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HUMAN PEROXI';OME PROLIFERATOR ACTIVATED RECEPTORS,
hPPAR gamma AND hPPAR gamma 2
Human Perox:isome Proliferator Activated Receptors
Field of the InventLon
This invention relates to screening for agents
active on peroxisome proliferator activated receptors
(PPAR). This invention also relates to the cloning and
uses of human peroxisome proliferator activated receptor
subtypes.
~3ackground of the Invention
Perox:isomes are subcellular organelles found
in animals and plants. Peroxisomes contain enzymes for
cholesterol and lipid metabolism and respiration.
A var:iety of chemical agents called peroxisome
proliferators induce the proliferation of peroxisomes
and increase the capacity of peroxisomes to metabolize
fatty acids via increased expression of the enzymes
recIuired for the ~-oxidation cycle. Peroxisome
proliferators include unsaturated fatty acids,
hypolipidemic drugs (Reddy, et al., Nature 283:397-398,
1980), herbicides, leukotriene antagonists, and
plasticizers (for a review, see Green, S., Biochem.
Pharmacol. 43:3'33-400, 1992). Hypolipidemic drugs such
as clofibrates have been found to lower triglycerides
and cholesterol levels in plasma and to be beneficial in
the prevention of ischemic heart disease in individuals
with elevated levels of cholesterol (Havel, et al., Ann.
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Rev. Pharmac. 13:287-308, 1973). However, fibrate
hypolipidemic drugs are also rodent hepatocarcinogens
(Reddy,et al., ~3r. J. Cancer 40:476-482, 1979; Reddy et
al., Nature 283 397-398, 1980).
There are two hypotheses for peroxisome
proliferation. The "lipid overload hypothesis'~ suggests
that an increase in the intracellular concentration of
fatty acids is t:he main stimulus for peroxisome
proliferation (~estel, Ann. Rev. Nutr. 10:149-167, 1990,
and Phillipson, et al., N. Engld. J. Med. 312:1210-1216,
1985).
Another hypothesis postulates a receptor
mediated mechani.sm. Peroxisome proliferator activated
receptors (PPARs) have been isolated and cloned from
various species (Isseman, et al., Nature 347:645-650,
1990; Dreyer, et al., Cell 68:879-887, 1992; Gottlicher,
et al., Proc. Natl. Acad. Sci. USA. 89:4653-4657, 1992;
Sher, et al., Riochemistry 32:5598-5604, 1993; and
Schmidt, et al., Mol. Endocrinol. 6:1634-16414-8, 1992;
Tontonoz, et al . Genes & Development 8:1224-1234, 1994;
Kliewer, et al . Proc. Natl. Acad. Sci. 91:7355-7359,
1994; Chen, et al. Biochem. and Biophy. Res. Com.
196:671-677, 1993; Zhu, et al ., J. Biological Chemistry
268:26817-26820, 1993). The peroxisome proliferator
activated receptor subtypes are members of the
intracellular receptor superfamily. The ligand for
PPARs is still unidentified.
PPARy plays a key role in adipocyte differen-
tiation. Two isoforms of PPARy (PPARyl and PPARy2 that
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differ by 30 ami.no aclds at the N-terminus) have been
identified in mi.ce (Tontonoz, et al. Genes ~ Development
8:1224-1234, 19'~4, not admitted to be prior art).
PPARy2 is expressed at high levels specifically in
adipose tissue and is induced early in the course of
differentiation of 3T3-L1 preadipocytes to adipocytes.
Overexpression and activation of PPARy protein
stimulates adipose conversion in cultured fibroblasts
(Tontonoz, et a~.. Cell 79:1147-1156, 1994, not admitted
to be prior art). Activation of PPARy is sufficient to
turn on the enti.re program of adipocyte differentiation
(Lehm~nn, et al . J. Biol. Chemistry 270:12953-12956,
1995, not admitt:ed to be prior art).
Summary of the Invention
As shown in PCT applications PCT/US95/08328
and PCT/US94/11~197 (Publication No. WO95/11974),
applicant has isolated two human PPAR subtypes, i.e.,
hPPAR~ and hNUClB. However, the lack of a human PPARy
cDNA clone has hampered research such as an ~m;n~tion
of the expression patterns of the PPAR family of
receptors in human tissues and cell lines. To alleviate
this problem applicant cloned and characterized the cDNA
of two human PP~y subtypes, i.e. hPPARy and hPPARy2.
The present invention relates to hPPARy and
hPPARy2 polypept:ides, nucleic acids encoding such
polypeptides, cells, tissues and ~n;m~ls containing such
polypeptides ancl nucleic acids, antibodies to such
polypeptides, assays utilizing such polypeptides and
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nucleic acids, and methods relating to all of the
foregoing. The hPPARy and hPPARy2 polypeptides, nucleic
acids, and antibodies are useful for establishing the
tissue speci~ic expression pattern of hPPARy and hPPARy2
S genes. For example, a Northern blot can be used to
reveal tissue specific expression of the genes. They
are also useful for screening for agonists and
antagonists of hPPARy and hPPARy2 peptides for improved
pharmacological profiles for the treatment of diseases
with higher potency, efficacy, and fewer side effects.
The p:resent invention is based upon the
identification and isolation of two novel human
peroxisome prol:iferator activated receptor subtypes
termed hPPARy and hPPARy2.
Applicant has determined that hPPARy
polypeptides repress hPPAR~ (hPPAR~, referred to as
hPPAR1 in PCT application PCT/US94/11897 (Publication
No. W095/11974)~ is a subtype of PPAR) activity, and
that relief frorn such repression is therapeutically
useful. hPPARy polypeptides bind to peroxisome
proliferator response elements (PPREs) as a complex with
RXR polypeptides (e.g., RXR~, ~ or y). hPPARy
polypeptides are not significantly activated by
compounds that activate mPPARy polypeptides. hPPARy
polypeptides repress hPPAR~ polypeptides' transcription
activation activity by sequestering RXR polypeptides.
The present invention features methods for
identifying agonists and antagonists of hPPARy and
hPPARy2 polypept:ides. The present invention also
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features methods for identifying therapeutic agents that
alleviate the repressive effects of hPPARy polypeptides
on PPAR~ polypeptides' transcription activation
activity. These methods make it possible to screen
large collections of natural, semisynthetic, or
synthetic compounds for therapeutically useful profiles.
hPPARy and hPPARy2 agonists, antagonists, and agents
that alleviate l_he repressive effects of hPPARy
polypeptides on PPAR~ polypeptides may be used to treat
diseases and pa~hological conditions affected by the
level o~ hPPARy or hPPARy2 polypeptide activity, such
as, but not lim:ited to, obesity, diabetes,
hyperlipidemia, hypercholesteremia and hyper-
lipoproteinemia.
This :invention is also directed to compounds,
compositions, and methods for treating a patient
exhibiting a palhological condition affected by the
level of hPPARy or hPPARy2 polypeptide activity. More
particularly, the invention relates to hPPARy and
hPPARy2 agonists, antagonists, and compounds and
pharmaceutical compositions that relieve the repression
of PPAR~ activily by a hPPARy polypeptide.
Thus, in a first aspect the invention features
an isolated, pw-ified, enriched or recombinant nucleic
acid encoding a hPPARy or hPPARy2 polypeptide.
By "isolated" in reference to nucleic acid is
meant a polymer of 2 (preferably 21, more preferably 39,
most preferably 75) or more nucleotides conjugated to
each other, inc:Luding DNA or RNA that is isolated from a
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natural source or that is synthesized. The isolated
nucleic acid of the present invention is unique in the
sense that it is not found in a pure or separated state
in nature. Use of the term "isolated" indicates that a
naturally occurring sequence has been removed from its
normal cellular environment. Thus, the sequence may be
in a cell-free solution or placed in a different
cellular environment. The term does not imply that the
sequence is the only nucleotide chain present, but does
indicate that it: is the predominate sequence present (at
least 10 - 20% more than any other nucleotide sequence)
and is essentia]ly free (about 90 - 95~ pure at least)
of non-nucleotide material naturally associated with it.
Therefore, the t:erm does not encompass an isolated
chromosome encocling a hPPARy or hPPARy2 polypeptide.
By "purified" in reference to nucleic acid
does not require absolute purity (such as a homogeneous
preparation); instead, it represents an indication that
the sequence is relatively purer than in the natural
environment (co~lpared to the natural level this level
should be at least 2-5 fold greater, e.g., in terms of
mg/ml). IndivicLual clones isolated from a cDNA library
may be purified to electrophoretic homogeneity. The
claimed DNA molecules obtained from these clones could
be obtained directly from total DNA or from total RNA.
The cDNA clones are not naturally occurring, but rather
are preferably obtained via manipulation of a partially
purified naturally occurring substance (messenger RNA).
The construction of a cDNA library from mRNA involves
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the creation of a synthetic substance (cDNA) and pure
individual cDNA clones can be isolated from the
synthetic libra:ry by clonal selection of the cells
carrying the cDNA library. Thus, the process which
includes the construction of a cDNA library from ~IRNA
and isolation of distinct cDNA clones yields an
approximately l~6-fold purification of the native
message. Thus, purification of at least one order of
magnitude, preferably two or three orders, and more
preferably four or five orders of magnitude is expressly
contemplated.
By ~enriched" in reference to nucleic acid is
meant that the specific DNA or RNA sequence constitutes
a significantly higher fraction (2 - 5 fold) of the
total DNA or RNA present in the cells or solution of
interest than in normal or diseased cells or in the
cells from which the sequence was taken. This could be
caused by a person by preferential reduction in the
amount of other DNA or RNA present, or by a preferential
increase in the amount of the specific DNA or RNA se-
quence, or by a combination of the two. However, it
should be noted that enriched does not imply that there
are no other DNA or RNA sequences present, just that the
relative amount of the sequence of interest has been
significantly increased in a useful manner and
preferably separate from a sequence library. The term
"significantly" here is used to indicate that the level
of increase is useful to the person making such an
increase, and glenerally means an increase relative to
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other nucleic acids of about at least 2 fold, more
preferably at least 5 to 10 fold or even more. The term
also does not imply that there is no DNA or RNA from
other sources. The DNA from other sources may, for
example, comprise DNA from a yeast or bacterial genome,
or a cloning vector such as pUC19. This term
distinguishes from naturally occurring events, such as
viral infection, or tumor type growths, in which the
level of one mRNA may be naturally increased relative to
other species of mRN~. That is, the term is meant to
cover only those situations in which a person has
intervened to elevate the proportion of the desired
nucleic acid.
By "recombinant" in reference to a nucleic
acid is meant the nucleic acid is produced by
recombinant DNA techniques such that it is distinct from
a naturally occurring nucleic acid.
By "a hPPARy polypeptide" is meant two or more
contiguous amino acids set forth in the full length
amino acid sequence of SEQ ID NO:2, wherein said
contiguous amino acids have a sequence different from
those of mouse PPARy polypeptides. The hPPARy
polypeptide can be encoded by a full-length nucleic acid
sequence or any portion of the full-length nucleic acid
sequence, so long as a functional activity of the
polypeptide is retained.
In preferred embodiments the isolated nucleic
acid comprises, consists essentially of, or consists of
a nucleic acid sequence set forth in the full length
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nucleic acid sequence SEQ ID N0:1 or at least 27, 30,
35, 40 or 50 contiguous nucleotides thereof and the
hPPARy polypeptide comprises, consists essentially of,
or consists of at least 9, lo, 15, 20, or 30 contiguous
amino acids o~ a hPPARy polypeptide.
By "a hPPARy2 polypeptide" is meant two or
more contiguous amino acids set forth in the full length
amino acid sequence of SEQ ID N0:4, wherein said
contiguous amino acids have a sequence different from
those of mouse PPARy polypeptides. The hPPARy2
polypeptide can be encoded by a full-length nucleic acid
sequence or any portion of the full-length nucleic acid
sequence, so long as a functional activity of the
polypeptide is retained.
In preferred embodiments the isolated nucleic
acid comprises, consists essentially of, or consists of
a nucleic acid sequence set ~orth in the ~ull length
nucleic acid sequence SEQ ID NO:3 or at least 27, 30,
35, 40 or 50 contiguous nucleotides thereof and the
hPPARy2 polypeptide comprises, consists essentially of,
or consists of at least 9, 10, 15, 20, or 30 contiguous
amino acids of a hPPARy2 polypeptide.
By "comprising" is meant including, but not
limited to, whatever follows the word "comprising".
Thus, use of the term "comprising" indicates that the
listed elements are required or mandatory, but that
other elements are optional and may or may not be
present. By "consisting of" is meant including, and
limited to, whatever follows the phrase "consisting of~.
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Thus, the phrase "consisting of" indicates that the
listed elements are required or mandatory, and that no
other elements may be present. By "consisting
essentially of" is meant including any elements listed
after the phrase, and limited to other elements that do
not interfere with or contribute to the activity or
action specified in the disclosure for the listed
elements. Thus, the phrase "consisting essentially of"
indicates that the listed elements are required or
lo mandatory, but that other elements are optional and may
or may not be present depending upon whether or not they
affect the activity or action of the listed elements.
In other preferred embodiments, the nucleic
acid comprises no less than 60 contiguous nucleotides
from sequence numbers 157 to 1641 or 214 to 1641 of SEQ.
ID. NO.l.
Compositions and probes of the present
invention may contain human nucleic acid encoding a
hPPARy or hPPARy2 polypeptide but are substantially free
of nucleic acid not encoding a human hPPARy or hPPARy2
polypeptide. The human nucleic acid encoding a hPPARy
or hPPARy2 polypeptide is at least 18 contiguous bases
of the nucleotid,- sequence set forth in SEQ. ID NO. 1 or
3 and will selectively hybridize to human genomic DNA
encoding a hPPARy or hPPARy2 polypeptide, or is
complementary to such a sequence. The nucleic acid may
be isolated from a natural source by cDNA cloning or
subtractive hybr:idization; the natural source may be
blood, semen, an~l tissue of humans; and the nucleic acid
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may be synthesized by the triester method or by using an
automated DNA synthesizer. In yet other preferred
embodiments the nucleic acid is a unique region, for
example those useful for the design of hybridization
probes to facili.tate identification and cloning of
additional polypeptides, the design of PCR probes to
facilitate cloni.ng of additional polypeptides, and
obtaining antibc,dies to polypeptide regions.
By "unique nucleic acid region" is meant a
sequence present in a full length nucleic acid coding
for a hPPARy or hPPARy2 polypeptide that is not present
in a sequence coding for any other naturally occurring
polypeptide. Such regions preferably comprise 12 or 20
contiguous nucleotides present in the full length
nucleic acid encoding a hPPARy or hPPARy2 polypeptide.
The invention also features a nucleic acid
probe for the detection of a hPPARy or hPPARy2
polypeptide or nucleic acid encoding a hPPARy or hPPARy2
polypeptide in a sample. The nucleic acid probe
contains nucleic acid that will hybridize to a sequence
set forth in SEQ ID NO:1 or 3, but not to a mouse PPARy
nucleic acid sequence under high stringency
hybridization conditions. In preferred embodiments the
nucleic acid probe hybridizes to nucleic acid encoding
at least 12, 27, 30, 35, 40 or 50 contiguous amino acids
of the full-length sequence set forth in SEQ ID N0:2 or
4.
By "high stringency hybridization conditions"
is meant those hybridizing conditions that (1) employ
--
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low ionic strength and high temperature for washing, ~or
example, 0.015 I~ NaCl/0.0015 M sodium citrate/0.1~ SDS
at 50~C; (2) employ during hybridization a denaturing
agent such as formamide, for example, 50~ (vol/vol)
formamide with ~ bovine serum albumin/0.1~
Ficoll/0.1~ polrvinylpyrrolidone/50 mM sodium phosphate
buffer at pH 6.!~ with 750 mM NaCl, 75 mM sodium citrate
at 42~C; or (3) employ 50~ formamide, 5 x Ssc (0.75 M
NaCl, 0.075 M Sodium pyrophosphate, 5 x Denhardt's
solution, sonicated salmon sperm DNA (50 g/ml), 0.1~
SDS, and 10~ dextran sulfate at 42~C, with washes at 42~C
in 0.2 x SSC an~ 0.1~ SDS. Under stringent
hybridization conditions only highly complementary
nucleic acid sequences hybridize. Preferably, such
conditions prevent hybridization of nucleic acids having
1 or 2 mismatches out of 20 contiguous nucleotides.
Methods for using the probes include detecting
the presence or amount hPPARy or hPPARy2 RNA in a sample
by contacting the sample with a nucleic acid probe under
conditions such that hybridization occurs and detecting
the presence or amount of the probe bound to hPPARy or
hPPARy2 RNA. The nucleic acid duplex formed between the
probe and a nucleic acid sequence coding for a hPPARy or
hPPARy2 polypeptide may be used in the identification o~
the sequence of the nucleic acid detected (for example
see, Nelson et al., in Nonisotopic DNA Probe Techniques,
p. 275 Academic Press, San Diego (Kricka, ed., 1992)
hereby incorporated by reference herein in its entirety,
including any drawings). Kits for performing such
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methods may be constructed to include a container means
having disposed therein a nucleic acid probe.
The in.vention features recombinant nucleic
acid comprising a contiguous nucleic acid sequence
encoding a hPPAR.y or hPPARy2 polypeptide, preferably in
a cell or an organism. The recombinant nucleic acid may
contain a sequence set forth in SEQ ID N0:1 or 3 and a
vector or a promoter effective to initiate transcription
in a host cell. The recombinant nucleic acid can
alternatively cc,ntain a transcriptional initiation
region functiona.l in a cell, a sequence complimentary to
an RNA sequence encoding a hPPARy or hPPARy2 polypeptide
and a transcriptional termination region functional in a
cell.
In preferred embodiments, the recombinant
nucleic acid com.prises no less than 60 contiguous
nucleotides from. sequence numbers 157 to 1641 or 214 to
1641 of SEQ. ID. N0.1.
In another aspect the invention features an
isolated, enrich.ed, purified or recombinant hPPARy or
hPPARy2 polypeptide.
By "isolated" in reference to a polypeptide is
meant a polymer of 2 (preferably 7, more preferably 13,
most preferably 25) or more amino acids conjugated to
each other, including polypeptides that are isolated
from a natural source or that are synthesized. The
isolated polypeptides of the present invention are
unique in the sense that they are not found in a pure or
separated state in nature. Use of the term "isolated"
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14
indicates that c~ naturally occurring sequence has been
removed from its normal cellular environment. Thus, the
sequence may be in a cell-free solution or placed in a
different cellu]ar environment. The term does not imply
that the sequence is the only amino acid chain present,
but that it is t:he predominate sequence present (at
least 10 - 20~ more than any other sequence) and is
essentially free (about so - 95% pure at least) of
non-amino acid material naturally associated with it.
By "enriched" in reference to a polypeptide is
meant that the ~3pecific amino acid sequence constitutes
a significantly higher fraction (2 - 5 fold) of the
total of amino acids present in the cells or solution of
interest than in normal or diseased cells or in the
cells from which the sequence was taken. This could be
caused by a person by preferential reduction in the
amount of other amino acids present, or by a
preferential increase in the amount of the specific
amino acid sequence of interest, or by a combination of
the two. However, it should be noted that enriched does
not imply that there are no other amino acid sequences
present, just that the relative amount of the sequence
of interest has been significantly increased. The term
~significantly~ here is used to indicate that the level
of increase is useful to the person making such an
increase, and generally means an increase relative to
other amino acids of about at least 2 fold, more prefer-
ably at least 5 to 10 fold or even more. The term also
does not imply that there is no amino acid from other
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sources. The amino acid from other sources may, for
example, comprise amino acid encoded by a yeast or
bacterial genome, or a cloning vector such as pUClg.
The term is meant to cover only those situations in
which man has in.tervened to elevate the proportion of
the desired amino acid.
By ~puri~ied~ in reference to a polypeptide
does not require absolute purity (such as a homogeneous
preparation); instead, it represents an indication that
lo the sequence is relatively purer than in the natural
environment (compared to the natural level this level
should be at least 2-5 fold greater, e.g., in terms of
mg/ml). Purification of at least one order of
magnitude, preferably two or three orders, and more
preferably four or five orders of magnitude is expressly
contemplated. The substance is preferably free of
contamination at a functionally significant level, for
example 90%, 95%, or 99% pure.
By "recombinant hPPARy or hPPARy2 polypeptide~'
is meant a hPPARy or hPPARy2 polypeptide produced by
recombinant DNA techniques such that it is distinct from
a naturally occurring polypeptide either in its location
(e.a., present in a different cell or tissue than found
in nature), purity or structure. Generally, such a
recombinant polypeptide will be present in a cell in an
amount different from that normally observed in nature.
This invention features recombinant hPPARy or hPPARy2
polypeptides obtainable using techni~ues known to those
skilled in the art, including those described in
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McDonnell et al., PCT application PCT/US94/03795
(Publication No. W094/23068), Evans et al., U.S. Patent
5,071,773, and PCT application, PCT/US91/00399 filed
January 22, 1991 (International Publication No. WO
91/12258), incorporated by re~erence herein.
In a F,referred embodiment, either vector
pBacPAK8 (Clontech) or vector pBacPAK9 (Clontech) is
used to express recombinant hPPARy or hPPARy2
polypeptide in insect cells. In another preferred
embodiment, vector pYES2 (Invitrogen) is used to express
recombinant hPPARy or hPPARy2 polypeptide in yeast
cells. In yet another preferred embodiment, pBKCMV
(Stratagene) is used to express recombinant hPPARy or
hPPARy2 polypeptide in m~mm~l ian cells.
In preferred embodiments the hPPARy or hPPARy2
polypeptide contains at least 9, lO, 15, 20, or 30
contiguous amino acids of the full-length sequence set
forth in SEQ ID NO:2 or 4.
In yet another aspect the invention ~eatures a
purified antibody (e.g., a monoclonal or polyclonal
antibody) having specific binding affinity to a hPPARy
or hPPARy2 polypeptide. The antibody contains a
sequence of amino acids that is able to specifically
bind to a hPPARy or hPPARy2 polypeptide. An antipeptide
antibody may be prepared with techniques known to those
skilled in the art, including, but not limited to, those
disclosed in Niman, PCT application PCT/US88/03921
(International Publication No. WO 89/04489),
incorporated by reference herein.
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By " specific binding affinity" is meant that
the antibody wi].l bind to a hPPARy or hPPARy2
polypeptide at a certain detectable amount but will not
bind other polypeptides to the same extent under
identical condit:ions.
Antibodies having specific binding af~inity to
a hPPARy or hPP~y2 polypeptide may be used in methods
for detecting the presence and/or amount of a hPPARy or
hPPARy2 polypept:ide in a sample by contacting the sample
with the antibocly under conditions such that an
immunocomplex forms and detecting the presence and/or
amount of the antibody conjugated to the hPPARy or
hPPARy2 polypeptide. Diagnostic kits for performing
such methods may be constructed to include a first
container means containing the antibody and a second
container means having a conjugate of a binding partner
of the antibody and a label.
In another aspect the invention features a
hybridoma which produces an antibody having specific
binding affinity to a hPPARy or hPPARy2 polypeptide.
By "hybridoma" is meant an immortalized cell
line which is capable of secreting an antibody, for
example a hPPARy or hPPARy2 antibody.
In preferred embodiments the hPPARy or hPPARy2
antibody comprises a sequence of amino acids that is
able to specifically bind a hPPARy or hPPARy2
polypeptide.
In other aspects, the invention provides
transgenic, nonhuman m~mm~ 1 s containing a transgene
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18
encoding a hPP~y or hPPARy2 polypeptide or a gene
effecting the e~pression of a hPPARy or hPPARy2
polypeptide. Sllch transgenic nonhllm~n m~mm~l S are
particularly useful as an in vivo test system for
studying the efiects of introducing a hPPARy or hPPARy2
polypeptide, regulating the expression of a hPPARy or
hPPARy2 polypept:ide (i.e., through the introduction of
additional genes, antisense nucleic acids, or
ribozymes).
A "transgenic animal" is an animal having
cells that contain DNA which has been artificially
inserted into a cell, which DNA becomes part of the
genome of the animal which develops from that cell.
Preferred transgenic animals are primates, mice, rats,
cows, pigs, horses, goats, sheep, dogs and cats. The
transgenic DNA may encode for a hPPARy or hPPARy2
polypeptide. Native expression in an animal may be
reduced by providing an amount of anti-sense RNA or DNA
effective to recluce expression of the receptor.
In another aspect, the invention describes a
recombinant cell. or tissue containing a purified nucleic
acid coding for a hPPARy or hPPARy2 polypeptide. In
such cells, the nucleic acid may be under the control of
its genomic regulatory elements, or may be under the
control of exogenous regulatory elements including an
exogenous promoter. By "exogenous" it is meant a
promoter that is not normally coupled in vivo
transcriptionally to the coding sequence for the hPPARy
or hPPARy2 polypeptide.
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19
In another aspect, the invention features a
method for screening for a therapeutic agent for
treatment of a pathological condition affected by a
hPPARy or hPPARy2 polypeptide by detecting an agonist or
antagonist of the hPPARy or hPPARy2 polypeptide.
A cell. or an ln vl tro system is transformed
with a vector expressing the hPPARy or hPPARy2
polypeptide and a reporter gene whose expression is
activated by the: hPPARy or hPPARy2 polypeptide. The
cell or in vi tro system is brought into contact with a
test compound. An increase in the expression of the
reporter gene would indicate that the test compound is
an agonist of th.e hPPARy or hPPARy2 polypeptide; a
decrease in the expression of the reporter gene would
indicate that the test compound is an antagonist of the
hPPARy or hPPARy-2 polypeptide.
In a preferred embodiment, the vector contains
translation initiation sequence operationally linked to
a sequence encoding the hPPARy or hPPARy2 polypeptide.
The hPPARy or hPPARy2 polypeptide begins with the third,
second or first methionine in SEQ. ID. NO. 2 or 4.
By "reporter gene" is meant a gene encoding a
product that is easily detected and assayed by
techniques known to those skilled in the art. A
reporter gene in this invention is driven by a promoter
that is responsive to hPPARy or hPPARy2 polypeptides, or
PPAR~ polypeptides, including, but not limited to, the
native promoter of a gene such as acylcoenzyme A
oxidase, enoyl-CoA hydratase/3-hydrosyacyl-CoA
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dehydrogenase bifunctional enzyme, 3-ketoacyl thiolase
or ApoA1.
In another preferred embodiment, the reporter
gene comprises a peroxisome proliferator responsive
element (PPRE element) that is responsive to hPPARy or
hppARy2 polypeptide activation. The hPPARy or hPPARy2
gene and the reporter gene are encoded in vectors and
introduced into the cell by co-transfection.
Co-transfection assays may be performed as
previously described (Heyman, et al. Cell 68:397-406,
(1992); Allegretto, et al. J. Biol. Chem.
268:26625-26633 (1993); Isseman, I., and Green, S.,
Nature 347:645-650, 1990). In an example, the DNA-
binding domain of hPPARy or hPPARy2 is replaced with the
DNA-binding domain of a well characterized nuclear
receptor, including, but not limited to, the
glucocorticoid or estrogen receptor, to create a
chimeric receptor able to activate a glucocorticoid- or
estrogen-responsive reporter gene in the presence of the
hPPARy or hPPARy.2-specific ligand (Giguere, V. and
Evans, RM 1990, "Identification of receptors for
retinoids as members of the steroid and thyroid hormone
receptor family", In : Packer L (ed) Retinoids. Part A:
Molecular and Metabolic Aspects. Methods in Enzymoloay.
Academic Press, ',an Diego, CA, 189:223-232, incorporated
by reference herein). The cell is transformed with the
chimeric receptor. The cell is also transformed with a
reporter vector which comprises a segment encoding a
reporter polypeptide under the control of a promoter and
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a segment of hormone response element (such as a
glucocorticoid- or estrogen-responsive element).
Co-transfection assays will also determine
what genes are regulated by hPPARy or hPPARy2 and gel
retardation assays will indicate the sequence
specificity of the binding of hPPARy or hPPARy2 to DNA.
The reporter gene may be expressed at a basal
level in the cell. When a suitable agonist is provided
to the cell, the hPPARy or hPPARy2 polypeptide is
transformed and delivered to an appropriate DNA-binding
region of the reporter gene to thereby activate the
hormone response element and increase the expression of
the reporter gene. On the other hand, when a suitable
antagonist is provided to the cell, the expression of
the reporter gene is decreased to less than the basal
level. Activation or inactivation of the reporter gene
is detected by st:andard procedures used for detecting
the product of a reporter gene. After introduction of
the chimeric receptor and report gene constructs in
recipient cells by transient transfection, the cells are
challenged with a battery of compounds until an
activation or inactivation response is observed.
BecaLLse PPARy has been implicated in adipose
cell function ancl development, hPPARy or hPPARy2
agonists and antagonists may be useful for treating
obesity, diabetes, anorexia, lipoprotein defects,
hyperlipidemia, hypercholesteremia and
hyperlipoproteine!mia and other related disorders. PPARy
is a key receptor in the dif~erentiation step from
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preadipocytes to adipocytes. PPARy is an adipocyte
specific-nuclear hormone receptor that has been
identified as a key regulator of certain fat cell
enhancers (Tontonez et al., Cell 79:1147-1156, 199~).
Overexpressing ]?PARy stimulates adipose differentiation
in non-adipogen:ic cell lines like fibroblasts.
PPARy antagonists may be used to block or
reverse the difierentiation step from preadipocytes to
adipocytes. RXR agonists or antagonists may also be
used to block or reverse this differentiation step since
PPARy binds to DNA as a heterodimer with RXR. Such
compounds would be useful in the treatment of obesity,
diabetes, anorexia, lipoprotein defects, hyperlipidemia,
hypercholesteremia and hyperlipoproteinemia and related
disorders.
In another aspect, the present invention
features a method for identifying therapeutic agents for
treatment of a ~)athological condition affected by a
hPPARy polypeptide, by screening for therapeutic agents
which, when added to a system containing the hPPARy
polypeptide and PPAR~ protein, relieve the repression of
PPAR~ protein activity by the hPPARy polypeptide.
In a preferred embodiment, a hPPARy
polypeptide, PPARcx protein and reporter gene are
provided in a cell or an in vitro system. The reporter
gene has a peroxisome proliferator responsive element
(PPRE) and can be activated by the PPAR~ protein. The
hPPARy polypeptide represses the expression of the
reporter gene. The reduction or relief of the
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repression of the PPAR~ protein by the hPPARy
polypeptide is measured by the expression level of the
reporter gene.
In a further preferred embodiment, hPPARy
gene, PPAR~ gene and a reporter gene are encoded in
vectors and introduced into a cell by transfection.
In another further preferred embodiment, a
PPAR activator is added to the screening assay.
By "PE'AR activator" is meant a chemical agent
that is capable of activating the transcription
activation activity of PPAR protein, such as, but not
limited to, CFA (clofibric acid), ETYA
(5,8,11,14-eicosatetraynoic acid) or WY-14, 643 ([4-
chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid).
In yet another preferred embodiment, the
reporter gene comprises a PPRE element.
In other preferred embodiments, this method
screens for agents that interfere with the formation of
a heterodimer between a hPPARy polypeptide and a RXR
polypeptide such as RXR~, RXR~, or RXRy, or the binding
of a heterodimer between a hPPARy polypeptide and a RXR
polypeptide to a PPRE element.
By boosting PPAR~ activity, the agents that
relieve the repression of PPAR~ protein activity by
hPPARy may enhance the effects of PPAR~ agonists and be
helpful for treating obesity, diabetes, hyperlipidemia,
hypercholesterem:ia and hyperlipoproteinemia.
In anolher aspect, this invention features a
method for treatrnent of a pathological condition
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24
affe~ted by the level of hPPARy activity by providing an
agonist, an antagonist, or an agent that represses or
reduces the rep~ession of PPAR~ protein activity by
hPPARy polypept:Ldes. The pathological conditions
treated by this method include, but are not limited to,
obesity, diabetes, anorexia, lipoprotein defects,
hyperlipidemia, hypercholesteremia, hyperlipoproteinemia
and other metabolic diseases.
The present invention also features novel or
unique compounds identified by methods described above
that are hPPARy or hPPARy2 agonists, hPPARy or hPPARy2
antagonists, or capable of repressing or reducing the
repression of PPAR~ protein activity by hPPARy
polypeptides. ~3y "novel or uniclue" is meant that the
compounds are not known per se or are not already known
for uses relating to treatment of a pathological
condition affected by the level of hPPARy or hPPARy2
polypeptides.
Applicant is particularly interested in the
identification of agents of low molecular weight (less
than lO,oO0 daltons, preferably less than 5,000, and
most preferably less than 1,000) which can be readily
formulated as useful therapeutic agents.
Such agents can then be screened to ensure
that they are specific to tissues with pathological
conditions induced or aggravated by hPPARy or hPPARy2
protein with little or no effect on healthy tissues such
that the agents can be used in a therapeutic or
prophylactic manner. If such agents have some effect on
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healthy tissues they may still be useful in therapeutic
treatment, parti.cularly in those diseases which are life
threatening.
By ant:agonizing hPPARy, the agents will be
helpful to reduce adipocyte differentiation for treating
obesity, diabetes and other lipoprotein defects.
The compounds identified by the method of this
invention are particularly useful in the treatment of
diseases and pat:hological conditions affected by the
level of hPPARy or hPPARy2 protein, including, without
limitation, obe~3ity, diabetes, anorexia, lipoprotein
defects, hyperlipidemia, hypercholesteremia,
hyperlipoproteinemia, cardiovascular diseases, coronary
diseases, hypertension, hyperglycemia,
hypercholesterolemia and other metabolic disorders.
The present invention also includes pharmaceu-
tically acceptable compositions prepared for storage and
subsequent administration which include a pharmaceuti-
cally effective amount of an above-described product in
a pharmaceutica:Lly acceptable carrier or diluent.
By "therapeutically effective amount" is meant
an amount of a pharmaceutical composition having a ther-
apeutically relevant effect. A therapeutically relevant
effect relieves to some extent one or more symptoms of
the disease or condition in the patient; or returns to
normal either partially or completely one or more physi-
ological or biochemical parameters associated with or
causative of the disease or condition.
Other features and advantages of the invention
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will be apparenl from the following description of the
preferred embod:iments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph showing induction of
hPPARy by various compounds.
CV-l cells were transfected with 0.1 ~g
pCMVhPPARy or the empty expression vector pBKCMV (no
receptor). LY-:L71,883, 9-cis-retinoic acid, ETYA and
gemfibrozil were added. Fold induction is defined as
lo the ratio of the m~i m~ 1 response observed in the
presence of the compound to that in its absence.
Figure 2 is a graph showing normalized
response of a reporter gene to steady dose of hPPARcx
coupled with increasing dose of hNUC or hPPARy.
CV-1 cells were transfected with o.1 ~g o~
pCMVhPPAR~ and ().1 or 0.4 ~g of pCMVhNUC1 or pCMVhPPARy.
Gemfibrozil was added to a final concentration of 100 M.
Figure 3 is a graph showing normalized
response of a reporter gene to mixing doses of hPPAR~,
hPPARy, hRXR~, and hNUC.
HepG2 cells were transfected with 0.1 ~g of
pCMVhPPARcx and ().4 ~g of hPPAR (A) or o.1 ~g of hNUC1
(B). Where indi.cated 0.4 ~g of pRShRXR (Kleiwer et al.,
Nature 358:771-774, 1992) was added. Gemfibrozil or
clofibric acid l:CFA) were added to a final concentration
of 100 ~M and ln~ respectively.
Figure 4 is a graph showing normalized
response of a reporter gene to thiazolidinedione.
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wos6l23884 PCT~S96101469
CV-1 cells were transfected with pCMVhPPARy3
or the empty expression vector pBKCMV (no receptor).
Thiazolidinedione was added. Fold induction is defined
as the ratio of the maximal response observed in the
presence of the compound to that in its absence.
In al:L the figures, hPPARg = hPPARy, hPPARa =
hPPARc~, RXRa = RXR~.
Description of the Preferred Embodiments
I. Adipocyte Differentiation and PPARy.
Adipocytes play a central role in lipid
homeostasis and the maintenance of energy balance in
humans. They functiol1 to store and release lipid in
response to the metabolic needs of an organism.
Pathological conditions associated with adipocyte
abnormity inclucle obesity and several lipodystrophy
syndromes. Obesity is associated with an increased risk
for cardiovascu]ar disease, diabetes and an increased
mortality rate ~see Grundy et al., Disease-a-Month
36:645-696, 1990). Current treatment for obesity
includes diet, exercise and surgery (Leibel, R.L. et
al., New Englancl Journal of Medicine 332:621-628, 1995).
Adipoc:yte differentiation involves dramatic
changes in gene expression. A number of transcription
factors have been identified as potential regulators of
this process, e.g., CCAATT enhancer-binding protein c~
(C/EBP~) binds to the promoters of several fat cell
genes (Christy et al., Genes Dev. 3:1325-1335, 1989),
and overexpression of this factor can promote
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28
adipogenesis in fibroblastic cell lines (Freytag et al.,
Genes Dev. 8:16';4-1663, 1994).
Mouse PPARy2 has been identified as a key
regulator of fat: cell enhancers (Tontonoz et al., Genes
& Development 8:1224-34, 1994, and Tontonoz et al., Cell
79:1147-1156, 1994). It is expressed at very high
levels specifically in adipose tissue and forms a
heterodimer with mouse RXR~ to activate the adipocyte-
speci~ic enhancer aP?. Forced expression of mouse
PPARy2 in fibrok,last cell lines that do not normally
differentiate into adipocytes is sufficient to cause
overt adipose differentiation of the cell line in the
presence of dexamethasone and PPAR activators,
suggesting a role in adipose differentiation and lipid
metabolism.
II. Cardio-protective effect of hPPAR~ and hPPARy.
The effect of hypolipidemic drugs like
gemfibrozil that have significant cardio-protective
effect are mediated via hPPAR~. Applicant determined
that hPPARy is a specific repressor of the
transcriptional activation effected by hPPAR~
polypeptide. The repressive action of hPPARy protein on
hPPAR~ may limit the clinical efficacy of hPPAR~
agonists (e.g., fibrates). Agents that relieve this
repression will increase activity of hPPAR~ and increase
the efficacy of ~_xisting drugs, or render these drugs
unnecessary because endogenous activators of PPAR~ can
then work with g:reater efficacy.
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Since hPPARy is shown by Applicant to be
present in the human heart, kidney, pancreas, skeletal
muscle, and liver tissues where hPPAR~ is also present,
the screening methods o~ this invention and agents
identified thereby may have widespread therapeutic
significance.
Applic-ant has demonstrated co-operative
binding of hPPA~y and RXR~, RXR~ or RXRy to a PPAR
response element:, PPRE. Without being bound by any
particular theory, applicant proposes that repression of
hPPAR~ by hPPARy likely occurs by sequestering RXR~,
thereby antagoni.zing transcription activation activity
o:E hppARol protei.n.
The present invention relates to hPPARy and
hPPARy2 polypept:ides, nucleic acids encoding such
polypeptides, ce~lls, tissues and animals containing such
nucleic acids, antibodies to such polypeptides, assays
utilizing such polypeptides, and methods relating to all
of the foregoinq. The above mentioned compositions are
used to screen f.or hPPARy or hPPARy2 agonists and
antagonists, whi.ch can be used as lead compounds to
designed drugs active on hPPARy or hPPARy2 related
pathological conditions, such as obesity. The above
mentioned compositions are also used to establish cell
cultures or An;mAl models to study adipocyte
differentiation or obesity in humans.
The invention will now be described in greater
detail by re~ere~nce to the ~ollowing examples regarding
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screening for hPPARy or hPPARy2 agonists and
antagonists. This invention, however, is not limited to
co-transfection assay, gel retardation assay and
immunoprecipitation assay described below. Other
methods known to those skilled in the art for assaying
an agent that relieve the repressive ef~ect o~ a protein
on a cellular activity may also be used.
III. Materials and Methods.
Experimental procedures and reagents employed
in the examples described herein are set ~orth below:
Reagen~s
ETYA, ~-estradiol, ATRA, LT3 (3, 3l, 5 -
triiodo - L - thyronine) and CFA were purchased from
Sigma, and WY-14,643 from Chemsyn Science Laboratories,
Lenexa, Kansas, USA. Stock solutions of these compounds
were made in ethanol, methanol or dimethyl sulfoxide
(ETYA, LY-171,883 and gemfibrozil in ethanol, 9-cis-
retinoic acid in dimethyl sulfoxide).
The recipes for buffers, mediums, and
solutions in the following examples are given in J.
Sambrook, E. F. Fritsch, and T. Maniatis, Molecular
Clonincr: A Labc,ratory Manual, 2 Ed., Cold Spring Harbor
Laboratory Precs, Cold Spring Harbor, New York, 1989.
Vector Construction
For m~mm~l ian expression studies, the entire
hPPARy cDNA was subcloned into the EcoRI site of pcDNA-1
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31
(Invitrogen, San Diego, CA) under the control of the CMV
promoter to make plasmid pCMVhPPARy. The hPPAR~ cDNA
was cloned into the NotI site of pBKCMV (Stratagene) to
give pCMVhPPAR~. The hNUClB cDNA was directionally
cloned into the SalI-SacII site of pBKCMV to give
pCMVhNUClB.
The reporter plasmid pPPREA3-tk-luc was
generated by inserting three copies of the synthetic
oligonucleotide (5'-CCCGAACGTGACCTTTGTCCTGGTCC-3')
containing the "A~ site of the Acyl-CoA oxidase gene
regulatory sequence (Osumi et al., Biochem. Biophys.
Res. Commun. 17!;:866-871, 1991) into the XhoI site 5' of
the tk promoter in the previously described
pBLtk-luciferase vector (Giguere et al., Cell
46:645-652, 198~).
pRSh~R~, pRShRXR~, MTV-TREp2-LUC, and
CRBPII-tk-LUC have been described in Giguere et al.,
Nature 330(2):6:24-629, 1987; Mangelsdorf et al., Nature
345:224-229, 19!30; Umesono et al., Nature 336:262-265,
1988 and Mangel~sdorf et al., Cell 66:555-561, 1991.
Co-transfection Assay
CV-1 or HepG2 cells were grown in Dulbecco's
modified Eagle"s medium (DMEM) supplemented with
10~(v/v) fetal bovine serum (Hyclone), 2 mM L-glutamine,
and 55 ~g/ml gentamicin (BioWhittaker). Cells were
plated at 2 x 105 cells per well for HepG2 in 12 well
cell culture dishes (Costar). The media was replaced
with fresh media 20 hours later. After 4 hours, DNA was
CA 02211713 1997-07-29
Wos6J23884 PCT~S961~1469
added by the calcium phosphate coprecipitation technique
(Berger, T. S., Parandosh, Z., Perry, B., and Stein,
R.B. (1992) J. '3teroid. Biochem. Molec. Biol. 41,
733-738). Typically, 0.1 ~g of expression plasmid, 0.5
~g of the ~-gal expression plasmid pCH110 (internal
control), and 0.5 mg of reporter plasmid were added to
each well.
Where indicated, 0-O.5 ~g of hNUClB plasmid or
hPPARy plasmid lrepressor) was added. Repressor plasmid
dosage was kept constant by the addition of appropriate
amounts of the empty expression vector pBKCMV. Total
amount of DNA was kept at 20 ~g by the addition of pGEM
DNA (Promega).
After 14 hours the cells were washed with lX
PBS and fresh media added (DMEM with 10~ charcoal
stripped fetal bovine serum (Hyclone) plus the above
supplements). I,igands or PPAR activators were added to
the final concentrations indicated. Control cells were
treated with vehicle.
After another 24 hours the cells were
harvested and the luciferase and ~-galactosidase
activities quantified on a Dynatech ML 1000 luminometer
and a Beckman Biomek 1000 workstation respectively. The
normalized response is the luciferase activity of the
extract divided by the ~-galactosidase activity of the
same. Each data point represents the mean of three
transfections. Error bars represent the standard
deviation from the mean. CAT assays were performed as
in Ausbel et al., (1987) in Current Protocols in
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33
Molecu~ar Biolo~y, Wiley Interscience.
Gel Retardation Assay
Gel retardation assays with PPRE sequences
were performed as described in Mukherjee et al., ~SBMB
51:157-166, 199:3, incorporated by reference herein.
hPPARy was translated in vi tro using the T3 coupled
reticulocyte lysate system (Promega). The
baculovirus/Sf2:L cell system was used to express hRXR~
(Allegretto et al., JBC 268:1-9, 1993, incorporated by
reference herein). The sequences of the
oligonucleotides containing PPREs from three genes are
5'-CTAGCGATATCATGACCTTTGTCCTAGGCCTC-3' (acyl coenzyme A
oxidase), 5'-GATCCCTTTGACCTATTGAACTATTACCTACATTA-3'
(hydratase) and 5'-GATCCCCACTGAACCCTTGACCCCTGCCCTGCAGCA-
3' (human ApoAl 'A' site).
COS cells were transfected with 5 ~g ofpCMVhNUClB or pRShRXR~ (Ptashne, Nature 335:683-689,
1988) per 100 mrn dish for 48 hours. Whole cell extracts
were made by four cycles of freeze-thawing in 0.4 M KCl
containing buffer followed by centrifugation. Gel
retardations were performed by incubating 5 ~g of cell
extract in buffer containing 10 mM Hepes (7.8), 50 mM
KCl, 1 mM DTT, .'.5 mM MgCl2, 0.5mg/ml dIdC and 20~
glycerol at 4~C for 5 minutes. About 100,000 cpm of
32P-end-labeled probe was then added and incubated at
25~C for another 5 minutes.
Protein-DNA complexes were resolved by
electrophoresis on 5~ polyacrylamide gels in 0.5X TBE.
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34
The PPRE sequence from the acyl-coenzymeA oxidase (AOX)
gene used as probe is
5'-CTAGCGATATCATGACCTTTGTCCTAGGCCTC-3' (upper strand)
and 5'-CTAGGAGGCCTAGGACAAAGGTCATGATATCG-3' (lower
strand).
IV. cDNA cloni~g of hPPARy and hPPARy2.
The cloning of a hPPARy and hPPARy2 ~rom a
human heart cDNA library is described below. Those o~
ordinary skill in the art will recognize that equivalent
procedures can be readily used to isolate hPPARy or
hPPARy2 from genomic libraries or cDNA libraries of
other tissues.
The recipes for buffers, mediums, and
solutions in the following experiments are given in J.
Sambrook, E. F. Fritsch, and T. Maniatis, Molecular
Cloning: A La~o:ratory Manual, 2 Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New ~ork, 1989.
A human heart cDNA library, Human Heart
5'-STRETCH in A-gtlO, was purchased from Clontech
Laboratories Inc., Palo Alto, California.
A fragment isolated from a mPPARy cDNA clone
(Chen et al., Biochem. Biophy. Res. Com. 196:671-677,
1993) by digestion with EcoRI, was labeled with
[32P]-dCTP by random priming and was utilized to identify
potential hPPAR~y cDNA clones.
Approximately 2X106 phage plaques from the
human heart cDNi~ library were screened with the mPPARy
probe at low st:ringency (35~ formamide, 5 x SSC, 0.1~
CA 022ll7l3 l997-07-29
W 096/23884 PCTnUS96J01469
SDS, 100 ~g/ml fish DNA at 37~C). Positive clones were
isolated and subcloned into pBKCMV (Strategene) or pCRII
(Invitrogen) for sec~encing. The hPPARy clone contains
an open reading frame of 1482 nucleotides (see SEQ. ID
NO. 1). There is an 89~ nucleotide identity (i.e.,
"homology") between the hPPARy clone and the mPPARy
sequence.
hPPARy may start ~rom any of the three
methionines identified in SEQ. ID NO. 2, i.e., Met (1),
Met (18) and Mel (20). The deduced amino acid sequence
of hPPARy predicts a protein of 494, 477 or 475 amino
acids. A compa:rison of the amino acid sequences between
human and mouse show 96~ amino acid sequence identity
(i.e., "homoloc~").
Another positive clone called 3L4 was
isolated. The :insert was isolated by PCR technique
using Clontech amplimers and subcloned into the pCRII
vector (Invitroc3en). Sequencing reactions were
performed with ',P6 and T7 primers. Comparing the
sequence obtained with the SP6 primer with that of
hPPARy indicated that 3L4 is a novel clone and encodes a
novel polypeptide.
This polypeptide is identical to hPPARy except
for an additional 30 amino acids at the N-terminus. 20
of the 30 amino acids are present at the same position
in mouse PPARy2 (Tontonoz et al., ~enes and Development
8:1224-1234, 19'34), indicating that the sequence encoded
by 3L4 corresponds to the human equivalent of mouse
PPARy2. The gene encoded by 3L4 is named hPPARy2.
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36
hPPARy2 may start from any of the three
methionines identified in SEQ. ID NO.4.
Included within the scope of this invention
are the functional equivalents of the herein-described
isolated nucleic acid molecules. The degeneracy of the
genetic code permits substitution of certain codons by
other codons which speci~y the same amino acid and hence
would give rise to the same protein. The nucleic acid
sequence can vary substantially since, with the
exception of methionine and tryptophan, the known amino
acids can be coded for by more than one codon. Thus,
portions or all of the hPPARy or hPPARy2 gene could be
synthesized to give a nucleic acid sec~ence
significantly di:Eferent from that shown in SEQ ID NO: 1
or 3. The encoded amino acid sequence thereof would,
however, be preserved.
In addltion, the nucleic acid sequence may
comprise a nucleotide sequence which results from the
addition, deletion or substitution of at least one nu-
cleotide to the 5'-end and/or the 3'-end of the nucleic
acid formula shown in SEQ ID NO: 1 or 3 or a derivative
thereof. Any nucleotide or polynucleotide may be used
in this regard, provided that its addition, deletion or
substitution does not alter the amino acid sequence of
SEQ ID NO:2 or 4 which is encoded by the nucleotide
sequence. For example, the present invention is
intended to include any nucleic acid sequence resulting
from the addition of ATG as an initiation codon at the
5'-end of the inventive nucleic acid sequence or its
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37
derivative, or ~rom the addition of TTA, TAG or TGA as a
termination codon at the 3'-end of the inventive
nucleotide sequence or its derivative. Moreover, the
nucleic acid mo].ecule of the present invention may, as
necessary, have restriction endonuclease recognition
sites added to its 5'-end and/or 3'-end.
Such functional alterations of a given nucleic
acid sequence af.ford an opportunity to promote secretion
and/or processing of heterologous proteins encoded by
foreign nucleic acid sequences fused thereto. All
variations of the nucleotide sequence of the hPPARy or
hPPARy2 genes a~ld fragments thereof permitted by the
genetic code are, therefore, included in this invention.
Further, it is possible to delete codons or to
substitute one or more codons by codons other than
degenerate codons to produce a structurally modified
polypeptide, but one which has substantially the same
utility or activity of the polypeptide produced by the
unmodified nucleic acid molecule. As recognized in the
art, the two pol.ypeptides are functionally equivalent,
as are the two nucleic acid molecules which give rise to
their production, even though the differences between
the nucleic acid molecules are not related to degeneracy
of the genetic code.
V. Petecting e!xpre~sion of human PPAR subtypes in
tis~ues.
Northern blots of mRNA from various human
tissues were hybridized with human PPAR subtype specific
CA 022ll7l3 l997-07-29
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probes to determine the expression pattern of human PPAR
subtypes.
A hum~an multiple tissue Northern blot
(Clontech Laboratories Inc.) containing 2 ~g of poly-A
plus mRNA isolated ~rom several human tissues was
hybridized with the full length hPPARy cDNA that had
been random prime labeled with [32P]-dCTP. The
hybridization and all washes were conducted under
high-stringency.
The result showed that the three human PPAR
subtypes are expressed dif~erently in di~erent human
tissues. hPPARcx is expressed predominantly in the
liver, kidney, heart and skeletal muscle, with lower
levels in the pancreas, placenta and lung, and
nondetectable in the brain. hNUCl is ubic~uitously
expressed in different tissues, with the highest
expression leve]s in ~he placenta and low levels in the
liver. hPPARy is expressed at the highest levels in the
liver, heart ancl skeletal muscle, with lower levels in
the kidney and pancreas, and nondetectable in the brain,
placenta, or lung.
A nucleic acid probe o~ the present invention
may be used to probe an appropriate chromosomal or cDNA
library by usual hybridization methods to obtain another
nucleic acid molecule of the present invention. A
chromosomal DNA or cDNA library may be prepared from
appropriate cells according to recognized methods in the
art (c~. Molecular Cloning: A Laboratory Manual, second
edition, edited by Sambrook, Fritsch, & Maniatis, Cold
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39
Spring Harbor L.~boratory, 1989).
In the alternative, chemical synthesis is
carried out in order to obtain nucleic acid probes
having nucleotide sequences which correspond to N-
terminal and C-~erminal portions of the amino acid
sequence of the polypeptide of interest. Thus, the
synthesized nuc:Leic acid probes may be used as primers
in a polymerase chain reaction (PCR) carried out in
accordance with recognized PCR techniques, essentially
according to PCR Protocols, A Guide to Methods and
Applications, edited by Michael et al., Academic Press,
1990, utilizing the appropriate chromosomal or cDNA
library to obta:in the ~ragment of the present invention.
One skilled in the art can readily design such
probes based on the sequence disclosed herein using
methods of computer alignment and sequence analysis
known in the art: (cf. Molecular Cloning: A Laboratory
Manual, second edition, edited by Sambrook, Fritsch, &
Maniatis, Cold ',pring Harbor Laboratory, 1989). The
hybridization probes of the present invention can be
labeled by stanclard labeling techniques such as with a
radiolabel, enz~me label, fluorescent label, biotin-
avidin label, chemiluminescence, and the like. After
hybridization, t:he probes may be visualized using known
methods.
The nucleic acid probes of the present
invention inclucle RNA, as well as DNA probes, such
probes being generated using techniques known in the
art. The nucleic acid probe may be immobilized on a
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solid support. Examples of such solid supports include,
but are not limited to, plastics such as polycarbonate,
complex carbohydrates such as agarose and sepharose, and
acrylic resins, such as polyacrylamide and latex beads.
Technic~es for coupling nucleic acid probes to such
solid supports are well known in the art.
The test samples suitable for nucleic acid
probing methods of the present invention include, for
example, cells or nucleic acid extracts of cells, or
biological flui~s. The sample used in the above-
described methods will vary based on the assay format,
the detection method and the nature of the tissues,
cells or extracl_s to be assayed. Methods for preparing
nucleic acid exlracts of cells are well known in the art
and can be read:ily adapted in order to obtain a sample
which is compat:Lble with the method utilized.
One method of detecting the presence of hPPARy
or hPPARy2 nucleic acid in a sample comprises a)
contacting said sample with the above-described nucleic
acid probe, under conditions such that hybridization
occurs, and b) cletecting the presence of said probe
bound to said nucleic acid molecule. One skilled in the
art would select the nucleic acid probe according to
technicrues known in the art as described above. Samples
to be tested include but should not be limited to RNA
samples of human tissue.
A kit for detecting the presence o~ hPPARy or
hPPARy2 nucleic acid in a sample comprises at least one
container means having disposed therein the above-
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41
described nucleic acid probe. The kit may further
comprise other containers comprising one or more of the
following: wash reagents and reagents capable of
detecting the presence of bound nucleic acid probe.
Examples of detection reagents include, but are not
limited to radi31abelled probes, enzymatic labeled
probes (horse radish peroxidase, alkaline phosphatase),
and affinity labeled probes (biotin, avidin, or
steptavidin).
In detail, a compartmentalized kit includes
any kit in whic:h reagents are contained in separate
containers. Such containers include small glass
containers, plastic containers or strips of plastic or
paper. Such containers allow the efficient transfer of
reagents from one compartment to another compartment
such that the samples and reagents are not cross-
contaminated and the agents or solutions of each
container can be added in a quantitative fashion from
one compartment to another. Such containers will
include a conta:iner which will accept the test sample, a
container which contains the probe or primers used in
the assay, containers which contain wash reagents (such
as phosphate buifered saline, Tris-buffers, and the
like), and containers which contain the reagents used to
detect the hybridized probe, bound antibody, amplified
product, or the like. One skilled in the art will
readily recognize that the nucleic acid probes described
in the present invention can readily be incorporated
into one of the established kit formats which are well
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42
known in the ar-t.
VI. Expression of rQcombinant hPPARy or hPPARy2
polypeptide.
Applicant expressed recombinant hPPARy ~n
vitro. One predominant band estimated to be about 50 kd
was observed. This is compatible with translation
initiation at the third ATG codon from the 5'-end
(position 214, SEQ. I.D. No. 1). A lower band is
observed in the in vi tro translated hPPARy polypeptides,
which could be a degraded hPPARy polypeptide or a hPPARy
polypeptide translation from an internal methionine.
Amino acid sequence comparison of hPPARy with
other PPAR subt~pes shows that human PPARy has 96~
identity to mPP,~Ryl and 55~ identity to both hPPAR~ and
hNUC. The closest homology among PPAR subtypes is in
the DNA binding domains, followed by the ligand binding
domains. The N-terminal A/B domain, which in the PPAR
family encodes a transactivation function, is very
different in the three human PPAR subtypes, suggesting
that these human PPAR subtypes may have different
transactivation properties.
The p~-esent invention also relates to a recom-
binant DNA molecule comprising, 5' to 3', a promoter
ef~ective to initiate transcription in a host cell and
the above-described nucleic acid molecules. In
addition, the present invention relates to a recombinant
DNA molecule co~lprising a vector and an above-described
nucleic acid mo}ecules. The present invention also
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43
relates to a nucleic acid molecule comprising a
transcriptional region functional in a cell, a sequence
complimentary to an RNA sequence encoding an amino acid
sequence corresponding to the above-described
polypeptide, and a transcriptional termination region
functional in said cell. The above-described molecules
may be isolated and/or purified DNA molecules.
The present invention also relates to a cell
or organism that contains an above-described nucleic
acid molecule. The peptide may be purified from cells
which have been altered to express the peptide. A cell
is said to be "altered to express a desired peptide"
when the cell, through genetic manipulation, is made to
produce a protein which it normally does not produce or
which the cell normally produces at lower levels. One
skilled in the art can readily adapt procedures for
introducing and expressing either genomic, cDNA, or
synthetic sequences into either eukaryotic or prokary-
otic cells.
A nucleic acid molecule, such as DNA, is said
to be "capable of expressing" a polypeptide if it
contains nucleol_ide sequences which contain
transcriptional and translational regulatory information
and such sequences are "operably linked" to nucleotide
sequences which encode the polypeptide. An operable
linkage is a linkage in which the regulatory DNA
sequences and the DNA sequence sought to be expressed
are connected in such a way as to permit gene sequence
expression. The precise nature of the regulatory
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44
regions needed for gene sequence expression may vary
from organism to organism, but shall in general include
a promoter region which, in prokaryotes, contains both
the promoter (which directs the initiation of RNA
transcription) as well as the DNA sequences which, when
transcribed into RNA, will signal synthesis initiation.
Such regions wi:Ll normally include those 5'-non-coding
sequences involved with initiation of transcription and
translation, suc~h as the TATA box, capping sequence,
CAAT sequence, cmd the like.
If de~,ired, the non-coding region 3' to the
sequence encoding a hPPARy or hPPARy2 gene may be
obtained by the above-described methods. This region
may be retained for its transcriptional termination
regulatory sequences, such as termination and
polyadenylation. Thus, by retaining the 3'-region
naturally contiguous to the DNA sequence encoding a
hPPARy or hPPARy2 gene, the transcriptional termination
signals may be provided. Where the transcriptional
termination signals are not satisfactorily functional in
the expression host cell, then a 3' region functional in
the host cell may be substituted.
Two DNA sequences (such as a promoter region
sequence and a hPPARy or hPPARy2 sequence) are said to
be operably linked if the nature of the linkage between
the two DNA sequences does not (l) result in the
introduction of a frame-shift mutation, (2) interfere
with the ability of the promoter region sequence to
direct the transcription of a hPPARy or hPPARy2 gene
=.
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sequence, or (3) interfere with the ability of a hPPARy
or hPPARy2 gene sequence to be transcribed by the
promoter region secIuence. Thus, a promoter region would
be operably linked to a DNA sequence if the promoter
were capable of effecting transcription of that DNA
sequence. Thus, to express a hPPARy or hPPARy2 gene,
transcriptional and translational signals recognized by
an appropriate host are necessary.
The p:resent invention encompasses the
expression of the hPPARy or hPPARy2 gene (or a
functional deri~ative thereof) in either prokaryotic or
eukaryotic cells. Prokaryotic hosts are, generally,
very efficient iLnd convenient for the production of
recombinant proleins and are, therefore, one type of
preferred expression system for the hPPARy or hPPARy2
gene. Prokaryotes most frequently are represented by
various strains of E. col i . However, other microbial
strains may also be used, including other bacterial
strains.
In prokaryotic systems, plasmid vectors that
contain replicat:ion sites and control sequences derived
from a species c:ompatible with the host may be used.
Examples of suit:able plasmid vectors may include pBR322,
pUCl18, pUCll9 and the like; suitable phage or
bacteriophage vectors may include AgtlO, Agtll and the
like; and suitable virus vectors may include pMAM-neo,
pKRC and the like. Preferably, the selected vector of
the present inve~ntion has the capacity to replicate in
the selected host cell.
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46
Recognized prokaryotic hosts include bacteria
such as E. coil, Bacillus, Streptomyces, Pseudomonas,
Salmonella, Ser:ratia, and the like. However, under such
conditions, the peptide will not be glycosylated. The
prokaryotic hosl must be compatible with the replicon
and control sec~1ences in the expression plasmid.
To express hPPARy or hPPARy2 (or a functional
derivative thereof) in a prokaryotic cell, it is
necessary to operably link the hPPARy or hPPARy2
sequence to a fllnctional prokaryotic promoter. Such
promoters may be either constitutive or, more
preferably, reg~llatable (i.e., inducible or
derepressible). Examples of constitutive promoters
include the int promo~er of bacteriophage ~, the bla
promoter of the ~-lactamase gene sequence of pBR322, and
the CAT promoter of the chloramphenicol acetyl transfer-
ase gene sequence of pPR325, and the like. Examples of
inducible prokaryotic promoters include the major right
and left promoters of bacteriophage A (PL and PR)/ the
trp, recA, lacZ, lacI, and gal promoters of E. coli, the
c~-amylase (Ulmanen et at., J. Bacteriol. 162:176-182,
1985) and the s-28-specific promoters of B. subtilis
(Gilman et at., Gene sequence 32:11-20, 1984), the
promoters of the bacteriophages of Bacillus (Gryczan,
In: The Molecular Biology of the Bacilli, Academic
Press, Inc., NY (1982)), and Streptomyces promoters
(Ward et at., Mcl. Gen. Genet. 203:468-478, 1986).
Prokaryotic pro~oters are reviewed by Glick, J. Ind.
Microbiot. 1:277-282, 1987; Cenatiempo, Biochimie
CA 02211713 1997-07-29
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47
68:505-516, 198~;; and Gottesman, Ann. Rev. Gçnet.
18:415-442, 198'~.
Prope] expression in a prokaryotic cell also
requires the presence of a ribosome binding site up-
stream of the gene sequence-encoding sequence. Such
ribosome bindinc~ sites are disclosed, for example, by
Gold et al. Ann. Rev. Microbiol. 35:365-404, 1981. The
selection of control sequences, expression vectors,
transformation rnethods, and the like, are dependent on
the type of host: cell used to express the gene. As used
herein, "cell", "cell line", and "cell culture" may be
used interchangeably and all such designations include
progeny. Thus, the words "transformants" or
"transformed cells" include the primary subject cell and
cultures derive~ therefrom, without regard to the number
of transfers. :[t is also understood that all progeny
may not be prec:Lsely identical in DNA content, due to
deliberate or inadvertent mutations. However, as
defined, mutant progeny have the same functionality as
that of the originally transformed cell.
Host cells which may be used in the expression
systems of the present invention are not strictly
limited, provided that they are suitable for use in the
expression of the hPPARy or hPPARy2 polypeptide of
interest. Suitable hosts may often include eukaryotic
cells. Preferred eukaryotic hosts include, for example,
- yeast, fungi, insect cells, m~mm~l ian cells either in
vivo, or in tissue culture. Mammalian cells which may
be useful as hosts include HeLa cells, cells of
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48
fibroblast origin such as VER0 or CHO-Kl, or cells of
lymphoid origin and their derivatives. Preferred
m~mm~l ian host cells include SP2/0 and J558L, as well as
neuroblastoma cell lines such as IMR 332 which may
provide better <apacities for correct post-translational
processing.
In addition, plant cells are also available as
hosts, and control sequences compatible with plant cells
are available, such as the cauliflower mosaic virus 35S
lo and l9S, and nopaline synthase promoter and
polyadenylation signal sequences. Another preferred
host is an insect cell, for example the Drosophila
larvae. Using insect cells as hosts, the Drosophila
alcohol dehydrogenase promoter can be used. Rubin,
Science 240:1453-1459, 1988. Alternatively, baculovirus
vectors can be engineered to express large amounts of
hPPARy or hPPARy2 in insects cells (Jasny, Science
238:1653, 1987; Miller et al., In: Genetic Engineering
(1986), Setlow, et al., eds., Plenum, Vol. 8, pp. 277-
297).
Any of a series of yeast gene sequenceexpression systems can be utilized which incorporate
promoter and termination elements from the actively
expressed gene sequences coding for glycolytic enzymes
are produced in :large quantities when yeast are grown in
mediums rich in qlucose. Known glycolytic gene
sequences can also provide very efficient
transcriptional control signals. Yeast provides
substantial advantages in that it can also carry out
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49
post-translational peptide modifications. A number of
recombinant DNA strategies exist which utilize strong
promoter sequences and high copy number of plasmids
which can be utilized for production of the desired
proteins in yeast. Yeast recognizes leader sequences on
cloned m~mm~l ian gene sequence products and secretes
peptides bearin(~ leader sequences (i.e., pre-peptides).
For a m~mm~l ian host, several possible vector systems
are available fc)r the expression of hPPARy or hPPARy2.
A wide variety of transcriptional and
translational regulatory sequences may be employed,
depending upon the nature of the host. The
transcriptional and translational regulatory signals may
be derived from viral sources, such as adenovirus,
bovine papilloma virus, cytomegalovirus, simian virus,
or the like, where the regulatory signals are associated
with a particular gene sequence which has a high level
of expression. Alternatively, promoters from m~mm~l ian
expression products, such as actin, collagen, myosin,
and the like, may be employed. Transcriptional
initiation regulatory signals may be selected which
allow for repression or activation, so that expression
of the gene sequences can be modulated. Of interest are
regulatory signa:Ls which are temperature-sensitive so
that by varying l_he temperature, expression can be
repressed or initiated, or are subject to chemical (such
as metabolite) regulation.
Expression of hPPARy or hPPARy2 in eukaryotic
hosts requires the use of eukaryotic regulatory regions.
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Such regions wi]l, in general, include a promoter region
sufficient to direct the initiation of RNA synthesis.
Preferred eukaryotic promoters include, for example, the
promoter of the mouse metallothionein I gene sequence
(Hamer et al., J. Mol. Appl. Gen. 1:273-288(1982)); the
TK promoter of E~erpes virus (McKnight, Cell 31:355-365
(1982)); the SV40 early promoter (Benoist et al., Nature
290:304-310(1981)); the yeast gal4 gene sequence
promoter (Johnst:on et al., Proc. Natl. Acad. Sci. USA
10 79:6971-6975(19~2); Silver et al., Proc. Natl. Acad.
Sci. USA 81:5951-5955 (1984)).
Trans].ation of eukaryotic mRNA is initiated at
the codon which encodes the first methionine. For this
reason, it is px-eferable to ensure that the linkage
between a eukaryotic promoter and a DNA sequence which
encodes hPPARy or hPPARy2 (or a functional derivative
thereof) does not contain any intervening codons which
are capable of encoding a methionine (i.e., AUG). The
presence of such codons results either in a formation of
a fusion protein (if the AUG codon is in the same
reading frame as the hPPARy or hPPARy2 coding sequence)
or a frame-shift: mutation (if the AUG codon is not in
the same reading frame as the hPPARy or hPPARy2 coding
sequence).
A hPP~y or hPPARy2 nucleic acid molecule and
an operably linked promoter may be introduced into a
recipient prokaryotic or eukaryotic cell either as a
nonreplicating I)NA (or RNA) molecule, which may either
be a linear molecule or, more preferably, a closed
CA 02211713 1997-07-29
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covalent circular molecule. Since such molecules are
incapable of autonomous replication, the expression of
the gene may oc,~ur through the transient expression of
the introduced sequence. Alternatively, permanent
expression may occur through the integration of the
introduced DNA sequence into the host chromosome.
A vec~or may be employed which is capable of
integrating the desired gene sequences into the host
cell chromosome. Cells which have stably integrated the
introduced DNA :into their chromosomes can be selected by
also introducin~ one or more markers which allow for
selection of host cells which contain the expression
vector. The ma:rker may provide for prototrophy to an
auxotrophic host, biocide resistance, e.g., antibiotics,
or heavy metals, such as copper, or the like. The
selectable marker gene sequence can either be directly
linked to the DI~A gene sequences to be expressed, or
introduced into the same cell by co-transfection.
Additional elements may also be needed for optimal
synthesis of single chain binding protein mRNA. These
elements may include splice signals, as well as
transcription promoters, enhancers, and termination
signals. cDNA expression vectors incorporating such
elements include those described by Okayama Molec.
Cell. Biol. 3:280(1983).
The introduced nucleic acid molecule can be
incorporated int:o a plasmid or viral vector capable of
autonomous replication in the recipient host. Any of a
wide variety of vectors may be employed for this
CA 02211713 1997-07-29
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purpose. Factors o~ importance in selecting a
particular plasn~id or viral vector include: the ease
with which recipient cells that contain the vector may
be recognized and selected from those recipient cells
which do not contain the vector; the number of copies of
the vector which are desired in a particular host; and
whether it is desirable to be able to "shuttle" the
vector between host cells of different species.
Preferred prokaryotic vectors include plasmids capable
of replication in E. coli, e.g., pBR322, ~olEl, pSC101,
pACYC 184, ~VX. Such plasmids are, for example,
disclosed in MQlecular Cloning: A Laboratory Manual,
second edition, edited by Sambrook, Fritsch, & Maniatis,
Cold Spring Harbor Laboratory, (1989). Bacillus
plasmids including pC194, pC221, pT127, and the like can
also be used. Such plasmids are disclosed by Gryczan,
In: The Moleculcr Biology of the Bacilli, Academic
Press, NY (1982), pp. 307-329). Other suitable vectors
include Streptomyces plasmids including plJ101 (Kendall
et al., J. sacteriol. 169:4177-4183 (1987)), and
streptomyces bacteriophages such as ~C31 (Chater et al.,
In: Sixth Interrational Symposium on Actinomycetales
Biology, Akademiai Kaido, Budapest, Hungary (1986), pp.
45-54). Pseudomonas plasmids are reviewed by John et
al. Rev. Infect. Dis. 8:693-704, 1986, and Izaki, Jpn.
J. Bacteriol. 33:729-742, 1978.
Preferred eukaryotic plasmids include, for
example, BPV, vaccinia, SV40, 2-micron circle, and the
like, or their derivatives. Such plasmids are well
CA 02211713 1997-07-29
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known in the art: (Botstein et al., Miami Wntr. Symp.
19:265-274(1982~; Broach, In: The Molecular Biology of
the Yeast Sacchclromyçes: Life Cycle and Inheritance,
Cold Spring Harbor Laboratory, t~old Spring Harbor, NY,
p. 445-470 (198~); Broach, Cell 28:203-204 (1982);
Bollon et at., ;r. ctin. HematQl. Oncol. 10:39-48 (1980);
Maniatis, In: Cell Biology: A Comprehensive Treatise,
Vol. 3, Gene Sequence Expression, Academic Press, NY,
pp. 563-608(198t)).
Once t:he vector or nucleic acid molecule
containing the construct(s) has been prepared for
expression, the DNA construct(s) may be introduced into
an appropriate host cell by any of a variety of suitable
means, i.e., transformation, transfection, conjugation,
protoplast fusion, electroporation, particle gun tech-
nology, calcium phosphate-precipitation, direct
microinjection, and the like. After the introduction of
the vector, recipient cells are grown in a selective
medium, which selects for the growth of vector-contain-
ing cells. Expression of the cloned gene molecule(s)results in the production of hPPARy or hPPARy2 or
fragments thereof. This can take place in the
transformed cells as such, or following the induction of
these cells to clifferentiate (for example, by
administration of bromodeoxyuracil to neuroblastoma
cells or the like). A variety of incubation conditions
can be used to form the peptide of the present
invention. The most preferred conditions are those
which mimic physiological conditions.
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54
VII. hPPARy or :hPPARy2 polypeptide~, antibodies and
hybridoma8.
A variety of methodologies known in the art
can be utilized to obtain the peptide of the present
invention. The peptide may be purified from tissues or
cells which naturally produce the peptide.
Alternatively, the above-described isolated nucleic acid
fragments could be used to expressed the hPPARy or
hPPARy2 protein in any organism. The samples of the
present invention include cells, protein extracts or
membrane extracts of cells, or biological fluids. The
sample will vary based on the assay format, the
detection method and the nature of the tissues, cells or
extracts used a's the sample.
Any eukaryotic organism can be used as a
source for the peptide of the invention, as long as the
source organism naturally contains such a peptide. As
used herein, "source organism" refers to the original
organism from which the amino acid sequence of the sub-
unit is derived, regardless of the organism the subunit
is expressed in and ultimately isolated from.
One s~illed in the art can readily follow
known methods for isolating proteins in order to obtain
the peptide free of natural conti~m;ni~nts. These
include, but are not limited to: size-exclusion
chromatography, HPLC, ion-exchange chromatography, and
immuno-affinity chromatography.
The p:resent invention relates to an antibody
having binding affinity to a hPPARy or hPPARy2
CA 02211713 1997-07-29
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polypeptide. The polypeptide may have the amino acid
sequence set forth in SEQ ID NO: 2 or 4, or mutant or
species variation thereof, or at least 9 contiguous
amino acids thereof (preferably, at least 10, 15, 20, or
30 contiguous amino acids thereof).
The present invention also relates to an
antibody having specific binding affinity to a hPPARy or
hPPARy2 polypeptide. Such an antibody may be isolated
by comparing its binding affinity to a hPPARy or hPPARy2
polypeptide with its binding affinity to another
polypeptide. Those which bind selectively to hPPARy or
hPPARy2 would be chosen for use in methods requiring a
distinction between hPPARy or hPPARy2 and other
polypeptides.
The hPPARy or hPPARy2 proteins of the present
invention can be used in a variety of procedures and
methods, such as for the generation of antibodies, for
use in identifying pharmaceutical compositions, and for
studying DNA/protein interaction.
The hPPARy or hPPARy2 peptide of the present
invention can be used to produce antibodies or
hybridomas. One skilled in the art will recognize that
if an antibody is desired, such a peptide would be
generated as described herein and used as an immunogen.
The antibodies o:E the present invention include
monoclonal and polyclonal antibodies, as well fragments
~ of these antibod:ies, and humanized forms. Humanized
forms of the ant:ibodies of the present invention may be
generated using one of the procedures known in the art
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such as chimerization or CDR grafting. The present
invention also :relates to a hybridoma which produces the
above-described monoclonal antibody, or binding fragment
thereof. A hyb:ridoma is an immortalized cell line which
is capable of secreting a specific monoclonal antibody.
In general, techniques for preparing
monoclonal antibodies and hybridomas are well known in
the art (Campbell, Monoclonal Antibody Technology:
Lahoratory Tech]liques in siochemistry and Molecular
Biology, Elsevier Science Publishers, Amsterdam, The
Netherlands (19~34); St. Groth et al., ~. Immunol.
Methods 35:1-21(1980)). Any ~n;m~l (mouse, rabbit, and
the like) which is known to produce antibodies can be
immunized with the selected polypeptide. Methods for
;mmnn;zation are well known in the art. Such methods
include subcutaneous or intraperitoneal injection of the
polypeptide. One skilled in the art will recognize that
the amount of polypeptide used for immunization will
vary based on the animal which is immunized, the
antigenicity of the polypeptide and the site of
injection.
The polypeptide may be modified or
administered in an adjuvant in order to increase the
peptide antigenicity. Methods of increasing the
antigenicity of a polypeptide are well known in the art.
Such procedures include coupling the antigen with a
heterologous protein (such as globulin or ~-
galactosidase) or through the inclusion of an adjuvant
during immunization.
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For monoclonal antibodies, spleen cells from
the immunized animals are removed, fused with myeloma
cells, such as SP2/0-Agl4 myeloma cells, and allowed to
become monoclonal antibody producing hybridoma cells.
Any one of a number of methods well known in the art can
be used to iden~ify the hybridoma cell which produces an
antibody with the desired characteristics. These
include screening the hybridomas with an ELISA assay,
western blot analysis, or radioimmunoassay (Lutz et al.,
Exp. Cell Res. :L75:109-124(1988)). Hybridomas secreting
the desired ant:ibodies are cloned and the class and
subclass is determined using procedures known in the art
(Campbell, Monoclonal Antibody Technology: Laboratory
Techniques in B:iochemistry and Molecular Biology, supra
(1984)).
For polyclonal antibodies, antibody containing
antisera is iso:Lated from the immunized animal and is
screened for the presence of antibodies with the desired
specificity using one of the above-described procedures.
The above-descr:Lbed antibodies may be detectably
labeled. Antibc~dies can be detectably labeled through
the use of radioisotopes, affinity labels (such as
biotin, avidin, and the like), enzymatic labels (such as
horse radish peroxidase, alkaline phosphatase, and the
like) fluorescent labels (such as FITC or rhodamine, and
the like), paramagnetic atoms, and the like. Procedures
for accomplishing such labeling are well-known in the
art, for example, see (Stemberger et al., J. Histochem.
Cytochem. 18:31'i(1970); Bayer et at., Meth. Enzym.
CA 02211713 1997-07-29
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58
6Z:308(1979); E~ngval et al., Immunot. 109:129(1972);
Goding, J. Immunol. Meth. 13:215(1976)). The labeled
antibodies of t:he present invention can be used for in
vi tro, in vivo, and in si tu assays to identify cells or
tissues which express a specific peptide.
The above-described antibodies may also be
immobilized on a solid support. Examples of such solid
supports inclucLe plastics such as polycarbonate, complex
carbohydrates such as agarose and sepharose, acrylic
resins and such as polyacrylamide and latex beads.
Techniques for coupling antibodies to such solid
supports are well known in the art (Weir et al.,
Handbook of Experimental Immunoloay 4th Ed., Blackwell
Scientific Publications, Oxford, England, Chapter
10(1986); Jacoby et al., Meth. Enzym. 34 Academic Press,
N.Y. (1974)). The immobilized antibodies of the present
invention can ke used for in vi tro, in vivo, and in si tu
assays as well as in immunochromotography.
Furthermore, one skilled in the art can
readily adapt currently available procedures, as well as
the techniques, methods and kits disclosed above with
regard to antibodies, to generate peptides capable of
binding to a specific peptide sequence in order to
generate rationally designed antipeptide peptides, for
example see Hurby et al., Application of Synthetic
Peptldes: Antisense Peptides, In Synthetic Peptides, A
User's Guide, W.H. Freeman, NY, pp. 289-307(1992), and
Kaspczak et al., Biochemistry 28:9230-8(1989).
Anti-peptide peptides can be generated by
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replacing the basic amino acid residues ~ound in the
hPPARy or hPPAR~2 peptide se~uence with acidic residues,
while maintaining hydrophobic and uncharged polar
groups. For example, lysine, arginine, and/or histidine
residues are replaced with aspartic acid or glutamic
acid and glutamic acid residues are replaced by lysine,
arginine or histidine.
The present invention encompasses a method of
detecting a hPPARy or hPPARy2 polypeptide in a sample,
comprising: a) contacting the sample with an above--
described antibody, under conditions such that
immunocomplexes ~orm, and b) detecting the presence of
said antibody bound to the polypeptide. In detail, the
methods comprise incubating a test sample with one or
more of the antibodies of the present invention and
assaying whether the antibody binds to the test sample.
Conditions for incubating an antibody with a
test sample vary. Incubation conditions depend on the
format employed in the assay, the detection methods
employed, and the type and nature of the antibody used
in the assay. One skilled in the art will recognize
that any one of the commonly available immunological
assay formats (such as radioimmunoassays, enzyme-linked
immunosorbent assays, diffusion based Ouchterlony, or
rocket immunofluorescent assays) can readily be adapted
to employ the antibodies of the present invention.
Examples of such assays can be found in Chard, An
Introduction to Radioimmunoassay and Related Techniques
Elsevier Science Publishers, Amsterdam, The Netherlands
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(1986); Bullock et al., ~echniques in
Immunocytochemistry, Academic Press, Orlando, F~ Vol.
1(1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, Practice
and Theory of Enzyme Immunoassays: Laboratory Techniques
in Biochemistry and Molecular Biology, Elsevier Science
Publishers, Amsterdam, The Netherlands (1985).
The immunological assay test samples of the
present invention include cells, protein or membrane
extracts of cells, or biological fluids such as blood,
serum, plasma, or urine. The test sample used in the
above-described method will vary based on the assay
format, nature of the detection method and the tissues,
cells or extracts used as the sample to be assayed.
Methods for preparing protein extracts or membrane
extracts of cells are well known in the art and can be
readily be adapted in order to obtain a sample which is
capable with the system utilized.
A kit contains all the necessary reagents to
carry out the previously described methods of detection.
The kit may comprise: i) a first container means
containing an above-described antibody, and ii) second
container means containing a conjugate comprising a
binding partner of the antibody and a label. In another
preferred embodiment, the kit further comprises one or
more other containers comprising one or more of the
following: wash reagents and reagents capable of
detecting the presence of bound antibodies.
Examples of detection reagents include, but
are not limited to, labeled secondary antibodies, or in
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61
the alternative, if the primary antibody is labeled, the
chromophoric, enzymatic, or antibody binding reagents
which are capabLe of reacting with the labeled antibody.
The compartmentalized kit may be as described above for
nucleic acid probe kits. One skilled in the art will
readily recognize that the antibodies described in the
present invention can readily be incorporated into one
of the established kit formats which are well known in
the art.
VIII.Transgenic animal~ and gene therapy.
A variety of methods are available for the
production of transgenic animals associated with this
invention. DNA can be injected into the pronucleus of a
fertilized egg before ~usion of the male and female
pronuclei, or injected into the nucleus of an embryonic
cell (e.g., the nucleus of a two-cell embryo) following
the initiation of cell division (Brinster et al., Proc.
Nat. Acad. Sci. USA 82:4438-4442, 1985). Embryos can be
infected with viruses, especially retroviruses, modified
to carry inorganic-ion receptor nucleotide sequences of
the invention.
Pluripotent stem cells derived from the inner
cell mass of the embryo and stabilized in culture can be
manipulated in culture to incorporate nucleotide
sequences of the invention. A transgenic animal can be
~ produced from such cells through implantation into a
blastocyst that is implanted into a foster mother and
allowed to come to term. Animals suitable for
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62
transgenic experiments can be obtained ~rom standard
commercial sources such as Charles River (Wilmington,
MA), Taconic (Germantown, NY), Harlan Sprague Dawley
(Indianapolis, IN), etc.
The procedures for manipulation of the rodent
embryo and for microinjection of DNA into the pronucleus
of the zygote are well known to those o~ ordinary skill
in the art (Hogan et al., supra). Microin~ection
procedures for fish, amphibian eggs and birds are
detailed in Houdebine and Chourrout, Experientia 47:897-
905 (1991). Other procedures for introduction of DNA
into tissues of animals are described in U.S. Patent
No., 4,945,050 (Sandford et al ., July 30, 1990).
By way of example only, to prepare a
transgenic mouse, female mice are induced to superovu-
late. Females are placed with males, and the mated
females are sacrificed by CO2 asphyxiation or cervical
dislocation and embryos are recovered from excised
oviducts. Surrounding cumulus cells are removed.
Pronuclear embryos are then washed and stored until the
time of injection. Randomly cycling adult female mice
are paired with vasectomized males. Recipient females
are mated at the same time as donor females. Embryos
then are transferred surgically. The procedure for
generating transgenic rats is similar to that of mice.
See ~mm~ et aL., Cell 63:1099-1112, 1990.
Methods for the culturing of embryonic stem
(ES) cells and the subsequent production of transgenic
animals by the introduction of DNA into ES cells using
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63
methods such as electroporation, calcium phosphate/DNA
precipitation and direct injection also are well known
to those of ordinary skill in the art. See, for
example, Teratoc-arcinomas and Embryonic Stem Cells A
Practical Approach, E.J. Robertson, ed., IRL Press
(1987).
In cac,es involving random gene integration, a
clone containin~ the sequence(s) of the invention is co-
transfected with a gene encoding resistance.
Alternatively, the gene encoding neomycin resistance is
physically linke!d to the sequence(s) of the invention.
Transfection ancL isolation of desired clones are carried
out by any one of several methods well known to those of
ordinary skill in the art (E.J. Robertson, supra).
DNA mc,lecules introduced into ES cells can
also be integrated into the chromosome through the pro-
cess of homologous recombination. Capecchi, science
244:1288-1292 (1989). Methods for positive selection of
the recombination event (i.e., neo resistance) and dual
positive-negative selection (i.e., neo resistance and
gancyclovir resistance) and the subsequent
identification of the desired clones by PCR have been
described by Capecchi, supra and Joyner et al., Nature
338:153-156 (1989), the teachings of which are
incorporated herein. The final phase of the procedure
is to inject targeted ES cells into blastocysts and to
transfer the blastocysts into pseudopregnant females.
The resulting chimeric animals are bred and the
offspring are analyzed by Southern blotting to identify
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64
individuals that carry the transgene. Procedures for
the production of non-rodent m~mm~l s and other animals
have been discussed by others. See Houdebine and
Chourrout, suprai Pursel et al., Science 244:1281-1288
(1989); and Simms et al., Bio/Technology 6:179-183
(1988).
hPPARy or hPPARy2 and its genetic sequences
will be useful in gene therapy (reviewed in Miller,
Nature 357:455-460, (1992). Miller states that advances
have resulted in practical approaches to human gene
therapy that have demonstrated positive initial results.
An in vivo model of gene therapy for human severe
combined immunodeficiency is described in Ferrari, et
al., Science 251:1363-1366, (1991). The basic science
of gene therapy is described in Mulligan, Science
260:926-931, (1993).
In one preferred embodiment, an expression
vector containing the hPPARy or hPPARy2 coding sequence
is inserted into cells, the cells are grown in vi tro and
then infused in large numbers into patients. In another
preferred embodiment, a DNA segment containing a
promoter of choice (for example a strong promoter) is
transferred into cells containing an endogenous hPPARy
or hPPARy2 in such a manner that the promoter segment
enhances expression of the endogenous hPPARy or hPPARy2
gene (for example, the promoter segment is transferred
to the cell such that it becomes directly linked to the
endogenous hPPARy or hPPARy2 gene).
The gene therapy may involve the use of an
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adenovirus containing hPPARy or hPPARy2 cDNA targeted to
a tumor, systemic hPPARy or hPPARy2 increase by
implantation of engineered cells, injection with virus
encoding hPPARy or hPPARy2, or injection of naked hPPARy
or hPPARy2 DNA into appropriate tissues.
Target cell populations (ç.a., hematopoietic
or nerve cells) may be modified by introducing altered
forms of hPPARy or hPPARy2 in order to modulate the
activity of such cells.
Expression vectors derived from viruses such
as retroviruses, vaccinia virus, adenovirus, adeno-asso-
ciated virus, herpes viruses, several RNA viruses, or
bovine papilloma virus, may be used for delivery o~
nucleotide sequences (e.q., cDNA) encoding recombinant
hPPARy or hPPARy2 protein into the targeted cell
population (~5L, tumor cells). Methods which are well
known to those skilled in the art can be used to
construct recombinant viral vectors containing coding
sequences. See, for example, the techniques described
in Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, N.Y. (1989), and
in Ausubel et al., Current Protocols in Molecular
Biology, Greene Publishing Associates and Wiley
Interscience, N.Y. (1989). Alternatively, recombinant
nucleic acid molecules encoding protein sequences can be
used as naked D~IA or in reconstituted system e.g.,
liposomes or other lipid systems for delivery to target
cells (See e.q., Felgner et al., Nature 337:387-8,
1989). Several other methods for the direct transfer of
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66
plasmid DNA into cells exist for use in human gene
therapy and involve targeting the DNA to receptors on
cells by complexing the plasmid DNA to proteins. See,
Miller, supra.
In its simplest form, gene transfer can be
performed by simply injecting minute amounts of DNA into
the nucleus of a cell, through a process of
microinjection. Capecchi MR, Cell 22:479-88 (1980).
Once recombinant genes are introduced into a cell, they
can be recognized by the cell's normal mechanisms for
transcription and translation, and a gene product will
be expressed. Other methods have also been attempted
for introducing DNA into larger numbers of cells. These
methods include: transfection, wherein DNA is
precipitated with CaPO4 and taken into cells by
pinocytosis (Chen C. and Okayama H, Mol. Cell Biol.
7:2745-52 (1987)!; electroporation, wherein cells are
exposed to large voltage pulses to introduce holes into
the membrane (Chu G. et al., Nucleic Acids Res.,
15:1311-26 (1987~); lipofection/liposome ~usion, wherein
DNA is packaged :into lipophilic vesicles which fuse with
a target cell (Felgner PL., et al., Proc. Natl. Acad.
Sci. USA. 84:74:L3-7 (1987)); and particle bombardment
using DNA bound to small projectiles (Yang NS. et al.,
Proc. Natl. Acad. Sci. USA 87:9568-72 (1990)). Another
method for introducing DNA into cells is to couple the
DNA to chemicall~ modified proteins.
It has also been shown that adenovirus
proteins are capable of destabilizing endosomes and
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67
enhancing the uptake of DNA into cells. The admixture
of adenovirus to solutions containing DNA complexes, or
the binding of ~NA to polylysine covalently attached to
adenovirus using protein crosslinking agents
substantially improves the uptake and expression of the
recombinant gene. Curiel DT et al., Am. J. Respir.
Cell. Mol. Biol., 6:247-52 (1992).
As used herein "gene transfer" means the
process of introducing a foreign nucleic acid molecule
into a cell. Gene transfer is commonly performed to
enable the expression of a particular product encoded by
the gene. The product may include a protein,
polypeptide, anti-sense DNA or RNA, or enzymatically
active RNA. Gene transfer can be performed in cultured
cells or by direct administration into animals.
Generally gene transfer involves the process of nucleic
acid contact with a target cell by non-specific or
receptor mediated interactions, uptake of nucleic acid
into the cell through the membrane or by endocytosis,
and release of nucleic acid into the cytoplasm from the
plasma membrane or endosome. Expression may require, in
addition, movement of the nucleic acid into the nucleus
of the cell and binding to appropriate nuclear factors
for transcription.
As used herein "gene therapy" is a form of
gene transfer and is included within the definition of
gene transfer as used herein and specifically refers to
gene transfer tc express a therapeutic product from a
cell in vivo or in vitro. Gene transfer can be
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68
performed ex vivo on cells which are then transplanted
into a patient, or can be performed by direct
administration of the nucleic acid or nucleic acid-
protein complex into the patient.
In another preferred embodiment, a vector
having nucleic acid sequences encoding hPPARy or hPPARy2
is provided in which the nucleic acid sequence is
expressed only in specific tissue. Methods of achieving
tissue-specific gene expression as set forth in
10 International Publication No. WO 93/09236, filed
November 3, 1992 and published May 13, 1993.
In all of the preceding vectors set forth
above, a further aspect of the invention is that the
nucleic acid sequence contained in the vector may
include additions, deletions or modifications to some or
all of the sequence of the nucleic acid, as defined
above.
In another preferred embodiment, a method of
gene replacement is set forth. "Gene replacement" as
used herein means supplying a nucleic acid sequence
which is capable of being expressed in vivo in an animal
and thereby providing or augmenting the function of an
endogenous gene which is missing or defective in the
anlmal.
IX. Isolation of Agonists and Antagonists of
hPPARy or hPPARy2.
The present invention also relates to a method
of detecting an agonist or antagonist of hPPARy or
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69
hPPARy2 polypeptide comprising incubating cells that
produce hPPARy or hPPARy2 polypeptide in the presence of
a compound and detecting changes in the level of hPPARy
or hPPARy2 activity. Standard techniques can be u~ed,
including, but not limited to, what is described in
Evans et al., U.S. Patent 5,071,773, Beaumont et al.,
U.S. Patent 5,2~4,372, and PCT applications
PCT/US94/03795 (publication no. WO 94/23068) and
PCT/US95/08328, incorporated by reference herein.
Various compounds were tested for their
ability to transactivate hPPARy (Figure 1). ~Y-171,883
and gemfibrozil showed marginal activation o~ hPPARy
above that seen in control cells. ETYA or 9-cis
retinoic acid showed the same fold activation as in
control transfections. Thus the response of hPPARy to
LY-171,883 and ETYA is different from that seen with
mPPARy, which is transcriptionally activated by these
compounds (Tontonoz et al., Genes and Devel. 8:1224-
1234, 1994; Tont:onoz et al., Cell 79:1147-1156, 1994;
and Kliewer et al., PNAS 91:7355-7359).
To inc:rease the level o~ hPPARy protein
synthesis, Applicant cleleted a region containing the two
inframe upstream ATG codons since these are absent in
mouse PPARy. pCMVhPPARy was digested with NcoI, blunt
ended with Klenow, and digested again with KpnI. The
insert was isolated and directionally cloned into pBKCMV
plasmid, which was digested with XbaI (blunt ended with
Klenow) and Kpn]:. In the ensuing plasmid pCMVhPPARy3,
the translation initiation codon is within the context
CA 02211713 1997-07-29
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of a stronger Kozak translation initiation sec~uence.
A cotransfection assay was performed in CV-l
cells with the pPREA3-tk-~UC and l ~M thiazolidinedione
(BRL 49 653 seeC Ibrahimi et al. Molecular Pharmacoloc~y
46:1070-1076 l'394). Thiazolidinedione is an insulin
sensitizer and has potential use in the treatment of
non-insulin dependant diabetes mellitus.
Thiazolidinedione activated hPPARy (Figure 4).
In cells transfected with pCMVhPPARy3 25 fold induction
was observed in the presence of the compound while only
7 fold activation was seen in cells transfected with the
empty expression vector.
The present invention also encompasses a
method of agonizing (stimulating) or antagonizing hPPARy
or hPPARy2 assoc~iated activity in a m~mm~ 1 comprising
administering to said mammal an agonist or antagonist to
hPPARy or hPPARy2 in an amount sufficient to effect said
agonism or antagonism.
X. Screening ior hPPARy Inhibitors.
Co-trcmsfection assay shows that hPPARy
polypeptides repress the activity of hPPARcx. Applicant
uses the follow-lng screening method to identify
compounds that clerepress the activity of hPPARcx.
hPPARcx is activated in the presence of
gemfibrozil. When hNUCl or hPPARy is contransfected
into cells along with hPPARcx a dose dependant
repression was observed (Fig. 2). Repression of hPPARcx
with hNUCl is st:ronger than with hPPARy. No repression
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with O.l ~g of hPPARy was observed while repression with
o.1 ~g o~ hNUCl was clearly seen. However, repression
with 0.4 ~g of hPPARy was observed. Using equal amounts
of transfected receptor, higher levels oi~ repression was
observed with h~Cl compared to hPPARy.
hPPARy and hNUCl repress hPPAR~ transcription
by sequestering RXR. The repression o~ hPPAR~
activity by 0.4 ~g of hPPARy (Fig. 3A) or O.l ~g of
hNUCl (Fig. 3B) was overcome by cotransfecting 0.4 ~g of
an RXR~ expression plasmid. Repression by hPPARy was
completely overcome. However, relief of repression was
intermediate in the case of hNUCl. This suggests that
hNUCl is a stronger repressor than hPPARy. The mere
presence of excess RXR in the cell is sufficient to
relieve repression.
Compo~mds were dissolved in ethanol (ETYA, LY-
171,883 and gemiibrozil) or DMS0 (9-cis-retinoic acid).
Control cells received an equivalent amount of vehicle.
In the repression assays repressor plasmid dosage was
kept constant b~ adding the appropriate amount oi- the
empty expression vector pBKCMV.
Applicant has determined that hPPARy is a
specific repres~30r of the transcriptional activation
effected by PPAR~. The repressive action of hPPARy on
PPAR~ may limit the clinical efficacy of PPAR~
activators (e.g., fibrates, synthroid). Agents that
relieve this repression will increase activity of PPAR~
increase the ef:Eicacy of existing drugs, or render these
drugs unnecessa:ry.
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Applicant has demonstrated co-operative
binding of hPP~Ry and RXR~ to a PPAR response element,
PPRE. Without being bound by any particular theory,
applicant proposes that hPPARy polypeptides repress
PPAR~ by sequestering RXR or competing for DNA binding.
Screening for hPPARy Inhibitors with Co-transfection
Assay
In order to screen for agents that relieve the
repression PPAR(~ activity by hPPARy, PPAR~ and hPPARy
expressing plastnids will be contransfected into CV-1 (a
monkey kidney cell line) or HepG2 (a human liver cell
line) cells along with a reporter containing PPAR
binding elements (such as PPREs) in the presence of a
PPAR activator (e.g., clofibiric acid, WY-14,643) or a
TR activator (e.g., LT3).
Clofibric acid or LT3 normally activate their
respective receptors and will therefore give a strong
signal. In the presence of hPPARy the signal will be
very weak because of repression of these receptors by
hNUClB. We will add compounds to the transfected cells
at various concentrations and select those that relieve
the repression by hPPARy.
The above screening strategy will also be
followed in a yeast based assay with appropriate vectors
and reporters.
Screening for hE'PARy Inhibitors by Gel Retardation Assay
Gel re~tardation assays showed that hPPARy
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binds to a PPAR element, PPRE, with hRXR~.
Gel shift assays performed with in vitro
translated hPPA~y pol~peptides and recombinant
baculovirus expressed RXR~ polypeptides showed that
hPPARy binds to PPREs as a heterodimer with RXR~.
hPPARy alone did not form a complex with
oligonucleotides containing PPRE sec~Lences from the Acyl
CoenzymeA oxida~e (Mukherjee et al., JSBMB 51:157-166,
1994), bifunctional enzyme (Zhang et al., JBC 268:12939-
12945, 1993) or the A site of the human ApoA1 genepromoters. However, a strong retarded complex was
formed when both hPPARy and RXR~ were present with oligo
containing PPRE sequences. No retarded complex was
observed with RXR~ alone. Retarded complexes were also
observed when hPPARy was mixed with mRXR~ or mRXRy.
XI. Pharmaceuti.cal Formulations and Modes of
~;strat:ion.
The particular compound or antibody that
affects the disorder of interest can be administered to
a patient either by themselves, or in pharmaceutical
compositions where it is mixed with suitable carriers or
excipient(s). In treating a patient exhibiting a
disorder of interest, a therapeutically effective amount
of a agent or aqents such as these is administered. A
therapeutically effective dose refers to that amount of
the compound that results in amelioration of symptoms or
a prolongation of survival in a patient.
Toxicity and therapeutic efficacy of such
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74
compounds can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals,
e.g., for determining the LDso (the dose lethal to 50~ of
the population) and the ED50 (the dose therapeutically
effective in 50~ o~ the population). The dose ratio
between toxic and therapeutic effects is the therapeutic
index and it can be expressed as the ratio ~D~o/ED50.
Compounds which exhibit large therapeutic indices are
preferred. The data obtained from these cell culture
assays and animal studies can be used in formulating a
range of dosage for use in human. The dosage of such
compounds lies preferably within a range of circulating
concentrations that include the ED50 with little or no
toxicity. The aosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized.
For any compound used in the method of the
invention, the therapeutically effective dose can be
estimated initially from cell culture assays. For
example, a dose can be formulated in animal models to
achieve a circulating plasma concentration range that
includes the IC5(, as determined in cell culture (i.e.,
the concentration of the test compound which achieves a
half-maximal disruption of the protein complex, or a
half-maximal inhibition of the cellular level and/or
activity of a complex component). Such information can
be used to more accurately determine useful doses in
humans. Levels in plasma may be measured, for example,
by HPLC.
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The exact formulation, route of administration
and dosage can be chosen by the individual physician in
view of the patient's condition. (See e . a . Fingl
et al., in The Pharmacoloaical Basis of Therapeutics,
1975, Ch. l p. l). It should be noted that the
attending physician would know how to and when to
terminate, interrupt, or adjust administration due to
toxicity, or to organ dysfunctions. Conversely, the
attending physician would also know to adjust treatment
to higher levels if the clinical response were not
adequate (precluding toxicity). The magnitude of an
administrated dose in the management of the oncogenic
disorder of interest will vary with the severity of the
condition to be treated and to the route of
administration. The severity of the condition may, for
example, be evaluated, in part, by standard prognostic
evaluation methods. Further, the dose and perhaps dose
frequency, will also vary according to the age, body
weight, and response of the individual patient. A
program comparable to that discussed above may be used
in veterinary m.edicine.
Depending on the specific conditions being
treated, such agents may be formulated and administered
systemically or locally. Techniques for formulation and
administration may be found in Remington's
Pharmaceutical Sciences, 18th ed., Mack Publishing Co.,
Easton, PA (l990). Suitable routes may include oral,
rectal, transdermal, vaginal, transmucosal, or
intestinal administration; parenteral delivery,
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including intranluscular, subcutaneous, intramedullary
injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal,
intranasal, or intraocular injections, just to name a
few.
For irljection, the agents of the invention may
be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks's
solution, Ringer's solution, or physiological saline
buffer. For suc:h transmucosal administration,
penetrants appropriate to the barrier to be permeated
are used in the formulation. Such penetrants are
generally known in the art.
Use of pharmaceutically acceptable carriers to
formulate the compounds herein disclosed for the
practice of the invention into dosages suitable for
systemic administration is within the scope of the
invention. With proper choice of carrier and suitable
manufacturing practice, the compositions of the present
invention, in particular, those formulated as solutions,
may be administered parenterally, such as by intravenous
injection. The compounds can be formulated readily
using pharmaceutically acceptable carriers well known in
the art into dosages suitable for oral administration.
Such carriers enable the compounds of the invention to
be formulated as tablets, pills, capsules, liquids r
gels, syrups, slurries, suspensions and the like, for
oral ingestion by a patient to be treated.
Agents intended to be administered
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intracellularly may be administered using techniques
well known to those of ordinary skill in the art. For
example, such ac~ents may be encapsulated into liposomes,
then administered as described above. Liposomes are
spherical lipid bilayers with aqueous interiors. All
molecules present in an aqueous solution at the time of
liposome formati.on are incorporated into the aqueous
interior. The ]iposomal contents are both protected
from the external microenvironment and, because
liposomes fuse with cell membranes, are e~ficiently
delivered into the cell cytoplasm. Additionally, due to
their hydrophobicity, small organic molecules may be
directly adminictered intracellularly.
Pharmaceutical compositions suitable for use
in the present invention include compositions wherein
the active ingredients are contained in an effective
amount to achieve its intended purpose. Determination
of the effective amounts is well within the capability
of those skilled in the art, especially in light of the
detailed disclosure provided herein. In addition to the
active ingredients, these pharmaceutical compositions
may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries which
facilitate processing of the active compounds into
preparations which can be used pharmaceutically. The
preparations ~ormulated for oral administration may be
in the form of tablets, dragees, capsules, or solutions.
The pharmaceutical compositions of the present invention
may be manufactured in a manner that is itself known,
CA 02211713 1997-07-29
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78
e.g., by means of conventional mixlng, dissolving,
granulating, dragee-making, levitating, emulsifying,
encapsulating, entrapping or lyophilizing processes.
Pharmaceutical formulations for parenteral
administration include aqueous solutions of the active
compounds in water-soluble form. Additionally,
suspensions of the active compounds may be prepared as
appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such
as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposo~es. Aqueous
injection suspensions may contain substances which
increase the viscosity of the suspension, such as sodium
carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable
stabilizers or agents whi~h increase the solubility of
the compounds to allow for the preparation of highly
concentrated solutions.
Pharmaceutical preparations for oral use can
be obtained by combining the active compounds with solid
excipient, optionally grinding a resulting mixture, and
processing the mixture of granules, after adding
suitable auxiliaries, if desired, to obtain tablets or
dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose,
mannitol, or sorbitol; cellulose preparations such as,
for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium
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carboxymethylce:Llulose, and/or polyvinylpyrrolidone
(PvP). If desired, disintegrating agents may be added,
such as the cross-linked polyvinyl pyrrolidone, agar, or
alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable
coatings. For l:his purpose, concentrated sugar
solutions may be used, which may optionally contain gum
arabic, talc, polyvinyl pyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent
mixtures. Dyest:uffs or pigments may be added to the
tablets or dragee coatings for identification or to
characterize different combinations of active compound
doses.
Pharmaceutical preparations which can be used
orally include push-fit capsules made of gelatin, as
well as soft, sealed capsules made of gelatin and a
plasticizer, such as glycerol or sorbitol. The push-fit
capsules can contain the active ingredients in admixture
with filler SUC]l as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate
and, optionally, stabilizers. In soft capsules, the
active compounds may be dissolved or suspended in
suitable liquids" such as fatty oils, liquid paraffin,
or liquid polyel_hylene glycols. In addition,
stabilizers may be added.
Some methods of delivery that may be used
include:
a. encapsulation in liposomes,
CA 02211713 1997-07-29
wos6~23ss4 PCT~S96~1469
b. t;ransduction by retroviral vectors,
c. localization to nuclear compartment
utilizing nuclear targeting site found on
nnost nuclear proteins,
d. t:ransfection of cells ex vivo with
c,ubsequent reimplantation or
administration of the trans~ected cells,
e. a DNA transporter system.
A hPPZ~y or hPPARy2 nucleic acid sequence may
be administered utili~ing an ex vivo approach whereby
cells are removed from an animal, transduced with the
hPPARy or hPPAR~2 nucleic acid sequence and reimplanted
into the animal The liver can be accessed by an ex
vivo approach bv removing hepatocytes from an animal,
transducing the hepatocytes in vitro with the hPPARy or
hPPARy2 nucleic acid sequence and reimplanting them into
the animal ( e . g ., as described for rabbits by Chowdhury
et al, Science .'54: 1802-1805, 1991, or in humans by
Wilson, Hum. Gene Ther. 3: 179-222, 1992) incorporated
herein by reference.
Many nonviral technigues for the delivery of a
hPPARy or hPP~R~2 nucleic acid sequence into a cell can
be used, includ:Lng direct naked DNA uptake (e.g., Wolff
et al., Science 247: 1465-1468, 1990), receptor-mediated
DNA uptake, e.g., using DNA coupled to asialoorosomucoid
which is taken up by the asialoglycoprotein receptor in
the liver (Wu and Wu, J. Biol. Chem. 262: 4429-4432,
1987; Wu et al., J. Biol. Chem. 266: 14338-14342, 1991),
and liposome-mediated delivery ( e . g., Kaneda et al.,
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~xpt. Cell Res. 173: 56-69, 1987; Kaneda et al., Science
243: 375-378, 1989; Zhu et al., Science 261: 209-211,
1993). Many o~ these physical methods can be combined
with one another and with viral technic~ues; enhancement
of receptor-med:Lated DNA uptake can be effected, for
example, by co~ining its use with adenovirus (Curiel et
al., Proc. Natl Acad. Sci. USA 88: 8850-8854, 1991;
Cristiano et al., Proc. Na~l. Acad. Sci. USA 90: 2122-
2126, 1993).
The h]?PARy or hPPARy2 polypeptides or nucleic
acid encoding h]?PARy or hPPARy2 polypeptides may also be
administered via an implanted device that provides a
support for gro~ing cells. Thus, the cells may remain
in the implantec~ device and still provide the useful and
therapeutic agents of the present invention.
All publications referenced are incorporated by
reference herein, including the nucleic acid sequences
and amino acid sequences listed in each publication.
Other embodiments are within the following
claims.
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SEOUENCE LI STING
(1) GENERAL INFC)RMATION:
(i) APPLICANT: Mukherjee, Ranjan
(ii) TITLE OF INVENTION: Human Peroxisome
Proliferator Activat-
ed Receptors
(iii) NUMBER OE~' SEQUENCES: 4
(iv) coRREspoNr)ENcE ADDRESS:
(A) ADDRESSEE: Lyon ~ Lyon
(B) STREET: 633 West Fifth Street
(C) CITY: Los Angeles
(D) STATE: California
(E) COUNTRY: USA
(F) 7.IP: 90071
2 O (V) COM~U'1'~K READABLE FORM:
(A) MEDIUM TYPE~: 3.5ll Diskette, 1.44 Mb
(B) COM~ U'l'~;K: IBM compatible
(C) OPERATING ',YSTEM: Microsoft Windows 3.1
(D) SOFTWARE: WordPerfect (Version 6.1)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Warburg, Richard J.
(B) REGISTRATION NUMBER: 32,327
(C) REFERENCE/DOCKET NUMBER: 210/100 PCT
(ix) TELECOMMUN]:CATION INFORMATION:
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83
(A) TELEPHONB: (213) 489-1600
~ (B) TELEFAX: (213) 955-0440
(C) TELEX: 67-3510
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1936
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION : SEQ ID NO: 1:
GAATTCCGGA CCCTCAACAC CACTCCCTCT TAGCCAATAT TGTGCCTATT 50
GCCATACTAG TCTTTGCGCC TGCGAAGCAG CGGTGGCCTA GCCCTACTAG 100
TCTCAATCTC CAACl~TATAT CGGCCTAGAC TACGTACATA ACCTA~ACCT 150
ACTCCAATGC TAAA~CTAAT CGTCCCTTTT CTCAAACGAG AGTCAGCCTT 200
TAACGAAATG ACCA5.~GGTTG ACACAGAGAT GCCATTCTGG CCCACCAACT 250
TTGGGATCAG CTCCGTGGAT CTCTCCGTAA TGGAAGACCA CTCCCACTCC 300
TTTGATATCA AGCCCTTCAC TACTGTTGAC TTCTCCAGCA TTTCTACTCC 350
ACATTACGAA GACATTCCAT TCACAAGAAC AGATCCAGTG GTTGCAGATT 400
ACAAGTATGA CCTGAAACTT CAAGAGTACC A~AGTGCAAT CA~AGTGGAG 450
CCTGCATCTC CACCTTATTA TTCTGAGAAG ACTCAGCTCT ACAATAAGCC 500
TCATGAAGAG CCTT(_CAACT CCCTCATGGC AATTGAATGT CGTGTCTGTG 550
GAGATAAAGC TTCT(,GATTT CACTATGGAG TTCATGCTTG TGAAGGATGC 600
AAGGGTTTCT TCCGGAGAAC AATCAGATTG AAGCTTATCT ATGACAGATG 650
TGATCTTAAC TGTC(.GATCC ACA~AAAAAG TAGAAATAAA TGTCAGTACT 700
GTCGGTTTCA GAAATGCCTT GCAGTGGGGA TGTCTCATAA TGCCATCAGG 750
TTTGGGCGGA TGCC~CAGGC CGAGAAGGAG AAGCTGTTGG CGGAGATCTC 800
CAGTGATATC GACC~GCTGA ATCCAGAGTC CGCTGACCTC CGGGCCCTGG 850
CAAAACATTT GTATGACTCA TACATAAAGT CCTTCCCGCT GACCA~AGCA 900
AAGGCGAGGG CGAT~TTGAC AGGAAAGACA ACAGACAAAT CACCATTCGT 950
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TATCTATGAC ATGAi~TTCCT TAATGATGGG AGAAGATAAA ATCAAGTTCA1000
AACACATCAC CCCCGTGCAG GAGCAGAGCA AAGAGGTGGC CATCCGCATC1050
TTTCAGGGCT GCCAGTTTCG CTCCGTGGAG GCTGTGCAGG AGATCACAGA1100
GTATGCCAAA AGCATTCCTG GTTTTGTAAA TCTTGACTTG AACGACCAAG1150
TAACTCTCCT CAAA'rATGGA GTCCACGAGA TCATTTACAC AATGCTGGCC1200
TCCTTGATGA ATAAZ~GATGG GGTTCTCATA TCCGAGGGCC AAGGCTTCAT1250
GACAAGGGAG TTTCTAAAGA GCCTGCGAAA GCCTTTTGGT GACTTTATGG1300
AGCCCAAGTT TGAGTTTGCT GTGAAGTTCA ATGCACTGGA ATTAGATGAC1350
AGCGACTTGG CAAT1~TTTAT TGCTGTCATT ATTCTCAGTG GAGACCGCCC1400
AGGTTTGCTG AATGTGAAGC CCATTGAAGA CATTCAAGAC AACCTGCTAC1450
AAGCCCTGGA GCTCCAGCTG AAGCTGAACC ACCCTGAGTC CTCACAGCTG1500
TTTGCCAAGC TGCTCCAGAA AATGACAGAC CTCAGACAGA TTGTCACGGA1550
ACACGTGCAG CTACTGCAGG TGATCAAGAA GACGGAGACA GACATGAGTC1600
TTCACCCGCT CCTGCAGGAG ATCTACAAGG ACTTGTACTA GCAGAGAGTC1650
CTGAGCCACT GCCA~CATTT CCCTTCTTCC AGTTGCACTA TTCTGAGCCG1700
GAATTCTTTT G~11L11ACC CTGGAAGAAA TACTCATAAA AGCCGAATTC1750
CAGCACACTG GCGGCCGTTA CTAGTGGATC CGAGCTCGGT ACCAAGCTTG1800
ATGCATAGCT TGAGTATCTA TAGTGTCACC TAAATAGCTT GGCGTAATCA1850
TGGTCATAGC TGTTl'CCTGT GTGAAATTGT TATCCGCTCA CAATTCCACA1900
20 CAACATACGA GCCGGAAGCA TAAGTGTAAA GCCTGG 1936
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 494
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLEC'ULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION : SEQ ID NO: 2
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Met Leu Lys Leu Ile Val Pro Phe Leu Lys Arg Glu Ser Ala Phe
Asn Glu Met Thr Met Val Asp Thr Glu Met Pro Phe Trp Pro Thr
Asn Phe Gly Ile Ser Ser Val Asp Leu Ser Val Met Glu Asp His
Ser His Ser Phe Asp Ile Lys Pro Phe Thr Thr Val Asp Phe Ser
Ser Ile Ser Thr Pro His Tyr Glu Asp Ile Pro Phe Thr Arg Thr
1065 70 75
Asp Pro Val Val Ala Asp Tyr Lys Tyr Asp Leu Lys Leu Gln Glu
80 85 90
Tyr Gln Ser Ala Ile Lys Val Glu Pro Ala Ser Pro Pro Tyr Tyr
95 100 105
15Ser Glu Lys Thr Gln Leu Tyr Asn Lys Pro His Glu Glu Pro Ser
110 115 120
Asn Ser Leu Met Ala Ile Glu Cys Arg Val Cys Gly Asp Lys Ala
125 130 135
Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly
20140 145 150
Phe Phe Arg Arg Thr Ile Arg Leu Lys Leu Ile Tyr Asp Arg Cys
155 160 165
Asp Leu Asn Cys Arg Ile His Lys Lys Ser Arg Asn Lys Cys Gln
170 175 180
25 Tyr Cys Arg Phe Gln Lys Cys Leu Ala Val Gly Met Ser His Asn
185 190 195
- Ala Ile Arg Phe Gly Arg Met Pro Gln Ala Glu Lys Glu Lys Leu
200 205 210
30Leu Ala Glu Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser
215 220 225
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Ala Asp Leu Arg Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile
230 235 240
Lys Ser Phe Pro Leu Thr Lys Ala Lys Ala Arg Ala Ile Leu Thr
245 250 255
Gly Lys Thr Thr Asp Lys Ser Pro Phe Val Ile Tyr Asp Met Asn
260 265 270
Ser Leu Met Met Gly Glu Asp Lys Ile Lys Phe Lys His Ile Thr
275 280 285
Pro Leu Gln Glu Gln Ser Lys Glu Val Ala Ile Arg Ile Phe Gln
10290 295 300
Gly Cys Gln Phe Arg Ser Val Glu Ala Val Gln Glu Ile Thr Glu
305 310 315
Tyr Ala Lys Ser Ile Pro Gly Phe Val Asn Leu Asp Leu Asn Asp
320 325 330
Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu Ile Ile Tyr Thr
335 340 345
Met Leu Ala Ser Leu Met Asn Lys Asp Gly Val Leu Ile Ser Glu
350 355 360
Gly Gln Gly Phe Met Thr Arg Glu Phe Leu Lys Ser Leu Arg Lys
365 370 375
Pro Phe Gly Asp Phe Met Glu Pro Lys Phe Glu Phe Ala Val Lys
380 385 390
Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Ile Phe Ile
25395 400 405
Ala Val Ile Ile Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val
410 415 420
Lys Pro Ile Glu Asp Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu
425 430 435
Leu Gln Leu Lys Leu Asn His Pro Glu Ser Ser Gln Leu Phe Ala
440 445 450
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hys Leu Leu Gln Lys Met Thr Asp Leu Arg Gln Ile Val Thr Glu
455 460 465
His Val Gln Leu Leu Gln Val Ile Lys Lys Thr Glu Thr Asp Met
470 475 480
Ser Leu His Pro Leu Leu Gln Glu Ile Tyr Lys Asp Leu Tyr
485 490
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) I,ENGTH: 1647 base pairs
(B) l'YPE: nucleic acid
(C) STRANDEDNESS: single
(D) l'OPOLOGY: linear
(xi) SEQUEMCE DESCRIPTION: SEQ ID NO:3:
CGGCTTAGCA AGTTCAGCCT GGTTAAGTCC AAGCTGAATT CCG~llllll 50
T~~ AAcG GATTGATCTT TTGCTAGATA GAGACA~,AAT ATCAGTGTGA l00
ATTACAGCAA ACCCC'TATTC CATGCTGTTA TGGGTGAAAC TCTGGGAGAT 150
TCTCCTATTG ACCCAGA~.AG CGATTCCTTC ACTGATACAC TGTCTGCAAA 200
CATATCACAA GA~ATGACCA TGGTTGACAC AGAGATGCCA TTCTGGCCCA 250
CCAACTTTGG GATCAGCTCC GTGGATCTCT CCGTAATGGA AGACCACTCC 300
CACTCCTTTG ATATC'AAGCC CTTCACTACT GTTGACTTCT CCAGCATTTC 350
TACTCCACAT TACG~,AGACA TTCCATTCAC AAGAACAGAT CCAGTGGTTG 400
CAGATTACAA GTATGACCTG A~ACTTCAAG AGTACCA~AG TGCAATCAAA 450
GTGGAGCCTG CATCI'CCACC TTATTATTCT GAGAAGACTC AGCTCTACAA 500
~ 25 TAAGCCTCAT GAAG~,GCCTT CCAACTCCCT CATGGCA~,TT GAATGTCGTG 550
TCTGTGGAGA TA~AG'CTTCT GGATTTCACT ATGGAGTTCA TGCTTGTGAA 600
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GGATGCAAGG GTTTCTTCCG GAGAACAATC AGATTGAAGC TTATCTATGA 650
CAGATGTGAT CTTAACTGTC GGATCCACAA AAAAAGTAGA AATAAATGTC 700
AGTACTGTCG GTTTCAGAAA TGCCTTGCAG TGGGGATGTC TCATAATGCC 750
ATCAGGTTTG GGCG(GATGCC ACAGGCCGAG AAGGAGAAGC TGTTGGCGGA 800
GATCTCCAGT GATATCGACC AGCTGAATCC AGAGTCCGCT GACCTCCGGG 850
CCCTGGCA~A ACATTTGTAT GACTCATACA TAAAGTCCTT CCCGCTGACC 900
AAAGCAAAGG CGAGGGCGAT CTTGACAGGA AAGACAACAG ACAAATCACC 950
ATTCGTTATC TATGACATGA ATTCCTTAAT GATGGGAGAA GATAAAATCA1000
AGTTCAAACA CATCACCCCC CTGCAGGAGC AGAGCAAAGA GGTGGCCATC1050
CGCATCTTTC AGGGCTGCCA GTTTCGCTCC GTGGAGGCTG TGCAGGAGATllO0
CACAGAGTAT GCCA~AAGCA TTCCTGGTTT TGTAAATCTT GACTTGAACG1150
ACCAAGTAAC TCTCCTCAAA TATGGAGTCC ACGAGATCAT TTACACAATG1200
CTGGCCTCCT TGAT(GAATAA AGATGGGGTT CTCATATCCG AGGGCCAAGG1250
CTTCATGACA AGGG~GTTTC TAAAGAGCCT GCGA~AGCCT TTTGGTGACT1300
TTATGGAGCC CAAGTTTGAG TTTGCTGTGA AGTTCAATGC ACTGGAATTA1350
GATGACAGCG ACTT(,GCAAT ATTTATTGCT GTCATTATTC TCAGTGGAGA1400
CCGCCCAGGT TTGCTGAATG TGAAGCCCAT TGAAGACATT CAAGACAACC1450
TGCTACAAGC CCTG(GAGCTC CAGCTGAAGC TGAACCACCC TGAGTCCTCA1500
CAGCTGTTTG CCAA(CTGCT CCAGAAAATG ACAGACCTCA GACAGATTGT1550
CACGGAACAC GTGCAGCTAC TGCAGGTGAT CAAGAAGACG GAGACAGACA1600
TGAGTCTTCA CCCGCTCCTG CAGGAGATCT ACAAGGACTT GTACTAG 1647
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(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 505
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) ~IOLECULE TYPE: peptide
10 (xi) ',EQUENCE DESCRIPTION : SEQ ID NO: 4
Me t Gly Glu Thr Leu Gly Asp Ser Pro Ile Asp Pro Glu Ser
Asp Ser Phe Thr Asp Thr Leu Ser Ala Asn Ile Ser Gln Glu
Met Thr Met Val Asp Thr Glu Met Pro Phe Trp Pro Thr Asn
30 35 40
Phe Gly Ile Ser Ser Val Asp Leu Ser Val Met Glu Asp His
45 50 55
Ser His Ser Phe Asp Ile Lys Pro Phe Thr Thr Val Asp Phe
60 65 70
Ser Ser Ile Ser Thr Pro His Tyr Glu Asp Ile Pro Phe Thr
Arg Thr Asp Pro Val Val Ala Asp Tyr Lys Tyr Asp Leu Lys
Leu Gln Glu Tyr Gln Ser Ala Ile Lys Val Glu Pro Ala Ser
100 105 110
Pro Pro Tyr Tyr Ser Glu Lys Thr Gln Leu Tyr Asn Lys Pro
115 120 125
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His Glu Glu Pro Ser Asn Ser Leu Met Ala Ile Glu Cys Arg
130 135 140
Val Cys Gly Asp Lys Ala Ser Gly Phe His Tyr Gly Val His
145 150
Ala Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Thr Ile Arg
155 160 165
Leu Lys Leu Ile Tyr Asp Arg Cys Asp Leu Asn Cys Arg Ile
170 175 180
His Lys Lys Ser Arg Asn Lys Cys Gln Tyr Cys Arg Phe Gln
185 190 195
Lys Cys Leu A1OL Val Gly Met Ser His Asn Ala Ile Arg Phe
20C~ 205 210
Gly Arg Met Pro Gln Ala Glu Lys Glu Lys Leu Leu Ala Glu
215 220
Ile Ser Ser Asp Ile Asp Gln Leu Asn Pro Glu Ser Ala Asp
225 230 235
Leu Arg Ala Leu Ala Lys His Leu Tyr Asp Ser Tyr Ile Lys
240 245 250
Ser Phe Pro Leu Thr Lys Ala Lys Ala Arg Ala Ile Leu Thr
255 260 2~5
Gly Lys Thr Thr. Asp Lys Ser Pro Phe Val I le Tyr Asp Met
27~) 275 280
25 Asn Ser Leu Met: Met Gly Glu Asp Lys Ile Lys Phe Lys His
285 290
Ile Thr Pro Leu Gln Glu Gln Ser Lys Glu Val Ala Ile Arg
295 300 305
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Ile Phe Gln Gly Cys Gln Phe Arg Ser Val Glu Ala Val Gln
310 315 320
Glu Ile Thr Glu Tyr Ala Lys Ser Ile Pro Gly Phe Val Asn
325 330 335
5 Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val
340 345 350
His Glu Ile Ile Tyr Thr Met Leu Ala Ser Leu Met Asn Lys
355 360
Asp Gly Val Leu I le Ser Glu Gly Gln Gly Phe Met Thr Arg
10 365 370 375
Glu Phe Leu Lys Ser Leu Arg Lys Pro Phe Gly Asp Phe Met
380 385 390
Glu Pro Lys Phe Glu Phe Ala Val Lys Phe Asn Ala Leu Glu
395 400 405
~5 Leu Asp Asp Ser Asp Leu Ala Ile Phe Ile Ala Val Ile Ile
410 415 420
Leu Ser Gly Asp Arg Pro Gly Leu Leu Asn Val Lys Pro Ile
425 430
Glu Asp Ile Gln Asp Asn Leu Leu Gln Ala Leu Glu Leu Gln
20 435 440 445
Leu Lys Leu Asn His Pro Glu Ser Ser Gln Leu Phe Ala Lys
450 455 460
Leu Leu Gln Lys Met Thr Asp Leu Arg Gln I le Val Thr Glu
465 470 475
25 His Val Gln Leu Leu Gln Val Ile Lys Lys Thr Glu Thr Asp
480 485 490
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Met Ser Leu His Pro Leu Leu Gln Glu Ile Tyr Lys Asp Leu
495 500
Tyr
505
