~aC TACCTGCCCA TCA.GCGTCCT CAA1~'1'~L1-1' 960
AAGAGG~aTGT TCGGGATGTT CCGCCAAGCC AGTGACCGCG AAG~L~'1~1'A CGCCTGCTTC 1020
A~-11~-1CCC ACTGGCTGGT GTACGCCAAC AGCGCTGCCA ACCCCATCAT CTAcAAcTTc 10 80
CTCAGT~GCA AA'l lCCGGGA GCAGTTTAAG GCTGCCTTCT CCTGCTGCCT GCCTGGCCTG 1140
G~L~C~-'L~CG G~-1-~-L~-L~AA GGCCCCTAGT CCCCGCTCCT CTGCCAGCCA CAA~-lC~-l-L~ 1200~LC~-1-L~'1'AG 1209
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 402 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STR~NDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQ~ DESCRIPTION: SEQ ID NO:2:
Met Glu Pro Ser Ala Thr Pro Gly Ala Gln Met Gly Val Pro Pro
~5
Gly Ser Arg Glu Pro Ser Pro Val Pro Pro Asp Tyr Glu Asp Glu
Phe Leu Arg Tyr Leu Trp Arg Asp Tyr Leu Tyr Pro Lys Gln Tyr
Glu Trp Val Leu Ile Ala Ala Tyr Val Ala Val Phe Val Val Ala
l60
Leu Val Gly Asn Thr Leu Val Cys Leu Ala Val Trp Arg Asn His
His Met Arg Thr Val Thr Asn Tyr Phe Ile Val Asn Leu Ser L~eu
Ala ~,sp Val Leu Val Thr Ala Ile Cys Leu. Pro Ala Ser Leu Leu
100 105
Val Asp Ile Thr Glu Ser Trp Leu Phe Gly His Ala Leu Cys Lys
110 115 120
Val Ile Pro Tyr Leu Gln Ala Val Ser Val Ser Val Ala Val Leu
125 130 135
Thr Leu Ser Phe Ile Ala Leu Asp Arg Trp Tyr Ala Ile Cys His
140 145 150
Pro I,eu Leu Phe Lys Ser Thr Ala Arg Arg Ala Arg Gly Ser Ile
155 160 165
Leu C;ly Ile Trp Ala Val Ser Leu Ala Ile Met Val Pro Gln Ala
170 175 180
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~la Val Met Glu Cys Ser Ser Val Leu Pro Glu Leu Ala Asn Arg
185 190 195~hr Arg Leu Phe Ser Val Cys Asp Glu Arg Trp Ala Asp Asp Leu
200 205 210~yr Pro Lys Ile Tyr His Ser Cys Phe Phe I~e Val Thr Tyr Leu
215 220 225~la Pro Leu Gly Leu Met Ala Met Ala Tyr Phe Gln Ile Phe Arg
230 235 - 240~ys Leu Trp Gly Arg Gln Ile Pro Gly Thr Thr Ser Ala Leu Val
245 250 255~rg Asn Trp Lys Arg Pro Ser Asp Gln Leu Gly Asp Leu Glu Gln
260 265 270~ly Leu Ser Gly Glu Pro Gln Pro Arg Gly Arg Ala Phe Leu Ala
275 280 285~lu Val Lys Gln Met Arg Ala Arg Arg Lys Thr Ala Lys Met Leu
290 295 300~et Val Val Leu Leu Val Phe Ala Leu Cys Tyr Leu Pro Ile Ser
305 310 315~al Leu Asn Val Leu Lys Arg Val Phe Gly Met Phe Arg Gln Ala
320 325 330~er Asp Arg Glu Ala Val Tyr Ala Cys Phe Thr Phe Ser His Trp
335 340 345~eu Val Tyr Ala Asn Ser Ala Ala Asn Pro Ile Ile Tyr Asn Phe
350 355 360~eu Ser Gly Lys Phe Arg Glu Gln Phe Lys Ala Ala Phe Ser Cys
365 370 375~ys Leu Pro Gly Leu Gly Pro Cys Gly Ser Leu Lys Ala Pro Ser
380 385 390
Pro Arg Ser Ser Ala Ser His Lys Ser Leu Ser Leu
395 400
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1110 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ATGGAGCCCT CAGCCACCCC AGGGGCCCAG Al~GGG~lLC' C'C'C~-L~GCAG CAGAGAGCCG 60
lL-CCL-l~lGC CTCCAGACTA TGAAGATGAG l-l-lL-lLL~CT AlL-lLl-LGCG TGATTATCTG 120
TACCCA~AAC AGTATGAGTG GGl~-~-lLATC GCAGCCTATG TGG~-lL;lL-l-l C-~lL-L-LLiGCC 180
CTGGTGGGCA ACACGCTGGT CTGCCTGGCC GTGTGGCGGA ACCACCACAT GAGGACAGTC 240
ACCAACTACT TCATTGTCAA C~-1L;1CC~-1L; GCTGACGTTC TGGTGACTGC TATCTGCCTG 300
CCGGCCAGCC TG~-l-Li~GA CATCACTGAG TCCTGGCTGT TCGGCCATGC CCTCTGCAAG 360
GTCATCCCCT ATCTACAGGC lL;lLil~C'~lL TCAGTGGCAG TGCTAACTCT CAGCTTCATC 420
CCC~-lGGACC GCTGGTATGC CATCTGCCAC CCACTATTGT TCAAGAGCAC AGCCCGGCGG 480
GCC'~lGGCT CCAlC~-lGGG CATCTGGGCT GTGTCGCTGG CCATCATGGT GCCCCAGGCT 540
GCAGTCATGC AATCCAGCAG TGTGCTGCCT GAGCTAGCCA ACCGCACACG G~-1L-1-1L-1LA 600
L-lL-lL-lLATG AACGCTGGGC AGATGACCTC TATCCCAAGA TCTACCACAG TTG~-l-lL-l-l-l 660
ATTGTCACCT ACCTGGCCCC ACTGGGCCTC ATGGCCATGG CCTATTTCCA GATATTCCGC 720
AAGCTCTGGG GCCGCCAGAT CCCCGGCACC ACCTCAGCAC TGGTGCGGAA CTGGAAGCGC 780
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CCCTCAGACC AGCTGGGGGA CCTGGAGCAG GGCCTGAGTG GAGAGCCCCA GCCCC'GGGGC 840
CGCGCCTTCC TGGCTGAAGT GAAGCAGATG CGTGCACGGA GGAAGACAGC CAAGATGCTG 90O
Al w 1~71~C TG~-l~l~-l-l ~C~-l~-l~C TAC~lCCC~A TCAGCGTCCT CAAl~lC~-l-l 960
AAGAGGC;TGT TCGGGATGTT CCGCCAAGCC AGTGACCGCG AAG~-L~l~-lA CGCCTGCTTC 1020
AC~-l-l~-lCCC ACTGGCTGGT GTACGCCAAC AGCGCTGCCA ACCCCATCAT CTACAACTTC 1080
CTCAGTGGCC llcc~-l~AG TCTGCTCTAA 1110
(2) INFORMATION FOR SBQ ID NO:4:
~i) SE~ CHARACTERISTICS
(A) LENGTH: 369 BASE PAIRS
(B) TYPE: AMINO ACID
(C) STRAhv~vN~SS: SINGLE
(D) TOPOLOGY: LINEAR
(iiL) MOLECULE TYPE: cDNA
(x.L) SEQ~ DESCRIPTION: SEQ ID NO:4:
Met G:Lu Pro Ser Ala Thr Pro Gly Ala Gln Met Gly Val Pro Pro
Gly Ser Arg Glu Pro Ser Pro Val Pro Pro Asp Tyr Glu Asp Glu
Phe Leu Arg Tyr Leu Trp Arg Asp Tyr Leu Tyr Pro Lys Gln Tyr
Glu T:rp Val Leu Ile Ala Ala Tyr Val Ala Val Phe Val Val A].a
Leu V;~l Gly Asn Thr Leu Val Cys Leu Ala Val Trp Arg Asn Eis
His M~et Arg Thr Val Thr Asn Tyr Phe Ile Val Asn Leu Ser Leu
~0
Ala A,sp Val Leu Val Thr Ala Ile Cys Leu Pro Ala Ser Leu Leu
10.0 105
Val Asp Ile Thr Glu Ser Trp Leu Phe Gly His Ala Leu Cys Lys
110 115 120
Val Ile Pro Tyr Leu Gln Ala Val Ser Val Ser Val Ala Val Leu
125 130 - 135
Thr Leu Ser Phe Ile Ala Leu Asp Arg Trp Tyr Ala Ile Cys Hi.s
140 145 150
Pro Leu Leu Phe Lys Ser Thr Ala Arg Arg Ala Arg Gly Ser Ile
155 160 165
Leu Gly Ile Trp Ala Val Ser Leu Ala Ile Met Val Pro Gln Ala
170 175 1~0
Ala Val Met Glu Cys Ser Ser Val Leu Pro Glu Leu Ala Asn Arg
185 190 195
Thr Arg Leu Phe Ser Val Cys Asp Glu Arg Trp Ala Asp Asp Leu
200 205 210
Tyr Pro Lys Ile Tyr His Ser Cys Phe Phe Ile Val Thr Tyr Leu
215 220 225
Ala Pro Leu Gly Leu Met Ala Met Ala Tyr Phe Gln Ile Phe Arg
230 235 2~0
Lys L.eu Trp Gly Arg Gln Ile Pro Gly Thr Thr Ser Ala Leu Val
245 250 255
Arg A.sn Trp Lys Arg Pro Ser Asp Gln Leu Gly Asp Leu Glu Gln
260 265 270
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~ly Leu Ser Gly Glu Pro Gln Pro Arg Gly Arg Ala Phe Leu Ala
275 280 285~lu Val Lys Gln Met Arg Ala Arg Arg Lys Thr Ala Lys Met Leu
290 295 300~et Val Val Leu Leu Val Phe Ala Leu Cys Tyr Leu Pro Ile Ser
305 310 315~al Leu Asn Val Leu Lys Arg Val Phe Gly Met Phe Arg Gln Ala
320 325 330~er Asp Arg Glu Ala Val Tyr Ala Cys Phe Thr Phe Ser His Trp
335 340 345~eu Val Tyr Ala Asn Ser Ala Ala Asn Pro Ile Ile Tyr Asn Phe
350 355 360
Leu Ser Gly Leu Pro Trp Ser Leu Leu
365
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1133 BASE PAIRS
~B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) SEQu~N~ DESCRIPTION: SEQ ID NO:5:
ATGGAGCCCT CAGCCACCCC AGGGGCC~-AG ATGGGGGTCC CCC~-LGGCAG CAGAGACCCC 60
1-CCC.-1~1GC CTCCAGACTA TGAAGATGAG 'l-l-l'~'lC~-l Al~-L~LGGCG TGATTATCTG 120
TACCCAAAAC AGTATGAGTG G~1CL-1~ATC GCAGCCTATG TGG.-1~1~1-1 C~L~1GGCC 180
CTGGTGGGCA ACACGCTGGT CTGCCTGGCC GTGTGGCGGA ACCACCACAT GAGGACAGTC 240
ACCAACTACT TCATTGTCAA C~-1~LCC~-LG GCTGACGTTC TGGTGACTGC TATCTGCCTG 300
CCGGCCAGCC TG.-1~G1~GA CATCACTGAG TCCTGGCTGT TCGGCCATGC CCTCTGCAAG 360
GTCATCCCCT ATCTACAGGC 1~L~1C~1G TCAGTGGCAG TGCTAACTCT CAGCTTCATC 420
GCC~-1GGACC GCTGGTATGC CATCTGCCAC CCACTATTGT TCAAGAGCAC AGCCCGGCGG 480
GCCC~1GGCT CCAL~-1GGG CATCTGGGCT GTGTCGCTGG CCATCATGGT GCCCCAGGCT 540
GCAGTCATGG AATGCAGCAG TGTGCTGCCT GAGCTAGCCA ACCGCACACG G~-1~L1~-1~A 600
~1~-L~1~ATG AACGCTGGGC AGATGACCTC TATCCCAAGA TCTACCACAG TTG~-l~ Ll 660
ATTGTCACCT ACCTGGCCCC ACTGGGCCTC ATGGCCATGG CCTATTTCCA GATATTCCGC 720
AAGCTCTGGG GCCGCCAGAT CCCCGGCACC ACCTCAGCAC TGGTGCGGAA CTGGAAGCGC 780
CCCTCAGACC AG~-LGGGGGA CCTGGAGCAG GGCCTGAGTG GAGAGCCCCA GCCCCG&GGC 840
CGCGCCTTCC TGGCTGAAGT GAAGCAGATG CGTGCACGGA GGAAGACAGC CAAGATGCTG 900
A1G~L~1GC TGCTGGTCTT CGCC~-L~-1GC TACCTGCCCA TCAGCGTCCT CAA1~1C-11 960
AAGAGGGTGT TCGGGATGTT CCGCCAAGCC AGTGACCGCG AAG~-L~1~1A CGCCTGCTTC 1020
AC~-1-1~-1CCC ACTGGCTGGT GTACGCCAAC AGCGCTGCCA ACCCCATCAT CTACAACTTC 1080
CTCAGTGGAT GTAAAGAGAA GAGTCTAGTT ~-L~1C~-1GAC CATCGTGCCC CGG 1133
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 377 BASE PAIRS
(B) TYPE: AMINO ACID
(C) STR~NDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
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Met G:Lu Pro Ser Ala Thr Pro Gly Ala Gln Met Gly Val Pro Pro
15~ly Ser Arg Glu Pro Ser Pro Val Pro Pro Asp Tyr Glu Asp Glu
30~he Ll~u Arg Tyr Leu Trp Arg Asp Tyr Leu Tyr Pro Lys Gln Tyr
45~lu T:~p Val Leu Ile Ala Ala Tyr Val Ala Val Phe Val Val Ala
60~eu Val Gly Asn Thr Leu Val Cys Leu Ala Val Trp Ary Asn His
75~is Met Arg Thr Val Thr Asn Tyr Phe Ile Val Asn Leu Ser Leu
gO~la A~sp Val Leu Val Thr Ala Ile Cys Leu Pro Ala Ser Leu Leu
100 1()5~al Al~p Ile Thr Glu Ser Trp Leu Phe Gly Hi~ Ala Leu Cys Lys
110 115 120~al Ile Pro Tyr Leu Gln Ala Val Ser Val Ser Val Ala Val Leu
125 130 135~hr L,eu Ser Phe Ile Ala Leu Asp Arg Trp Tyr Ala Ile Cy~ Hi~
140 145 150~ro Lleu Leu Phe Lys Ser Thr Ala Arg Arg Ala Arg Gly Ser Ile
155 160 165~eu Gly Ile Trp Ala Val Ser Leu Ala Ile Met Val Pro Gln A~La
170 175 1~30~la V,al Met Glu Cys Ser Ser Val Leu Pro Glu Leu Ala Asn Arg
185 190 195~hr Arg Leu Phe Ser Val Cys Asp Glu Arg Trp Ala Asp Asp Leu
200 205 210~yr Pro Lys Ile Tyr His Ser Cys Phe Phe Ile Val Thr Tyr Leu
215 220 225~la Pro Leu Gly Leu Met Ala Met Ala Tyr Phe Gln Ile Phe Arg
230 235 2gO~ys Leu Trp Gly Arg Gln Ile Pro Gly Thr Thr Ser Ala Leu Val
245 250 255~rg Asn Trp Lys Arg Pro Ser Asp Gln Leu Gly Asp Leu Glu Gln
260 265 270~ly Leu Ser Gly Glu Pro Gln Pro Arg Gly Arg Ala Phe Leu Ala
275 280 2~35~lu Val Lys Gln Met Arg Ala Arg Arg Lys Thr Ala Lys Met Leu
290 295 300~et Val Val Leu Leu Val Phe Ala Leu Cys Tyr Leu Pro Ile Ser
305 310 315~al Leu Asn Val Leu Lys Arg Val Phe Gly Met Phe Arg Gln Ala
320 325 330~er A.sp Arg Glu Ala Val Tyr Ala Cys Phe Thr Phe Ser His Trp
335 340 345~eu Val Tyr Ala Asn Ser Ala Ala Asn Pro Ile Ile Tyr Asn Phe
350 355 360~eu Ser Gly Cys Lys Glu Lys Ser Leu Val Leu Ser Pro Ser Cy~;
365 370 375~ro G,ly
CA 02220036 1997-10-31
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~2) INFORMATION FOR SEQ ID NO:7:
(i) SEQU~N~h CHARACTERISTICS
(A) LENGTH: 30 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANv~N~SS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CACTA~AGCT TAATGGAGCC CTCAGCCACC 30
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 25 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQU~N~ DESCRIPTION: SEQ ID NO:8:
ACAAGTCCTT GTCCTTCTAG AGGGC 25
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 26 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CCTAGGATGC C~-l~-lGCTG CAGCGG 26
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUEN OE CHARACTERISTICS
(A) LENGTH: 25 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
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ACAAGTCCTT GTCCTTCTAG AGGGC 25
(2) INFORMATION FOR SEQ ID No~
(i) SEQUENOE CHARACTERISTICS
(A) LENGTH: 32 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STR~NDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CGGGATCCGC CATCATGGAG ~C~-L~AGCCA CC 32
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 25 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOhOGY: LINEAR
(i.i) MOLECULE TYPE Oligonucleotide
(xi) SEQU~N~ DESCRIPTION: SEQ ID NO:12:
ACAAGTCCTT GT~-l-L~-lAG AGGGC 25
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