Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
G-PROTEIN COUPLED GLYCOPROTEIN HORMONE RECEPTOR
AOMF05
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional
Application No. 60/059,868, filed 9/24/97, the contents of which are
incorporated herein by reference in their entirety.
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
is
FIELD OF THE INVENTION
This invention relates to a novel G-protein coupled
glycoprotein hormone receptor in substantially purified form, and also
to mutant or polymorphic forms of the receptor, recombinant nucleic
acids encoding the same, recombinant host cells transformed with the
nucleic acids, transgenic knockout animals lacking the receptor,
transgenic animals expressing a non-native receptor gene, antibodies
against the receptor and polypeptides thereof, and the uses of the
receptor, recombinant nucleic acids, recombinant host cells and
transgenic animals in drug screening and development, diagnosis and
therapeutic applications.
BACKGROUND OF THE INVENTION
The G-protein coupled receptor of the present invention is a
member of the glycoprotein hormone receptor family. Only three G-
protein coupled glycoprotein hormone receptors have been previously
reported: the Follicle Stimulating Hormone (FSH) Receptor (Minegish,
et. al., 1991. Biomed. Biochem. Res. Comm. 175:1125-1130; Sprengel, et.
acl., 1990. Mol. Endocrinol. 4:525-530); the Thyroid Stimulating Hormone
(TSH) Receptor (Frazier, et. al., 1990. Mol. Endocrinol. 4:1264-1276;
Parmentier, et. al., 1990. Science 246:1620-1622) and the Leutenizing
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Hormone/Placental Chorionic Gonadotropin Hormone (LH/hCG)
Receptor (Loosfelt, et. al., 1990. Science 245:525-528).
The structure and function of the known glycoprotein
hormone receptors has been reviewed (Pearce, et. acl., 1995. Q. J. ~Med.
88:3-8; Reichert, et. al., 1991. Trends in Pharmacol. Sci. 12:219-203). This
group of glycoprotein hormone receptors exhibit a structure of the
rhodopsin family G-protein coupled receptors. This class of.receptors
contains seven transmembrane domains with three extracellular loops
and three intracellular loops.
The large ligands, including the glycoprotein hormones,
bind the N-terminal domain while smaller peptides, amines and other
ligands can bind in a pocket formed by the extracellular loops. Upon
binding of an activating ligand a conformational change is believed to
occur which activates the associated G-protein. In this activation the
cytoplasmic loops, particularly the third loop, and the C-terminal
domain of the receptor are believed to interact with the G-protein.
The receptor associated G-protein can be associated with
several cellular signaling pathways. Moat common are the adenylate-
cyclase%AMP pathway, the phospholipase C-b/phosphoinositol
pathways and the elevation of intracellular Ca2'" . These second
messenger pathways mediate the action of the receptor ligand within
the cell. They also advantageously can be used to assess the activity of a
receptor in assays.
Receptor activity can be regulated at the cellular level.
Extensive activation of a receptor by agonists can result in
phosphorylation of the C-terminus and cytoplasmic loops resulting in a
rapid desensitization of the receptor. Further, receptors can be
regulated by modulators of transcriptional activity on the receptor gene.
cAMP responsive elements have been demonstrated within the promoter
regions of some G-protein coupled receptor genes. Again, these aspects
of cellular biochemistry can advantageously be used to monitor and
assess receptor activity in assays, e.g., by monitoring receptor
phosphorylation as an indication of the presence of an agonist of the
receptor or monitoring transcriptional activity as an indication of the
presence of a modulator of receptor gene expression.
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Mutations in the known G-protein coupled glycoprotein
receptors can lead to or indicate a disease state (Pearce, et. acl., 1995).
Given the importance of glycoprotein hormone receptors in the
endocrine system, AOMF05 is expected to play an important role-in the
development and function of skeletal muscle, spinal cord, placenta, and, to a
lesser extent, the brain..
SUMMARY OF THE INVENTION
Preferred aspects of the present invention are disclosed in
FIGS. lA-IC, 4A-4C and SEQ ID NOS:1 and 3, human cDNAs encoding
variants a & b of a G-protein coupled glycoprotein hormone receptor
protein, AOMF05.
Aspects of this invention are isolated nucleic acid
fragments of the AOMF05 G-protein coupled glycoprotein hormone
receptor (SEfa ID N0:1) which encode a biologically active novel human
receptor. Any such nucleic acid fragment will encode either a protein or
protein fragment comprising at least an intracellular G-protein
associating domain and/or extracellular ligand binding domain,
domains conserved throughout the G-coupled glycoprotein hormone
receptor family which exist in the amino acid sequence of AOMF05
variants a & b (SEI~ ID NOS:2 & 4). Any such polynucleotide includes
but is not necessarily limited to nucleotide substitutions, deletions,
additions, amino-terminal truncations and carboxy-terminal
truncations such that these mutations encode mRNA which express a
protein or protein fragment of diagnostic, therapeutic or prophylactic
use, or would be useful for screening for modulators of expression,
agonists and/or antagonists of AOMF05 function.
In particular embodiments, the isolated nucleic acid
molecule of the present invention can be a deoxyribonucleic acid
molecule (DNA), such as genomic DNA and complementary DNA
(cDNA), which can be single (coding or noncoding strand) or double
stranded, as well as synthetic DNA, such as a synthesized, single
stranded polynucleotide. The isolated nucleic acid molecule of the
present invention can also be a ribonucleic acid molecule (RNA). In
particualr embodiments, the nucleic acid can include the entire
sequence of SEQ ID NOS:1 or 3, a sequence encoding the open reading
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frame of SECT ID NOS:1 or 3, or smaller sequences useful for expressing
peptides, or polypeptides of AOMF05 protein. In particular
embodiments the nucleic acid can have natural, non-natural or
modified nucleotides or internucleotide linkages or mixtures of these.
Aspects of the present invention include nucleotide probes
and primers derived from the nucleotide sequences disclosed herein as
FIGS. lA-1C, 3A-3F, 4A-4C, fA-6F and SEf~ ID NOS: 1, & 3. In
particular embodiments of the invention, probes and primers are used to
identify or isolate polynucleotides encoding AOMF05 or mutant or
polymorphic forms of the AOMF05 receptor protein or gene. Probe and
primers can be highly specific for AOMF05 nucleotide sequences.
An aspect of this invention is a substantially purified form
of the novel G-protein coupled glycoprotein hormone receptor protein,
AOMF05, variant a, which is disclosed in FIG. 2 and as set forth in SEQ
ID N0:2.
An aspect of this invention is a substantially purified form
of the novel G-protein coupled glycoprotein hormone receptor protein,
AOMF05, variant b, which is disclosed in FIG. 8 and as set forth in SEla
ID NO:4.
Aspects of the present invention include biologically active
fragments and/or mutants of an AOMF05 protein, including but not
necessarily limited to amino acid substitutions, deletions, additions,
amino terminal truncations and carboxy-terminal truncations such
that these mutations provide for proteins or protein fragments of
diagnostic, therapeutic or prophylactic use and would be useful for
screening for modulators, agonists and/or antagonists of AOMF05
function. In a preferred embodiment, the fragment is a soluble N-
terminal fragment that can compete with the receptor for receptor
ligands.
Aspects of the present invention include recombinant
vectors and recombinant hosts which contain the nucleic acid molecules
disclosed throughout this specification. In particular embodiments, the
vectors and hosts can be prokaryotic or eukaryotic. In particular
embodiments the hosts express AOMF05 peptides, polypepetides,
proteins or fusion proteins. In further embodiments the host cells are
used as a source of expression products.
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Aspects of the invention are polyclonal and monoclonal
antibodies raised in response to either the entire human form of
AOMF05 disclosed herein, or only a fragment, or a single epitope
thereof. In a preferred embodiment antibodies are raised against
epitopes within the NH2-terminal domain of AOMF05. In another
preferred embodiment, antibodies are rasied to epitopes that are unique
to the AOMF05 receptor.
An Aspect of this invention is the use of the DNA
molecules, RNA molecules, recombinant protein and antibodies of the
present invention to screen and measure levels of human AOMF05. The
recombinant proteins, DNA molecules, RNA molecules and antibodies
lend themselves to the formulation of kits suitable for the detection and
typing of human AOMF05.
Aspects of this invention are assays to detect agonists and
antagonists of the AOMF05 receptor and modulators of the expression of
AOMF05. In particular embodiments of this aspect, cells comprising
AOMF05 are used in screening assays including the melanophore
system, yeast expressing mammalian adenylate cyclase, yeast
pheromone protein surrogate screening, phospholipase second signal
screening and the yeast two-hybrid system, all of which are well known
and simply adapted by one of skill in the art.
An aspect of this invention is tissue typing using probes or
antibodies of this invention. In a particular embodiment, polynucleotide
probes are used to identify tissues expressing AOMF05 RNA. In
another embodiment, probes or antibodies can be used to identify a type
of tissue based on AOMF05 expression or display of AOMF05 receptors
on the surface of one or more cells.
An aspect of this invention is isolated nucleic acid
molecules which are fusion constructions expressing fusion proteins
useful in assays to identify compounds which are modulators, agonist or
antagonists of wild-type human AOMF05 activity. A preferred
embodiment of this aspect of the invention includes, but is not limited to,
glutathione S-transferase GST-AOMF05 fusion constructs. These fusion
constructs include, but are not limited to, all or a portion of the ligand-
binding domain of AOMF05, as an in-frame fusion at the carboxy
terminus of the GST gene. The fusion protein is useful to isolate or
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identify ligands of the AOMF05 receptor. The disclosure of SEQ ID
NOS:1-4 allow the artisan of ordinary skill to construct any such nucleic
acid molecule encoding a GST-G-protein coupled glycoprotein hormone
receptor fusion protein. Soluble recombinant GST-G-protein coupled
glycoprotein hormone receptor fusion proteins can be expressed in
various expression systems, including Spodoptera frugiperda (Sf21)
insect cells (Invitrogen) using a baculovirus expression vector (e.g., Bac-
N-Blue DNA from Invitrogen or pAcG2T from Pharmingen).
An aspect of this invention is pharmaceutical compositions
including an AOMF05 protein, fragments thereof, agonists, antagonists
or modulators of AOMF05 or AOMF05 polynucleotides.
An aspect of this invention is using polynucleotides
according to the invention in methods of gene therapy, for instance in
treatment of individuals with the aim of preventing or curing (wholly or
partially) disease states associated with mutations in the AOMF05 gene.
This may ease one or more symptoms of the disease. Introduction of
nucleic acid may take place in vivo by way of gene therapy vectors and
methods.
An aspect of this invention is a transgenic animal useful
for the study of the tissue and temporal specific expression or activity of
the AOMF05 receptor in a non-human animal. The animal is also
useful for studying the ability of a variety of compounds to act as
modulators of AOMF05 receptor activity or expression in viUO or, by
providing cells for culture or assays, in vitro. In an embodiment of this
aspect of the invention, the animal is used in a method for the
preparation of a further animal which lacks a functional endogenous
AOMF05 gene. In another embodiment, the animal of this aspect is
used in a method to prepare an animal which expresses a non-native
AOMF05 gene in the absence of the expression of a endogenous gene. In
particular embodiments the non-human animal is a mouse. In further
embodiments the non-native AOMF05 gene is a wild-type human gene
or a mutant human AOMF05 gene.
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BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. lA-1B. Schematically depicts the nucleotide
sequence of a cDNA polynucleotide encoding the AOMF05 receptor,
variant a (SEQ ID NO:1). -
FIG. 2. Schematically depicts the full length amino acid
sequence of the AOMF05 receptor protein, variant a, (SEQ ID N0:2) in
single letter code.
FIGS. 3A-3F. Schematically depicts the nucleotide
sequence of a polynucleotide encoding AOMF05 (nucleotides 2-3950 of
SEQ ID NO:1) and the translation of the AOMF05 open reading frame
(SEQ ID N0:2).
FIGS. 4A-4B. Schematically depicts the nucleotide
sequence of a cDNA polynucleotide encoding the AOMF05 receptor,
variant b (SEQ ID N0:3).
FIG. 5. Schematically depicts the full length amino acid
sequence of the AOMF05 receptor protein, variant b, (SEQ ID N0:4) in
single letter code.
FIGS. 6A-6F. Schematically depicts the nucleotide
sequence of a polynucleotide encoding AOMF05 (nucleotides 2-3950 of
SEQ ID N0:3) and the translation of the AOMF05 open reading frame
(SEQ ID N0:4).
FIG. 7. Depicts nine predicted signal peptide cleavage sites
of the AOMF05 protein. The nine sequences depicted are amino acids 7-
49, 557-599, 12-54, 5-47, 6fi4-706, 634-675, 9-51, 666-708 and 553-595 of SEQ
ID N0:2 respectively, in single letter code. The predicted cleavage sites
apply to both variants a & b.
FIG. 8. Depicts a Multi-tissue Northern blot analysis of the
expression of the AOMF05 receptor gene.
DETAILED DESCRIPTION OF THE INVENTION
This invention provides polynucleotides and polypeptidea of
a human G-coupled glycoprotein hormone receptor, referred to herein
as AOMF05. The polynucleotides and polypeptides are used to further
provide expression vectors, host cells comprising the vectors, non-
human animals transgenic for the polynucleotides, knockout animals,
probes and primers, antibodies against the receptor and polypeptides
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thereof, assays for the presence or expression of AOMF05 and assays for
the identification of modulators, agonists and antagonists of the
AOMF05 receptor.
The AOMFOS gene, receptor and agonists, antagonists and modulators
thereof can be useful in the treatment of diseases of the pancreas. Further
uses include the treatment of obesity and diabetes. Further uses can include
to stimulate the growth or regeneration of cells of the skeletal muscles.
Each document mentioned in this specification is hereby
incorporated herein by reference in its entirety.
As used herein a "compound" or a "molecule" is an organic
or inorganic assembly of atoms of any size, and can include
macromolecules, e.g., peptides, polypeptides, whole proteins, and
polynucleotides. The terms are used interchangeable herein.
As used herein, a "candidate" is a molecule or compound
that may be an modulator, agonist or antagonist of an AOMF05 receptor.
As used herein an "agonist" is a compound or molecule that
interacts with and activates a polypeptide of an AOMF05 receptor. An
activated AOMF05 receptor polypeptide can stimulate the cleavage of
GTP by a G protein, activate the adenylate cyclase pathway or activate
the phospholipase b pathway.
As used herein an "antagonist" is a compound or molecule
that interacts with and inhibits or prevents a polypeptide of an AOMF05
receptor from becoming activated.
As used herein a "modulator" is a compound or molecule
that interacts-with an aspect of cellular biochemistry to effect an
increase or decrease in the amount of a polypeptide of an AOMF05
receptor present at the surface of a cell, or in the surrounding serum or
media. The change in amount of the receptor polypeptide can be
mediated by the effect of a modulator on the expression of the receptor,
e.g., the transcription, translation, post-translational processing,
translocation or folding of the receptor, or by affecting a components) of
cellular biochemistry that directly or indirectly participates in the
expression of the receptor. Alternatively, a modulator can act by
accelerating or decelerating the turnover of the receptor either by direct
interaction with the receptor or by interacting with another
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components) of cellular biochemistry which directly or indirectly effects
the change.
Polynucleotides
A preferred aspect of the present invention is disclosed in
FIGS. lA-1C and SECI ID NO:I, a human cDNA encoding a G-protein
coupled glycoprotein hormone receptor, AOMF05, disclosed.as follows: .
ACGCGGGCCC CAGTGTGGTG GAATTCTTTT GCATGTACCT AAGTGATTTG
CATAAGCCAG CGGCCGGGGG CTTGGGAACC AAAGCGTGCA ACCCTAGAAG
GGAAAAGGAC GGGAAGAGAT TGAGCCGCGG CTGGGAGACA GCGAGCCAGA
GTCTGGGTGT TTGTGCGAGA GCCACGGCGG GGGCTGGGGC GAGTGGCCGG
CATGGCTGAA GGCTGCGCTC TGCAACCTTG AAGAGCCGCT GCATTGAGAG
GCCAGGGACA GGGAGACCGG TGCGATGGCA GAGCGCGGCC CCCGCCGCTG
1S CGCCGGGCCG GCCCGGCTGG CCTGAGCCGC CGGAGGAGCG GGGCTGCCTC
TGCGCGTCCA TGGAGCAGCG GGAAGGGCGA AACTCCGGAG CGCCGCGTCC
CTGCGCCGCT GCGGCGGACT GCTGAAGGGG CCGAGCCCGC GCGGACCGCC
GAGGAAGAGA CCCCCGCTCC AGCCCGCAGG CCGGCTGCCC GGGGGCGGCG
GGGGACATCG GAGGGCAGCG GAGCGAGCAG CGCCGCGGCA GAGGCCGGCG
CGGGAGGCGG CCGCAGCAAT GCCGGGCCCG CTAGGGCTGC TCTGCTTCCT
CGCCCTGGGG CTGCTCGGCT CGGCCGGGCC CAGCGGCGCG GCGCCGCCTC
TCTGCGCGGC GCCCTGCAGC TGCGACGGCG ACCGTCGGGT GGACTGCTCC
GGGAAGGGGC TGACGGCCGT GCCCGAGGGG CTCAGCGCCT TCACCCAAGC
GCTGGATATC AGTATGAACA ACATTACTCA GTTGCCAGAA GATGCATTTA
2S AGAACTTTCC TTTTCTAGAA GAGCTACAAT TGGCGGGCAA CGACCTTTCT
TTTATCCACC CAAAGGCCTT GTCTGGGTTG AAAGAACTCA AAGTTCTAAC
GCTCCAGAAT AATCAGTTGA AAACAGTACC CAGTGAAGCC ATTCGAGGGC
TGAGTGCTTT GCAGTCTTTG CGTTTAGATG CCAACCATAT TACCTCAGTC
CCCGAGGACA GTTTTGAAGG ACTTGTTCAG TTACGGCATC TGTGGCTGGA
TGACAACAGC TTGACGGAGG TGCCTGTGCA CCCCCTCAGC AATCTGCCCA
CCCTACAGGC GCTGACCCTG GCTCTCAACA AGATCTCAAG TATCCCTGAC
TTTGCATTTA CCAACCTTTC AAGCCTGGTA GTTCTGCATC TTCATAACAA
TAAAATTAGA AGCCTGAGTC AACACTGTTT TGATGGACTA GATAACCTGG
AGACCTTAGA CTTGAATTAT AATAACTTGG GGGAATTTCC TCAGGCTATT
3S AAAGCCCTTC CTAGCCTTAA AGAGCTAGGA TTTCATAGTA ATTCTATTTC
TGTTATCCCT GATGGAGCAT TTGATGGTAA TCCACTCTTA AGAACTATAC
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ATTTGTATGA TAATCCTCTG TCTTTTGTGG GGAACTCAGC ATTTCACAAT
TTATCTGATC TTCATTCCCT AGTCATTCGT GGTGCAAGCA TGGTGCAGCA
GTTCCCCAAT CTTACAGGAA CTGTCCACCT GGAAAGTCTG ACTTTGACAG
GTACAAAGAT AAGCAGCATA CCTAATAATT TGTGTCAAGA ACAAAAGATG
S CTTAGGACTT TGGACTTGTC TTACAATAAT ATAAGAGACC TTCCAAGTTT
TAATGGTTGC CATGCTCTGG AAGAAATTTC TTTACAGCGT AATCAAATTT
ACCAAATAAA GGAAGGCACC TTTCAAGGCC TGATATCTCT AAGGATTCTA
GATGTGAGTA GAAACCTGAT ACATGAAATT CACAGTAGAG CTTTTGCCAC
ACTTGGGCCA ATAACTAACC TAGATGTAAG TTTCAATGAA TTAACTTCCT
TTCCTACGGA AGGCCTGAAT GGGCTAAATC AACTGAAACT TGTGGGCAAC
TTCAAGCTGA AAGAAGCCTT AGCAGCAAAA GACTTTGTTA ACCTCAGGTC
TTTATCAGTA CCATATGCTT ATCAGTGCTG TGCATTTTGG GGTTGTGACT
CTTATGCAAA TTTAAACACA GAAAATAACA GCCTCCAGGA CCACAGTGTG
GCACAGGAGA AAGGTACTGC TGATGCAGCA AATGTCACAA GCACTCTTGA
1S AAATGAAGAA CATAGTCAAA TAATTATCCA TTGTACACCT TCAACAGGTG
CTTTTAAGCC CTGTGAATAT TTACTGGGAA GCTGGATGAT TCGTCTTACT
GTGTGGTTCA TTTTCTTGGT TGCATTATTT TTCAACCTGC TTGTTATTTT
AACAACATTT GCATCTTGTA CATCACTGCC TTCGTCCAAA TTGTTTATAG
GCTTGATTTC TGTGTCTAAC TTATTCATGG GAATCTATAC TGGCATCCTA
2O ACTTTTCTTG ATGCTGTGTC CTGGGGCAGA TTCGCTGAAT TTGGCATTTG
GTGGGAAACT GGCAGTGGCT GCAAAGTAGC TGGGTTTCTT GCAGTTTTCT
CCTCAGAAAG TGCCATATTT TTATTAATGC TAGCAACTGT CGAAAGAAGC
TTATCTGCAA AAGATATAAT GAAAAATGGG AAGAGCAATC ATCTCAAACA
GTTCCGGGTT GCTGCCCTTT TGGCTTTCCT AGGTGCTACA GTAGCAGGCT
2S GTTTTCCCCT TTTCCATAGA GGGGAATATT CTGCATCACC CCTTTGTTTG
CCATTTCCTA CAGGTGAAAC GCCATCATTA GGATTCACTG TAACGTTAGT
GCTATTAAAC TCACTAGCAT TTTTATTAAT GGCCGTTATC TACACTAAGC
TATACTGCAA CTTGGAAAAA GAGGACCTCT CAGAAAACTC ACAATCTAGC
ATGATTAAGC ATGTCGCTTG GCTAATCTTC ACCAATTGCA TCTTTTTCTG
3O CCCTGTGGCG TTTTTTTCAT TTGCACCATT GATCACTGCA ATCTCTATCA
GCCCCGAAAT AATGAAGTCT GTTACTCTGA TATTTTTTCC ATTGCCTGCT
TGCCTGAATC CAGTCCTGTA TGTTTZ'CTTC AACCCAAAGT TTAAAGAAGA
CTGGAAGTTA CTGAAGCGAC GTGTTACCAA GAAAAGTGGA TCAGTTTCAG
TTTCCATCAG TAGCCAAGGT GGTTGTCTGG AACAGGATTT CTACTACGAC
3S TGTGGCATGT ACTCACATTT GCAGGGCAAC CTGACTGTTT GCGACTGCTG
CGAATCGTTT CTTTTAACAA AGCCAGTATC ATGCAAACAC TTGATAAAAT
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CACACAGCTG TCCTGCATTG GCAGTGGCTT CTTGCCAAAG ACCTGAGGGC
TACTGGTCCG ACTGTGGCAC ACAGTCGGCC CACTCTGATT ATGCAGATGA
AGAAGATTCC TTTGTCTCAG ACAGTTCTGA CCAGGTGCAG GCCTGTGGAC
GAGCCTGCTT CTACCAGAGT AGAGGATTCC CTTTTGGTGC GCTATGCTTA
S CAATCTACCA AGAGTTAAAG ACTGAACTAC TGTGTGTGTA ACCGTTTCCC
CCGTCAACCA AAATCAGTGT TTATAGAGTG AACCCTATTC TCATCTTTCA
TCTGGGAAGC ACTTCTGTAA TCACTGCCTG GTGTCACTTA GAAGAAGGAG
AGGTGGCAGT TTATTTCTCA AACCAGTCAT TTTCAAAGAA CAGGTGCCTA
AATTATAAAT TGGTGAAAAA TGCAATGTCC AAGCAATGTA TGATCTGTTT
lO GAAACAAATA TATGACTTGA AAAGGATCTT AGGTGTAGTA GAGCAATATA
ATGTTAGTTT TTTCTGATCC ATAAGAAGCA AATTTATACC TATTTGTGTA
TTAAGCACAA GATAAAGAAC AGCTGTTAAT ATTTTTTAAA AATCTATTTT
AAAATGTGAT TTTCTATAAC TGAAGAAAAT ATCTTGCTAA TTTTACCTAA
TGTTTCATCC TTAATCTCAG GGACAACTTA CTGGCAGGGC CAAAAAAGGG
15 GACTGTCCCA GGCTAGGAAC TGTGAGGGGT ATTACATAGG GCCTTACTTT
ATTGNTGTTT TCCACTTGGC CCTCCTTGGA CNTAGGNGGA CCA(SEQ ID NO:1)
We refer to polynucleotides having a DNA or RNA sequence
corresponding to the sequence shown above as 'variant a'
20 polynucleotides. A variant of AOMF05 can be naturally occurring or
mana-made.
A most preferred aspect of the present invention is disclosed
in FIGS. 4A-4C and SEQ ID N0:3, a human cDNA encoding a G-protein
coupled glycoprotein hormone receptor, AOMF05, disclosed as follows:
ACGCGGGCCC CAGTGTGGTG GAATTCTTTT GCATGTACCT AAGTGATTTG
CATAAGCCAG CGGCCGGGGG CTTGGGAACC AAAGCGTGCA ACCCTAGAAG
GGAAAAGGAC GGGAAGAGAT TGAGCCGCGG CTGGGAGACA GCGAGCCAGA
GTCTGGGTGT TTGTGCGAGA GCCACGGCGG GGGCTGGGGC GAGTGGCCGG
3O CATGGCTGAA GGCTGCGCTC TGCAACCTTG AAGAGCCGCT GCATTGAGAG
GCCAGGGACA GGGAGACCGG TGCGATGGCA GAGCGCGGCC CCCGCCGCTG
CGCCGGGCCG GCCCGGCTGG CCTGAGCCGC CGGAGGAGCG GGGCTGCCTC
TGCGCGTCCA TGGAGCAGCG GGAAGGGCGA AACTCCGGAG CGCCGCGTCC
CTGCGCCGCT GCGGCGGACT GCTGAAGGGG CCGAGCCCGC GCGGACCGCC
GAGGAAGAGA CCCCCGCTCC AGCCCGCAGG CCGGCTGCCC GGGGGCGGCG
GGGGACATCG GAGGGCAGCG GAGCGAGCAG CGCCGCGGCA GAGGCCGGCG
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CGGGAGGCGG CCGCAGCAAT GCCGGGCCCG CTAGGGCTGC TCTGCTTCCT
CGCCCTGGGG CTGCTCGGCT CGGCCGGGCC CAGCGGCGCG GCGCCGCCTC
TCTGCGCGGC GCCCTGCAGC TGCGACGGCG ACCGTCGGGT GGACTGCTCC
GGGAAGGGGC TGACGGCCGT GCCCGAGGGG CTCAGCGCCT TCACCCAAGC
S GCTGGATATC AGTATGAACA ACATTACTCA GTTGCCAGAA GATGCA~TA
AGAACTTTCC TTTTCTAGAA GAGCTACAAT TGGCGGGCAA CGACCTTTCT
TTTATCCACC CAAAGGCCTT GTCTGGGTTG AAAGAACTCA AAGTTCTAAC
GCTCCAGAAT AATCAGTTGA AAACAGTACC CAGTGAAGCC ATTCGAGGGC
TGAGTGCTTT GCAGTCTTTG CGTTTAGATG CCAACCATAT TACCTCAGTC
lO CCCGAGGACA GTTTTGAAGG ACTTGTTCAG TTACGGCATC TGTGGCTGGA
TGACAACAGC TTGACGGAGG TGCCTGTGCA CCCCCTCAGC AATCTGCCCA
CCCTACAGGC GCTGACCCTG GCTCTCAACA AGATCTCAAG TATCCCTGAC
TTTGCATTTA CCAACCTTTC AAGCCTGGTA GTTCTGCATC TTCATAACAA
TAAAATTAGA AGCCTGAGTC AACACTGTTT TGATGGACTA GATAACCTGG
1S AGACCTTAGA CTTGAATTAT AATAACTTGG GGGAATTTCC TCAGGCTATT
AAAGCCCTTC CTAGCCTTAA AGAGCTAGGA TTTCATAGTA ATTCTATTTC
TGTTATCCCT GATGGAGCAT TTGATGGTAA TCCACTCTTA AGAACTATAC
ATTTGTATGA TAATCCTCTG TCTTTTGTGG GGAACTCAGC ATTTCACAAT
TTATCTGATC TTCATTCCCT AGTCATTCGT GGTGCAAGCA TGGTGCAGCA
ZO GTTCCCCAAT CTTACAGGAA CTGTCCACCT GGAAAGTCTG ACTTTGACAG
GTACAAAGAT AAGCAGCATA CCTAATAATT TGTGTCAAGA ACAAAAGATG
CTTAGGACTT TGGACTTGTC TTACAATAAT ATAAGAGACC 'M'CCAAGTTT
TAATGGTTGC CATGCTCTGG AAGAAATTTC TTTACAGCGT AATCAAATTT
ACCAAATAAA GGAAGGCACC TTTCAAGGCC TGATATCTCT AAGGATTCTA
ZS GATGTGAGTA GAAACCTGAT ACATGAAATT CACAGTAGAG CTTTTGCCAC
ACTTGGGCCA ATAACTAACC TAGATGTAAG TTTCAATGAA TTAACTTCCT
TTCCTACGGA AGGCCTGAAT GGGCTAAATC AACTGAAACT TGTGGGCAAC
TTCAAGCTGA AAGAAGCCTT AGCAGCAAAA GACTTTGTTA ACCTCAGGTC
TTTATCAGTA CCATATGCTT ATCAGTGCTG TGCATTTTGG GGTTGTGACT
30 CTTATGCAAA TI'TAAACACA GAAAATAACA GCCTCCAGGA CCACAGTGTG
GCACAGGAGA AAGGTACTGC TGATGCAGCA AATGTCACAA GCACTCTTGA
AAATGAAGAA CATAGTCAAA TAATTATCCA TTGTACACCT TCAACAGGTG
CTTTTAAGCC CTGTGAATAT TTACTGGGAA GCTGGATGAT TCGTCTTACT
GTGTGGTTCA TTTI'CTTGGT TGCATTATTT TTCAACCTGC TTGTTATTTT
3S AACAACATTT GCATCTTGTA CATCACTGCC TTCGTCCAAA TTGTTTATAG
GCTTGAT~ TGTGTCTAAC TTATTCATGG GAATCTATAC TGGCATCCTA
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ACTTTTCTTG ATGCTGTGTC CTGGGGCAGA TTCGCTGAAT TTGGCATZTG
GTGGGAAACT GGCAGTGGCT GCAAAGTAGC TGGGTTTCTT GCAGTTTTCT
CCTCAGAAAG TGCCATATTT TTATTAATGC TAGCAACTGT CGAAAGAAGC
TTATCTGCAA AAGATATAAT GAAAAATGGG AAGAGCAATC ATCTCAAACA
S GTTCCGGGTT GCTGCCCTTT TGGCTTTCCT AGGTGCTACA GTAGCAGGCT
GTTTTCCCCT TTTCCATAGA GGGGAATATT CTGCATCACC CCTTTGTTTG
CCATTTCCTA CAGGTGAAAC GCCATCATTA GGATTCACTG TAACGTTAGT
GCTATTAAAC TCACTAGCAT TTTTATTAAT GGCCGTTATC TACACTAAGC
TATACTGCAA CTTGGAAAAA GAGGACCTCT CAGAAAA.CTC ACAATCTAGC
lO ATGATTAAGC ATGTCGCTTG GCTAATCTTC ACCAATTGCA TCTTTTTCTG
CCCTGTGGCG TTTTTTTCAT TTGCACCATT GATCACTGCA ATCTCTATCA
GCCCCGAAAT AATGAAGTCT GTTACTCTGA TATTTTTTCC ATTGCCTGCT
TGCCTGAATC CAGTCCTGTA TGTTTTCTTC AACCCAAAGT TTAAAGAAGA
CTGGAAGTTA CTGAAGCGAC GTGTTACCAA GAAAAGTGGA TCAGTTTCAG
1S TTTCCATCAG TAGCCAAGGT GGTTGTCTGG AACAGGATTT CTACTACGAC
TGTGGCATGT ACTCACATTT GCAGGGCAAC CTGACTGTTT GCGACTGCTG
CGAATCGTTT CTTTTAACAA AGCCAGTATC ATGCAAACAC TTGATAAAAT
CACACAGCTG TCCTGCATTG GCAGTGGCTT CTTGCCAAAG ACCTGAGGGC
TACTGGTCCG ACTGTGGCAC ACAGTCGGCC CACTCTGATT ATGCAGATGA
2O AGAAGATTCC TTTGTCTCAG ACAGTTCTGA CCAGGTGCAG GCCTGTGGAC
GAGCCTGCTT CTACCAGAGT AGAGGATTCC CTTTGGTGCG CTATGCTTAC
AATCTACCAA GAGTTAAAGA CTGAACTACT GTGTGTGTAA CCGTTTCCCC
CGTCAACCAA AATCAGTGTT TATAGAGTGA ACCCTATTCT CATCTTTCAT
CTGGGAAGCA CTTCTGTAAT CACTGCCTGG TGTCACTTAG AAGAAGGAGA
2S GGTGGCAGTT TATTTCTCAA ACCAGTCATT TTCAAAGAAC AGGTGCCTAA
ATTATAAATT GGTGAAAAAT GCAATGTCCA AGCAATGTAT GATCTGTTTG
AAACAAATAT ATGACTTGAA AAGGATCTTA GGTGTAGTAG AGCAATATAA
TGTTAGTTTT TTCTGATCCA TAAGAAGCAA ATTTATACCT ATTTGTGTAT
TAAGCACAAG ATAAAGAACA GCTGTTAATA TTTTTTAAAA ATCTATTTTA
3O AAATGTGATT TTCTATAACT GAAGAAAATA TCTTGCTAAT TTTACCTAAT
GTTTCATCCT TAATCTCAGG GACAACTTAC TGGCAGGGCC AAAAAAGGGG
ACTGTCCCAG GCTAGGAACT GTGAGGGGTA TTACATAGGG CCTTACTTTA
(SEQ ID N0:3)
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We refer to polynucleotides having a DNA or RNA sequence
corresponding to the sequence shown above as 'variant b'
polynucleotides.
The isolated nucleic acid molecule of the present invention
can include a deoxyribonucleic acid molecule (DNA), such as genomic
DNA and complementary DNA (cDNA), which can be single (coding or
noncoding strand) or double stranded, as well as synthetic DNA, such
as a synthesized, single stranded polynucleotide. The isolated nucleic
acid molecule of the present invention can also include a ribonucleic
acid molecule (RNA).
The present invention also relates to recombinant vectors
and recombinant hosts, both prokaryotic and eukaryotic, which contain
the substantially purified nucleic acid molecules disclosed throughout
this specification.
As used herein a "polynucleotide" is a nucleic acid of more
than one nucleotide. A polynucleotide can be made up of multiple
polynucleotide units that are referred to by description of the unit. For
example, a polynucleotide can comprise within its bounds a
polynucleotide(s) having a coding sequence(s), a polynucleotide(s) that is
a regulatory regions) and/or other polynucleotide units commonly used
in the art.
An "expression vector" is a polynucleotide having
regulatory regions operably linked to a coding region such that, when in
a host cell, the vector can direct the expression of the coding sequence.
The use of expression vectors is well known in the art. Expression
vectors can be used in a variety of host cells and, therefore, the
regulatory regions are preferably chosen as appropriate for the
particular host cell.
A "regulatory region" is a polynucleotide that can promote
or enhance the initiation or termination of transcription or translation
of a coding sequence. A regulatory region includes a sequence that is
recognized by the RNA polymerase, ribosome, or associated
transcription or translation initiation or termination factors of a host
cell. Regulatory regions that direct the initiation of transcription or
translation can direct constitutive or inducible expression of a coding
sequence.
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Polynucleotides of this invention contain full length or
partial length sequences of the mammalian AOMF05 receptor gene.
Polynucleotides of this invention can be single or double stranded. If
single stranded, the polynucleotides can be a coding, "sense," strand or a
complementary, "antisense," strand. Antisense strands can be useful
as modulators of the receptor by interacting with RNA encoding the
receptor. Antisense strands are preferably less than full length strands
having sequences unique or highly specific for RNA encoding the
receptor.
The polynucleotides can include deoxyribonucleotides,
ribonucleotides or mixtures of both. The polynucleotides can be
produced by cells, in cell-free biochemical reactions or through chemical
synthesis. Non-natural or modified nucleotides, including inosine,
methyl-cytosine, deaza-guanosine, etc., can be present. Natural
phosphodiester internucleotide linkages can be appropriate. However,
polynucleotides can have non-natural linkages between the nucleotides.
Non-natural linkages are well known in the art and include, without
limitation, methylphosphonates, phosphorothioates,
phosphorodithionates, phosphoroamidites and phosphate ester
linkages. Dephospho-linkages are also known, as bridges between
nucleotides. Examples of these include siloxane, carbonate,
carboxymethyl ester, acetamidate, carbamate, and thioether bridges.
"Plastic DNA," having, for example, N-vinyl, methacryloxytethyl,
methacrylamide or ethyleneimine internucleotide linkages, can be used.
"Peptide Nucleic Acid" (PNA) is also useful and resists degradation by
nucleases. These linkages can be mixed in a polynucleotide.
As used herein, "purified" and "isolated" are utilized
interchangeably to stand for the proposition that the poiynucleotides,
proteins and polypeptides, or respective fragments thereof in question
has been removed from its in vivo environment so that it can be
manipulated by the skilled artisan, such as but not limited to
sequencing, restriction digestion, site-directed mutagenesis, and
subcloning into expression vectors for a nucleic acid fragment as well as
obtaining the protein or protein fragment in pure quantities so as to
afford the opportunity to generate polyclonal antibodies, monoclonal
antibodies, amino acid sequencing, and peptide digestion. Therefore,
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the nucleic acids claimed herein can be present in whole cells or in cell
lysates or in a partially purified or substantially purified form. A
polynucleotide is considered purified when it is purified away from
environmental contaminants. Thus, a polynucleotide purifed and
isolated from cells is considered to be substantially purified when
purified from cellular components by standard methods while a
chemically synthesized nucleic acid sequence is considered to be
substantially purified when purified from its chemical precursors.
Polypeptides
The present invention also relates to a substantially purified
and isolated form of the novel G-protein coupled glycoprotein hormone
receptor protein, AOMF05. A preferred embodiment is a protein of the
sequence which is shown in FIG. 2, set forth in SE6Z ID N0:2, and
disclosed as follows in single letter code:
MPGPLGLLCF LALGLLGSAG PSGAAPPLCA APCSCDGDRR VDCSGKGLTA
VPEGLSAFTQ ALDISMNNIT QLPEDAFKNF PFLEELQLAG NDLSFIHPKA
LSGLKELKVL TLQNNQLKTV PSEAIRGLSA LQSLRLDANH ITSVPEDSFE
2O GLVQLRHLWL DDNSLTEVPV HPLSNLPTLQ ALTLALNKIS SIPDFAFTNL
SSLVVLHLHN NKIRSLSQHC FDGLDNLETL DLNYNNLGEF PQAIKALPSL
KELGFHSNSI SVIPDGAFDG NPLLRTIHLY DNPLSFVGNS AFHNLSDLHS
LVIRGASMVQ QFPNLTGTVH LESLTLTGTK ISSIPNNLCQ EQKMLRTLDL
SYNNIRDLPS FNGCHALEEI SLQRNQIYQI KEGTFQGLIS LRILDVSRNL
2S IHEIHSRAFA TLGPITNLDV SFNELTSFPT EGLNGLNQLK LVGNFKLKEA
LAAKDFVNLR SLSVPYAYQC CAFWGCDSYA NLNTENNSLQ DHSVAQEKGT
ADAANVTSTL ENEEHSQIII HCTPSTGAFK PCEYLLGSWM IRLTVWFIFL
VALFFNLLVI LTTFASCTSL PSSKLFIGLI SVSNLFMGIY TGILTFLDAV
SWGRFAEFGI WGJETGSGCKV AGFLAVFSSE SAIFLLMLAT VERSLSAKDI
30 MKNGKSNHLK QFRVAALLAF LGATVAGCFP LFHRGEYSAS PLCLPFPTGE
TPSLGFTVTL VLLNSLAFLL MAVIYTKLYC NLEKEDLSEN SQSSMIKHVA
WLIFTNCIFF CPVAFFSFAP LITAISISPE IMKSVTLIFF PLPACLNPVL
YVFFNPKFKE DWKLLKRRVT KKSGSVSVSI SSQGGCLEQD FYYDCGMYSH
LQGNLTVCDC CESFLLTKPV SCKHLIKSHS CPALAVASCQ RPEGYWSDCG
35 TQSAHSDYAD EEDSFVSDSS DQVQACGRAC FYQSRGFPFG ALCLQSTKS
( SEQ ID N0:2)
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We refer to proteins and polypeptides having a sequence corresponding
to the sequence shown above as 'variant a' proteins and polypeptides.
A more preferred embodiment is a protein of the sequence
S which is shown in FIG. 5, set forth in SEQ ID N0:4, and disclosed as
follows in single letter code:
MPGPLGLLCF LALGLLGSAG PSGAAPPLCA APCSCDGDRR VDCSGKGLTA
VPEGLSAFTQ ALDISMNNIT QLPEDAFKNF PFLEELQLAG NDLSFIHPKA
lO LSGLKELKVL TLQNNQLKTV PSEAIRGLSA LQSLRLDANH ITSVPEDSFE
GLVQLRHLWL DDNSLTEVPV HPLSNLPTLQ ALTLALNKIS SIPDFAFTNL
SSLVVLHLHN NKIRSLSQHC FDGLDNLETL DLNYNNLGEF PQAIKALPSL
KELGFHSNSI SVIPDGAFDG NPLLRTIHLY DNPLSFVGNS AFHNLSDLHS
LVIRGASMVQ QFPNLTGTVH LESLTLTGTK ISSIPNNLCQ EQKMLRTLDL
1S SYNNIRDLPS FNGCHALEEI SLQRNQIYQI KEGTFQGLIS LRILDVSRNL
IHEIHSRAFA TLGPITNLDV SFNELTSFPT EGLNGLNQLK LVGNFKLKEA
LAAKDFVNLR SLSVPYAYQC CAFWGCDSYA NLNTENNSLQ DHSVAQEKGT
ADAANVTSTL ENEEHSQIII HCTPSTGAFK PCEYLLGSWM IRLTVWFIFL
VALFFNLLVI LTTFASCTSL PSSKLFIGLI SVSNLFMGIY TGILTFLDAV
2O SWGRFAEFGI WWETGSGCKV AGFLAVFSSE SAIFLLMLAT VERSLSAKDI
MKNGKSNHLK QFRVAALLAF LGATVAGCFP LFHRGEYSAS PLCLPFPTGE
TPSLGFTVTL VLLNSLAFLL MAVIYTKLYC NLEKEDLSEN SQSSMIKHVA
WLIFTNCIFF CPVAFFSFAP LITAISISPE IMKSVTLIFF PLPACLNPVL
YVFFNPKFKE DWKLLKRRVT KKSGSVSVSI SSQGGCLEQD FYYDCGMYSH
2S LQGNLTVCDC CESFLLTKPV SCKHLIKSHS CPALAVASCQ RPEGYWSDCG
TQSAHSDYAD EEDSFVSDSS DQVQACGRAC FYQSRGFPLV RYAYNLPRVK
D ( SEQ ID N0:4)
We refer to proteins and polypeptides having a sequence corresponding
30 to the sequence shown above as 'variant b' proteins and polypeptides.
The present invention also relates to biologically active
fragments and mutant or polymorphic forms of AOMFO~ as set forth as
SEG.~ ID NOS:2 & 4, including but not necessarily limited to amino acid
substitutions, deletions, additions, amino terminal truncations and
3S carboxy-terminal truncations such that these mutations provide for
proteins or protein fragments of diagnostic, therapeutic or prophylactic
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use and would be useful for screening for modulators, agonists and/or
antagonists of AOMF05 function.
In a preferred embodiment, the biologically active fragment
of AOMF05 is a soluble N-terminal fragment that can compete with the
complete AOMF05 receptor for ligands of the receptor. Such soluble
forms of receptors are well known in the art and can be derived from the
polypeptides disclosed herein. It is preferred that soluble N-terminal
fragments lack the signal sequence, that is that lack about the first 20
amino acids of SEQ ID N0:2 or 4. By "about" it is meant that the
fragment need not lack exactly 20 amino acids as it is expected that
deletion or removal of more or less can be useful. The important point is
not so much the amount deleted but that the N-terminal fragment
retains ligand binding activity. Any AOMF05 fragment can be simply
tested for competition with the AOMF05 receptor using an antagonist
assay described herein. The length can also vary. Soluble N-terminal
fragments having the sequence of SEh,I ID N0:2 or 4 up to but not
including the seven hydrophobic domains are preferred. For example, it
is preferred that soluble N-terminal fragments extend up to about amino
acid 539 of SEQ ID NOS:2 or 4. Again, this need not be an exact
endpoint, as other appropriate endpoints can be determined by simple
testing, e.g., for binding activity compared to the wild-type.
Using the disclosure of polynucleotide and polypeptide
sequences provided herein to isolate polynucleotides encoding naturally
occurring forms of AOMF05, one of skill in the art can determine
whether such naturally occurring forms are mutant or polymorphic
forms of AOMF05 by sequence comparison. One can further determine
whether the encoded protein, or fragments of any AOMF05 protein, is
biologically active by routine testing of the protein of fragment in a in
vitro or in vivo assay for the biological activity of the AOMF05 receptor.
For example, one can express N-terminal or C-terminal truncations, or
internal additions or deletions, in host cells and test for their ability to
stimulate the cleavage of GTP by a G protein, activate the adenylate
cyclase pathway or activate the phospholipase b pathway.
It is known that there is a substantial amount of
redundancy in the various codons which code for specific amino acids.
Therefore, this invention is also directed to those DNA sequences encode
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RNA comprising alternative codons which code for the eventual
translation of the identical amino acid, as shown below:
A=Ala=Alanine: codons GCA, GCC, GCG, GCU
C=Cys=Cysteine: codons UGC, UGU
D=Asp=Aspartic acid: codons GAC, GAU
E=Giu=Glutamic acid: codons GAA, GAG
F=Phe=Phenylalanine: codons UUC, UUU
G=Gly=Glycine: codons GGA, GGC, GGG, GGU
H=His=Histidine: codons CAC, CAU
I=Ile=Isoleucine: codons AUA, AUC, AUU
K=Lys=Lysine: codons AAA, AAG
L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU
M=Met=Methionine: codon AUG
N=Asp=Asparagine: codons AAC, AAU
P=Pro=Proline: codons CCA, CCC, CCG, CCU
Q=Gln=Glutamine: codons CAA, CAG
R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU
S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU
T=Thr=Threonine: codons ACA, ACC, ACG, ACU
V=Val=Valine: codons GUA, GUC, GUG, GUU
W=Trp=Tryptophan: codon UGG
Y=Tyr=Tyrosine: codons UAC, UAU
Therefore, the present invention discloses codon redundancy which can
result in differing DNA molecules expressing an identical protein. For
purposes of this specification, a sequence bearing one or more replaced
codons will be defined as a degenerate variation. Also included within
the scope of this invention are mutations either in the DNA sequence or
the translated protein which do not substantially alter the ultimate
physical properties of the expressed protein. For example, substitution
of valine for leucine, arginine for lysine, or asparagine for glutamine
may not cause a change in functionality of the polypeptide.
It is known that DNA sequences coding for a peptide can be
altered so as to code for a peptide having properties that are different
than those of the naturally occurring peptide. Methods of altering the
DNA sequences include but are not limited to site directed mutagenesis.
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Examples of altered properties include but are not limited to changes in
the affinity of an enzyme for a substrate or a receptor for a ligand.
As used herein, a "biologically active equivalent" or
"functional derivative" of a wild-type human AOMF05 possesses a
biological activity that is substantially similar to the biological activity
of
the wild type human AOMF05. The term "functional derivative" is
intended to include the "fragments," "mutants," "variants," "degenerate
variants," "analogs" and "homologues" or to "chemical derivatives" of
the wild type human AOMF05 protein. The term "fragment" is meant to
refer to any polypeptide subset of wild-type human AOMF05. The term
"mutant" is meant to refer to a molecule that may be substantially
similar to the wild-type form but possesses distinguishing biological
characteristics. Such altered characteristics include but are in no way
limited to altered substrate binding, altered substrate affinity and
1S altered sensitivity to chemical compounds affecting biological activity of
the human AOMF05 or human AOMF05 functional derivative. The
term "variant" is meant to refer to a molecule substantially similar in
structure and function to either the entire wild-type protein or to a
fragment thereof. A molecule is "substantially similar" to a wild-type
human AOMF05-like protein if both molecules have substantially
similar structures or if both molecules possess similar biological
activity. Therefore, if the two molecules possess substantially similar
activity, they are considered to be variants even if the structure of one of
the molecules is not found in the other or even if the two amino acid
sequences are not identical. The term "analog" refers to a molecule
substantially similar in function to either the foil-length human
AOMF05 protein or to a biologically active fragment thereof.
As used herein in reference to a human AOMF05 gene or
encoded protein, a "polymorphic" AOMF05 is an AOMF05 that is
naturally found as an allele in the population at large. A polymorphic
form of AOMF05 can have a different nucleotide sequence from the
particular human AOMF05 allele disclosed herein. However, because of
silent mutations, a polymorphic AOMF05 gene can encode the same or
different amino acid sequence as that disclosed herein. Further, some
polymorphic forms AOMF05 will exhibit biological characteristics that
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distinguish the form from wild-type receptor activity, in which case the
polymorphic form is also a mutant.
A protein or fragment thereof is considered purified or
isolated when it is obtained at a concentration at least about five-fold to
ten-fold higher than that found in nature. A protein or fragment thereof
is considered substantially pure if it is obtained at a concentration of at
least about 100-fold higher than that found in nature. A protein or
fragment thereof is considered essentially pure if it is obtained at a
concentration of at least about 1000-fold higher than that found in
nature.
Probes and Primers
The AOMF05 receptor disclosed herein shows a tissue
specific pattern of expression. Therefore, polynucleatides of this
invention can be used as probes for tissue typing. Polynucleotide probes
comprising full length or partial sequences of SEQ ID NOS:1 or 3 can be
used to determine whether a tissue expresses AOMF05 RNA. The
temporal and tissue specific expression of AOMF05 RNA throughout an
animal can also be studied using polynucleotide probes. The effect of
modulators that effect the transcription of the AOMF05 receptor gene
can be studied via the use of these probes. A preferred probe is a single
stranded antisense probe having at least the full length of the coding
sequence of AOMF05. It is also preferred to use probes that have Iess
than the full length sequence, but at least 14 contiguous nucleotides,
preferably at least 15 or lfi nucleotides and more preferably at least 20
contiguous nucleotides, wherein the nucleotide sequences are highly
specific for AOMF05 DNA or RNA.
A nucleotide probe is "highly specific" for AOMF05 DNA or
RNA if one of skill in the art can use standard techniques to determine
hybridization and washing conditions through which one can detect an
AOMF05 encoding DNA in a Southern Blot of total human genomic
DNA (digested with a restriction enzyme to an average size of about 4000
nucleotides) without visually detectable nonspecific background
hybridization. A probe is specific if one can detect the AOMF05 DNA
despite any visually detectable nonspecific backgound hybridization that
may be present. The identification of a sequences) for use as a specific
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probe is well known in the art and involves choosing a sequences) that
is unique to the target sequence, or is specific or highly specific thereto.
It is preferred that polynucleotides that are probes have at least about 14
nucleotides, more preferably at least about 20-25 nucleotides, and also
preferably about 30 to 35 nucleotides or longer. The longer probes are
believed to be more specific for AOMF05 genes and RNAs and can be
used under more stringent hybridization conditions. Longer probes can
be used but can be more difficult to prepare synthetically, or can result
in lower yields from a synthesis. Examples of sequences within SECI ID
NOS:1 & 3 that are useful as probes or primers are the AOMF05 series of
primers given in Example 1. However, one skilled in the art will
recognize that these are only a few of the useful probe or primer
sequences that can be derived from SEQ ID NOS:I & 3.
Polynucleotides having sequences that are unique or highly
specific for AOMF05 can be used as primers in amplification reaction
assays: These assays can be used in tiasue typing as described herein.
Additionally, amplification reactions employing primers derived from
AOMF05 sequences can be used to obtain amplified AOMF05 DNA using
the AOMF05 DNA of the cells as an initial template. The AOMF05 DNA
so obtained can be a mutant or polymorphic form of AOMF05 that differ
from SEI~ ID NOS:1 or 3 by one or more nucleotides of the AOMF05 open
reading frame or sequences flanking the ORF. The differences can be
associated with a non-defective naturally occurring allele or with a
defective form of AOMF05. Thus, polynucleotides of this invention can
be used in allelic identification of various AOMF05 genes or the detection
of a defective AOMF05 gene.
Probes can be labeled by any number of ways known in the
art including isotopes, enzymes, substrates, chemilumineacent,
electrochemiluminescent, biotin and fret pairs among many others. A
probe so labeled can generate a detectable signal directly (e.g., isotopes),
or upon hybridization (fret pairs), or indirectly after a chemical (e.g.,
luminescence) or biochemical reaction (e.g., enzyme-substrate) or after
binding a strepavidin linked moiety that can generate a detectable signal
direclty or indirectly. The labeling of probes and the generation of
detectable signals are well known techniques in the art.
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A primer is specific for the amplification of AOMF05
sequences if one of skill in the art can use standard techniques to
determine conditions under which an amplification reaction yields a
predominant amplified product corresponding to the AOMF05
S sequences. A primer is highly specific if no background amplification
products are visually detectable.
Many types of amplification reactions are known in the art
and include Polymerase Chain Reaction and Reverse Transcriptase
Polymerase Chain Reaction (See e.g., PCR Primer, edited by
C.W.Diefl'enbach and G.S.Dveksler, ( 1995). Cold Spring Harbor
Laboratory Press.), Strand Displacement Amplification, Self Sustained
Sequence Reaction, and any other amplification known to one of skill in
the art that uses primers. Any of these or like reactions can be used
with primers derived from SEQ ID NOS:1 or 3.
Polynucleotide Cloning
The AOMF05 nucleotide and annino acid sequences
provided herein can be used to isolate and/or clone AOMF05
polynucleotides. Any of a variety of procedures can be used to clone
AOMF05. These methods include, but are not limited to, (1) a RACE
PCR cloning technique {Frohman, et al., 1988, Proc. Nutl. Acacd. Sca.85:
8998-9002). 5' and/or 3' RACE can be performed to generate a full length
cDNA sequence. This strategy involves using gene-specific
oligonucleotide primers for PCR amplification of AOMF05 cDNA. These
gene-specific primers are designed through identification of an
expressed sequence tag (EST) nucleotide sequence which has been
identified by searching any number of publicly available nucleic acid
and protein databases; (2) direct functional expression of the AOMF05
cDNA following the construction of an AOMF05-containing cDNA
library in an appropriate expression vector system; (3) screening a
AOMF05-containing cDNA library constructed in a bacteriophage or
plasmid shuttle vector with a labeled degenerate oligonucleotide probe
designed from the amino acid sequence of the AOMF05 protein; (4)
screening a AOMF05-containing cDNA library constructed in a
bacteriophage or plasmid shuttle vector with a partial cDNA encoding
the AOMF05 protein. This partial cDNA is obtained by the specific PCR
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amplification of AOMF05 DNA fragments through the design of
degenerate oligonucleotide primers from the amino acid sequence
known for other receptors which are related to the AOMF05 protein
(e.g., leutenizing, follicle-stimulating and thyroid stimulating hormone
receptors); (5) screening an AOMF05-containing cDNA library
constructed in a bacteriophage or plasmid shuttle vector with a partial
cDNA encoding the AOMF05 protein. This strategy can also involve
using gene-specific oligonucleotide primers for PCR amplification of
AOMF05 cDNA identified as an EST as described herein; or (6)
designing 5' and 3' gene specific oligonucleotides using SEIa ID N0:1 as
a template so that either the full length cDNA can be generated by
known PCR techniques, or a portion of the coding region can be
generated by these same known PCR techniques to generate and isolate
a portion of the coding region to use as a probe to screen one of
numerous types of cDNA and/or genomic libraries in order to isolate a
full length version of the nucleotide sequence encoding AOMF05.
It is readily apparent to those skilled in the art that other
types of libraries, as well as libraries constructed from other cells types
or species types, can be useful for isolating a human AOMF05-encoding
DNA, a mammalian AOMF05 homologue, or mutant or polymorphic
forms of AOMF05 receptor DNA or RNA. Other types of libraries
include, but are not limited to, cDNA libraries derived from other cells
or cell lines other than human cells or tissue such as primate, marine,
rodent, porcine and bovine cells or any other such vertebrate host which
contains AOMF05-encoding DNA. Additionally, an AOMF05 gene can
be isolated by oligonucleotide- or polynucleotide- based hybridization
screening of a vertebrate genomic library, including but not limited to
primate, marine, rodent, porcine or bovine genomic libraries, as well as
concomitant human genomic DNA libraries.
It is readily apparent to those skilled in the art that suitable
cDNA libraries can be prepared from cells or cell lines which express an
AOMF05 receptor. The selection of cells or cell lines for use in
preparing a cDNA library to isolate a AOMF05 cDNA can be done by first
detecting cell associated AOMF05 receptors using an assay for AOMF05,
e.g., an assay using antibodies disclosed herein or a PCR assay using
AOMF05-specific primers.
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Preparation of cDNA libraries can be performed by
standard techniques well known in the art. Well known cDNA library
construction techniques can be found for example, in Sambrook, et al.,
1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York. Complementary DNA
libraries can also be obtained from numerous commercial sources,
including but not limited to Clontech Laboratories, Inc., Palo Alta, CA,
USA and Stratagene, Inc., La Jolla, CA, USA.
It is also readily apparent to those skilled in the art that
DNA encoding AOMF05 can also be isolated from a suitable genomic
DNA library. Construction of genomic DNA libraries can be performed
by standard techniques well known in the art. Well known genomic
DNA library construction techniques can be found in Sambrook, et al.,
supra.
In order to clone the AOMF05 gene by one of the preferred
methods, the amino acid sequence or DNA sequence of AOMF05 or a
homologous protein may be necessary. To accomplish this, the AOMF05
or a homologous protein can be purified, e.g., through cross reaction
with the anti-AOMF05 antibodies taught herein, and partial amino acid
sequences) determined by automated sequenators. It is not necessary to
determine the entire amino acid sequence, but the linear sequence of two
regions of fi to 8 amino acids can be determined for the PCR
amplification of a partial AOMF05 DNA fragment. Once suitable amino
acid sequences have been identified, the DNA sequences capable of
encoding them are synthesized. Because the genetic code is degenerate,
more than one codon can be used to encode a particular amino acid, and
therefore, the amino acid sequence can be encoded by any of a set of
similar, degenerate, DNA oligonucleotides. Only one member of the
degenerate set will be identical to the AOMF05 sequence but others in the
set will be capable of hybridizing to AOMF05 DNA even in the presence
of DNA oligonucleotides with mismatches. The mismatched DNA
oligonucleotides can still sufficiently hybridize to the AOMF05 DNA to
permit identification and isolation of AOMF05 encoding DNA.
Alternatively, the nucleotide sequence of a region of an expressed
sequence can be identified by searching one or more available genomic
databases. Gene-specific primers can be used to perform PCR
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amplification of a cDNA of interest from either a cDNA library or a
population of cDNAs. As noted herein, the appropriate nucleotide
sequence for use in a PCR-based method can be obtained from SEf~,'1 ID
N0:1, either for the purpose of isolating overlapping 5' and 3' PCR
products for generation of a full-length sequence coding for AOMF05, or
to isolate a portion of the nucleotide sequence coding for AOMF05 for use
as a probe to screen one or more cDNA- or genomic-based libraries to
isolate a full-length sequence encoding AOMF05 or AOMF05-like
proteins.
In a method used in Example 1, the AOMF05 full length
cDNA of the present invention was generated by a method of cDNA
screening called Reduced Complexity cDNA Analysis (RCCA). Briefly,
the extension of partial cDNA sequences have historically been achieved
with one or both of the two commonly used methods: filter screening of
cDNA libraries by hybridization with labeled probes, and 5'- and 3'-
RACE with total cellular mRNA by PCR. The first method is effective
but laborious and slow while the latter method is fast but limited in
efficiency. This RACE protocol is hindered by limited length of
extension due to the use of the entire cellular mR,NA population in a
single reaction. Since smaller fragments are amplified much more
efficiently than larger fragments by PCR in the same reaction, PCR
products obtained using the second method are often quite small.
The RCCA method improves upon known methods of cDNA
library screening by initially constructing and subdividing cDNA
libraries followed by isolating 5'- and 3'- flanking fragments by PCR.
Since each pool is unlikely to contain more than one clone for a given
gene which is low to moderately expressed, competition between large
and small PCR products in one pool does not exist, making it possible to
isolate fragments of various sizes. One definite advantage of the method
as described herein is the ei~ciency, throughput, and its potential to
isolate alternatively spliced cDNA forms.
The RCCA process provides for rapid extension of a partial
cDNA sequence based on subdividing a primary cDNA library and DNA
amplification by polymerase chain reaction (PCR). A cDNA library is
constructed with cDNA primed by random, oligo-dT or a combination of
both random and oligo-dT primers and then subdivided into pools at
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approximately 10,000 -20,000 clones per pool ("superpools"). Each
superpool is amplified separately and therefore represents an
independent portion of the cDNA molecules from the original mRNA
source. Samples from all the superpools are collected and transferred
into 96-well plates. To extend a partial cDNA sequence, such as SEQ ID
NO:1, positive pools containing the partial cDNA sequence are first
identified by PCR with a pair of primers complementary to the cDNA
sequence. Each positive pool in the library contains an independent
clone of the cDNA sequence; within each clone are embedded the partial
cDNA sequence and its flanking fragments. The flanking fragments
are isolated by PCR with primers complementary to the known vector
and cDNA sequences and then sequenced directly. The DNA sequences
from these fragments plus the original partial cDNA sequence are
assembled into a continuous fragment, resulting in the extension of the
partial cDNA sequence and the eventual determination of its full-length
gene sequence by repeating the process, if necessary, until a complete
open reading frame is obtained.
The fundamental principle of this process is to subdivide a
complex library into superpools of about 10,000 to about 20,000 clones. A
library of two million primary clones, a number large enough to cover
most mRNA transcripts expressed in the tissue, can be subdivided into
188 pools and stored in two 96-well plates. Since the number of
transcripts for most genes is fewer than one copy per 10,000 transcripts
in total cellular mRNA, each pool is unlikely to contain more than one
clone for a given cDNA sequence. Such reduced complexity makes it
possible to use PCR to isolate flanking fragments of partial cDNA
sequences larger than those obtained by known methods.
The skilled artisan, aided with this specification, will
understand the far reaching cDNA cloning process disclosed herein:
multiple primer combinations from an EST or other partial cDNA
sequence, in combination with flanking vector primer oligonucleotides
can be used to "walk" in both directions away from the internal, gene
specific, sequence, and respective primers, such that a contig
representing a full length cDNA can be constructed. This procedure
relies on the ability to screen multiple pools which comprise a
representative portion of the total cDNA library. This procedure is not
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dependent upon using a cDNA library with directionally cloned inserts.
Instead, both 5' and 3' vector and gene specific primers are added and a
contig map is constructed from additional screening of positive pools
using both vector primers and gene specific primers. Of course, these
gene specific primers are initially constructed from a known nucleic
acid fragment such as an expressed sequence tag. However, as the walk
continues, gene specific primers are utilized from the 5' and 3'
boundaries of the newly identified regions of the cDNA. As the walk
continues, there is still no requirement that the vector orientation of a
yet unidentified fragment be known. Instead, all combinations are
tested on a positive pool and the actual vector orientation is determined
by the ability of certain vector/gene specific primers to generate the
predicted PCR fragment. A full-length cDNA can then be easily
constructed by known subcloning procedures.
Isolation of other species homologs of the AOMF05 gene
The AOMF05 gene from different species, e.g. mouse, rat,
dog, are isolated by screening of a cDNA library with portions of the gene
that have been obtained from cDNA of the species of interest using PCR
primers designed from the human AOMF05 sequence. Degenerate PCR
is performed by designing primers of 1?-20 nucleotides with 32-128 fold
degeneracy by selecting regions that code for amino acids that have low
codon degeneracy e.g. Met and Trp. When selecting these primers
preference is given to regions that are conserved in the protein. PCR
products are analyzed by DNA sequence analysis to confirm their
similarity to human AOMF05. The correct product is used to screen
cDNA libraries by colony or plaque hybridization at high stringency.
Alternatively, probes derived directly from the human AOMF05 gene are
utilized to isolate the cDNA sequence of AOMF05 from different species
by hybridization at reduced stringency. A cDNA library can be
generated as known in the art or as described herein.
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Transgenic Animals
In reference to the transgenic animals of this invention, we
refer to transgenes and genes. As used herein, a "transgene" is a
genetic construct including a gene. The transgene is integrated into one
or more chromosomes in the cells in an animal or its ancestor by
methods known in the art. Once integrated, the transgene is carried in
at least one place in the chromosomes of a transgenic animal. A gene is
a nucleotide sequence that encodes a protein. The gene and/or
transgene can also include genetic regulatory elements and/or
structural elements known in the art.
The term "animal" is used herein to include all mammals,
except humans. It also includes an individual animal in all stages of
development, including embryonic and fetal stages. Preferably the
animal is a rodent, and most preferably mouse or rat. A "transgenic
animal" is an animal containing one or more cells bearing genetic
information received, directly or indirectly, by deliberate genetic
manipulation at a subcellular level, such as by microinjection or
infection with recombinant virus. This introduced DNA molecule can
be integrated within a chromosome, or it can be extra-chromosomally
replicating DNA. Unless otherwise noted or understood from the
context of the description of an animal, the term "transgenic animal" as
used herein refers to a transgenic animal in which the genetic
information was introduced into a germ line cell, thereby conferring the
ability to transfer the information to offspring. If offspring in fact
possess some or all of the genetic information, then they, too, are
transgenic animals. The genetic information is typically provided in the
form of a transgene carried by the transgenic animal.
The genetic information received by the non-human animal
can be foreign to the species of animal to which the recipient belongs, or
foreign only to the particular individual recipient. In the last case, the
information can be altered or it can be expressed differently than the
native gene. Alternatively, the altered or introduced gene can cause the
native gene to become non-functional to produce a "knockout" animal.
As used herein, a "targeted gene" or "Knockout" (KO)
transgene is a DNA sequence introduced into the germline of a non-
human animal by way of human intervention, including but not limited
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to, the methods described herein. The targeted genes of the invention
include nucleic acid sequences which are designed to specifically alter
cognate endogenous alleles of the non-human animal.
An altered AOMF05 receptor gene should not fully encode
the same receptor endogenous to the host animal, and its expression
product can be altered to a minor or great degree, or absent altogether.
In cases where it is useful to express a non-native AOMF05 receptor in a
transgenic animal in the absence of a endogenous AOMF05 receptor we
prefer that the altered AOMF05 gene induce a null, "knockout,"
phenotype in the animal. However a more modestly modified AOMF05
gene can also be useful and is within the scope of the present invention.
A type of target cell for transgene introduction is the
embryonic stem cell (ES). ES cells can be obtained from pre-
implantation embryos cultured in vitro and fused with embryos (M. J.
Evans et al., Nature 292:154-156 (1981); Bradley et ad., Nature 309:255-258
( 1984); Gossler et al. Proc. Natl. Acad. Sci. USA 83:9065-9069 ( 1986); and
Robertson et al., Nature 322:445-448 (1986)). Transgenes can be
efficiently introduced into the ES cells by a variety of standard techniques
such as DNA transfection, microinjection, or by retrovirus-mediated
transduction. The resultant transformed ES cells can thereafter be
combined with blastocysts from a non-human animal. The introduced
ES cells thereafter colonize the embryo and contribute to the germ line of
the resulting chimeric animal (R. Jaenisch, Science 240: 1468-1474
(1988)). Animals are screened for those resulting in germline
transformants. These are crossed to produce animals homozygous for
the transgene.
Methods for evaluating the targeted recombination events
as well as the resulting knockout mice are readily available and known
in the art. Such methods include, but are not limited to DNA (Southern)
hybridization to detect the targeted allele, polymerase chain reaction
(PCR), polyacrylamide gel electrophoresis (PAGE) and Western blots to
detect DNA, RNA and protein.
This may have a therapeutic aim. (Gene therapy is
discussed below.) The presence of a mutant, allele or variant sequence
within cells of an organism, particularly when in place of a homologous
endogenous sequence, may allow the organism to be used as a model in
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testing and/or studying the role of the AOMF05 gene or substances
which modulate activity of the encoded polypeptide and/or promoter in
vitro or are otherwise indicated to be of therapeutic potential.
Expression of AOMF05
The present invention also relates to recombinant vectors
and recombinant hosts, both prokaryotic and eukaryotic, which contain
the substantially purified nucleic acid molecules disclosed throughout
this specification.
Therefore, the present invention also relates to methods of
expressing AOMF05 and biological equivalents disclosed herein, assays
employing these recombinantly expressed gene products, cells
expressing these gene products, and modulators, agonistic and/or
antagonistic compounds identified through the use of assays utilizing
these recombinant forms, including, but not limited to, one or more
compounds or molecules that act through direct contact with the
receptor, particularly with the ligand binding domain, or through direct
or indirect contact with a ligand which either interacts with the receptor
or with the transcription or translation of AOMF05, thereby modulating
AOMF05 expression.
A variety of expression vectors can be used to express
recombinant AOMF05 in host cells. Expression vectors are defined
herein as DNA sequences that are required for the transcription of
cloned DNA and the translation of their mRNAs in an appropriate host.
Such vectors can be used to express eukaryotic DNA in a variety of hosts
such as bacteria, bluegreen algae, plant cells, insect cells and animal
cells. Specifically designed vectors allow the shuttling of DNA between
hosts such as bacteria-yeast or bacteria-animal cells. An appropriately
constructed expression vector should contain: an origin of replication for
autonomous replication in host cells, selectable markers, a limited
number of useful restriction enzyme sites, a potential for high copy
number, and active promoters. A promoter is defined as a DNA
sequence that directs RNA polymerase to bind to DNA and initiate RNA
synthesis. A strong promoter is one which causes mRNAs to be
initiated at high frequency. Expression vectors can include, but are not
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limited to, cloning vectors, modified cloning vectors, specifically
designed plasmida or viruses.
Commercially available mammalian expression vectors
which can be suitable for recombinant human AOMF05 expression,
include but are not limited to, pcDNA3.1 (Invitrogen), pLITMUS28,
pLITMUS29, pLITMUS38 and pLITMUS39 (New England Biolaba),
pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo
(Stratagene), pXT1 (Stratagene), pSGS (Stratagene), EBO-pSV2-neo
(ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12)
(ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-
dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565).
A variety of bacterial expression vectors can be used to
express recombinant human AOMF05 in bacterial cells. Commercially
available bacterial expression vectors which are suitable for
recombinant human AOMF05 expression include, but are not limited to
pCIE (fgliagen), pETlla {Novagen), lambda gtll (Invitrogen), and pKK223-
3 (Pharmacia).
A variety of fungal cell expression vectors can be used to
express recombinant human AOMF05 in fungal cells. Commercially
available fungal cell expression vectors which are suitable for
recombinant human AOMF05 expression include but are not limited to
pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).
A variety of insect cell expression vectors can be used to
express recombinant receptor in insect cells. Commercially available
insect cell expression vectors which are suitable for recombinant
expression of human AOMF05 include but are not limited to
pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T
(Pharmingen).
An expression vector containing DNA encoding a human
AOMF05-like protein can be used for expression of human AOMF05 in a
recombinant host cell. Recombinant host cells can be prokaryotic or
eukaryotic, including but not limited to bacteria such as E. coli, fungal
cells such as yeast, mammalian cells including but not limited to cell
lines of human, bovine, porcine, monkey and rodent origin, and insect
cells including but not limited to Drosophila- and silkworm-derived cell
lines. Cell lines derived from mammalian species which can be suitable
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and which are commercially available, include but are not limited to, L
cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2
(ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1
(ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651),
CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL
1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL
26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL 209).
The expression vector can be introduced into host cells via
any one of a number of techniques including but not limited to
transformation, transfection, protoplast fusion, and electroporation.
The expression vector-containing cells are individually analyzed to
determine whether they produce human AOMF05 protein.
Identification of human AOMF05 expressing cells can be done by several
means, including but not limited to immunological reactivity with anti-
human AOMF05 antibodies, labeled ligand binding and the presence of
host cell-associated human AOMF05 activity.
The cloned human AOMF05 cDNA obtained through the
methods described herein can be recombinantly expressed by molecular
cloning into an expression vector (such as pcDNA3.1, pQE,
pBlueBacHis2 and pLITMUS28) containing a suitable promoter and
other appropriate transcription regulatory elements, and transferred
into prokaryotic or eukaryotic host cells to produce recombinant human
AOMF05. Techniques for such manipulations can be found described in
Sambrook, et al., suprac , and are well known and easily available to the
one of ordinary skill in the art.
Expression of human AOMF05 DNA can also be performed
using in vitro produced synthetic mRNA. Synthetic mRNA can be
efficiently translated in various cell-free systems, including but not
limited to wheat germ extracts and reticulocyte extracts, as well as
e~ciently translated in cell based systems, including but not limited to
microinjection into frog oocytes, with microinjection into frog oocytes
being preferred.
To determine the human AOMF05 cDNA sequences) that
yields optimal levels of human AOMF05, cDNA molecules including but
not limited to the following can be constructed: a cDNA fragment
containing the full-length open reading frame for human AOMF05 as
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well as various constructs containing portions of the cDNA encoding
only specific domains of the protein or rearranged domains of the
protein. All constructs can be designed to contain none, all or portions
of the 5' and/or 3' untranslated region of a human AOMF05 cDNA.- The
expression levels and activity of human AOMF05 can be determined
following the introduction, both singly and in combination, of these
constructs into appropriate host cells. Following determination of the
human AOMF05 cDNA cassette yielding optimal expression in
transient assays, this AOMF05 cDNA construct is transferred to a
variety of expression vectors (including recombinant viruses), including
but not limited to those for mammalian cells, plant cells, insect cells,
oocytes, bacteria, and yeast cells.
Following expression of AOMF05 in a host cell, AOMF05
polypeptides can be recovered. Several AOMF05 protein purification
procedures are available and suitable for use. AOMF05 protein and
polypeptides can be purified from cell lysates and extracts, or from
conditioned culture medium, by various combinations of, or individual
application of methods including ultrafiltration, acid extraction, alcohol
precipitation, salt fractionation, ionic exchange chromatography,
phosphocellulose chromatography, lecithin chromatography, affinity
(e.g., antibody or His-Ni) chromatography, size exclusion
chromatography, hydroxylapatite adsorption chromatography and
chromatography based on hydrophobic or hydrophillic interactions. In
some instances, protein denaturation and refolding steps can be
employed. High performance liquid chromatography (HPLC) and
reversed phase HPLC can also be useful. Dialysis can be used to adjust
the final buffer composition.
Anti-AOMF05 Antibodies
The present invention also relates to polyclonal and
monoclonal antibodies raised in response to either the human form of
AOMF05 disclosed herein, or a biologically active fragment thereof. It
will be especially preferable to raise antibodies against epitopes within
the NH2-terminal domain or the extracellular inter-membrane domains
of AOMF05. It is also preferable to raise antibodies to epitopes which
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show the least homology to other known glycoprotein hormone receptor
proteins.
An antibody is specific far an AOMF05 epitope if one of skill
in the art can use standard techniques to determine conditions under
which one can detect AOMF05 in a Western Blot of a sample from a host
cell that displays AOMF05 on its surface. The blot can be of a native or
denaturing gel as appropriate for the epitope. An antibody is highly
specific for an AOMF05 epitope if no nonspecific background binding is
visually detectable. An antibody can also be considered highly specific
for AOMF05 if the binding of the antibody to AOMF05 can not be
competed by non-AOMF05 peptides, polypepetides or proteins.
Recombinant AOMF05 protein can be separated from other
cellular proteins by use of an immunoaffinity column made with
monoclonal or polyclonal antibodies specific for full-length AOMF05
protein, or polypeptide fragments of AOMF05 protein. Additionally,
polyclonal or monoclonal antibodies can be raised against a synthetic
peptide (usually from about 9 to about 25 amino acids in length) from a
portion of the protein as disclosed in SEQ ID N0:2. Monospecific
antibodies to human AOMF05 are purified from mammalian antisera
containing antibodies reactive against human AOMF05 or axe prepared
as monoclonal antibodies reactive with human AOMF05 using the
technique of Kohler and Milstein (1975, Nature 256: 495-497).
Monospecific antibody as used herein is defined as a single antibody
species or multiple antibody species with homogenous binding
characteristics for human AOMF05. Homogenous binding as used
herein refers to the ability of the antibody species to bind to a specific
antigen or epitope, such as those associated with human AOMF05, as
described herein. Human AOMF05-specific antibodies are raised by
immunizing animals such as mice, rats, guinea pigs, rabbits, goats,
horses and the like, with an appropriate concentration of human
AOMF05 protein or a synthetic peptide generated from a portion of
human AOMF05 with or without an immune adjuvant.
Preimmune serum is collected prior to the first
immunization. Each animal receives between about 0.1 mg and about
1000 mg of human AOMF05 protein associated with an acceptable
immune adjuvant. Such acceptable adjuvants include, but are not
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limited to, Freund's complete, Freund's incomplete, alum-precipitate,
water in oil emulsion containing Corynebcacterium pcxrvum and tRNA.
The initial immunization consists of human AOMF05 protein or peptide
fragment thereof in, preferably, Freund's complete adjuvant at multiple
sites either subcutaneously (SC), intraperitoneally {IP) or both. Each
animal is bled at regular intervals, preferably weekly, to determine
antibody titer. The animals may or may not receive booster injections
following the initial immunization. Those animals receiving booster
injections are generally given an equal amount of human AOMF05 in
Freund's incomplete adjuvant by the same route. Booster injections are
given at about three week intervals until maximal titers are obtained.
At about 7 days after each booster immunization or about weekly after a
single immunization, the animals axe bled, the serum collected, and
aliquots are stored at about -20°C.
Monoclonal antibodies (mAb) reactive with human
AOMF05 are prepared by immunizing inbred mice, preferably Balb/c,
with human AOMF05 protein. The mice are immunized by the IP or SC
route with about 1 mg to about 100 mg, preferably about 10 mg, of human
AOMF05 protein in about 0.5 ml buffer or saline incorporated in an
equal volume of an acceptable adjuvant, as discussed herein. Freund's
complete adjuvant is preferred. The mice receive an initial
immunization on day 0 and are rested for about 3 to about 30 weeks.
Immunized mice are given one or more booster immunizations of about
1 to about 100 mg of human AOMF05 in a buffer solution such as
phosphate buffered saline by the intravenous {IV) route. Lymphocytes,
from antibody positive mice, preferably splenic lymphocytes, are
obtained by removing spleens from immunized mice by standard
procedures known in the art. Hybridoma cells are produced by mixing
the splenic lymphocytes with an appropriate fusion partner, preferably
myeloma cells, under conditions which will allow the formation of stable
hybridomas. Fusion partners can include, but are not limited to: mouse
myelomas P3/NSl/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being
preferred. The antibody producing cells and myeloma cells are fused in
polyethylene glycol, about 1000 mol. wt., at concentrations from about
30% to about 50%. Fused hybridoma cells are selected by growth in
hypoxanthine, thymidine and aminopterin supplemented Dulbecco's
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Modified Eagles Medium (DMEM) by procedures known in the art.
Supernatant fluids are collected form growth positive wells on about
days 14, 18, and 21 and are screened for antibody production by an
immunoassay such as solid phase immunoradioassay (SPIRA) using
human AOMF05 as the antigen. The culture fluids are also tested in the
Ouchterlony precipitation assay to determine the isotype of the mAb.
Hybridoma cells from antibody positive wells are cloned by a technique
such as the soft agar technique of MacPherson, 1973, Soft Agar
Techniques, in Tissue Culture Methods cznd Applacactions, Kruse and
Paterson, Eds., Academic Press.
Monoclonal antibodies are produced in uiuo by injection of
pristine primed Balb/c mice, approximately 0.5 ml per mouse, with
about 2 x 106 to about 6 x 106 hybridoma cells about 4 days after priming.
Ascites fluid is collected at approximately 8-12 days after cell transfer
and the monoclonal antibodies are purified by techniques known in the
art.
In uitro production of anti-human AOMF05 mAb is carried
out by growing the hybridoma in DMEM containing about 2% fetal calf
serum to obtain sufficient quantities of the specific mAb. The mAb are
purified by techniques known in the art.
Antibody titers of ascites or hybridoma culture fluids are
determined by various serological or immunological assays which
include, but are not limited to, precipitation, passive agglutination,
enzyme-linked immunosorbent antibody (ELISA) technique and
radioimmunoassay (RIA) techniques. Similar assays are used to detect
the presence of human AOMF05 in body fluids or tissue and cell
extracts.
It is readily apparent to those skilled in the art that the
herein described methods for producing monospeaific antibodies can be
utilized to produce antibodies specific for human AOMF05 peptide
fragments, or full-length human AOMF05.
Human AOMF05 antibody affinity columns are made, for
example, by adding the antibodies to Affigel-10 (Biorad), a gel support
which is pre-activated with N-hydroxysuccinimide esters such that the
antibodies form covalent linkages with the agarose gel bead support.
The antibodies are then coupled to the gel via amide bonds with the
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spacer arm. The remaining activated esters are then quenched with 1M
ethanolamine HCl (pH 8). The column is washed with water followed by
0.23 M glycine HCl (pH 2.6) to remove any non-conjugated antibody or
extraneous protein. The column is then equilibrated in phosphate-
s buffered saline (pH 7.3) and the cell culture supernatants or cell extracts
containing full-length human AOMF05 or human AOMF05 protein
fragments are slowly passed through the column. The column is then
washed with phosphate buffered saline until the optical density (A280)
falls to background, then the protein is eluted with 0.23 M glycine-HCl
(pH 2.6). The purified human AOMF05 protein is then dialyzed against
phosphate buffered saline.
Levels of human AOMF05 in host cells is quantified by a
variety of techniques including, but not limited to, immunoaf~nity
andlor ligand affinity techniques. AOMF05-specific affinity beads or
AOMF05-specific antibodies are used to isolate 36S-methionine labeled or
unlabelled AOMF05. Labeled AOMF05 protein is analyzed by SDS-
PAGE. Unlabelled AOMF05 protein is detected by Western blotting,
ELISA or RIA assays employing either AOMF05 protein specific
antibodies and/or antiphosphotyrosine antibodies.
Modulators, Agonists and Antagonists of AOMF05
The present invention is also directed to methods for
screening for compounds or molecules which modulate the expression
of DNA or RNA encoding a human AOMF05 protein. Compounds or
molecules which modulate these activities can be DNA, RNA, peptides,
proteins, or non-proteinaceous organic molecules. They can modulate
by increasing or attenuating the expression of DNA or RNA encoding
human AOMF05. Compounds that modulate the expression of DNA or
RNA encoding human AOMF05 or are agonists or antagonists of the
biological function thereof can be detected by a variety of assays. The
assay can be a simple "yes/no" assay to determine whether there is a
change in expression or function. The assay can be made quantitative
by comparing the expression or function of a test sample with the levels
of expression or function in a standard sample. Kits containing human
AOMF05, antibodies to human AOMF05, or modified human AOMF05
can be prepared by known methods for such uses.
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The DNA molecules, RNA molecules, recombinant protein
and antibodies of the present invention can be used to screen and
measure levels of human AOMF05. The recombinant proteins, DNA
molecules, RNA molecules and antibodies lend themselves to the -
formulation of lilts suitable for the detection and typing of human
AOMF05. Such a kit would comprise a compartmentalized carrier
suitable to hold in close confinement at least one container. The carrier
would further comprise reagents such as recombinant AOMF05 or anti-
AOMF05 antibodies suitable for detecting human AOMF05. The carrier
can also contain a means for detection such as labeled antigen or
enzyme substrates or the like.
Pharmaceutical Compositions
Pharmaceutically useful compositions comprising
agonists, antagonist or modulators of human AOMF05 can be
formulated according to known methods such as by the admixture of a
pharmaceutically acceptable carrier. Examples of such carriers and
methods of formulation can be found in Remington's Pharmaceutical
Sciences. To form a pharmaceutically acceptable composition suitable
for effective administration, such compositions will contain an effective
amount of the protein, DNA, RNA, modified human AOMF05, or either
AOMF05 modulators, agonsits or antagonists.
Therapeutic or diagnostic compositions of the invention are
administered to an individual in amounts suf~.cient to treat or diagnose
disorders. The effective amount can vary according to a variety of
factors such as the individual's condition, weight, sex and age. Other
factors include the mode of administration.
The pharmaceutical compositions can be provided to the
individual by a variety of routes such as subcutaneous, topical, oral and
intramuscular.
The term "chemical derivative" describes a molecule that
contains additional chemical moieties which are not normally a part of
the base molecule. Such moieties can improve the solubility, half life,
absorption, etc. of the base molecule. Alternatively the moieties can
3 5 attenuate undesirable side effects of the base molecule or decrease the
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toxicity of the base molecule. Examples of such moieties are described in
a variety of texts, such as Remington's Pharmaceutical Sciences.
Compounds identified according to the methods disclosed
herein can be used alone at appropriate dosages. Alternatively, cc~-
administration or sequential administration of other agents can be
desirable.
The present invention also provides a means to obtain
suitable topical, oral, systemic and parenteral pharmaceutical
formulations for use in the methods of treatment of the present
invention. The compositions containing compounds or molecules
identified according to this invention as the active ingredient can be
administered in a wide variety of therapeutic dosage forms in
conventional vehicles for administration. For example, the compounds
can be administered in such oral dosage forms as tablets, capsules (each
including timed release and sustained release formulations}, pills,
powders, granules, elixirs, tinctures, solutions, suspensions, syrups
and emulsions, or by injection. Likewise, they can also be administered
in intravenous (both bolus and infusion), intraperitoneal, subcutaneous,
topical with or without occlusion, or intramuscular form, all using
forma well known to those of ordinary skill in the pharmaceutical arts.
Advantageously, compounds of the present invention can be
administered in a single daily dose, or the total daily dosage can be
administered in divided doses of two, three or four times daily.
Furthermore, compounds for the present invention can be administered
in intranasal form via topical use of suitable intranasal vehicles, or via
transdermal routes, using those forms of transdermal skin patches well
known to those of ordinary skill in that art. To be administered in the
form of a transdermal delivery system, the dosage administration will,
of course, be continuous rather than intermittent throughout the dosage
regimen.
For combination treatment with more than one active
agent, where the active agents are in separate dosage formulations, the
active agents can be administered concurrently, or they each can be
administered at separately staggered times.
The dosage regimen utilizing the compounds of the present
invention is selected in accordance with a variety of factors including
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type, species, age, weight, sex and medical condition of the patient; the
severity of the condition to be treated; the route of administration; the
renal, hepatic and cardiovascular function of the patient; and the
particular compound thereof employed. A physician or veterinarian of
ordinary skill can readily determine and prescribe the effective amount
of the drug required to prevent, counter or arrest the progress of the
condition. Optimal precision in achieving concentrations of drug within
the range that yields efficacy without toxicity requires a regimen based
on the kinetics of the drug's availability to target sites. This involves a
consideration of the distribution, equilibrium, and elimination of a drug.
The following examples are presented by the way of
illustration and, because various other embodiments will be apparent to
those in the art, the following is not to be construed as a limitation on the
scope of the invention:
EXAMPLE 1
Identification of a partial cDNA for the AOMF05 receptor
Polypeptide sequences of human G-protein coupled
glycoprotein hormone receptors were used as probes to search the EST
database dbEST of NCBI (National Center for Biotechnology Information)
using the search program tFASTA. The sequences chosen were the protein
sequences of known human receptors, i.e., receptors for FSH (Follicle-
stimulating hormone), TSH (thyroid-stimulating hormone}, LH (leutinizing
hormone). An EST (GenBank accession #'I"73957) was found to encode a
polypeptide that is approximately 30% identical to these receptors at the
amino acid level. This EST, containing a sequence of 350 base pairs, was
sequenced from the 5' end of a clone from a total human liver cDNA library
(the LM.A.G.E. ID of this clone = 84521).
The DNA sequence information of this EST was used to isolate
cDNA fragments containing the original EST. DNA sequences of these
fragments were then determined and analyzed, resulting in the identification
of the full-length coding sequence of the AOMFOS gene. The full-length
cDNA sequence was then cloned into a mammalian expression vector.
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PrlmerS
The following primers were used for the isolation of AOMF05 as
described below. For convenience and clarity, the SEQ ID NOS are presented
here. In the following description, primers can be referred to by the
numerical
component of their designation.
871 GGCCATTAATAAAAATGCTAGTGA (SEQ ID N0:5)
F77 GCATTTTTATTAATGGCCGTTATC (SEQ ID N0:6)
F30 GCCATCATTAGGATTCACTGTAAC (SEQ ID N0:7)
8117 GGTCCCTTTTTCCAAGTTGC (SEQ ID N0:8)
8175 TGGATAAAAGAAAGGTCGTTGC (SEQ ID N0:9)
8167 AGAAAGGTCGTTGCCCGCCAAT (SEQ ID N0:10)
F31 ACTGCTCCGGGAAGGGGCTGAC (SEQ ID N0:11)
R104s GAGTCACAACCCCAAAATGC (SEQ ID N0:12)
R126s GGCAACCATTAAAACTTGGA (SEQ ID N0:13)
F1803s AGACAGTTCTGACCAGGTGC (SEQ ID N0:14)
F210s GGCCTGATATCTCTAAGGATTC (SEQ ID N0:15)
869 GCTTGGGTGAAGGCGCTGAG (SEQ ID N0:16)
2O F16 CCTGTGAGCCCCTGAGGTTCA (SEQ ID N0:17)
82289 ATAAACTGCCACCTCTCCTTCTT (SEQ ID N0:18)
NNheMF05-1569
CTAGCTAGCGCCATCATGCCGGGCCCGCTAGGGCTG (SEQ ID N0:19)
CNheMF05-2479 GAACTGTTTGAGATGATTGCTCTT (SEQ ID N0:20)
PBS.838F TTGTGTGGAATTGTGAGCGGATAAC (SEQ ID N0:21)
PBS.873F CCCAGGCTTTACACTTTATGCTTCC (SEQ ID N0:22)
PBS.543R GGGGATGTGCTGCAAGGCGA (SEQ ID N0:23)
PBS.578R CCAGGGTTTTCCCAGTCACGAC (SEQ ID N0:24)
Cloning and sequencing of AOMF05
The full-length sequence of AOMF05 was isolated from a fetal
brain cDNA library by multiple rounds RCCA (Reduced Complexity cDNA
Analysis, described herein). A random and oligo dT primed fetal brain cDNA
library consisting of approximately 4 million primary clones each was
constructed in the plasmid vector pBluescript SK- (Stratagene, La Jolla, CA).
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The primary clones were subdivided into 188 superpools with each pool
containing about 20,000 clones.
For the initial scanning of the fetal brain cDNA library, 5' and 3'
primers predicted to be specific for the AOMF05 EST T73957, (primers F30
and R 117), as well as oligonucleodde primers both 5' and 3' of the polylinker
sequence of the vector (primers PBS.873F and PBS.543R) were used. PCR
reactions were carried out with Amplitaq Gold (Perkin Elmer-Roche,
Branchberg, NJ, U.S.A) using standard PCR conditions as suggested by the
enzyme supplier.
After positive pools were identified, nested insert-vector PCRs
were carried out on the positive pools with the following combinations:
primary reactions, F30+PBS.543R, F30+ PBS.873F; 8117+ PBS.543R,
8117+ FBS.873F. Secondary (nested) reactions, F77+ PBS.578R, F77+
PBS.838F, 871+ PBS.578R, 871+ PBS.838F. PCR products were then
sequenced and assembled. Two new sequencing primers R126s and F1803s
for the 3' and 5' direction were synthesized and used to sequence the
previous nested PCR products. The assembled sequence contained an open
reading frame.
The sequence containing the open reading frame was amplified
using two primers F16 and 82289 and cloned into the vector pCR2.1
(Invitrogen, San Diego, CA) by TA cloning. The AOMF05 sequence was
excised with KpnI+NotI digestion and ligated into pcDNA3.1 (Invitrogen,
San Diego, CA) digested with the same enzymes. This plasmid was named
pMF053.1.A. Later, new 5' sequences were obtained that contained a longer
open reading frame as described below.
Based on the sequence of AOMF05 as assembled, two new
primers F210s and R104s were synthesized and used to scan the fetal brain
and prostate cDNA libraries. After positive superpools were identified, 5'
extension was carried out on these pools using the following primer
combination: 104s+ PBS.578R, R104s+ PBS.838F. The products were
sequenced and assembled into the contig.
From the new contig a walking primer 8175 for the 5' direction
was synthesized. This primer and vector specific primer PBS.538R was used
to scan the superpooled libraries. After positive rows were identified 5'
extension was performed on these rows and the product sequenced and
assembled. From the new sequence two primers F31 and 8167 were picked to
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identify new pools in the fetal brain and prostrate cDNA libraries. After
positive pools were identified, 5' extension was carried with the following
primer combinations: 8167+ PBS 5788, 8167+ PBS.838F. PCR products
were then sequenced and assembled into the contig.
Based on the new sequence, another 5' primer R69 was
synthesized. This primer was then used to amplify with PBS.838F or
PBS.543R on the positive pools in the presence of 5% DMSO. The PCR
products were then sequenced and assembled into a single contig. This
sequence contains an open reading frame of 2850 base pairs, encoding a
polypeptide of 949 amino acids. Two PCR primers NNheMF05-1569 and
CNheMF05-2479 were synthesized and used to amplify the 5' end. The PCR
fragment was digested with NheI and ligated with NheI-digested
pMF053.1.A. The resulting plasmid was verified by physical mapping and
sequencing, and named pcDNA3.1 MFOS.
EXAMPLE 2
The sequence of the two variants of the full length AOMFOS
cDNA are provided in FIGS. lA-1B (SEQ ID NO:1) and FIGS. 4A-4C (SEQ ID
N0:3. The amino acid sequence of the variants of this receptor are provided
in FIG. 2 (SEQ )D N0:2) and FIG. 5 (SEQ II7 N0:4}. FASTA searches and
phylogenetic analysis were performed using the program Pepplot of GCG
(Genetics Computer Group, Madison, Wisconsin, USA). The analysis revealed
that AOMFOS is a member of the G-protein coupled glycoprotein hormone
receptor family. Hydropathy analysis was performed using the program
Pepplot of GCG (Genetics Computer Group, Madison, Wisconsin, USA) and
showed that AOMFOS has 7 transmembrane domains typical of the rhodopsin
family of G-protein coupled receptors. The domains begin at about amino acid
539 of SEQ ID N0:2 or 4. The deduced polypeptide sequence of AOMFOS
contains several sites for cleavage of a signal peptide from the N-terminus of
the protein (FIG. 7).
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EXAMPLE 3
Anal of the pattern of ex~~ression of AOM~,15
Mufti-tissue Northern blot analysis was performed as follows.
Ready-to-use human mufti-tissue Northern blots were purchased from
Clontech (Clontech, Palo Alto, CA, USA). A total of six blots were used to
analyze the expression of AOMFOS in human tissues.
Random Priming
Fragments of the AOMFOS cDNA were labeled with 32P by
random priming using the REDDY-PRIME~ labeling kit (Amersham, Inc.,
Chicago, IL, USA). Reactions were carried using the protocol of the kit
supplier. Approximately 50 ng of DNA in 45 pl of H20 was boiled for 3
minutes., and then quickly chilled to 0°C for 5 minutes. The DNA
solution
was transferred to REDDY-PRIME~ tube and mixed with the lyophilized
reagents in the tube. Then, 5.0 ul of a-32P-dCTP (5000 Ci/mM) was added
and the tube was incubated at 37°C for 15 minutes. The reaction was
stopped
by adding 5.0 pl of 0.5 M EDTA (pH8.0). Unincorporated nucleotides were
removed by gel-filtration using a spun column.
Northern Hybridization.
The labeled fragments were used as probes for AOMFOS RNA.
Hybridizations were carried out in the ExpressHyb buffer of Clontech
following the protocol provided by the membrane supplier Clontech (Palo
Alto, CA, USA). The membranes were prehybridized at 68°C for 1 hr
in the
Expresshyb buffer with gentle agitation. The 32P-labeled probe was
denatured by adding NaOH to a final concentration of 0.2 nM and then
added into the hybridization solution. Hybridizations were performed for 3
hours at 68°C. The membranes were removed from the hybridization buffer
and washed once in 2x SSC, 0.1 % SDS, for 10 min. at room temperature. The
membranes were then washed at 0.1 xSSC, 0.1 % SDS for 30 minutes at
50°C.
The blots were analyzed using a Phosphaimager (Molecular Dynamics,
Sunnyvale, CA, USA).
Analysis.
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AOMFOS was most abundantly expressed in pancreas and
moderately expressed in heart, brain, liver, kidney, skeletal muscle,
placenta,
adrenal medulla, adrenal cortex, thyroid, stomach, and testis (FIG. 8). In all
of
these tissues, AOMFOS was detected as a transcript of ~5.5 kb, except in
placenta where an additional ~4.5 kb messenger was also detected.
EXAMPLE 4
Isolation of ~enomic DNA encoding AOMF05
The AOMF05 cDNA is used as a probe to isolate human
genomic DNA encoding the receptor. The cDNA can be used in its
entirety or portions of the sequence can be used. If portions of the
sequence less than 100 nucleotides are used as a probe, one should
perform homology analysis of the selected probe sequence against
human sequences in general to assess the uniqueness of the chosen
sequence in human DNA. If the chosen sequence exhibits high
homology to a variety of human DNA sequences, then that sequence will
not perform well as a probe specific for AOMF05 genomic DNA. For
example, portions of the cDNA encoding amino acid sequences that are
highly conserved among G-protein coupled receptors can be used.
However, in that case one should expect to identify receptor genes in
addition to AOMF05, and a large number of identified fragments should
be studied further. Thereafter, one will be required to determine which
of the identified DNAs encodes AOMF05. This can be accomplished
simply by sequencing the identified genomic DNA fragments and
comparing the sequences to AOMF05 sequence provided herein (SEQ ID
NOS:1 & 3).
Once a probe sequence has been selected the probe is labeled
by any means known in the art, including but not limited to
incorporation of radioisotopes or biotin. Under appropriately stringent
conditions, the probe is hybridized against a library of human genomic
DNA fragments. The stringency of the hybridization reaction can be
adjusted by means known in the art, e.g., varying salt concentrations
and temperature, to obtain appropriately specific hybridization of the
probe to the target sequence. The fragments identified by the probe can
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be sequenced or subjected to restriction enzyme digestion to confirm that
they contain AOMF05 genomic DNA.
It is possible that the entire genomic gene may not be
contained within any one identified fragment. In that case, one will be
required to perform chromosome walking, e.g., using an identified
fragment as a probe to isolate additional fragments that overlap in the
chromosome, to isolate the entire gene. If the isolation of overlapping
fragments is required, one can use known methods of manipulation of
DNA to construct a contiguous DNA fragment encoding the entire
AOMF05 genomic DNA.
EXAMPLE 5
Transgenic animals
Transgenic animals expressing AOMF05 as a transgene
are provided as follows. A polynucleotide having an AOMF05 nucleotide
sequence, e.g., the nucleotide sequence of a cDNA or genomic DNA
encoding a full length AOMF05 receptor, or a polynucleotide encoding a
partial sequence of the receptor, sequences flanking the coding
sequence, or both, can be combined into a vector for the integration of the
polynucl~otide into the genome of an animal. The AOMF05 sequence
can be from a human AOMF05 or from the animal's AOMF05.
In this example, the target cell for transgene introduction
is a marine embryonic stem cell (ES). ES cells can be obtained from pre-
implantation embryos of a variety of non-human animals cultured in
vitro and fused with embryos (M. J. Evans et al., Nature 292:154-156
(1981); Bradley et al., Nature 309:255-258 (I984); Gossler et al. Proc. Natl.
Acad. Sci. USA 83:9065-9069 (1986); and Robertson et al., Nature 322:445-
448 (198fi)).
The transgene is introduced into the marine ES cells by
mieroinjection, however, a variety of standard techniques such as DNA
transfection, or retrovirus-mediated transduction can be used. The
injected ES cells are then combined with blastocysts from a non-human
animal. The introduced ES cells colonize the embryo and contribute to
the germ line of the resulting chimeric animal (R. Jaenisch, Science
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240: 1468-1474 ( 1988)). The chimeric mice are screened for individuals in
which germline transformation has occurred. These are crossed to
produce animals homozygous for the transgene.
The targeted recombination events as well as the resulting
mice are evaluated by techniques well known in the art, including but
not limited to DNA (Southern) hybridization to detect the targeted allele,
polymerase chain reaction (PCR), polyacrylamide gel electrophoresis
(PAGE) and Western blots to detect DNA, RNA and protein.
Three basis types of transgenic animals are created
depending on the construction of the transgene vector. If the vector is
designed to include a nucleotide sequence that encodes a full length
human AOMF05 receptor and to integrate at a site other than the
animal's endogenous AOMF05 gene, the resultant transgenic animal
will express both a native and human AOMF05 receptors. If the vector
is designed without a cognate AOMF05 gene and to integrate at the site
of the animal's endogenous AOMF05 gene such that after integration
the endogenous gene is altered to such an extent that the animal lacks a
functional AOMF05 receptor, then a knockout animal is produced.
Finally, if the vector is designed to replace the endogenous AOMF05
gene with a human gene, or is designed to change the sequence of the
endogenous gene to encode the amino acid sequence of the human gene,
i.e., is humanized, then the resultant animal lacks a native AOMF05
receptor and expresses a human AOMF05 receptor. Animals having a
human gene and lacking an endogenous gene can also be created by
crossing the first type of animal with a knockout animal to obtain
animals homozygous for the knockout and homozygous for the added
human AOMF05 gene. This can be facilitated if the human gene
integrates in a chromosome different from the chromosome carrying
the endogenous AOMF05 gene.
Transgenic animals are a source of cells and tissues for use
in assays of AOMF05 modulation, activation or inhibition. Cells can be
removed from the animals, established as cell lines and maintained in
culture as convenient.
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EXAMPLE 6
Glutathione S-transferase ("GST") AOMF05 receptor fusion
constructs.
Polypeptide fusion constructs are made by inframe fusion of
all or a portion of the N-terminal ligand-binding domain of the AOMF05
G-protein coupled glycoprotein hormone receptor and the carboxy
terminus of the GST gene. The disclosure of SE(a ID NOS:1-4 allow the
artisan of ordinary skill to construct any such nucleic acid molecule
encoding a GST-AOMF05 fusion protein. In particular, fusions can be
constructed using a polynucleotide that encodes the N-terminal
fragment of AOMF05 from about amino acid 20 to about 539, or from
about 20 to the end of the sequence of SEla ID N0:2, fused to GST C-
terminus.
Soluble recombinant AOMF05 fusion proteins can be
expressed in various expression systems, some of which are described
herein, including Spodoptera frugiperdu (Sf21) insect cells using a
baculovirus expression vector (e.g., Bac-N-Blue DNA from Invitrogen or
pAcG2T from Pharmingen).
The fusion protein is then loaded onto a glutathione
column. The C-terminal domain of GST binds to the glutathione and the
N-terminal region of AOMF05 is exposed to the buffer phase. After
washing the column, a sample that may contain a ligand of the
AOMF05 receptor is passed over the column. The sample can be cell or
tissue extracts, bodily fluids or compounds or molecules that are
purified or synthesized. The sample can be applied directly or after
dilution or dialysis in a buffer approximating physiological conditions.
Ligands of the receptor are bound by the N-terminal domain of AOMF05.
After washing the column the Iigands are eluted. This can be achieved,
for example, by applying a gradient of NaCl to the column in wash
buffer. Unknown ligands present in biological extracts or fluids can be
characterized by standard chemical and biochemical methods. Ligands
identified in this method can be used as candidates in assays for
agonists or antagonists of the AOMF05 receptor.
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Assays for ligands can also be conducted as described below
for assays far agonist and antagonists of AOMF05. A candidate
compound or molecule that shows agonist or antagonist activity can also
be a ligand for AOMF05. -
EXAMPLE 7
Assavs for agonists and antagonists of the rece
In any assay using recombinant host cells it is first
necessary to produce the cells as described elsewhere herein. Briefly, a
polynucleotide of the present invention is used to transform or transfect
the appropriate cells, or cells can be obtained and cultured from an
appropriate transgenic animal.
Melanophore system.
The melanophore screening system is described in WO
92/01810, published February 6; 1992. Briefly, melanophores are
transfected to express the AOMF05 G-protein coupled receptor. In an
assay for antagonists, the transformed melanophores are exposed to
both an activating ligand and a candidate compound. Inhibition of the
signal generated by the ligand indicates that the candidate is a potential
antagonist of the receptor. In an assay for an agonist, the cells are
contacted with candidate compounds and it is determined whether any
compound activates the receptor to generate a signal. Activation of the
receptor indicates that the candidate is a potential agonist of the
receptor.
Yeast expressing mammalian adenylate cyclase.
Screening methods employing yeast that express
mammalian adenylate cyclase are described in WO 95/30012, published
November 9, 1995. These yeast can be engineered to co-express the
AOMF05 receptor in the presence of an appropriate G-protein. In an
assay for antagonists, the transformed yeast are exposed to both an
activating ligand of AOMF05 and a candidate compound. Inhibition of
the signal generated by the ligand indicates that the candidate is a
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potential antagonist of the receptor. In an assay for an agonist, the cells
are contacted with candidate compounds and it is determined whether
any~compound activates the receptor to generate a signal. Activation of
the receptor indicates that the candidate is a potential agonist of the
receptor.
Yeast pheromone protein surrogate screening.
Yeast cells engineered to produce pheromone system
protein surrogates can be used to screen for the ability of the surrogate to
substitute for the cognate yeast pheromone receptor as described in WO
94/23025, published October 13, 1994. Generally, the method involves
expressing the AOMF05 G-protein coupled receptor in Saccharomyces
cerevisiae in which the receptor is linked to pheromone pathway. In
this system, the yeast Ga subunit is generally deleted and replaced with
a mammalian Ga protein so that the mammalian G protein-coupled
receptor can be coupled to the yeast pheromone pathway. Members of a
plasmid library capable of expressing peptides of random sequences are
introduced into an appropriate yeast strain. Clones encoding agonist
ligands for the AOMF05 receptor can be selected for their stimulation of
the pheromone pathway. Clones encoding antagonist ligands for the
AOMF05 receptor can be selected for their inhibition of the pheromone
pathway in the presence of an AOMF05 agonist. Alternatively, libraries
of chemicals can be screened for their agonist or antagonist activity by
testing the chemicals directly.
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Phospholipase second signal screening
Another screening technique involves expressing the
AOMF05 receptor wherein the receptor is linked to a phospholipase C or
D. Cells including CHO, endothelial, embryonic kidney and other -cells
can be used. As in other screens, ligand and candidates are screened
for agonist or antagonist activities by detecting the activation or
inhibition or the receptor's activation of the phospholipase second
signal. An example of one such system using yeast cells expressing a
heterologous phospholipase is found in WO 96/40939, published
December 19, 1996.
Yeast two-hybrid system
The yeast two-hybrid system expressing the AOMF05 G-
protein coupled receptor can be used for screening for agonists and
antagonists of the receptor (Fields and Song, 1989, Nature 340:245-246).
In particular, the entire or portions of the extracellular domain of the G-
protein coupled receptor can be fused to the DNA binding domain of
transcription factor Gal4 or LexA. Yeast cells expressing these
constructs are used to carry out screening for molecules that interact
with the G-protein coupled receptor by using standard protocols such as
those described previously (Fields and Song, 1989) of the two-hybrid
screening method. Such molecules represent potential agonists or
antagonists of the receptor.
E~;.AMPLE 8
Compounds or molecules that are modulators of the
receptor can be detected in assay described or as follows. An antibody
specific for the extracellular domain of the receptor is obtained by
standard techniques. The antibody can be polyclonal or monoclonal.
The amity of the antibody for the extracellular domain of the receptor
should preferably be at least 106, and more preferably at least 108, to
simplify conducting the assay. A cell culture that expresses the receptor
is provided. The cell culture can be one that naturally expresses the
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receptor, a cell line stably or transiently transfected with an expression
vector including the receptor gene, or derived from a transgenic animal
having a transgene including the receptor gene.
Two samples of the culture are used in the assay. Cane
sample is used as a control and is treated with a placebo, i.e., a
compound or molecule determined to have no modulatory effects on the
receptor in the assay. The second sample is treated with a candidate
modulator. At various times after or during treatment a portion of the
culture can be withdrawn. The antibody can then be used to qualify or
quantify the amount of receptor present on the surface of the cell. This
can be done by numerous techniques known in the art including using
antibody detectably labeled with '25I, gold, enzyme or other known labels.
Alternatively, a detectable label can be carried on a second antibody
specific for the first. The amount of receptor found on the cells treated
with a potential modulator is quantitatively or qualitatively compared to
the amount of receptor found on the control cells. A change in the
former relative to the latter is indicative of the whether or not the test
compound is a modulator of the receptor.
In an alternative form of the assay one can treat cells as
described herein and then isolate the receptors present in treated and
control cells. The receptor preparations can be made as crude cell
extracts, membrane or intracellular fractions of the cells or after
purification steps, e.g., chromatography, precipitation or affinity
isolation steps. Crude, partially or highly purified preparations of
receptors can be analyzed for receptor content, e.g., by using antibodies
specific for the receptor.
In any assay it can be advantageous to devise an internal
control so that the results of different runs of assays can be compared to
each other. A cellular protein that is unrelated to the receptor and
present in relatively constant amounts in the cells used in the assay can
serve as an internal control.
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EXAMPLE 9
The present invention includes methods of identifying
compounds that specifically bind to an AOMF05 protein, as well as
compounds identified by such methods. The specificity of binding of
compounds having affinity for an AOMF05 protein is shown by measuring
the affinity of the compounds for recombinant cells expressing the cloned
receptor or for membranes from these cells. Expression of the cloned receptor
and screening for compounds that bind to an AOMF05 protein or that inhibit
the binding of a known, radiolabeled ligand of AOMF05 to these cells, or
membranes prepared from these cells, provides an effective method for the
rapid selection of compounds with high affinity for an AOMF05 protein.
Such ligands need not necessarily be radiolabeled but can also be nonisotopic
compounds that can be used to displace bound radiolabeled compounds or
that can be used as activators in functional assays. Compounds identified by
the herein method are likely to be agonists or antagonists of AOMF05 and
may be peptides, proteins, or non-proteinaceous organic molecules.
Therefore, the present invention includes assays by which
AOMF05 agonists and antagonists may be identified. Methods for
identifying agonists and antagonists of other receptors are well known in the
art and can be adapted to identify agonists and antagonists of AOMF05.
Accordingly, the present invention includes a method for determining whether
a candidate compound is a potential agonist or antagonist of AOMF05 that
comprises:
(a) transfecting cells with an expression vector encoding an
AOMF05 protein;
(b} allowing the transfected cells to grow for a time sufficient
to allow the AOMF05 protein to be expressed;
(c) exposing the cells to a labeled ligand of an AOMF05
protein in the presence and in the absence of the candidate compound;
(d) measuring the binding of the labeled ligand to the
AOMF05 protein; where if the amount of binding of the labeled ligand is less
in the presence of the candidate compound than in the absence of the
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candidate compound, then the candidate compound is a potential agonist or
antagonist of an AOMFOS protein.
The conditions under which step (c) of the method is practiced
are conditions that are typically used in the art for the study of protein-
ligand
interactions: e.g., physiological pH; salt conditions such as those
represented
by such commonly used buffers as PBS or in tissue culture media; a
temperature of about 4°C to about 55°C.
The present invention also includes a method for determining
whether a candidate compound is capable of binding to an AOMFOS protein,
i.e., whether the candidate compound is a potential agonist or an antagonist
of an AOMFOS protein, where the method comprises:
(a) providing test cells by transfecting cells with an
expression vector that directs the expression of an AOMFOS protein in the
cells;
(b) exposing the test cells to the candidate compound;
(c) measuring the amount of binding of the candidate
compound to the AOMFOS protein;
(d) comparing the amount of binding of the candidate
compound to the AOMFOS protein in the test cells with the amount of
binding of the candidate compound to control cells that have not been
transfected with an AOMFOS protein;
wherein if the amount of binding of the candidate compound is
greater in the test cells as compared to the control cells, the candidate
compound is capable of binding to an AOMFOS protein. Determining
whether the candidate compound is actually an agonist or antagonist can
then be accomplished by the use of functional assays such as, e.g., the assay
involving the use of promiscuous G-proteins described herein.
The conditions under which step (b) of the method is practiced
are conditions that are typically used in the art for the study of protein-
ligand
interactions: e.g., physiological pH; salt conditions such as those
represented
by such commonly used buffers as PBS or in tissue culture media; a
temperature of about 4°C to about SS°C.
In a particular embodiment of the herein-described methods, the
cells are eukaryotic cells. In another embodiment, the cells are mammalian
cells. In other embodiments, the cells are L cells L-M(TK-) (ATCC CCL 1.3), L
cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86),
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CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651),
CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658),
HeLa (ATCC CCL 2}, C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) or
MRC-5 (ATCC CCL 171 ). -
The assays described herein can be carried out with cells that
have been transiently or stably transfected with an AOMFOS protein.
Transfection is meant to include any method known in the art for introducing
an AOMFOS protein into the test cells. For example, transfection includes
calcium phosphate or calcium chloride mediated transfecdon, lipofection,
infection with a retroviral construct containing an AONIFOS protein, and
electroporation.
Where binding of the candidate compound or agonist to
AOMFOS is measured, such binding can be measured by employing a labeled
candidate compound or agonist. The candidate compound or agonist can be
labeled in any convenient manner known to the art, e.g., radioactively,
fluorescently, enzymatically.
In particular embodiments of the herein-described methods, the
AOMFOS protein has an amino acid sequence of SEQ ID NOS:2 or 4.
The herein-described methods can be modified in that, rather
than exposing the test cells to the candidate compound, membranes can be
prepared from the test cells and those membranes can be exposed to the
candidate compound. Such a modification utilizing membranes rather than
cells is well known in the art and is described in, e.g., Hess et al., 1992,
Biochem. Biophys. Res. Comm. 184:260-268.
Accordingly, the present invention provides a method for
determining whether a candidate compound is capable of binding to an
AOMFOS protein comprising:
(a) providing test cells by transfecting cells with an
expression vector that directs the expression of an AOMFOS protein in the
cells;
(b) preparing membranes containing the AOMFOS protein
from the test cells and exposing the membranes to a ligand of an AOMFOS
protein under conditions such that the ligand binds to the AOMFOS protein in
the membranes;
(c) subsequently or concurrently to step (b), exposing the
membranes from the test cells to a candidate compound;
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(d) measuring the amount of binding of the ligand to the
AOMFOS protein in the membranes in the presence and the absence of the
candidate compound;
(e) comparing the amount of binding of the ligand to an
AOMFOS protein in the membranes in the presence and the absence of the
candidate compound where a decrease in the amount of binding of the
ligand to an AOMFOS protein in the membranes in the presence of the
candidate compound indicates that the candidate compound is capable of
binding to an AOMFOS protein;
The present invention provides a method for determining
whether a candidate compound is capable of binding to an AOMFOS protein
comprising:
(a} providing test cells by transfecting cells with an
expression vector that directs the expression of an AOMFOS protein in the
cells;
(b) preparing membranes containing the AOMFOS protein
from the test cells and exposing the membranes from the test cells to the
candidate compound;
(c) measuring the amount of binding of the candidate
compound to the AOMFOS protein in the membranes from the test cells;
(d) comparing the amount of binding of the candidate
compound to the AOMFOS protein in the membranes from the test cells with
the amount of binding of the candidate compound to membranes from
control cells that have not been transfected with an AOMFOS protein;
where if the amount of binding of the candidate compound to
the AOMFOS protein in the membranes from the test cells is greater than the
amount of binding of the candidate compound to the membranes from the
control cells, then the candidate compound is capable of binding to an
AOMFOS protein
EXAMPLE 10
IJse of AOMF05 sequence fn_r gene ther,~pv
Nucleic acid according to the present invention, e.g.
encoding the authentic biologically active AOMF05 polypeptide or a
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functional fragment thereof, can be used in a method of gene therapy, to
treat a patient who is unable to synthesize the active polypeptide or
unable to synthesize it at the normal level, thereby providing the effect
provided by the wild-type with the aim of treating and/or preventing one
or more symptoms of one or more other diseases.
Vectors such as viral vectors have been used to introduce
genes into a wide variety of different target cells. Typically the vectors
are exposed to the target cells so that transfection can take place in a
sufficient proportion of the cells to provide a useful therapeutic or
prophylactic effect from the expression of the desired polypeptide. The
transfected nucleic acid can be permanently incorporated into the
genome of each of the targeted cells, providing long lasting effect, or
alternatively the treatment may have to be repeated periodically.
A variety of vectors, both viral vectors and plasmid vectors,
are known in the art, see e.g. US Patent No. 5,252,479 and WO 93/07282.
In particular, a number of viruses have been used as gene transfer
vectors, including adenovirus, papovaviruses, such as SV40, vaccinia
virus, herpesviruses, including HSV and EBV, and retroviruses,
including gibbon ape leukemia virus, Rous Sarcoma Virus,
Venezualian equine enchephalitis virus, Moloney marine leukemia
virus and marine mammary tumorvirus. Many gene therapy protocols
have used disabled marine retroviruses.
Disabled virus vectors are produced in helper cell lines in
which genes required for production of infectious viral particles are
expressed. Helper cell lines are generally missing a sequence which is
recognised by the mechanism which packages the viral genome and
produce virions which contain no nucleic acid. A viral vector which
contains an intact packaging signal along with the gene or other
sequence to be delivered (e.g. encoding the AOMF05 polypeptide or a
fragment thereof) can be packaged in the helper cells into infectious
virion particles, which can then be used for the gene delivery.
Other known methods of introducing nucleic acid into cells
include electroporation, calcium phosphate co-precipitation,
mechanical techniques such as microinjection, transfer mediated by
liposomes and direct DNA uptake and receptor-mediated DNA transfer.
Liposomes can encapsulate RNA, DNA and virions for delivery to cells.
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Depending on factors such as pH, ionic strength and divalent cations
being present, the composition of liposomes can be tailored for targeting
of particular cells or tissues. Liposomes include phospholipids and may
include lipids and steroids and the composition of each such component
can be altered. Targeting of liposomes can also be achieved using a
specific binding pair member such as an antibody or binding fragment
thereof, a protein, a sugar or a glycolipid.
The aim of gene therapy using nucleic acid encoding the
polypeptide, or an active portion thereof, is to increase the amount of the
expression product of the nucleic acid in cells in which the level of the
wild-type polypeptide is absent or present only at reduced levels. Such
treatment can be therapeutic or prophylactic, particularly in the
treatment of individuals known through screening or testing to have an
AOMF05 allele associated with a disease state and hence a
predisposition to the disease.
Similar techiques can be used for anti-sense regulation of
gene expression, e.g. targeting an antisense nucleic acid molecule to
cells in which a mutant form of the gene is expressed, the aim being to
reduce production of the mutant gene product. Other approaches to
specific down-regulation of genes are well known, including the use of
ribozymes designed to cleave specific nucleic acid sequences. Ribozymes
are nucleic acid molecules, actually RNA, which specifically cleave
single-stranded RNA, such as mRNA, at defined sequences, and their
specificity can be engineered. Hammerhead ribozymes can be preferred
because they recognize base sequences of about 11-18 bases in length,
and so have greater specificity than ribozymes of the Tetrahymenac type
which recognise sequences of about 4 bases in length, though the latter
type of ribozymes can also be useful in certain circumstances as will be
recognized by one of skill in the art. References on the use of ribozymes
include Marschall, et al. 1994. Cellular and Molecular Neurobiology
14(5):523; Hasselhoff, 1988. Nature 334:585 and Cech, 1988. J. Amer.
Med. Assn. 260;3030.
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EXAMPLE 11
Construction of po]~mucleotides pnroding an AOMF05 recestor rotei
Two examples of the full length amino acid sequence of the
AOMF05 receptor protein is provided in SEQ ID NOS:2 & 4. A native
human cDNA sequence including an open reading frame encoding the
amino acid sequence of AOMF05, is provided in SEQ ID NOS:1 & 3.
Because of the degeneracy of the genetic code, the sequence of the open
reading frame provided in SE(a ID NOS:1 & 2 are anly examples of the
many nucleotide sequences that can encode the amino acid sequence of
variant a and b of AOMF05. One of ordinary skill in the art is familiar
with the genetic code and can, using standard techniques of molecular
biology, can generate polynucleotides having alternative nucleotide
sequences that encode the same amino acid sequences provided in SEfa
ID NOS:2 or 4.
IS Alternative nucleotide sequences can be DNA, RNA,
mixtures of DNA and RNA or can include alternative linkages between
nucleotides as described herein.
_ 6p _
