Note: Descriptions are shown in the official language in which they were submitted.
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RNA METABOLISM PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of RNA
metabolism proteins
and to the use of these sequences in the diagnosis, treatment, and prevention
of nervous system,
autoimmune/inflammatory, and cell proliferative disorders, including cancer.
BACKGROUND OF THE INVENTION
Ribonucleic acid (RNA) is a linear single-stranded polymer of four
nucleotides, ATP, CTP,
1Q UTP, and GTP. In most organisms, RNA is transcribed as a copy of
deoxyribonucleic acid (DNA),
the genetic material of the organism. In retroviruses RNA rather than DNA
serves as the genetic
material. RNA copies of the genetic material encode proteins or serve various
structural, catalytic, or
regulatory roles in organisms. RNA is classified according to its cellular
localization and function.
Messenger RNAs (mRNAs) encode polypeptides. Ribosomal RNAs (rRNAs) are
assembled, along
with ribosomal proteins, into ribosomes, which are cytoplasmic particles that
translate mRNA into
polypeptides. Transfer RNAs (tRNAs) are cytosolic adaptor molecules that
function in mRNA
translation by recognizing both an mRNA codon and the amino acid that matches
that codon.
Heterogeneous nuclear RNAs (hnRNAs) include mRNA precursors and other nuclear
RNAs of
various sizes. Small nuclear RNAs (snRNAs) are a part of the nuclear
spliceosome complex that
removes intervening, non-coding sequences (introns) and rejoins exons in pre-
mRNAs.
Proteins are associated with RNA during its transcription from DNA, RNA
processing, and
translation of mRNA into protein. Proteins are also associated with RNA as it
is used for structural,
catalytic, and regulatory purposes.
Various proteins are necessary for processing of transcribed RNAs in the
nucleus. Pre-
mRNA processing steps include capping at the 5' end with methylguanosine,
polyadenylating the 3'
end, and splicing to remove introns. The spliceosomal complex is comprised of
five small nuclear
ribonucleoprotein particles (snRNPs) designated U1, U2, U4, U5, and U6. Each
snRNP contains a
single species of snRNA and about ten proteins. The RNA components of some
snRNPs recognize
and base-pair with intron consensus sequences. The protein components mediate
spliceosome
assembly and the splicing reaction.
An early step in pre-mRNA cleavage involves the cleavage factor Im (CF Im).
The human
CF Im protein aids in the recruitment and assembly of processing factors that
make up the 3' end
processing complex (Ruegsegger, U. et al (1998) Mol. Cell. 1:243-253). The
marine formin binding
proteins (FBP's) FBP11 and FBP12 are components of pre-mRNA splicing complexes
that facilitate
the bridging of 5' and 3' ends of the intron. These proteins function through
bridging interactions
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invloving U1 and U2 snRNPs. Autoantibodies to snRNP proteins are found in the
blood of patients
with systemic lupus erythematosus (Stryer, L. (1995) Biochemistry W.H. Freeman
and Company,
New York NY, p. 863).
Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified that
have roles in
splicing, exporting of the mature RNAs to the cytoplasm, and mRNA translation
(Biamonti, G. et al.
(1998) Clin. Exp. Rheumatol. 16:317-326). Some examples of hnRNPs include the
yeast proteins
Hrplp, involved in cleavage and polyadenylation at the 3' end of the RNA;
Cbp80p, involved in
capping the 5' end of the RNA; and Npl3p, a homolog of mammalian hnRNP A1,
involved in export
of mRNA from the nucleus (Shen, E.C. et al. (1998) Genes Dev. 12:679-691).
HnRNPs have been
shown to be important targets of the autoimmune response in rheumatic diseases
(Biamonti, su ra .
Many snRNP and hnRNP proteins are characterized by an RNA recognition motif
(RRM).
(Reviewed in Birney, E. et al. (1993) Nucleic Acids Res. 21:5803-5816.) The
RRM is about 80 amino
acids in length and forms four ~i-strands and two a-helices arranged in an
a/(3 sandwich. The RRM
contains a core RNP-1 octapeptide motif along with surrounding conserved
sequences. In addition to
snRNP proteins, examples of RNA-binding proteins which contain the above
motifs include
heteronuclear ribonucleoproteins which stabilize nascent RNA and factors which
regulate alternative
splicing. Alternative splicing factors include developmentally regulated
proteins, specific examples of
which have been identified in lower eukaryotes such as Drosophila melano~aster
and Caenorhabditis
elegans. These proteins play key roles in developmental processes such as
pattern formation and sex
determination, respectively. (See, for example, Hodgkin, J. et al. (1994)
Development 120:3681-3689.)
Ribonucleases (RNases) catalyze the hydrolysis of phosphodiester bonds in RNA
chains, thus
cleaving the RNA. For example, RNase P is a ribonucleoprotein enzyme which
cleaves the 5' end of
pre-tRNAs as part of their maturation process. RNase H digests the RNA strand
of an RNA/DNA
hybrid. Such hybrids occur in cells invaded by retroviruses, and RNase H is an
important enzyme in
the retroviral replication cycle. RNase H domains are often found as a domain
associated with
reverse transcriptases. RNase activity in serum and cell extracts is elevated
in a variety of cancers and
infectious diseases (Schein, C.H. (1997) Nat. Biotechnol. 15:529-536).
Regulation of RNase activity
is being investigated as a means to control tumor angiogenesis, allergic
reactions, viral infection and
replication, and fungal infections.
Degradation of mRNAs having premature termination or nonsense codons is
accomplished
through a surveillance mechanism that has been termed nonsense-mediated mRNA
decay (NMD).
This mechanism helps eliminate flawed mRNAs that might code for nonfunctional
or deleterious
polypeptides. Various NMD components are linked to both yeast and human RNA
metabolism
disorders (Hentze, M. and Kulozik, A. (1999) Cell 96:307-310).
The conversion of information in the form of mRNA to protein involves the many
ribosomal
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proteins of the translation machinery of the cell. The eukaryotic ribosome is
composed of a 60S
(large) subunit and a 40S (small) subunit, which together form the 80S
ribosome. In addition to the
18S, 28S, SS, and 5.8S rRNAs, the ribosome also contains more than fifty
proteins. The ribosomal
proteins have a prefix which denotes the subunit to which they belong, either
L (large) or S (small).
Initiation of translation requires the participation of several initiation
factors, many of which
contain multiple subunits. One eukaryotic initiation factor (EIF~ EIFSA is an
18-kD protein
containing the unique amino acid residue, hypusine (N epsilon-(4-amino-2-
hydroxybutyl)lysine)
(Rinaudo, M. et al. (1993) Gene 137:303-307).
The release factor eRF carries out termination of translation. eRF recognizes
stop codons in
the mRNA, leading to the release of the polypeptide chain from the ribosome.
The discovery of new RNA metabolism proteins and the polynucleotides encoding
them
satisfies a need in the art by providing new compositions which are useful in
the diagnosis, prevention,
and treatment of nervous system, autoimmune/inflammatory, and cell
proliferative disorders,
including cancer.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, RNA metabolism proteins,
referred to collectively
as "RMEP" and individually as "RMEP-1 ", "RMEP-2", "RMEP-3", "RMEP-4", "RMEP-
5",
"RMEP-6", "RMEP-7", "RMEP-8", "RMEP-9", "RMEP-10", "RMEP-11", "RMEP-12" and
"RMEP-13". In one aspect, the invention provides an isolated polypeptide
comprising an amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-13, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-13, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
NO:1-13, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-13. In one alternative, the invention provides an
isolated polypeptide
comprising the amino acid sequence of SEQ ID NO:l-13.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising an
amino acid sequence selected from the group consisting of a) an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-13, b) a naturally occurring amino acid
sequence having at least
90% sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
13, c) a biologically active fragment of an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-13, and d) an immunogenic fragment of an amino acid sequence
selected from the group
consisting of SEQ ID NO:l-13. In one alternative, the polynucleotide encodes a
polypeptide selected
from the group consisting of SEQ ID NO:1-13. In another alternative, the
polynucleotide is selected
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from the group consisting of SEQ ID N0:14-26.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising
an amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-13, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-13, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
NO:1-13, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:l-13. In one alternative, the invention provides a
cell transformed with the
recombinant polynucleotide. In another alternative, the invention provides a
transgenic organism
comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising an
amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-13, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-13, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
NO:1-13, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-13. The method comprises a) culturing a cell under
conditions suitable for
expression of the polypeptide, wherein said cell is transformed with a
recombinant polynucleotide
comprising a promoter sequence operably linked to a polynucleotide encoding
the polypeptide, and b)
recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino acid
sequence selected from the group consisting of SEQ ID NO:1-13, b) a naturally
occurring amino acid
sequence having at least 90% sequence identity to an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-13, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-13, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-13.
The invention further provides an isolated polynucleotide comprising a
polynucleotide sequence
selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID N0:14-26, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleodde sequence selected from the group consisting of SEQ
ID N0:14-26, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to b), and e)
an RNA equivalent of a)-d). In one alternative, the polynucleotide comprises
at least 60 contiguous
nucleotides.
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Additionally, the invention provides a method for detecting a target
polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide comprising a
polynucleotide sequence
selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID N0:14-26, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:14-26, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to b), and e)
an RNA equivalent of a)-d). The method comprises a) hybridizing the sample
with a probe comprising
at least 20 contiguous nucleotides comprising a sequence complementary to said
target polynucleotide
in the sample, and which probe specifically hybridizes to said target
polynucleotide, under conditions
whereby a hybridization complex is formed between said probe and said target
polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and
optionally, if present, the amount thereof. In one alternative, the probe
comprises at least 60 contiguous
nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample, said
target polynucleotide having a sequence of a polynucleotide comprising a
polynucleotide sequence
selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID N0:14-26, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:14-26, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to b), and e)
an RNA equivalent of a)-d). The method comprises a) amplifying said target
polynucleotide or
fragment thereof using polymerase chain reaction amplification, and b)
detecting the presence or
absence of said amplified target polynucleotide or fragment thereof, and,
optionally, if present, the
amount thereof.
The invention further provides a pharmaceutical composition comprising an
effective amount
of a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:l-13, b) a
naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-13, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-13, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-13, and a
pharmaceutically acceptable
excipient. In one embodiment, the pharmaceutical composition comprises an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-13. The invention
additionally provides a method
of treating a disease or condition associated with decreased expression of
functional RMEP, comprising
administering to a patient in need of such treatment the pharmaceutical
composition.
The invention also provides a method for screening a compound for
effectiveness as an
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agonist of a polypeptide comprising an amino acid sequence selected from the
group consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO:1-13, b) a
naturally occurring
amino acid sequence having at least 90% sequence identity to an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-13, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-13, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-13. The method
comprises a)
exposing a sample comprising the polypeptide to a compound, and b) detecting
agonist activity in the
sample. In one alternative, the invention provides a pharmaceutical
composition comprising an agonist
compound identified by the method and a pharmaceutically acceptable excipient.
In another
alternative, the invention provides a method of treating a disease or
condition associated with
decreased expression of functional RMEP, comprising administering to a patient
in need of such
treatment the pharmaceutical composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide comprising an amino acid sequence selected from
the group consisting
of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-
13, b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-13, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-13, and d) an
immunogenic fragment
of an amino acid sequence selected from the group consisting of SEQ ID NO:1-
13. The method
comprises a) exposing a sample comprising the polypeptide to a compound, and
b) detecting
antagonist activity in the sample. In one alternative, the invention provides
a pharmaceutical
composition comprising an antagonist compound identified by the method and a
pharmaceutically
acceptable excipient. In another alternative, the invention provides a method
of treating a disease or
condition associated with overexpression of functional RMEP, comprising
administering to a patient
in need of such treatment the pharmaceutical composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-13, b) a
naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-13, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:l-13, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-13. The method
comprises a) combining
the polypeptide with at least one test compound under suitable conditions, and
b) detecting binding
of the polypeptide to the test compound, thereby identifying a compound that
specifically binds to the
polypepdde.
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The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-13,
b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-13, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-13, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-13. The
method comprises a) combining the polypeptide with at least one test compound
under conditions
permissive for the activity of the polypeptide, b) assessing the activity of
the polypeptide in the
presence of the test compound, and c) comparing the activity of the
polypeptide in the presence of the
test compound with the activity of the polypepdde in the absence of the test
compound, wherein a
change in the activity of the polypeptide in the presence of the test compound
is indicative of a
compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected from the group consisting of SEQ ID N0:14-26, the method
comprising a)
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs),
clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments
used to assemble full-
length sequences encoding RMEP.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of RMEP.
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-
specific expression
patterns of each nucleic acid sequence as determined by northern analysis;
diseases, disorders, or
conditions associated with these tissues; and the vector into which each cDNA
was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA clones
encoding RMEP were isolated.
Table 5 shows the tools, programs> and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
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that this invention is not limited to the particular machines> materials and
methods described, as these
may vary. It is also fo be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth
Unless defined otherwise, all technical and scientific terms used herein have
the same meanings
as commonly understood by one of ordinary skill in the art to which this
invention belongs. Although
any machines, materials, and methods similar or equivalent to those described
herein can be used to
practice or test the present invention, the preferred machines, materials and
methods are now described.
All publications mentioned herein are cited for the purpose of describing and
disclosing the cell lines,
protocols, reagents and vectors which are reported in the publications and
which might be used in
connection with the invention. Nothing herein is to be construed as an
admission that the invention is
not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"RMEP" refers to the amino acid sequences of substantially purified RMEP
obtained from any
species, particularly a mammalian species, including bovine, ovine, porcine,
marine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
RMEP. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of RMEP either by
directly interacting with
RMEP or by acting on components of the biological pathway in which RMEP
participates.
An "allelic variant" is an alternative form of the gene encoding RMEP. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times in
a given sequence.
"Altered" nucleic acid sequences encoding RMEP include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as RMEP or a
polypeptide with at least one functional characteristic of RMEP. Included
within this definition are
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polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding RMEP, and improper or unexpected hybridization to
allelic variants, with a
locus other than the normal chromosomal locus for the polynucleotide sequence
encoding RMEP. The
encoded protein may also be "altered," and may contain deletions, insertions,
or substitutions of amino
acid residues which produce a silent change and result in a functionally
equivalent RMEP. Deliberate
amino acid substitutions may be made on the basis of similarity in polarity,
charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues,
as long as the biological
or immunological activity of RMEP is retained. For example, negatively charged
amino acids may
include aspartic acid and glutamic acid, and positively charged amino acids
may include lysine and
arginine. Amino acids with uncharged polar side chains having similar
hydrophilicity values may
include: asparagine and glutamine; and serine and threonine. Amino acids with
uncharged side chains
having similar hydrophilicity values may include: leucine, isoleucine, and
valine; glycine and alanine;
and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid sequence
to the complete native amino acid sequence associated with the recited protein
molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well known
in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity of
RMEP. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of RMEP either by
directly interacting with RMEP or by acting on components of the biological
pathway in which RMEP
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments thereof,
such as Fab, F(ab')2, and Fv fragments, which are capable of binding an
epitopic determinant.
Antibodies that bind RMEP polypepddes can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used
to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from
the translation of RNA, or
synthesized chemically, and can be conjugated to a carrier protein if desired.
Commonly used carriers
that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and keyhole
limpet hemocyanin (KLH). The coupled peptide is then used to immunize the
animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
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makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies which
bind specifically to antigenic determinants (particular regions or three-
dimensional structures on the
protein). An antigenic determinant may compete with the intact antigen (i.e.,
the immunogen used to
elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic RMEP, or,of
any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising a
given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution
Compositions comprising polynucleotide sequences encoding RMEP or fragments of
RMEP may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCl), detergents
(e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(PE Biosystems,
Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which
has been assembled from
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one or more overlapping cDNA, EST, or genomic DNA fragments using a computer
program for
fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison
WI) or Phrap
(University of Washington, Seattle WA). Some sequences have been both extended
and assembled to
produce the consensus sequence.
S "Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
~s Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val - Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of the
side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide. Chemical
modifications of a polynucleotide sequence can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
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immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
A "fragment" is a unique portion of RMEP or the polynucleotide encoding RMEP
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25 % or 50% of a polypeptide)
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:14-26 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:14-26, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:14-26 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:14-26 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:14-26 and the region of SEQ ID N0:14-26 to which the fragment
corresponds are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID NO:1-13 is encoded by a fragment of SEQ ID N0:14-26. A
fragment
of SEQ ID NO:1-13 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:1-13. For example, a fragment of SEQ ID NO:l-13 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:1-13.
The precise length of
a fragment of SEQ ID NO:1-13 and the region of SEQ ID NO:1-13 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
A "full-length" polynucleodde sequence is one containing at least a
translation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full-
length" polynucleodde sequence encodes a "full-length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer to
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the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps in
the sequences being compared in order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence
alignment program. This program is part of the LASERGENE software package, a
suite of molecular
biological analysis programs (DNASTAR, Madison Wn. CLUSTAL V is described in
Higgins, D.G.
and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992)
CABIOS 8:189-191.
For pairwise alignments of polynucleotide sequences, the default parameters
are set as follows:
Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted"
residue weight table is
selected as the default. Percent identity is reported by CLUSTAL V as the
"percent similarity" between
aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms is
provided by the National Center for Biotechnology Information (NCBn Basic
Local Alignment Search
Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which
is available from several
sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.govBLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off.' S0
Expect: 10
Word Size: 1l
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example, as
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defined by a particular SEQ ID number, or may be measured over a shorter
length, for example, over
the length of a fragment taken from a larger, defined sequence, for instance,
a fragment of at least 20, at
least 30, at least 40, at least 50, at least 70, at least 100, or at least 200
contiguous nucleotides. Such
lengths are exemplary only, and it is understood that any fragment length
supported by the sequences
shown herein, in the tables, figures, or Sequence Listing, may be used to
describe a length over which
percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes in
a nucleic acid sequence can be made using this degeneracy to produce multiple
nucleic acid sequences
that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default parameters
of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e
sequence alignment
program (described and referenced above). For pairwise alignments of
polypeptide sequences using
CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3,
window=5, and
"diagonals saved"=5. The PAM250 matrix is selected as the default residue
weight table. As with
polynucleotide alignments, the percent identity is reported by CLUSTAL V as
the "percent similarity"
between aligned polypeptide sequence pairs.
Aiternadvely the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12
(Apr-21-2000) with blastp set at default parameters. Such default parameters
may be, for example:
Matrix: BLOSUM62
Open Gap: 1l and Extension Gap: 1 penalties
Gap x drop-off.' S0
Expect: l0
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence, for
example, as defined by a particular SEQ ID number, or may be measured over a
shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for instance,
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a fragment of at least 15, at least 20, at least 30, at least 40, at least 50,
at least 70 or at least 150
contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment length
supported by the sequences shown herein, in the tables, figures or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the stringency
of the hybridization process, with more stringent conditions allowing less non-
specific binding, i.e.,
binding between pairs of nucleic acid strands that are not perfectly matched.
Permissive conditions for
annealing of nucleic acid sequences are routinely determinable by one of
ordinary skill in the art and
may be consistent among hybridization experiments, whereas wash conditions may
be varied among
experiments to achieve the desired stringency, and therefore hybridization
specificity. Permissive
annealing conditions occur, for example, at 68°C in the presence of
about 6 x SSC, about 1 % (w/v)
SDS, and about 100 ~g~ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (T"~ for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of the
target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and conditions
for nucleic acid hybridization are well known and can be found in Sambrook, J.
et al., 1989, Molecular
Clonine: A Laboratory Manual, 2°d ed., vol. 1-3, Cold Spring Harbor
Press, Plainview NY; specifically
see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1 % SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used SSC concentration may
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
CA 02375407 2001-11-28
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sheared and denatured salmon sperm DNA at about 100-200 ~g/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizadons. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A hybridization
complex may be formed in solution (e.g., Cot or Rot analysis) or formed
between one nucleic acid
sequence present in solution and another nucleic acid sequence immobilized on
a solid support (e.g.,
paper, membranes, filters, chips, pins or glass slides, or any other
appropriate substrate to which cells
or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide sequence
resulting in the addition of one or more amino acid residues or nucleotides,
respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression of
various factors, e.g., cytokines, chemokines, and other signaling molecules,
which may affect cellular
and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of RMEP
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment of
RMEP which is useful in any of the antibody production methods disclosed
herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides, polypeptides,
or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of RMEP. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
functional, or immunological properties of RMEP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
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linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an RMEP may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in the
art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary by
cell type depending on the enzymatic milieu of RMEP.
"Probe" refers to nucleic acid sequences encoding RMEP, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule. Typical
labels include radioactive isotopes, ligands, chemiluminescent agents, and
enzymes. "Primers" are
short nucleic acids, usually DNA oligonucleotides, which may be annealed to a
target polynucleotide by
complementary base-pairing. The primer may then be extended along the target
DNA strand by a DNA
polymerase enzyme. Primer pairs can be used for amplification (and
identification) of a nucleic acid
sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers may
be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al., 1989, Molecular Cloning: A Laborato~ Manual, 2"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et a1.,1987, Current
Protocols in Molecular
Biolo~v, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al., 1990, PCR
Protocols. A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
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purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas South
West Medical Center, Dallas TX) is capable of choosing specific primers from
megabase sequences
and is thus useful for designing primers on a genome-wide scope. The Primer3
primer selection
program (available to the public from the Whitehead Institute/MIT Center for
Genome Research,
Cambridge MA) allows the user to input a "mispriming library," in which
sequences to avoid as primer
binding sites are user-specified. Primer3 is useful, in particular, for the
selection of oligonucleotides for
microarrays. (The source code for the latter two primer selection programs may
also be obtained from
their respective sources and modified to meet the user's specific needs.) The
PrimeGen program
(available to the public from the UK Human Genome Mapping Project Resource
Centre, Cambridge
UK) designs primers based on multiple sequence alignments, thereby allowing
selection of primers that
hybridize to either the most conserved or least conserved regions of aligned
nucleic acid sequences.
Hence, this program is useful for identification of both unique and conserved
oligonucleotides and
polynucleotide fragments. The oligonucleotides and polynucleotide fragments
identified by any of the
above selection methods are useful in hybridization technologies, for example,
as PCR or sequencing
primers, microarray elements, or specific probes to identify fully or
partially complementary
polynucleotides in a sample of nucleic acids. Methods of oligonucleotide
selection are not limited to
those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, su ra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for
example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions (UTRs).
Regulatory elements interact with host or viral proteins which control
transcription, translation, or RNA
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stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding RMEP, or fragments thereof, or RMEP itself, may comprise a
bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a cell; a cell;
genomic DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recognized by the
binding molecule. For
example, if an antibody is specific for epitope "A," the presence of a
polypeptide comprising the epitope
A, or the presence of free unlabeled A, in a reaction containing free labeled
A and the antibody will
reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides by
different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" refers to the collective pattern of gene expression by a
particular cell type
or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods well
known in the art, and may rely on any known method for the insertion of
foreign nucleic acid sequences
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into a prokaryotic or eukaryotic host cell. The method for transformation is
selected based on the type
of host cell being transformed and may include, but is not limited to,
bacteriophage or viral infection,
electroporation, heat shock, lipofection, and particle bombardment. The term
"transformed" cells
includes stably transformed cells in which the inserted DNA is capable of
replication either as an
autonomously replicating plasmid or as part of the host chromosome, as well as
transiently transformed
cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants, and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook et al. ( 1989),
su ra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having at
least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of the
nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
set at default parameters. Such a pair of nucleic acids may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or
at least 98% or greater
sequence identity over a certain defined length A variant may be described as,
for example, an "allelic"
(as defined above), "splice," "species," or "polymorphic" variant. A splice
variant may have significant
identity to a reference molecule, but will generally have a greater or lesser
number of polynucleotides
due to alternative splicing of exons during mRNA processing. The corresponding
polypeptide may
possess additional functional domains or lack domains that are present in the
reference molecule.
Species variants are polynucleotide sequences that vary from one species to
another. The resulting
polypeptides generally will have significant amino acid identity relative to
each other. A polymorphic
variant is a variation in the polynucleotide sequence of a particular gene
between individuals of a given
species. Polymorphic variants also may encompass "single nucleotide
polymorphisms" (SNPs) in
which the polynucleotide sequence varies by one nucleotide base. The presence
of SNPs may be
indicative of, for example, a certain population, a disease state, or a
propensity for a disease state.
CA 02375407 2001-11-28
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A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having at
least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of the
polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
set at default parameters. Such a pair of polypeptides may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human RNA metabolism proteins
(RMEP), the
polynucleotides encoding RMEP, and the use of these compositions for the
diagnosis, treatment, or
prevention of nervous system, autoimmune/inflammatory, and cell proliferative
disorders, including
cancer.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
RMEP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of
the polypeptide
and nucleotide sequences, respectively. Column 3 shows the clone IDs of the
Incyte clones in which
nucleic acids encoding each RMEP were identified, and column 4 shows the cDNA
libraries from which
these clones were isolated Column 5 shows Incyte clones and their
corresponding cDNA libraries.
Clones for which cDNA libraries are not indicated were derived from pooled
cDNA libraries. The
Incyte clones in column 5 were used to assemble the consensus nucleotide
sequence of each RMEP and
are useful as fragments in hybridization technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; column 6
shows homologous sequences as identified by BLAST analysis; and column 7 shows
analytical methods
and in some cases, searchable databases to which the analytical methods were
applied. The methods of
column 7 were used to characterize each polypeptide through sequence homology
and protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding RMEP. The first column of Table
3 lists the nucleotide
SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1.
These fragments are
useful, for example, in hybridization or amplification technologies to
identify SEQ ID N0:14-26 and
to distinguish between SEQ ID N0:14-26 and related polynucleotide sequences.
The polypeptides
encoded by these fragments are useful, for example, as immunogenic peptides.
Column 3 lists tissue
categories which express RMEP as a fraction of total tissues expressing RMEP.
Column 4 lists
diseases, disorders, or conditions associated with those tissues expressing
RMEP as a fraction of total
tissues expressing RMEP. Column 5 lists the vectors used to subclone each cDNA
library. Of
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WO 00/78952 PCT/US00/16644
particular note is the expression of SEQ ID N0:24 in muscle tumor and fetal
brain.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
from which cDNA clones encoding RMEP were isolated. Column 1 references the
nucleotide SEQ ID
NOs, column 2 shows the cDNA libraries from which these clones were isolated,
and column 3 shows
the tissue origins and other descriptive information relevant to the cDNA
libraries in column 2.
SEQ ID N0:14 maps to chromosome 3 within the interval from 176.40 to 179.80
centiMorgans. SEQ ID NO:15 maps to chromosome 22 within the interval from
24.30 to 36.60
centiMorgans, to chromosome 16 within the interval from 19.70 to 33.30
centiMorgans, and to
chromosome 5 within the interval from 174.30 centiMorgans to q-terminus. SEQ
ID N0:17 maps to
chromosome 11 within the interval from 70.90 to 72.10 centiMorgans. SEQ ID
N0:26 maps to
chromosome 8 within the interval from 64.60 to 78.80 centiMorgans.
The invention also encompasses RMEP variants. A preferred RMEP variant is one
which has
at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid sequence
identity to the RMEP amino acid sequence, and which contains at least one
functional or structural
characteristic of RMEP.
The invention also encompasses polynucleotides which encode RMEP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected from
the group consisting of SEQ ID N0:14-26, which encodes RMEP. The
polynucleotide sequences of
SEQ ID N0:14-26, as presented in the Sequence Listing, embrace the equivalent
RNA sequences,
wherein occurrences of the nitrogenous base thymine are replaced with uracil,
and the sugar backbone
is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
RMEP. In
particular, such a variant polynucleotide sequence will have at least about
80%, or alternatively at least
about 90%, or even at least about 95% polynucleotide sequence identity to the
polynucleotide sequence
encoding RMEP. A particular aspect of the invention encompasses a variant of a
polynucleotide
sequence comprising a sequence selected from the group consisting of SEQ ID
N0:14-26 which has at
least about 80%, or alternatively at least about 90%, or even at least about
95% polynucleotide
sequence identity to a nucleic acid sequence selected from the group
consisting of SEQ ID N0:14-26.
Any one of the polynucleotide variants described above can encode an amino
acid sequence which
contains at least one functional or structural characteristic of RMEP.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the genetic
code, a multitude of polynucleotide sequences encoding RMEP, some bearing
minimal similarity to the
polynucleodde sequences of any known and naturally occurring gene, may be
produced. Thus, the
invention contemplates each and every possible variation of polynucleotide
sequence that could be made
by selecting combinations based on possible codon choices. These combinations
are made in
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CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
accordance with the standard triplet genetic code as applied to the
polynucleotide sequence of naturally
occurring RMEP, and all such variations are to be considered as being
specifically disclosed
Although nucleotide sequences which encode RMEP and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring RMEP under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding RMEP or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
occurs in a particular prokaryotic or eukaryodc host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding RMEP and its derivatives without altering the encoded amino
acid sequences include
the production of RNA transcripts having more desirable properties, such as a
greater half life, than
transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode RMEP
and
RMEP derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell systems
using reagents well known in the art. Moreover, synthetic chemistry may be
used to introduce
mutations into a sequence encoding RMEP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:14-26 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of the
embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment of
DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (PE
Biosystems,
Foster City CA), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno N~,
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler (PE
Biosystems). Sequencing is then carried out using either the ABI 373 or 377
DNA sequencing system
(PE Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics,
Sunnyvale
CA), or other systems known in the art. The resulting sequences are analyzed
using a variety of
algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997)
Short Protocols in
23
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WO 00/78952 PCT/US00/16644
Molecular Biolo~v, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
(1995) Molecular
Bioloey and Biotechnology, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding RMEP may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unlmown
sequence from a circularized template. The template is derived from
restriction fragments comprising a
known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al.
(1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent
to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al.
(1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction
enzyme digestions and
ligations may be used to insert an engineered double-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unlmown sequences
are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids
Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 Primer Analysis software
(National Biosciences,
Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides
in length, to have a
GC content of about 50% or more, and to anneal to the template at temperatures
of about 68°C to
72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T) library
does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence into S'
non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze the
size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
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CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode RMEP may be cloned in recombinant DNA molecules that direct expression
of RMEP, or
fragments or functional equivalents thereof, in appropriate host cells. Due to
the inherent degeneracy of
the genetic code, other DNA sequences which encode substantially the same or a
functionally equivalent
amino acid sequence may be produced and used to express RMEP.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter RMEP-encoding sequences for a variety of
purposes including, but not
limited to, modification of the cloning, processing, and/or expression of the
gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction sites,
alter glycosylation patterns, change codon preference, produce splice
variants, and so forth
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of RMEP, such as its biological or enzymatic
activity or its ability
to bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding RMEP may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively,
RMEP itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide
synthesis can be performed using various solution-phase or solid-phase
techniques. (See, e.g.,
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
Creighton, T. (1984) Proteins. Structures and Molecular Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated
synthesis may be achieved
using the ABI 431A peptide synthesizer (PE Biosystems). Additionally, the
amino acid sequence of
RMEP, or any part thereof, may be altered during direct synthesis and/or
combined with sequences
from other proteins, or any part thereof, to produce a variant polypeptide or
a polypeptide having a
sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active RMEP, the nucleotide sequences
encoding RMEP or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in a
suitable host. These elements include regulatory sequences, such as enhancers,
constitutive and
inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotide sequences
encoding RMEP. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
RMEP. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding RMEP and its initiation codon and upstream regulatory
sequences are inserted into
the appropriate expression vector, no additional transcriptional or
translational control signals may be
needed. However, in cases where only coding sequence, or a fragment thereof,
is inserted, exogenous
translational control signals including an in-frame ATG initiation codon
should be provided by the
vector. Exogenous translational elements and initiation codons may be of
various origins, both natural
and synthetic. The efficiency of expression may be enhanced by the inclusion
of enhancers appropriate
for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994)
Results Probl. Cell Differ.
20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding RMEP and appropriate transcriptional and
translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. (1995)
Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, ch 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding RMEP. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
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WO 00/78952 PCT/US00/16644
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Bitter, G.A. et al. (1987) Methods
Enzymol. 153:516-544;
Scorer, C.A. et al. (1994) Bio/Technology 12:181-184; Engelhard, E.K. et al.
(1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-
1945; Takamatsu,
N. (1987) EMBO J. 6:307-311; Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;
Brogue, R. et al.
(1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105; The
McGraw Hill Yearbook of Science and Technology ( 1992) McGraw Hill, New York
NY, pp.
191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-
3659; and Harrington,
J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from
retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al., (1993)
Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.)
The invention is not limited by the host cell employed
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleodde sequences encoding RMEP. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding RMEP can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1 plasmid
(Life Technologies). Ligation of sequences encoding RMEP into the vector's
multiple cloning site
disrupts the lacZ gene, allowing a colorimetric screening procedure for
identification of transformed
bacteria containing recombinant molecules. In addition, these vectors may be
useful for in vitro
transcription, dideoxy sequencing, single strand rescue with helper phage, and
creation of nested
deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster
(1989) J. Biol. Chem
264:5503-5509.) When large quantities of RMEP are needed, e.g. for the
production of antibodies,
vectors which direct high level expression of RMEP may be used. For example,
vectors containing the
strong, inducible T5 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of RMEP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
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WO 00/78952 PCT/US00/16644
Bitter, supra; and Scorer, supra.)
Plant systems may also be used for expression of RMEP. Transcription of
sequences encoding
RMEP may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV
used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO
J. 6:307-311).
Alternatively, plant promoters such as the small subunit of RUBISCO or heat
shock promoters may be
used. (See, e.g., Coruzzi, supra; Brogue, supra; and Winter, supra.) These
constructs can be
introduced into plant cells by direct DNA transformation or pathogen-mediated
transfection. (See, e.g.,
The McGraw Hill Yearbook of Science and Technolo y (1992) McGraw Hill, New
York NY, pp.
191-196.)
In mammalian cells, a number of viral-based expression systems may be utilized
In cases
where an adenovirus is used as an expression vector, sequences encoding RMEP
may be ligated into an
adenovirus transcription/translation complex consisting of the late promoter
and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses RMEP in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino polymers,
or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
RMEP in cell lines is preferred. For example, sequences encoding RMEP can be
transformed into cell
lines using expression vectors which may contain viral origins of replication
and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include,
but are not limited to, the herpes simplex virus thymidine kinase and adenine
phosphoribosyltransferase
genes, for use in tk- and apr cells, respectively. (See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232;
Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or
herbicide resistance can be
used as the basis for selection. For example, dhfr confers resistance to
methotrexate; neo confers
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CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
resistance to the aminoglycosides neomycin and G-418; and als and pat confer
resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g.,
Wigler, M. et al. (1980)
Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J.
Mol. Biol. 150:1-14.)
Additional selectable genes have been described, e.g., trpB and hisD, which
alter cellular requirements
for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins
(GFP; Clontech), B
glucuronidase and its substrate B-glucuronide, or luciferase and its substrate
luciferin may be used
These markers can be used not only to identify transformants, but also to
quantify the amount of
transient or stable protein expression attributable to a specific vector
system (See, e.g., Rhodes, C.A.
(1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding RMEP is inserted within a marker gene sequence, transformed
cells containing
sequences encoding RMEP can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding RMEP under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually indicates
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding RMEP
and that express
RMEP may be identified by a variety of procedures known to those of skill in
the art. These procedures
include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR
amplification, and
protein bioassay or immunoassay techniques which include membrane, solution,
or chip based
technologies for the detection and/or quantification of nucleic acid or
protein sequences.
Immunological methods for detecting and measuring the expression of RMEP using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques include
enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and
fluorescence
activated cell sorting (FACS). A two-site, monoclonal-based immunoassay
utilizing monoclonal
antibodies reactive to two non-interfering epitopes on RMEP is preferred, but
a competitive binding
assay may be employed. These and other assays are well known in the art. (See,
e.g., Hampton, R. et
al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN,
Sect. IV; Coligan, J.E.
et al. (1997) Current Protocols in Immunoloay, Greene Pub. Associates and
Wiley-Interscience, New
York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa
NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled hybridization
or PCR probes for detecting sequences related to polynucleotides encoding RMEP
include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
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WO 00/78952 PCT/US00/16644
Alternatively, the sequences encoding RMEP, or any fragments thereof, may be
cloned into a vector for
the production of an mRNA probe. Such vectors are known in the art, are
commercially available, and
may be used to synthesize RNA probes in vitro by addition of an appropriate
RNA polymerise such as
T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of
commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for ease
of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or
chromogenic agents, as
well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding RMEP may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used As will be understood by those of skill in the art,
expression vectors containing
polynucleoddes which encode RMEP may be designed to contain signal sequences
which direct
secretion of RMEP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of the
polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which cleaves a
"prepro" or "pro" form of the
protein may also be used to specify protein targeting, folding, and/or
activity. Different host cells
which have specific cellular machinery and characteristic mechanisms for post-
translational activities
(e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type
Culture
Collection (ATCC, Manassas VA) and may be chosen to ensure the correct
modification and processing
of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding RMEP may be ligated to a heterologous sequence resulting in
translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric RMEP protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of RMEP activity.
Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available affinity
matrices. Such moieties include, but are not limited to, glutathione S-
transferase (GST), maltose
binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-
His, FLAG, c-myc, and
hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their
cognate fusion
proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
and metal-chelate resins,
respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
proteins using commercially available monoclonal and polyclonal antibodies
that specifically recognize
CA 02375407 2001-11-28
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these epitope tags. A fusion protein may also be engineered to contain a
proteolytic cleavage site
located between the RMEP encoding sequence and the heterologous protein
sequence, so that RMEP
may be cleaved away from the heterologous moiety following purification.
Methods for fusion protein
expression and purification are discussed in Ausubel (1995, supra, ch. 10). A
variety of commercially
available kits may also be used to facilitate expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled RMEP may
be achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These systems
couple transcription and translation of protein-coding sequences operably
associated with the T7, T3, or
SP6 promoters. Translation takes place in the presence of a radiolabeled amino
acid precursor, for
example, 35S-methionine.
RMEP of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to RMEP. At least one and up to a plurality of test
compounds may be screened
for specific binding to RMEP. Examples of test compounds include antibodies,
oligonucleotides,
proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
RMEP, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, Coligan, J.E. et al. (1991) Current Protocols
in Immunoloev 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which RMEP
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express RMEP,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coll. Cells expressing RMEP or cell membrane fractions which contain RMEP are
then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either RMEP or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
RMEP, either in
solution or affixed to a solid support, and detecting the binding of RMEP to
the compound.
Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carried out using cell-free
preparations, chemical
libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to a
solid support.
RMEP of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of RMEP. Such compounds may include agonists,
antagonists, or partial or
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inverse agonists. In one embodiment, an assay is performed under conditions
permissive for RMEP
activity, wherein RMEP is combined with at least one test compound, and the
activity of RMEP in
the presence of a test compound is compared with the activity of RMEP in the
absence of the test
compound. A change in the activity of RMEP in the presence of the test
compound is indicative of a
compound that modulates the activity of RMEP. Alternatively, a test compound
is combined with an
in vitro or cell-free system comprising RMEP under conditions suitable for
RMEP activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of RMEP
may do so indirectly and need not come in direct contact with the test
compound. At least one and up
to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding RMEP or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem
(ES) cells. Such techniques are well known in the art and are useful for the
generation of animal
models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent
No. 5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding
region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding RMEP may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding RMEP can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a
region of a polynucleotide encoding RMEP is injected into animal ES cells, and
the injected sequence
integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the
blastulae are implanted as described above. Transgenic progeny or inbred lines
are studied and
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treated with potential pharmaceutical agents to obtain information on
treatment of a human disease.
Alternatively, a mammal inbred to overexpress RMEP, e.g., by secreting RMEP in
its milk, may also
serve as a convenient source of that protein (Janne, J. et al. (1998)
Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of RMEP and RNA metabolism proteins. In addition, the
expression of RMEP is
closely associated with cell proliferation, cancer, and inflammation.
Therefore, RMEP appears to
play a role in nervous system, autoimmune/inflammatory, and cell proliferative
disorders, including
cancer. In the treatment of disorders associated with increased RMEP
expression or activity, it is
desirable to decrease the expression or activity of RMEP. In the treatment of
disorders associated
with decreased RMEP expression or activity, it is desirable to increase the
expression or activity of
RMEP.
Therefore, in one embodiment, RMEP or a fragment or derivative thereof may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of RMEP. Examples of such disorders include, but are not limited to,
a nervous system
disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral
neoplasms, Alzheimer's
disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease
and other extrapyramidal
disorders, amyotrophic lateral sclerosis and other motor neuron disorders,
progressive neural muscular
atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and
other demyelinating diseases,
bacterial and viral meningitis, brain abscess, subdural empyema, epidural
abscess, suppurative
intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous
system disease; prion
diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-
Scheinker syndrome;
fatal familial insomnia, nutritional and metabolic diseases of the nervous
system, neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental
retardation and other developmental disorder of the central nervous system,
cerebral palsy, a
neuroskeletal disorder, an autonomic nervous system disorder, a cranial nerve
disorder, a spinal cord
disease, muscular dystrophy and other neuromuscular disorder, a peripheral
nervous system disorder,
dermatomyositis and polymyositis; inherited, metabolic, endocrine, and toxic
myopathy; myasthenia
gravis, periodic paralysis; a mental disorder including mood, anxiety, and
schizophrenic disorders;
seasonal affective disorder (SAD); akathesia, amnesia, catatonia, diabetic
neuropathy, tardive
dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and
Tourette's disorder, an
autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS), Addison's
disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
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dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema,
episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema
nodosum, atrophic
gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis,
psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's
syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and
extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic
infections, and trauma; and a
cell proliferative disorder such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal
nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas, parathyroid,
penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and
uterus.
In another embodiment, a vector capable of expressing RMEP or a fragment or
derivative
thereof may be administered to a subject to treat or prevent a disorder
associated with decreased
expression or activity of RMEP including, but not limited to, those described
above.
In a firrttrer embodiment, a pharmaceutical composition comprising a
substantially purified
RMEP in conjunction with a suitable pharmaceutical carrier may be administered
to a subject to treat
or prevent a disorder associated with decreased expression or activity of RMEP
including, but not
limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of RMEP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or activity
of RMEP including, but not limited to, those listed above.
In a further embodiment, an antagonist of RMEP may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of RMEP.
Examples of such
disorders include, but are not limited to, those nervous system,
autoimmune/inflammatory, and cell
proliferative disorders, including cancer described above. In one aspect, an
antibody which specifically
binds RMEP may be used directly as an antagonist or indirectly as a targeting
or delivery mechanism
for bringing a pharmaceutical agent to cells or tissues which express RMEP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding RMEP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of RMEP including, but not limited to, those
described above.
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WO 00/78952 PCT/US00/16644
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made by
one of ordinary skill in the art,. according to conventional pharmaceutical
principles. The combination
of therapeutic agents may act synergistically to effect the treatment or
prevention of the various
disorders described above. Using this approach, one may be able to achieve
therapeutic efficacy with
lower dosages of each agent, thus reducing the potential for adverse side
effects.
An antagonist of RMEP may be produced using methods which are generally known
in the art.
In particular, purified RMEP may be used to produce antibodies or to screen
libraries of
pharmaceutical agents to identify those which specifically bind RMEP.
Antibodies to RMEP may also
be generated using methods that are well known in the art. Such antibodies may
include, but are not
limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab
fragments, and fragments
produced by a Fab expression library. Neutralizing antibodies (i.e., those
which inhibit dimer
formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with RMEP or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral gels
such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants
used in humans, BCG
(bacilli Calmette-Guerin) and Corvnebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to RMEP
have an amino acid sequence consisting of at least about 5 amino acids, and
generally will consist of at
least about 10 amino acids. It is also preferable that these oligopeptides,
peptides, or fragments are
identical to a portion of the amino acid sequence of the natural protein.
Short stretches of RMEP amino
acids may be fused with those of another protein, such as KLH, and antibodies
to the chimeric molecule
may be produced.
Monoclonal antibodies to RMEP may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not limited
to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-
hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
RMEP-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g., Burton,
D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter,
G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for RMEP may also be
generated For
example, such fragments include, but are not limited to, F(ab~2 fragments
produced by pepsin digestion
of the antibody molecule and Fab fragments generated by reducing the disulfide
bridges of the F(ab~2
fragments. Alternatively, Fab expression libraries may be constructed to allow
rapid and easy
identification of monoclonal Fab fragments with the desired specificity. (See,
e.g., Huse, W.D. et al.
(1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
RMEP and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two
non-interfering RMEP epitopes is generally used, but a competitive binding
assay may also be
employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for RMEP. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of RMEP-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The Ka determined
for a preparation of polyclonal antibodies, which are heterogeneous in their
affinities for multiple
RMEP epitopes, represents the average affinity, or avidity, of the antibodies
for RMEP. The Ka
determined for a preparation of monoclonal antibodies, which are monospecific
for a particular RMEP
epitope, represents a true measure of amity. High-affinity antibody
preparations with Ka ranging from
about 109 to 10'2 L/mole are preferred for use in immunoassays in which the
RMEP-antibody complex
must withstand rigorous manipulations. Low-amity antibody preparations with Ka
ranging from
about 106 to 10' L/mole are preferred for use in immunopurification and
similar procedures which
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WO 00/78952 PCT/US00/16644
ultimately require dissociation of RMEP, preferably in active form, from the
antibody (Catty, D. (1988)
Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; Liddell,
J.E. and A. Cryer
( 1991 ) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New
York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
the quality and suitability of such preparations for certain downstream
applications. For example, a
polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of RMEP-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, swpra, and
Coligan et al., su ra.)
In another embodiment of the invention, the polynucleotides encoding RMEP, or
any fragment
or complement thereof, may be used for therapeutic purposes. In one aspect,
modifications of gene
expression can be achieved by designing complementary sequences or antisense
molecules (DNA, RNA,
PNA, or modified oligonucleotides) to the coding or regulatory regions of the
gene encoding RMEP.
Such technology is well known in the art, and antisense oligonucleotides or
larger fragments can be
designed from various locations along the coding or control regions of
sequences encoding RMEP.
(See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. ( 1990) Blood
76:271; Ausubel, su ra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Moms, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding RMEP may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-linked
inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe
combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
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cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and Somia, N. (1997) Nature
389:239-242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D. (1988)
Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA.
93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
TrYpanosoma cruzi). In the
case where a genetic deficiency in RMEP expression or regulation causes
disease, the expression of
RMEP from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in RMEP
are treated by constructing mammalian expression vectors encoding RMEP and
introducing these
vectors by mechanical means into RMEP-deficient cells. Mechanical transfer
technologies for use with
cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii) ballistic gold
particle delivery, (iii) liposome-mediated transfection, (iv) receptor-
mediated gene transfer, and (v) the
use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev.
Biochem. 62:191-217;
Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon (1998) Curr.
Opin Biotechnol. 9:445-
450).
Expression vectors that may be effective for the expression of RMEP include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen,
Carlsbad CA),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). RMEP may be
expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV),
Rous sarcoma virus
(RSV), SV40 virus, thymidine kinase (TK), or ~i-actin genes), (ii) an
inducible promoter (e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. U.S.A.
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998)
Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the
ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M. V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding RMEP from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
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WO 00/78952 PCT/US00/16644
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of these
standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to RMEP expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding RMEP under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. U.S.A. 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses a
method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ T-cells), and the
return of transduced cells to a patient are procedures well known to persons
skilled in the art of gene
therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et
al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al.
(1998) Proc. Natl. Acad. Sci. U.S.A. 95:1201-1206; Su, L. (1997) Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding RMEP to cells which have one or more genetic
abnormalities with respect to
the expression of RMEP. The construction and packaging of adenovirus-based
vectors are well known
to those with ordinary skill in the art. Replication defective adenovirus
vectors have proven to be
versatile for importing genes encoding immunoregulatory proteins into intact
islets in the pancreas
(Csete, M.E, et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999) Annu.
Rev. Nutr. 19:511-544; and Verma, LM. and N. Somia (1997) Nature 18:389:239-
242, both
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incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding RMEP to target cells which have one or more genetic
abnormalities with
respect to the expression of RMEP. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing RMEP to cells of the central nervous
system, for which HSV has a
tropism The construction and packaging of herpes-based vectors are well known
to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye
Res.169:385-395). The construction of a HSV-1 virus vector has also been
disclosed in detail in U.S.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is
hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use
of recombinant HSV
d92 which consists of a genome containing at least one exogenous gene to be
transferred to a cell under
the control of the appropriate promoter for purposes including human gene
therapy. Also taught by this
patent are the construction and use of recombinant HSV strains deleted for
ICP4, ICP27 and ICP22.
For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and
Xu, H. et al. (1994) Dev.
Biol. 163:152-161, hereby incorporated by reference. The manipulation of
cloned herpesvirus
sequences, the generation of recombinant virus following the transfection of
multiple plasmids
containing different segments of the large herpesvirus genomes, the growth and
propagation of
herpesvirus, and the infection of cells with herpesvirus are techniques well
known to those of ordinary
skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding RMEP to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based on
the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotech. 9:464-
469). During alphavirus
RNA replication, a subgenomic RNA is generated that normally encodes the viral
capsid proteins. This
subgenomic RNA replicates to higher levels than the full-length genomic RNA,
resulting in the
overproduction of capsid proteins relative to the viral proteins with
enzymatic activity (e.g., protease
and polymerase). Similarly, inserting the coding sequence for RMEP into the
alphavirus genome in
place of the capsid-coding region results in the production of a large number
of RMEP-coding RNAs
and the synthesis of high levels of RMEP in vector transduced cells. While
alphavirus infection is
typically associated with cell lysis within a few days, the ability to
establish a persistent infection in
hamster normal kidney cells (BHK-21 ) with a variant of Sindbis virus (SIN)
indicates that the lytic
replication of alphaviruses can be altered to suit the needs of the gene
therapy application (Dryga, S.A.
et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will
allow the introduction of
RMEP into a variety of cell types. The specific transduction of a subset of
cells in a population may
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
require the sorting of cells prior to transduction. The methods of
manipulating infectious cDNA clones
of alphaviruses, performing alphavirus cDNA and RNA transfections, and
performing alphavirus
infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions -10
and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can
be achieved using triple helix base-pairing methodology. Triple helix pairing
is useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in
Huber, B.E. and B.I. Carr,
Molecular and Immunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-
177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA by
preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding RMEP.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared by
any method known in the art for the synthesis of nucleic acid molecules. These
include techniques for
chemically synthesizing oligonucleotides such as solid phase phosphoramidite
chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA sequences
encoding RMEP. Such DNA sequences may be incorporated into a wide variety of
vectors with
suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA
constructs that
synthesize complementary RNA, constitutively or inducibly, can be introduced
into cell lines, cells, or
tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
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WO 00/78952 PCT/US00/16644
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine, and
wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms
of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding RMEP. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming
oligonucleotides,
transcription factors and other polypeptide transcriptional regulators, and
non-macromolecular
chemical entities which are capable of interacting with specific
polynucleotide sequences. Effective
compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
polynucleotide expression. Thus, in the treatment of disorders associated with
increased RMEP
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding RMEP may be therapeutically useful, and in the treament of disorders
associated with
decreased RMEP expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding RMEP may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding RMEP is exposed to at least one test compound thus
obtained. The sample
may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
RMEP are assayed
by any method commonly known in the art. Typically, the expression of a
specific nucleotide is
detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
of the polynucleotide encoding RMEP. The amount of hybridization may be
quantified, thus
forming the basis for a comparison of the expression of the polynucleotide
both with and without
exposure to one or more test compounds. Detection of a change in the
expression of a polynucleotide
exposed to a test compound indicates that the test compound is effective in
altering the expression of
the polynucleotide. A screen for a compound effective in altering expression
of a specific
polynucleotide can be carried out, for example, using a Schizosaccharomvces
pombe gene expression
system (Atkins, D. et al. (1999) U.S. Patent No. 5.932,435; Arndt, G.M. et al.
(2000) Nucleic Acids
42
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WO 00/78952 PCT/US00/16644
Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al.
(2000) Biochem. Biophys.
Res. Commun. 268:8-13). A particular embodiment of the present invention
involves screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000} U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable for
use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells taken
from the patient and clonally propagated for autologous transplant back into
that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of such
therapy, including, for example, mammals such as humans, dogs, cats, cows,
horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
pharmaceutical
composition which generally comprises an active ingredient formulated with a
pharmaceutically
acceptable excipient. Excipients may include, for example, sugars, starches,
celluloses, gums, and
proteins. Various formulations are commonly known and are thoroughly discussed
in the latest edition
of ReminQton's Pharmaceutical Sciences (Maack Publishing, Euston PA). Such
pharmaceutical
compositions may consist of RMEP, antibodies to RMEP, and mimetics, agonists,
antagonists, or
inhibitors of RMEP.
The pharmaceutical compositions utilized in this invention may be administered
by any number
of routes including, but not limited to, oral, intravenous, intramuscular,
infra-arterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal, enteral,
topical, sublingual, or rectal means.
Pharmaceutical compositions for pulmonary administration may be prepared in
liquid or dry
powder form. These compositions are generally aerosolized immediately prior to
inhalation by the
patient. In the case of small molecules (e.g. traditional low molecular weight
organic drugs), aerosol
delivery of fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g. larger
peptides and proteins), recent developments in the field of pulmonary delivery
via the alveolar region of
the lung have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g.,
Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the
advantage of administration
without needle injection, and obviates the need for potentially toxic
penetration enhancers.
Pharmaceutical compositions suitable for use in the invention include
compositions wherein the
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active ingredients are contained in an effective amount to achieve the
intended purpose. The
determination of an effective dose is well within the capability of those
skilled in the art.
Specialized forms of pharmaceutical compositions may be prepared for direct
intracellular
delivery of macromolecules comprising RMEP or fragments thereof. For example,
liposome
S preparations containing a cell-impermeable macromolecule may promote cell
fusion and intracellular
delivery of the macromolecule. Alternatively, RMEP or a fragment thereof may
be joined to a short
cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus
generated have been
found to transduce into the cells of all tissues, including the brain, in a
mouse model system (Schwarze,
S.R. et al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, monkeys,
or pigs. An animal model may also be used to determine the appropriate
concentration range and route
of administration. Such information can then be used to determine useful doses
and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example RMEP
or fragments thereof, antibodies of RMEP, and agonists, antagonists or
inhibitors of RMEP, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio.
Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained from cell
culture assays and animal
studies are used to formulate a range of dosage for human use. The dosage
contained in such
compositions is preferably within a range of circulating concentrations that
includes the EDso with little
or no toxicity. The dosage varies within this range depending upon the dosage
form employed, the
sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the subject
requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the active
moiety or to maintain the desired effect. Factors which may be taken into
account include the severity
of the disease state, the general health of the subject, the age, weight, and
gender of the subject, time
and frequency of administration, drug combination(s), reaction sensitivities,
and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3 to 4 days,
every week, or
biweekly depending on the half life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 /cg, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
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WO 00/78952 PCT/US00/16644
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind RMEP may be used for
the
diagnosis of disorders characterized by expression of RMEP, or in assays to
monitor patients being
treated with RMEP or agonists, antagonists, or inhibitors of RMEP. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for RMEP include methods which utilize the antibody and a label to detect RMEP
in human body fluids
or in extracts of cells or tissues. The antibodies may be used with or without
modification, and may be
labeled by covalent or non-covalent attachment of a reporter molecule. A wide
variety of reporter
molecules, several of which are described above, are known in the art and may
be used.
A variety of protocols for measuring RMEP, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
RMEP expression. Normal
or standard values for RMEP expression are established by combining body
fluids or cell extracts taken
from normal mammalian subjects, for example, human subjects, with antibody to
RMEP under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of RMEP
expressed in subject,
control, and disease samples from biopsied tissues are compared with the
standard values. Deviation
between standard and subject values establishes the parameters for diagnosing
disease.
In another embodiment of the invention, the polynucleotides encoding RMEP may
be used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleoddes may be used
to detect and
quantify gene expression in biopsied tissues in which expression of RMEP may
be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
RMEP, and to monitor regulation of RMEP levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding RMEP or closely related
molecules may be used to
identify nucleic acid sequences which encode RMEP. The specificity of the
probe, whether it is made
from a highly specific region, e.g., the 5'regulatory region, or from a less
specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding RMEP, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
sequence identity to any of the RMEP encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:14-26 or from
genomic sequences including promoters, enhancers, and introns of the RMEP
gene.
Means for producing specific hybridization probes for DNAs encoding RMEP
include the
cloning of polynucleotide sequences encoding RMEP or RMEP derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a variety
of reporter groups, for example, by radionuclides such as 32P or 35S, or by
enzymatic labels, such as
alkaline phosphatase coupled to the probe via avidin/biodn coupling systems,
and the like.
Polynucleotide sequences encoding RMEP may be used for the diagnosis of
disorders
associated with expression of RMEP. Examples of such disorders include, but
are not limited to, a
nervous system disorder such as epilepsy, ischemic cerebrovascular disease,
stroke, cerebral
neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease,
dementia, Parkinson's disease
and other extrapyramidal disorders, amyotrophic lateral sclerosis and other
motor neuron disorders,
progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias,
multiple sclerosis and
other demyelinating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis,
viral central nervous system
disease; prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-Straussler-Scheinker
syndrome; fatal familial insomnia, nutritional and metabolic diseases of the
nervous system,
neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal
syndrome, mental retardation and other developmental disorder of the central
nervous system, cerebral
palsy, a neuroskeletal disorder, an autonomic nervous system disorder, a
cranial nerve disorder, a spinal
cord disease, muscular dystrophy and other neuromuscular disorder, a
peripheral nervous system
disorder, dermatomyositis and polymyositis; inherited, metabolic, endocrine,
and toxic myopathy;
myasthenia gravis, periodic paralysis; a mental disorder including mood,
anxiety, and schizophrenic
disorders; seasonal affective disorder (SAD); akathesia, amnesia, catatonia,
diabetic neuropathy,
tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and
Tourette's disorder, an
autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS), Addison's
disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystids, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema,
episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema
nodosum, atrophic
gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's
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thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, osteoarthrids, osteoporosis,
pancreatitis, polymyositis,
psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's
syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and
extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic
infections, and trauma; and a
cell proliferative disorder such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal
nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas, parathyroid,
penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and
uterus . The polynucleotide
sequences encoding RMEP may be used in Southern or northern analysis, dot
blot, or other
membrane-based technologies; in PCR technologies; in dipstick, pin, and
multiformat ELISA-like
assays; and in microarrays utilizing fluids or tissues from patients to detect
altered RMEP expression.
Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding RMEP may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding RMEP may be labeled by standard methods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a suitable
incubation period, the sample is washed and the signal is quantified and
compared with a standard
value. If the amount of signal in the patient sample is significantly altered
in comparison to a control
sample then the presence of altered levels of nucleotide sequences encoding
RMEP in the sample
indicates the presence of the associated disorder. Such assays may also be
used to evaluate the efficacy
of a particular therapeutic treatment regimen in animal studies, in clinical
trials, or to monitor the
treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of RMEP,
a normal or standard profile for expression is established. This may be
accomplished by combining
body fluids or cell extracts taken from normal subjects, either animal or
human, with a sequence, or a
fragment thereof, encoding RMEP, under conditions suitable for hybridization
or amplification.
Standard hybridization may be quantified by comparing the values obtained from
normal subjects with
values from an experiment in which a known amount of a substantially purified
polynucleotide is used.
Standard values obtained in this manner may be compared with values obtained
from samples from
patients who are symptomatic for a disorder. Deviation from standard values is
used to establish the
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presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding RMEP
may involve the use of PCR. These oligomers may be chemically synthesized,
generated enzymatically,
or produced in vitro. Oligomers will preferably contain a fragment of a
polynucleotide encoding
RMEP, or a fragment of a polynucleotide complementary to the polynucleotide
encoding RMEP, and
will be employed under optimized conditions for identification of a specific
gene or condition.
Oligomers may also be employed under less stringent conditions for detection
or quantification of
closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding RMEP may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired generic disease
in humans. Methods of SNP detection include, but are not limited to, single-
stranded conformation
polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP,
oligonucleodde primers
derived from the polynucleotide sequences encoding RMEP are used to amplify
DNA using the
polymerase chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the secondary
and tertiary structures of PCR products in single-stranded form, and these
differences are detectable
using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such as
DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of
individual
overlapping DNA fragments which assemble into a common consensus sequence.
These computer-
based methods filter out sequence variations due to laboratory preparation of
DNA and sequencing
errors using statistical models and automated analyses of DNA sequence
chromatograms. In the
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alternative, SNPs may be detected and characterized by mass spectrometry
using, for example, the high
throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
Methods which may also be used to quantify the expression of RMEP include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C. et
al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple
samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described in Seilhamer, J.J. et al.,
"Comparative Gene Transcript
Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference. The
microarray may also be
used to identify genetic variants, mutations, and polymorphisms. This
information may be used to
determine gene function, to understand the genetic basis of a disorder, to
diagnose a disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic profile.
In another embodiment, antibodies specific for RMEP, or RMEP or fragments
thereof may be
used as elements on a microarray. The microarray may be used to monitor or
measure protein-protein
interactions, drug-target interactions, and gene expression profiles, as
described above.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalon, D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are well
known and thoroughly described in DNA Microarrays: A Practical Approach, M.
Schena, ed. (1999)
Oxford University Press, London, hereby expressly incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding RMEP
may be used to
generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
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of a mufti-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop genetic
linkage maps, for example, which correlate the inheritance of a disease state
with the inheritance of a
particular chromosome region or restriction fragment length polymorphism
(RFLP). (See, e.g.,
Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic map
data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.)
Examples of genetic map
data can be found in various scientific journals or at the Online Mendelian
Inheritance in Man (OMIM)
World Wide Web site. Correlation between the location of the gene encoding
RMEP on a physical map
and a specific disorder, or a predisposition to a specific disorder, may help
define the region of DNA
associated with that disorder and thus may further positional cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse, may
reveal associated markers even if the exact chromosomal locus is not known.
This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely localized
by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia
to l 1q22-23, any sequences
mapping to that area may represent associated or regulatory genes for further
investigation. (See, e.g.,
Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the
instant invention may
also be used to detect differences in the chromosomal location due to
translocation, inversion, etc.,
among normal, carrier, or affected individuals.
In another embodiment of the invention, RMEP, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between RMEP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
CA 02375407 2001-11-28
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synthesized on a solid substrate. The test compounds are reacted with RMEP, or
fragments thereof,
and washed. Bound RMEP is then detected by methods well known in the art.
Purified RMEP can also
be coated directly onto plates for use in the aforementioned drug screening
techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and immobilize
it on a solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding RMEP specifically compete with a test compound
for binding RMEP. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with RMEP.
In additional embodiments, the nucleotide sequences which encode RMEP may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U.S. Ser. No. 60/139,922, are hereby expressly incorporated by
reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some tissues
were homogenized and lysed in guanidinium isothiocyanate, while others were
homogenized and lysed
in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life
Technologies), a monophasic
solution of phenol and guanidine isothiocyanate. The resulting lysates were
centrifuged over CsCl
cushions or extracted with chloroform. RNA was precipitated from the lysates
with either isopropanol
or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
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vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-1000
bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies),
pcDNA2.1 plasmid
(Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA).
Recombinant
plasmids were transformed into competent E. coli cells including XL,1-Blue,
XL1-BlueMRF, or SOLR
from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo excision
using the UN1ZAP vector system (Stratagene) or by cell lysis. Plasmids were
purified using at least
one of the following: a Magic or WIZARD Minipreps DNA purification system
(Promega); an AGTC
Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8
Plasmid, QIAWELL
8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L.
PREP 96 plasmid
purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1 ml of
distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in 384-
well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSICA1V II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (PE Biosystems) thermal cycler or the PTC-200
thermal cycler (MJ
Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or
the MICROLAB
2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were
prepared using reagents
provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such
as the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
Electrophoredc
separation of cDNA sequencing reactions and detection of labeled
polynucleotides were carried out
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using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI
PRISM 373 or
377 sequencing system (PE Biosystems) in conjunction with standard ABI
protocols and base calling
software; or other sequence analysis systems known in the art. Reading frames
within the cDNA
sequences were identified using standard methods (reviewed in Ausubel, 1997,
supra, unit 7.7). Some
of the cDNA sequences were selected for extension using the techniques
disclosed in Example VI.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilled in the
art. Table 5 summarizes the tools, programs, and algorithms used and provides
applicable descriptions,
references, and threshold parameters. The first column of Table 5 shows the
tools, programs, and
algorithms used, the second column provides brief descriptions thereof, the
third column presents
appropriate references, all of which are incorporated by reference herein in
their entirety, and the fourth
column presents, where applicable, the scores, probability values, and other
parameters used to evaluate
the strength of a match between two sequences (the higher the score, the
greater the homology between
two sequences). Sequences were analyzed using MACDNASIS PRO software (Hitachi
Software
Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and
polypeptide sequence alignments were generated using the default parameters
specified by the clustal
algorithm as incorporated into the MEGALIGN multisequence alignment program
(DNASTAR), which
also calculates the percent identity between aligned sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA sequences
and by masking ambiguous bases, using algorithms and programs based on BLAST,
dynamic
programing, and dinucleotide nearest neighbor analysis. The sequences were
then queried against a
selection of public databases such as the GenBank primate, rodent, mammalian,
vertebrate, and
eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation
using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled
into full
length polynucleotide sequences using programs based on Phred, Phrap, and
Consed, and were screened
for open reading frames using programs based on GeneMark, BLAST, and FASTA.
The full length
polynucleotide sequences were translated to derive the corresponding full
length amino acid sequences,
and these full length sequences were subsequently analyzed by querying against
databases such as the
GenBank databases (described above), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM,
Prosite,
and Hidden Markov Model (HMM)-based protein family databases such as PFAM. HMM
is a
probabilistic approach which analyzes consensus primary structures of gene
families. (See, e.g.,
Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide and
amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:14-26. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
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amplification technologies were described in The Invention section above.
IV. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a gene
and involves the hybridization of a labeled nucleotide sequence to a membrane
on which RNAs from a
particular cell type or tissue have been bound (See, e.g., Sambrook, supra, ch
7; Ausubel, 1995,
supra, ch 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics~. This
analysis 1S
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
search can be modified to determine whether any particular match is
categorized as exact or similar.
The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum { length(Seq. 1 ), length(Seq. 2) }
The product score takes into account both the degree of similarity between two
sequences and the length
of the sequence match The product score is a normalized value between 0 and
100, and is calculated
as follows: the BLAST score is multiplied by the percent nucleotide identity
and the product is divided
by (5 times the length of the shorter of the two sequences). The BLAST score
is calculated by
assigning a score of +5 for every base that matches in a high-scoring segment
pair (HSP), and -4 for
every mismatch Two sequences may share more than one HSP (separated by gaps).
If there is more
than one HSP, then the pair with the highest BLAST score is used to calculate
the product score. The
product score represents a balance between fractional overlap and quality in a
BLAST alignment. For
example, a product score of 100 is produced only for 100% identity over the
entire length of the shorter
of the two sequences being compared. A product score of 70 is produced either
by 100% identity and
70% overlap at one end, or by 88% identity and 100% overlap at the other. A
product score of 50 is
produced either by 100% identity and 50% overlap at one end, or 79% identity
and 100% overlap.
The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding RMEP occurred. Analysis involved the categorization of
cDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease/condition categories included cancer,
inflammation, trauma,
cell proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
sequence of interest was counted and divided by the total number of libraries
across all categories.
Percentage values of tissue-specific and disease- or condition-specific
expression are reported in Table
3.
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V. Chromosomal Mapping of RMEP Encoding Polynucleotides
The cDNA sequences which were used to assemble SEQ ID N0:14 through 26 were
compared with sequences from the Incyte LIFESEQ database and public domain
databases using
BLAST and other implementations of the Smith-Waterman algorithm. Sequences
from these
databases that matched SEQ ID N0:14 through 26 were assembled into clusters of
contiguous and
overlapping sequences using assembly algorithms such as Phrap (Table 5).
Radiation hybrid and
genetic mapping data available from public resources such as the Stanford
Human Genome Center
(SHGC), Whitehead Institute for Genome Research (WIGR), and G6n~thon were used
to determine if
any of the clustered sequences had been previously mapped. Inclusion of a
mapped sequence in a
cluster resulted in the assignment of all sequences of that cluster, including
its particular SEQ ID
NO:, to that map location.
The genetic map locations of SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:17, and SEQ
ID
N0:26 are described in The Invention as ranges, or intervals, of human
chromosomes. More than one
map location is reported for SEQ ID N0:15, indicating that previously mapped
sequences having
similarity, but not complete identity, to SEQ ID N0:15 were assembled into
their respective clusters.
The map position of an interval, in centiMorgans, is measured relative to the
terminus of the
chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement based on
recombination
frequencies between chromosomal markers. On average, 1 cM is roughly
equivalent to 1 megabase
(Mb) of DNA in humans, although this can vary widely due to hot and cold spots
of recombination.)
The cM distances are based on genetic markers mapped by Genethon which provide
boundaries for
radiation hybrid markers whose sequences were included in each of the
clusters.
VI. Extension of RMEP Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:14-26 were produced by
extension of an
appropriate fragment of the full length molecule using oligonucleotide primers
designed from this
fragment. One primer was synthesized to initiate 5' extension of the known
fragment, and the other
primer, to initiate 3' extension of the known fragment. The initial primers
were designed using OLIGO
4.06 software (National Biosciences), or another appropriate program, to be
about 22 to 30 nucleotides
in length, to have a GC content of about 50% or more, and to anneal to the
target sequence at
temperatures of about 68 ° C to about 72 ° C. Any stretch of
nucleotides which would result in hairpin
structures and primer-primer dimerizations was avoided
Selected human cDNA libraries were used to extend the sequence. If more than
one extension
was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)ZS04,
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
and ~i-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~1 to 10 ~1 aliquot of the reaction mixture was
analyzed by electrophoresis
on a 1 % agarose mini-gel to determine which reactions were successful in
extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%) agarose
gels, fragments were excised, and agar digested with Agar ACE (Promega).
Extended clones were
religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector
(Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site overhangs,
and transfected into competent E. coli cells. Transformed cells were selected
on antibiotic-containing
media, and individual colonies were picked and cultured overnight at
37°C in 384-well plates in LB/2x
carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following parameters:
Step l: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C,
1 min; Step 4: 72°C, 2 min; Step 5: steps 2,
3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at
4°C. DNA was quantified by
PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries were
reamplified using the same conditions as described above. Samples were diluted
with 20%
dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing primers and
the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (PE Biosystems).
In like manner, the polynucleotide sequences of SEQ ID N0:14-26 are used to
obtain 5'
regulatory sequences using the procedure above, along with oligonucleotides
designed for such
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WO 00/78952 PCT/US00/16644
extension, and an appropriate genomic library.
VII. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:14-26 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide fragments.
Oligonucleotides are designed using state-of the-art software such as OLIGO
4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~Ci of ['y-
32P] adenosine
triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase
(DuPont NEN, Boston
MA). The labeled oligonucleotides are substantially purified using a SEPHADEX
G-25 superfine size
exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot
containing 10' counts per
minute of the labeled probe is used in a typical membrane-based hybridization
analysis of human
genomic DNA digested with one of the following endonucleases: Ase I, Bgl II,
Eco RI, Pst I, Xba I, or
Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared
VIII. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink-jet printing, See, e.g.,
Baldeschweiler, su ra , mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), su ra . Suggested
substrates include silicon, silica, glass slides, glass chips, and silicon
wafers. Alternatively, a procedure
analogous to a dot or slot blot may also be used to arrange and link elements
to the surface of a
substrate using thermal, UV, chemical, or mechanical bonding procedures. A
typical array may be
produced using available methods and machines well known to those of ordinary
skill in the art and may
contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470;
Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson
(1998) Nat. Biotechnol.
16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The array
elements are hybridized with polynucleotides in a biological sample. The
polynucleoddes in the
57
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described in
detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(d'I~ cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/Eil oligo-(d'I~
primer (2lmer), 1X first
strand buffer, 0.03 units/~.Q RNase inhibitor, 500 E.iM dATP, 500 ~M dGTP, 500
~M dTTP, 40 ~M
dCTP, 40 EiM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37 °C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium
hydroxide and
incubated for 20 minutes at 85 °C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
N~ and
resuspended in 14 ~.~1 SX SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
fig. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
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WO 00/78952 PCT/US00/16644
oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522, incorporated herein by reference. 1 ~.il of the array
element DNA, at an average
concentration of 100 ng/Erl, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microatrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60
°C followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 ~1 of sample mixture consisting of 0.2 ~g
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65 °C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of
140 ~1 of SX SSC in a corner of the chamber. The chamber containing the arrays
is incubated for
about 6.5 hours at 60 °C. The arrays are washed for 10 min at 45
°C in a first wash buffer (1X SSC,
0.1% SDS), three times for 10 minutes each at 45 °C in a second wash
buffer (0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater N.>] corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
59
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are differentially
expressed, the calibration is done by labeling samples of the calibrating cDNA
with the two
fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
IX. Complementary Polynucleotides
Sequences complementary to the RMEP-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring RMEP. Although
use of oligonucleotides
comprising from about 15 to 30 base pairs is described, essentially the same
procedure is used with
smaller or with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of RMEP. To
inhibit transcription, a
complementary oligonucleotide is designed from the most unique 5' sequence and
used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is
designed to prevent ribosomal binding to the RMEP-encoding transcript.
X. Expression of RMEP
Expression and purification of RMEP is achieved using bacterial or virus-based
expression
systems. For expression of RMEP in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express RMEP upon induction with isopropyl beta-
D-
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
thiogalactopyranoside (IPTG). Expression of RMEP in eukaryotic cells is
achieved by infecting insect
or mammalian cell lines with recombinant AutoQraphica californica nuclear
polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding RMEP by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera fru~iperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional generic modifications to
baculovirus. (See Engelhard, E.K. et
al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)
Hum. Gene Ther.
7:1937-1945.)
In most expression systems, RMEP is synthesized as a fusion protein with,
e.g., glutathione S-
transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Schistosoma ianonicum, enables the purification of fusion proteins
on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Pharmacia
Biotech). Following purification, the GST moiety can be proteolytically
cleaved from RMEP at
specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity purification
using commercially available monoclonal and polyclonal anti-FLAG antibodies
(Eastman Kodak). 6-
His, a stretch of six consecutive histidine residues, enables purification on
metal-chelate resins
(QIAGEN). Methods for protein expression and purification are discussed in
Ausubel (1995, supra,
ch. 10 and 16). Purified RMEP obtained by these methods can be used directly
in the assays shown in
Examples X and XIV.
XI. Demonstration of RMEP Activity
RMEP RNA-binding activity is demonstrated by a polyacrylamide gel mobility-
shift assay.
In preparation for this assay, RMEP is expressed by transforming a mammalian
cell line such as
COS7, HeLa or CHO with a eukaryotic expression vector containing RMEP cDNA.
The cells are
incubated for 48-72 hours after transformation under conditions appropriate
for the cell line to allow
expression and accumulation of RMEP. Extracts containing solubilized proteins
can be prepared
from cells expressing RMEP by methods well known in the art. Portions of the
extract containing
RMEP are added to [32P]-labeled RNA. Radioactive RNA can be synthesized in
vitro by techniques
well known in the art. The mixtures are incubated at 25 °C in the
presence of RNase inhibitors under
buffered conditions for 5-10 minutes. After incubation, the samples are
analyzed by polyacrylamide
gel electrophoresis followed by autoradiography. The presence of a band on the
autoradiogram
indicates the formation of a complex between RMEP and the radioactive
transcript. A band of similar
mobility will not be present in samples prepared using control extracts
prepared from untransformed
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WO 00/78952 PCT/US00/16644
cells.
Alternatively, RMEP, or biologically active fragments thereof, are labeled
with "~I
Bolton-Hunter reagent and tested for interaction with candidate RNA metabolism
molecules. (See, e.g.,
Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously
arrayed in the wells of a
multi-well plate are incubated with the labeled RMEP, washed, and any wells
with labeled RMEP
complex are assayed. Data obtained using different concentrations of RMEP are
used to calculate
values for the number, affinity, and association of RMEP with the candidate
molecules.
Alternatively, molecules interacting with RMEP are analyzed using the yeast
two-hybrid
system as described in Fields, S. and Song, O. (1989) Nature 340:245-246, or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(CLONTECH).
XII. Functional Assays
RMEP function is assessed by expressing the sequences encoding RMEP at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid
(Invitrogen), both of which
contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are
transiently transfected into a
human cell line, for example, an endothelial or hematopoietic cell line, using
either liposome
formulations or electroporation. 1-2 ~g of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate the
apoptotic state of the cells and other cellular properties. FCM detects and
quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events include
changes in nuclear DNA content as measured by staining of DNA with propidium
iodide; changes in
cell size and granularity as measured by forward light scatter and 90 degree
side light scatter; down-
regulation of DNA synthesis as measured by decrease in bromodeoxyuridine
uptake; alterations in
expression of cell surface and intracellular proteins as measured by
reactivity with specific antibodies;
and alterations in plasma membrane composition as measured by the binding of
fluorescein-conjugated
Annexin V protein to the cell surface. Methods in flow cytometry are discussed
in Ormerod, M.G.
(1994) Flow Cvtometry, Oxford, New York NY.
The influence of RMEP on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding RMEP and either CD64 or CD64-GFP.
CD64 and CD64-
GFP are expressed on the surface of transfected cells and bind to conserved
regions of human
62
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success N~.
mRNA can be purified from the cells using methods well known by those of skill
in the art. Expression
of mRNA encoding RMEP and other genes of interest can be analyzed by northern
analysis or
microarray techniques.
XIII. Production of RMEP Specific Antibodies
RMEP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the RMEP amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with
the oligopeptide-KLH
complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-RMEP
activity by, for example, binding the peptide or RMEP to a substrate, blocking
with 1 % BSA, reacting
with rabbit antisera, washing, and reacting with radio-iodinated goat anti-
rabbit IgG.
XIV. Purification of Naturally Occurring RMEP Using Specific Antibodies
Naturally occurring or recombinant RMEP is substantially purified by
immunoaffinity
chromatography using antibodies specific for RMEP. An immunoaffinity column is
constructed by
covalently coupling anti-RMEP antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing RMEP are passed over the immunoaffinity column, and the
column is washed
under conditions that allow the preferential absorbance of RMEP (e.g., high
ionic strength buffers in the
presence of detergent). The column is eluted under conditions that disrupt
antibody/RMEP binding
(e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such
as urea or thiocyanate ion),
and RMEP is collected.
XV. Identification of Molecules Which Interact with RMEP
RMEP, or biologically active fragments thereof, are labeled with'ZSI Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
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WO 00/78952 PCT/US00/16644
previously arrayed in the wells of a mufti-well plate are incubated with the
labeled RMEP, washed, and
any wells with labeled RMEP complex are assayed. Data obtained using different
concentrations of
RMEP are used to calculate values for the number, affinity, and association of
RMEP with the
candidate molecules.
Alternatively, molecules interacting with RMEP are analyzed using the yeast
two-hybrid
system as described in Fields, S. and O. Song ( 1989, Nature 340:245-246), or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
RMEP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S. Patent
No. 6,057,1 O 1 ).
Various modifications and variations of the described methods and systems of
the invention will
be apparent to those skilled in the art without departing from the scope and
spirit of the invention.
Although the invention has been described in connection with certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which are obvious
to those skilled in molecular biology or related fields are intended to be
within the scope of the following
claims.
64
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
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CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
BANDMAN, Olga
YUE, Henry
LAL, Preeti
TANG, Y. Tom
REDDY, Roopa
BAUGHN, Mariah R.
AZIMZAI, Yalda
<120> RNA METABOLISM PROTEINS
<130> PF-0712 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/139,922
<151> 1999-06-17
<160> 26
<170> PERL Program
<210> 1
<211> 503
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 046926
<400> 1
Met Glu Tyr Met Ala Glu Ser Thr Asp Arg Ser Pro Gly His Ile
1 5 10 15
Leu Cys Cys Glu Cys Gly Val Pro Ile Ser Pro Asn Pro Ala Asn
20 25 30
Ile Cys Val Ala Cys Leu Arg Ser Lys Val Asp Ile Ser Gln Gly
35 40 45
Ile Pro Lys Gln Val Ser Ile Ser Phe Cys Lys Gln Cys Gln Arg
50 55 60
Tyr Phe Gln Pro Pro Gly Thr Trp Ile Gln Cys Ala Leu Glu Ser
65 70 75
Arg Glu Leu Leu Ala Leu Cys Leu Lys Lys Ile Lys Ala Pro Leu
80 85 90
Ser Lys Val Arg Leu Val Asp Ala Gly Phe Val Trp Thr Glu Pro
95 100 105
His Ser Lys Arg Leu Lys Val Lys Leu Thr Ile Gln Lys Glu Val
110 115 120
Met Asn Gly Ala Ile Leu Gln Gln Val Phe Val Val Asp Tyr Val
125 130 135
Val Gln Ser Gln Met Cys Gly Asp Cys His Arg Val Glu Ala Lys
140 145 150
Asp Phe Trp Lys Ala Val Ile Gln Val Arg Gln Lys Thr Leu His
155 160 165
1/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
Lys Lys Thr Phe Tyr Tyr Leu Glu Gln Leu Ile Leu Lys Tyr Gly
170 175 180
Met His Gln Asri Thr Leu Arg Ile Lys Glu Ile His Asp Gly Leu
185 190 195
Asp Phe Tyr Tyr Ser Ser Lys Gln His Ala Gln Lys Met Val Glu
200 205 210
Phe Leu Gln Cys Thr Val Pro Cys Arg Tyr Lys Ala Ser Gln Arg
215 220 225
Leu Ile Ser Gln Asp Ile His Ser Asn Thr Tyr Asn Tyr Lys Ser
230 235 240
Thr Phe Ser Val Glu Ile Val Pro Ile Cys Lys Asp Asn Val Val
245 250 255
Cys Leu Ser Pro Lys Leu Ala Gln Ser Leu Gly Asn Met Asn Gln
260 265 270
Ile Cys Val Cys Ile Arg Val Thr Ser Ala Ile His Leu Ile Asp
275 280 285
Pro Asn Thr Leu Gln Val Ala Asp Ile Asp Gly Ser Thr Phe Trp
290 295 300
Ser His Pro Phe Asn Ser Leu Cys His Pro Lys Gln Leu Glu Glu
305 310 315
Phe Ile Val Met Glu Cys Ser Ile Val Gln Asp Ile Lys Arg Ala
320 325 330
Ala Gly Ala Gly Met Ile Ser Lys Lys His Thr Leu Gly Glu Val
335 340 345
Trp Val Gln Lys Thr Ser Glu Met Asn Thr Asp Lys Gln Tyr Phe
350 355 360
Cys Arg Thr His Leu Gly His Leu Leu Asn Pro Gly Asp Leu Val
365 370 375
Leu Gly Phe Asp Leu Ala Asn Cys Asn Leu Asn Asp Glu His Val
380 385 390
Asn Lys Met Asn Ser Asp Arg Val Pro Asp Val Val Leu Ile Lys
395 400 405
Lys Ser Tyr Asp Arg Thr Lys Arg Gln Arg Arg Arg Asn Trp Lys
410 415 420
Leu Lys Glu Leu Ala Arg Glu Arg Glu Asn Met Asp Thr Asp Asp
425 430 435
Glu Arg Gln Tyr Gln Asp Phe Leu Glu Asp Leu Glu Glu Asp Glu
440 445 450
Ala Ile Arg Lys Asn Val Asn Ile Tyr Arg Asp Ser Ala Ile Pro
455 460 465
Val Glu Ser Asp Thr Asp Asp Glu Gly Ala Pro Arg Ile Ser Leu
470 475 480
Ala Glu Met Leu Glu Asp Leu His Ile Ser Gln Asp Ala Thr Gly
485 490 495
Glu Glu Gly Ala Ser Met Leu Thr
500
<210> 2
<211> 594
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 618791
<400> 2
2/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
Met Ser Ala Gly Glu Val Glu Arg Leu Val Ser Glu Leu Ser Gly
1 5 10 15
Gly Thr Gly Gly Asp Glu Glu Glu Glu Trp Leu Tyr Gly Gly Pro
20 25 30
Trp Asp Val His Val His Ser Asp Leu Ala Lys Asp Leu Asp Glu
35 40 45
Asn Glu Val Glu Arg Pro Glu Glu Glu Asn Ala Ser Ala Asn Pro
50 55 60
Pro Ser Gly Ile Glu Asp Glu Thr Ala Glu Asn Gly Val Pro Lys
65 70 75
Pro Lys Val Thr Glu Thr Glu Asp Asp Ser Asp Ser Asp Ser Asp
80 85 90
Asp Asp Glu Asp Asp Val His Val Thr Ile Gly Asp Ile Lys Thr
95 100 105
Gly Ala Pro Gln Tyr Gly Ser Tyr Gly Thr Ala Pro Val Asn Leu
110 115 120
Asn Ile Lys Thr Gly Gly Arg Val Tyr Gly Thr Thr Gly Thr Lys
125 130 135
Val Lys Gly Val Asp Leu Asp Ala Pro Gly Ser Ile Asn Gly Val
140 145 150
Pro Leu Leu Glu Val Asp Leu Asp Ser Phe Glu Asp Lys Pro Trp
155 160 165
Arg Lys Pro Gly Ala Asp Leu Ser Asp Tyr Phe Asn Tyr Gly Phe
170 175 180
Asn Glu Asp Thr Trp Lys Ala Tyr Cys Glu Lys Gln Lys Arg Ile
185 190 195
Arg Met Gly Leu Glu Val Ile Pro Val Thr Ser Thr Thr Asn Lys
200 205 210
Ile Thr Ala Glu Asp Cys Thr Met Glu Val Thr Pro Gly Ala Glu
215 220 225
Ile Gln Asp Gly Arg Phe Asn Leu Phe Lys Val Gln Gln Gly Arg
230 235 240
Thr Gly Asn Ser Glu Lys Glu Thr Ala Leu Pro Ser Thr Lys Ala
245 250 255
Glu Phe Thr Ser Pro Pro Ser Leu Phe Lys Thr Gly Leu Pro Pro
260 265 270
Ser Arg Asn Ser Thr Ser Ser Gln Ser Gln Thr Ser Thr Ala Ser
275 280 285
Arg Lys Ala Asn Ser Ser Val Gly Lys Trp Gln Asp Arg Tyr Gly
290 295 300
Arg Ala Glu Ser Pro Asp Leu Arg Arg Leu Pro Gly Ala Ile Asp
305 310 315
Val Ile Gly Gln Thr Ile Thr Ile Ser Arg Val Glu Gly Arg Arg
320 325 330
Arg Ala Asn Glu Asn Ser Asn Ile Gln Val Leu Ser Glu Arg Ser
335 340 345
Ala Thr Glu Val Asp Asn Asn Phe Ser Lys Pro Pro Pro Phe Phe
350 355 360
Pro Pro Gly Ala Pro Pro Thr His Leu Pro Pro Pro Pro Phe Leu
365 370 375
Pro Pro Pro Pro Thr Val Ser Thr Ala Pro Pro Leu Ile Pro Pro
380 385 390
Pro Gly Phe Pro Pro Pro Pro Gly Ala Pro Pro Pro Ser Leu Ile
395 400 405
Pro Thr Ile Glu Ser Gly His Ser Ser Gly Tyr Asp Ser Arg Ser
410 415 420
Ala Arg Ala Phe Pro Tyr Gly Asn Val Ala Phe Pro His Leu Pro
425 430 435
Gly Ser Ala Pro Ser Trp Pro Ser Leu Val Asp Thr Ser Lys Gln
440 445 450
Trp Asp Tyr Tyr Ala Arg Arg Glu Lys Asp Arg Asp Arg Glu Arg
3/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
455 460 465
Asp Arg Asp Arg Glu Arg Asp Arg Asp Arg Asp Arg Glu Arg Glu
470 475 480
Arg Thr Arg Glu Arg Glu Arg Glu Arg Asp His Ser Pro Thr Pro
485 490 495
Ser Val Phe Asn Ser Asp Glu Glu Arg Tyr Arg Tyr Arg Glu Tyr
500 505 510
Ala Glu Arg Gly Tyr Glu Arg His Arg Ala Ser Arg Glu Lys Glu
515 520 525
Glu Arg His Arg Glu Arg Arg His Arg Glu Lys Glu Glu Thr Arg
530 535 540
His Lys Ser Ser Arg Ser Asn Ser Arg Arg Arg His Glu Ser Glu
545 550 555
Glu Gly Asp Ser His Arg Arg His Lys His Lys Lys Ser Lys Arg
560 565 570
Ser Lys Glu Gly Lys Glu Ala Gly Ser Glu Pro Ala Pro Glu Gln
575 580 585
Glu Ser Thr Glu Ala Thr Pro Ala Glu
590
<210> 3
<211> 413
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1240366
<400> 3
Met Ser Glu Thr Gln Tyr Ser Ser Leu Thr Gln Thr Leu Ile Met
1 5 10 15
Thr Met Lys Leu Ser Gly Phe Gly Val Ala Asp Ser Met Arg Ile
20 25 30
Ser Gly Cys Ser Ile Gln Lys Gln Ser Arg Ile Ile Ile Thr Asp
35 40 45
Arg Gln Ala Glu Pro Pro Lys Lys Glu Ala Ala Thr Thr Gly Pro
50 55 60
Gln Val Lys Arg Ala Asp Glu Trp Lys Asp Pro Trp Arg Arg Ser
65 70 75
Lys Ser Pro Lys Lys Lys Leu Gly Val Ser Val Ser Pro Ser Arg
80 85 90
Ala Arg Arg Arg Arg Lys Thr Ser Ala Ser Ser Ala Ser Ala Ser
95 100 105
Asn Ser Ser Arg Ser Ser Ser Arg Ser Ser Ser Tyr Ser Gly Ser
110 115 120
Gly Ser Ser Arg Ser Arg Ser Arg Ser Ser Ser Tyr Ser Ser Tyr
125 130 135
Ser Ser Arg Ser Ser Arg His Ser Ser Phe Ser Gly Ser Arg Ser
140 145 150
Arg Ser Arg Ser Phe Ser Ser Ser Pro Ser Pro Ser Pro Thr Pro
155 160 165
Ser Pro His Arg Pro Ser Ile Arg Thr Lys Gly Glu Pro Ala Pro
170 175 180
Pro Pro Gly Lys Ala Gly Glu Lys Ser Val Lys Lys Pro Ala Pro
185 190 195
Pro Pro Ala Pro Pro Gln Ala Thr Lys Thr Thr Ala Pro Val Pro
4/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
200 205 210
Glu Pro Thr Lys Pro Gly Asp Pro Arg Glu Ala Arg Arg Lys Glu
215 220 225
Arg Pro Ala Arg Thr Pro Pro Arg Arg Arg Thr Leu Ser Gly Ser
230 235 240
Gly Ser Gly Ser Gly Ser Ser Tyr Ser Gly Ser Ser Ser Arg Ser
245 250 255
Arg Ser Leu Ser Val Ser Ser Val Ser Ser Val Ser Ser Ala Thr
260 265 270
Ser Ser Ser Ser Ser Ala His Ser Val Asp Ser Glu Asp Met Tyr
275 280 285
Ala Asp Leu Ala Ser Pro Val Ser Ser Ala Ser Ser Arg Ser Pro
290 295 300
Ala Pro Ala Gln Thr Arg Lys Glu Lys Gly Lys Ser Lys Lys Glu
305 310 315
Asp Gly Val Lys Glu Glu Lys Arg Lys Arg Asp Ser Ser Thr Gln
320 325 330
Pro Pro Lys Ser Ala Lys Pro Pro Ala Gly Gly Lys Ser Ser Gln
335 340 345
Gln Pro Ser Thr Pro Gln Gln Ala Pro Pro Gly Gln Pro Gln Gln
350 355 360
Gly Thr Phe Val Ala His Lys Glu Ile Lys Leu Thr Leu Leu Asn
365 370 375
Lys Ala Ala Asp Lys Gly Ser Arg Lys Arg Tyr Glu Pro Ser Asp
380 385 390
Lys Asp Arg Gln Ser Pro Pro Pro Ala Lys Arg Pro Asn Thr Ser
395 400 405
Pro Asp Arg Gly Ser Arg Asp Arg
410
<210> 4
<211> 219
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1295773
<400> 4
Met His Val Gln Leu Ser Thr Ser Arg Leu Arg Thr Ala Pro Gly
1 5 10 15
Met Gly Asp Gln Ser Gly Cys Tyr Arg Cys Gly Lys Glu Gly His
20 25 30
Trp Ser Lys Glu Cys Pro Val Asp Arg Thr Gly Arg Val Ala Asp
35 40 45
Phe Thr Glu Gln Tyr Asn Glu Gln Tyr Gly Ala Val Arg Thr Pro
50 55 60
Tyr Thr Met Gly Tyr Gly Glu Ser Met Tyr Tyr Asn Asp Ala Tyr
65 70 75
Gly Ala Leu Asp Tyr Tyr Lys Arg Tyr Arg Val Arg Ser Tyr Glu
80 85 90
Ala Val Ala Ala Ala Ala Ala Ala Ser Ala Tyr Asn Tyr Ala Glu
95 100 105
Gln Thr Met Ser His Leu Pro Gln Val Gln Ser Thr Thr Val Thr
110 115 120
Ser His Leu Asn Ser Thr Ser Val Asp Pro Tyr Asp Arg His Leu
125 130 135
5/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
Leu Pro Asn Ser Gly Ala Ala Ala Thr Ser Ala Ala Met Ala Ala
140 145 150
Ala Ala Ala Thr Thr Ser Ser Tyr Tyr Gly Arg Asp Arg Ser Pro
155 160 165
Leu Arg Arg Ala Ala Ala Met Leu Pro Thr Val Gly Glu Gly Tyr
170 175 180
Gly Tyr Gly Pro Glu Ser Glu Leu Ser Gln Ala Ser Ala Ala Thr
185 190 195
Arg Asn Ser Leu Tyr Asp Met Ala Arg Tyr Glu Arg Glu Gln Tyr
200 205 210
Val Asp Arg Ala Arg Tyr Ser Ala Phe
215
<210> 5
<211> 641
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1858421
<400> 5
Met Gly Arg Arg Ser Thr Ser Ser Thr Lys Ser Gly Lys Phe Met
1 5 10 15
Asn Pro Thr Asp Gln Ala Arg Lys Glu Ala Arg Lys Arg Glu Leu
20 25 30
Lys Lys Asn Lys Lys Gln Arg Met Met Val Arg Ala Ala Val Leu
35 40 45
Lys Met Lys Asp Pro Lys Gln Ile Ile Arg Asp Met Glu Lys Leu
50 55 60
Asp Glu Met Glu Phe Asn Pro Val Gln Gln Pro Gln Leu Asn Glu
65 70 75
Lys Val Leu Lys Asp Lys Arg Lys Lys Leu Arg Glu Thr Phe Glu
80 85 90
Arg Ile Leu Arg Leu Tyr Glu Lys Glu Asn Pro Asp Ile Tyr Lys
95 100 105
Glu Leu Arg Lys Leu Glu Val Glu Tyr Glu Gln Lys Arg Ala Gln
110 115 120
Leu Ser Gln Tyr Phe Asp Ala Val Lys Asn Ala Gln His Val Glu
125 130 135
Val Glu Ser Ile Pro Leu Pro Asp Met Pro His Ala Pro Ser Asn
140 145 150
Ile Leu Ile Gln Asp Ile Pro Leu Pro Gly Ala Gln Pro Pro Ser
155 160 165
Ile Leu Lys Lys Thr Ser Ala Tyr Gly Pro Pro Thr Arg Ala Val
170 175 180
Ser Ile Leu Pro Leu Leu Gly His Gly Val Pro Arg Leu Pro Pro
185 190 195
Gly Arg Lys Pro Pro Gly Pro Pro Pro Gly Pro Pro Pro Pro Gln
200 205 210
Val Val Gln Met Tyr Gly Arg Lys Val Gly Phe Ala Leu Asp Leu
215 220 225
Pro Pro Arg Arg Arg Asp Glu Asp Met Leu Tyr Ser Pro Glu Leu
230 235 240
Ala Gln Arg Gly His Asp Asp Asp Val Ser Ser Thr Ser Glu Asp
245 250 255
Asp Gly Tyr Pro Glu Asp Met Asp Gln Asp Lys His Asp Asp Ser
6/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
260 265 270
Thr Asp Asp Ser Asp Thr Asp Lys Ser Asp Gly G1u Ser Asp Gly
275 280 285
Asp Glu Phe Val His Arg Asp Asn Gly Glu Arg Asp Asn Asn Glu
290 295 300
Glu Lys Lys Ser Gly Leu Ser Val Arg Phe Ala Asp Met Pro Gly
305 310 315
Lys Ser Arg Lys Lys Lys Lys Asn Met Lys Glu Leu Thr Pro Leu
320 325 330
Gln Ala Met Met Leu Arg Met Ala Gly Gln Glu Ile Pro Glu Glu
335 340 345
Gly Arg Glu Val Glu Glu Phe Ser Glu Asp Asp Asp Glu Asp Asp
350 355 360
Ser Asp Asp Ser Glu Ala Glu Lys Gln Ser Gln Lys Gln His Lys
365 370 375
Glu Glu Ser His Ser Asp Gly Thr Ser Thr Ala Ser Ser Gln Gln
380 385 390
Gln Ala Pro Pro Gln Ser Val Pro Pro Ser Gln Ile Gln Ala Pro
395 400 405
Pro Met Pro Gly Pro Pro Pro Leu Gly Pro Pro Pro Ala Pro Pro
410 415 420
Leu Arg Pro Pro Gly Pro Pro Thr Gly Leu Pro Pro Gly Pro Pro
425 430 435
Pro Gly Ala Pro Pro Phe Leu Arg Pro Pro Gly Met Pro Gly Leu
440 445 450
Arg Gly Pro Leu Pro Arg Leu Leu Pro Pro Gly Pro Pro Pro Gly
455 460 465
Arg Pro Pro Gly Pro Pro Pro Gly Pro Pro Pro Gly Leu Pro Pro
470 475 480
Gly Pro Pro Pro Arg Gly Pro Pro Pro Arg Leu Pro Pro Pro Ala
485 490 495
Pro Pro Gly Ile Pro Pro Pro Arg Pro Gly Met Met Arg Pro Pro
500 505 510
Leu Val Pro Pro Leu Gly Pro Ala Pro Pro Gly Leu Phe Pro Pro
515 520 525
Ala Pro Leu Pro Asn Pro Gly Val Leu Ser Ala Pro Pro Asn Leu
530 535 540
Ile Gln Arg Pro Lys Ala Asp Asp Thr Ser Ala Ala Thr Ile Glu
545 550 555
Lys Lys Ala Thr Ala Thr Ile Ser Ala Lys Pro Gln Ile Thr Asn
560 565 570
Pro Lys Ala Glu Ile Thr Arg Phe Val Pro Thr Ala Leu Arg Val
575 580 585
Arg Arg Glu Asn Lys Gly Ala Thr Ala Ala Pro Gln Arg Lys Ser
590 595 600
Glu Asp Asp Ser Ala Val Pro Leu Ala Lys Ala Ala Pro Lys Ser
605 610 615
Gly Pro Ser Val Pro Val Ser Val Gln Thr Lys Asp Asp Val Tyr
620 625 630
Glu Ala Phe Met Lys Glu Met Glu Gly Leu Leu
635 640
<210> 6
<211> 153
<212> PRT
<213> Homo Sapiens
7/23
CA 02375407 2001-11-28
WO 00/78952 PCT/LTS00/16644
<220>
<221> misc_feature
<223> Incyte Clone No: 2152431
<400> 6
Met Ala Asp Glu Ile Asp Phe Thr Thr Gly Asp Ala Gly Ala Ser
1 5 10 15
Ser Thr Tyr Pro Met Gln Cys Ser Ala Leu Arg Lys Asn Gly Phe
20 25 30
Val Val Leu Lys Gly Arg Pro Cys Lys Ile Val Glu Met Ser Thr
35 40 45
Ser Lys Thr Gly Lys His Gly His Ala Lys Val His Leu Val Gly
50 55 60
Ile Asp Ile Phe Thr Gly Lys Lys Tyr Glu Asp Ile Cys Pro Ser
65 70 75
Thr His Asn Met Asp Val Pro Asn Ile Lys Arg Asn Asp Tyr Gln
80 85 90
Leu Ile Cys Ile Gln Asp Gly Tyr Leu Ser Leu Leu Thr Glu Thr
95 100 105
Gly Glu Val Arg Glu Asp Leu Lys Leu Pro Glu Gly Glu Leu Gly
110 115 120
Lys Glu Ile Glu Gly Lys Tyr Asn Ala Gly Glu Asp Val Gln Val
125 130 135
Ser Val Met Cys Ala Met Ser Glu Glu Tyr Ala Val Ala Ile Lys
140 145 150
Pro Cys Lys
<210> 7
<211> 194
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2641494
<400> 7
Met G1n Ala Val Arg Asn Ala Gly Ser Arg Phe Leu Arg Ser Trp
1 5 10 15
Thr Trp Pro Gln Thr Ala Gly Arg Val Val Ala Arg Thr Pro Ala
20 25 30
Gly Thr Ile Cys Thr Gly Ala Arg Gln Leu Gln Asp Ala Ala Ala
35 40 45
Lys Gln Lys Val Glu Gln Asn Ala Ala Pro Ser His Thr Lys Phe
50 55 60
Ser Ile Tyr Pro Pro Ile Pro Gly Glu Glu Ser Ser Leu Arg Trp
65 70 75
Ala Gly Lys Lys Phe Glu Glu Ile Pro Ile Ala His Ile Lys Ala
80 85 90
Ser His Asn Asn Thr Gln Ile Gln Val Val Ser Ala Ser Asn Glu
95 100 105
Pro Leu Ala Phe Ala Ser Cys Gly Thr Glu Gly Phe Arg Asn Ala
110 115 120
Lys Lys Gly Thr Gly Ile Ala Ala Gln Thr Ala Gly Ile Ala Ala
125 130 135
Ala Ala Arg Ala Lys Gln Lys Gly Val Ile His Ile Arg Val Val
140 145 150
Val Lys Gly Leu Gly Pro Gly Arg Leu Ser Ala Met His Gly Leu
8/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
155 160 165
Ile Met Gly Gly Leu Glu Val Ile Ser Ile Thr Asp Asn Thr Pro
170 175 180
Ile Pro His Asn Gly Cys Arg Pro Arg Lys Ala Arg Lys Leu
185 190
<210> 8
<211> 629
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 3803409
<400> 8
Met Gly Lys Pro Pro Gly Ser Ile Val Arg Pro Ser Ala Pro Pro
1 5 10 15
Ala Arg Ser Ser Val Pro Val Thr Arg Pro Pro Val Pro Ile Pro
20 25 30
Pro Pro Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Val Ile
35 40 45
Lys Pro Gln Thr Ser Ala Val Glu Gln Glu Arg Trp Asp Glu Asp
50 55 60
Ser Phe Tyr Gly Leu Trp Asp Thr Asn Asp Glu Gln Gly Leu Asn
65 70 75
Ser Glu Phe Lys Ser Glu Thr Ala Ala Ile Pro Ser Ala Pro Val
80 85 90
Leu Pro Pro Pro Pro Val His Ser Ser Ile Pro Pro Pro Gly Pro
95 100 105
Val Pro Met Gly Met Pro Pro Met Ser Lys Pro Pro Pro Val Gln
110 115 120
Gln Thr Val Asp Tyr Gly His Gly Arg Asp Ile Ser Thr Asn Lys
125 130 135
Val Glu Gln Ile Pro Tyr Gly Glu Arg Ile Thr Leu Arg Pro Asp
140 145 150
Pro Leu Pro Glu Arg Ser Thr Phe Glu Thr Glu His Ala Gly Gln
155 160 165
Arg Asp Arg Tyr Asp Arg Glu Arg Asp Arg Glu Pro Tyr Phe Asp
170 175 180
Arg Gln Ser Asn Val Ile Ala Asp His Arg Asp Phe Lys Arg Asp
185 190 195
Arg Glu Thr His Arg Asp Arg Asp Arg Asp Arg Gly Val Ile Asp
200 205 210
Tyr Asp Arg Asp Arg Phe Asp Arg Glu Arg Arg Pro Arg Asp Asp
215 220 225
Arg Ala Gln Ser Tyr Arg Asp Lys Lys Asp His Ser Ser Ser Arg
230 235 240
Arg Gly Gly Phe Asp Arg Pro Ser Tyr Asp Arg Lys Ser Asp Arg
245 250 255
Pro Val Tyr Glu Gly Pro Ser Met Phe Gly Gly Glu Arg Arg Thr
260 265 270
Tyr Pro Glu Glu Arg Met Pro Leu Pro Ala Pro Ser Leu Ser His
275 280 285
Gln Pro Pro Pro Ala Pro Arg Val Glu Lys Lys Pro Glu Ser Lys
290 295 300
Asn Val Asp Asp Ile Leu Lys Pro Pro Gly Arg Glu Ser Arg Pro
305 310 315
9/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
Glu Arg Ile Val Val Ile Met Arg Gly Leu Pro Gly Ser Gly Lys
320 325 330
Thr His Val Ala Lys Leu Ile Arg Asp Lys Glu Val Glu Phe Gly
335 340 345
Gly Pro Ala Pro Arg Val Leu Ser Leu Asp Asp Tyr Phe Ile Thr
350 355 360
Glu Val Glu Lys Glu Glu Lys Asp Pro Asp Ser Gly Lys Lys Val
365 370 375
Lys Lys Lys Val Met Glu Tyr Glu Tyr Glu Ala Glu Met Glu Glu
380 385 390
Thr Tyr Arg Thr Ser Met Phe Lys Thr Phe Lys Lys Thr Leu Asp
395 400 405
Asp Gly Phe Phe Pro Phe Ile Ile Leu Asp Ala Ile Asn Asp Arg
410 415 420
Val Arg His Phe Asp Gln Phe Trp Ser Ala Ala Lys Thr Lys Gly
425 430 435
Phe Glu Val Tyr Leu Ala Glu Met Ser Ala Asp Asn Gln Thr Cys
440 445 450
Gly Lys Arg Asn Ile His Gly Arg Lys Leu Lys Glu Ile Asn Lys
455 460 465
Met Ala Asp His Trp Glu Thr Ala Pro Arg His Met Met Arg Leu
470 475 480
Asp Ile Arg Ser Leu Leu Gln Asp Ala Ala Ile Glu Glu Val Glu
485 490 495
Met Glu Asp Phe Asp Ala Asn Ile Glu Glu Gln Lys Glu Glu Lys
500 505 510
Lys Asp Ala Glu Glu Glu Glu Ser Glu Leu Gly Tyr Ile Pro Lys
515 520 525
Ser Lys Trp Glu Met Asp Thr Ser Glu Ala Lys Leu Asp Lys Leu
530 535 540
Asp Gly Leu Arg Thr Gly Thr Lys Arg Lys Arg Asp Trp Glu Ala
545 550 555
Ile Ala Ser Arg Met Glu Asp Tyr Leu Gln Leu Pro Asp Asp Tyr
560 565 570
Asp Thr Arg Ala Ser Glu Pro Gly Lys Lys Arg Val Arg Trp Ala
575 580 585
Asp Leu Glu Glu Lys Lys Asp Ala Asp Arg Lys Arg Ala Ile Gly
590 595 600
Phe Val Val Gly Gln Thr Asp Trp Glu Lys Ile Thr Asp Glu Ser
605 610 615
Gly His Leu Ala Glu Lys Ala Leu Asn Arg Thr Lys Tyr Ile
620 625
<210> 9
<211> 445
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 3979009
<400> 9
Met Asn Arg His Leu Cys Val Trp Leu Phe Arg His Pro Ser Leu
1 5 10 15
Asn Gly Tyr Leu Gln Cys His Ile Gln Leu His Ser His Gln Phe
20 25 30
Arg Gln Ile His Leu Asp Thr Arg Leu Gln Val Phe Arg Gln Asn
10/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
35 40 45
Arg Asn Cys Ile Leu His Leu Leu Ser Lys Asn Trp Ser Arg Arg
50 55 60
Tyr Cys His Gln Asp Thr Lys Met Leu Trp Lys His Lys Ala Leu
65 70 75
Gln Lys Tyr Met Glu Asn Leu Ser Lys Glu Tyr Gln Thr Leu Glu
80 85 90
Gln Cys Leu Gln His Ile Pro Val Asn Glu Glu Asn Arg Arg Ser
95 100 105
Leu Asn Arg Arg His Ala Glu Leu Ala Pro Leu Ala Ala Ile Tyr
110 115 120
Gln Glu Ile Gln Glu Thr Glu Gln Ala Ile Glu Glu Leu Glu Ser
125 130 135
Met Cys Lys Ser Leu Asn Lys Gln Asp Glu Lys Gln Leu Gln Glu
140 145 150
Leu Ala Leu Glu Glu Arg Gln Thr Ile Asp Gln Lys Ile Asn Met
155 160 165
Leu Tyr Asn Glu Leu Phe Gln Ser Leu Val Pro Lys Glu Lys Tyr
170 175 180
Asp Lys Asn Asp Val Ile Leu Glu Val Thr Ala Gly Arg Thr Thr
185 190 195
Gly Gly Asp Ile Cys Gln Gln Phe Thr Arg Glu Ile Phe Asp Met
200 205 210
Tyr Gln Asn Tyr Ser Cys Tyr Lys His Trp Gln Phe Glu Leu Leu
215 220 225
Asn Tyr Thr Pro Ala Asp Tyr Gly Gly Leu His His Ala Ala Ala
230 235 240
Arg Ile Ser Gly Asp Gly Val Tyr Lys His Leu Lys Tyr Glu Gly
245 250 255
Gly Ile His Arg Val Gln Arg Ile Pro Glu Val Gly Leu Ser Ser
260 265 270
Arg Met Gln Arg Ile His Thr Gly Thr Met Ser Val Ile Val Leu
275 280 285
Pro Gln Pro Asp Glu Val Asp Val Lys Leu Asp Pro Lys Asp Leu
290 295 300
Arg Ile Asp Thr Phe Arg Ala Lys Gly Ala Gly Gly Gln His Val
305 310 315
Asn Lys Thr Asp Ser Ala Val Arg Leu Val His Ile Pro Thr Gly
320 325 330
Leu Val Val Glu Cys Gln Gln Glu Arg Ser Gln Ile Lys Asn Lys
335 340 345
Glu Ile Ala Phe Arg Val Leu Arg Ala Arg Leu Tyr Gln Gln Ile
350 355 360
Ile Glu Lys Asp Lys Arg Gln Gln Gln Ser Ala Arg Lys Leu Gln
365 370 375
Val Gly Thr Arg Ala Gln Ser Glu Arg Ile Arg Thr Tyr Asn Phe
380 385 390
Thr Gln Asp Arg Val Ser Asp His Arg Ile Ala Tyr Glu Val Arg
395 400 405
Asp Ile Lys Glu Phe Leu Cys Gly Gly Lys Gly Leu Asp Gln Leu
410 415 420
Ile Gln Arg Leu Leu Gln Ser Ala Asp Glu Glu Ala Ile Ala Glu
425 430 435
Leu Leu Asp Glu His Leu Lys Ser Ala Lys
440 445
<210> 10
<211> 280
11 /23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 3992058
<400> 10
Met Val Ala Arg Arg Arg Lys Cys Ala Ala Arg Asp Pro Glu Asp
1 5 10 15
Arg Ile Pro Ser Pro Leu Gly Tyr Ala Ala Ile Pro Ile Lys Phe
20 25 30
Ser Glu Lys Gln Gln Ala Ser His Tyr Leu Tyr Val Arg Ala His
35 40 45
Gly Val Arg Gln Gly Thr Lys Ser Thr Trp Pro Gln Lys Arg Thr
50 55 60
Leu Phe Val Leu Asn Val Pro Pro Tyr Cys Thr Glu Glu Ser Leu
65 70 75
Ser Arg Leu Leu Ser Thr Cys Gly Leu Val Gln Ser Ile Glu Leu
80 85 90
Gln Glu Lys Pro Asp Leu Ala Glu Ser Pro Lys Glu Ser Arg Ser
95 100 105
Lys Phe Phe His Pro Lys Pro Val Pro Gly Phe Gln Val Ala Tyr
110 115 120
Val Val Phe Gln Lys Pro Ser Gly Val Ser Ala Ala Leu Ala Leu
125 130 135
Lys Gly Pro Leu Leu Val Ser Thr Glu Ser His Pro Val Lys Ser
140 145 150
Gly Ile His Lys Trp Ile Ser Asp Tyr Ala Asp Ser Val Pro Asp
155 160 165
Pro Glu Ala Leu Arg Val Glu Val Asp Thr Phe Met Glu Ala Tyr
170 175 180
Asp Gln Lys Ile Ala Glu Glu Glu Ala Lys Ala Lys Glu Glu Glu
185 190 195
Gly Val Pro Asp Glu Glu Gly Trp Val Lys Val Thr Arg Arg Gly
200 205 210
Arg Arg Pro Val Leu Pro Arg Thr Glu Ala Ala Ser Leu Arg Val
215 220 225
Leu Glu Arg Glu Arg Arg Lys Arg Ser Arg Lys Glu Leu Leu Asn
230 235 240
Phe Tyr Ala Trp Gln His Arg Glu Ser Lys Met Glu His Leu Ala
245 250 255
Gln Leu Arg Lys Lys Phe Glu Glu Asp Lys Gln Arg Ile Glu Leu
260 265 270
Leu Arg Ala Gln Arg Lys Phe Arg Pro Tyr
275 280
<210> 11
<211> 130
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 4011179
<400> 11
Met Ala Arg Gly Val Val Ser Ala Lys Gly Gly Ala Val Ala Gly
12/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
1 5 10 15
Lys Lys Lys Gly Ser Val Ser Phe Thr Ile Asp Cys Thr Lys Pro
20 25 30
Val Glu Asp Lys Ile Met Glu Val Ala Ser Leu Glu Lys Phe Leu
35 40 45
Gln Glu Arg Ile Lys Val Ala Gly Gly Lys Ala Gly Asn Leu Gly
50 55 60
Asp Ser Val Thr Ile Ser Arg Glu Lys Thr Lys Val Thr Val Thr
65 70 75
Ser Asp Gly Pro Phe Ser Lys Arg Tyr Leu Lys Tyr Leu Thr Lys
80 85 90
Lys Tyr Leu Lys Lys His Asn Val Arg Asp Trp Leu Arg Val Val
95 100 105
Ala Ala Asn Lys Asp Arg Asn Val Tyr Glu Leu Arg Tyr Phe Asn
110 115 120
Ile Ala Glu Asn Glu Gly Glu Glu Glu Asp
125 130
<210> 12
<211> 226
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 5425219
<400> 12
Met Ser Asn Tyr Val Asn Asp Met Trp Pro Gly Ser Pro Gln Glu
1 5 10 15
Lys Asp Ser Pro Ser Thr Ser Arg Ser Gly Gly Ser ser Arg Leu
20 25 30
Ser Ser Arg Ser Arg Ser Arg Ser Phe Ser Arg Ser Ser Arg Ser
35 40 45
His Ser Arg Val Ser Ser Arg Phe Ser Ser Arg Ser Arg Arg Ser
50 55 60
Lys Ser Arg Ser Arg Ser Arg Arg Arg His Gln Arg Lys Tyr Arg
65 70 75
Arg Tyr Ser Arg Ser Tyr Ser Arg Ser Arg Ser Arg Ser Arg Ser
80 85 90
Arg Arg Tyr Arg Glu Arg Arg Tyr Gly Phe Thr Arg Arg Tyr Tyr
95 100 105
Arg Ser Pro Ser Arg Tyr Arg Ser Arg Ser Arg Ser Arg Ser Arg
110 115 120
Ser Arg Gly Arg Ser Tyr Cys Gly Arg Ala Tyr Ala Ile Ala Arg
125 130 135
Gly Gln Arg Tyr Tyr Gly Phe Gly Arg Thr Val Tyr Pro Glu Glu
140 145 150
His Ser Arg Trp Arg Asp Arg Ser Arg Thr Arg Ser Arg Ser Arg
155 160 165
Thr Pro Phe Arg Leu Ser Glu Lys Asp Arg Met Glu Leu Leu Glu
170 175 180
Ile Ala Lys Thr Asn Ala Ala Lys Ala Leu Gly Thr Thr Asn Ile
185 190 195
Asp Leu Pro Ala Ser Leu Arg Thr Val Pro Ser Ala Lys Glu Thr
200 205 210
Ser Arg Gly Ile Gly Val Ser Ser Asn Gly Ala Lys Pro Glu Lys
215 220 225
13/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
Ser
<210> 13
<211> 296
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 5522684
<400> 13
Met Ala Gly Pro Leu Gln Gly Gly Gly Ala Arg Ala Leu Asp Leu
1 5 10 15
Leu Arg Gly Leu Pro Arg Val Ser Leu Ala Asn Leu Lys Pro Asn
20 25 30
Pro Gly Ser Lys Lys Pro Glu Arg Arg Pro Arg Gly Arg Arg Arg
35 40 45
Gly Arg Lys Cys Gly Arg Gly His Lys Gly Glu Arg Gln Arg Gly
50 55 60
Thr Arg Pro Arg Leu Gly Phe Glu Gly Gly Gln Thr Pro Phe Tyr
65 70 75
Ile Arg Ile Pro Lys Tyr Gly Phe Asn Glu Gly His Ser Phe Arg
80 85 90
Arg Gln Tyr Lys Pro Leu Ser Leu Asn Arg Leu Gln Tyr Leu Ile
95 100 105
Asp Leu Gly Arg Val Asp Pro Ser Gln Pro Ile Asp Leu Thr Gln
110 115 120
Leu Val Asn Gly Arg Gly Val Thr Ile Gln Pro Leu Lys Arg Asp
125 130 135
Tyr Gly Val Gln Leu Val Glu Glu Gly Ala Asp Thr Phe Thr Ala
140 145 150
Lys Val Asn Ile Glu Val Gln Leu Ala Ser Glu Leu Ala Ile Ala
155 160 165
Ala Ile Glu Lys Asn Gly Gly Val Val Thr Thr Ala Phe Tyr Asp
170 175 180
Pro Arg Ser Leu Asp Ile Val Cys Lys Pro Val Pro Phe Phe Leu
185 190 195
Arg Gly Gln Pro Ile Pro Lys Arg Met Leu Pro Pro Glu Glu Leu
200 205 210
Val Pro Tyr Tyr Thr Asp Ala Lys Asn Arg Gly Tyr Leu Ala Asp
215 220 225
Pro Ala Lys Phe Pro Glu Ala Arg Leu Glu Leu Ala Arg Lys Tyr
230 235 240
Gly Tyr Ile Leu Pro Asp Ile Thr Lys Asp Glu Leu Phe Lys Met
245 250 255
Leu Cys Thr Arg Lys Asp Pro Arg Gln Ile Phe Phe Gly Leu Ala
260 265 270
Pro Gly Trp Val Val Asn Met Ala Asp Lys Lys Ile Leu Lys Pro
275 280 285
Thr Asp Glu Asn Leu Leu Lys Tyr Tyr Thr Ser
290 295
<210> 14
<211> 2297
14/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 046926
<400> 14
ttctctgtgg cggagacagc caggttggca gctgacggga cagccggggt ctattttgtt 60
gcgggttttc agcaaatcca gggctggtct ggaggcgcga aaacttaagg catacagaac 120
gatggagtat atggcagaat ccaccgaccg cagccctgga cacatcttgt gctgtgagtg 180
tggtgttccg ataagtccaa atcctgccaa tatttgtgtg gcctgtttgc gaagtaaagt 240
ggacatcagc caaggtattc cgaaacaagt ctcgatttcg ttctgcaaac aatgtcaaag 300
gtattttcaa ccaccaggaa cttggataca gtgtgcttta gaatccaggg aacttcttgc 360
tttgtgcttg aaaaaaatca aagcccctct gagtaaggta cggcttgtag atgcaggctt 420
tgtttggact gagcctcatt ctaagagact taaagttaaa ctgactattc agaaagaggt 480
gatgaatggt gctatccttc aacaagtgtt tgtggtggat tatgttgttc agtcccaaat 540
gtgtggagat tgccatagag tagaagctaa ggatttctgg aaggctgtga ttcaagtgag 600
gcaaaagact ttgcataaaa aaactttcta ctatctggaa cagttaattc tgaaatatgg 660
aatgcatcag aatacacttc gtatcaaaga gattcatgat ggtctggatt tttattattc 720
ctcaaaacaa catgctcaga agatggtcga atttcttcag tgtacagttc cctgtagata 780
caaagcatca caaagactga tctctcaaga tatccatagt aacacataca attacaaaag 840
cactttttct gtggaaattg ttccaatatg caaggataat gttgtctgtc tgtctccaaa 900
actggcacaa agcctgggaa atatgaacca gatttgtgtg tgtattcgag taaccagtgc 960
cattcacctc attgatccaa acaccctaca agtggcagat attgatggga gcactttctg 1020
gagtcaccct ttcaatagtt tatgtcatcc caaacagcta gaggagttta ttgtgatgga 1080
atgcagcata gtccaagata taaaacgtgc tgcaggtgct ggaatgatat caaaaaagca 1140
taccctcggg gaagtctggg tacagaagac atctgaaatg aatacagata aacagtattt 1200
ttgtcgtact catttgggac atcttctaaa tcccggagac ctggtgttag ggtttgattt 1260
ggccaactgt aacttaaatg atgagcatgt caacaaaatg aactcagata gagttccaga 1320
tgtggtatta atcaagaaga gctatgaccg gaccaaacgt cagcgtcgta gaaactggaa 1380
attgaaagag cttgcaagag agagagaaaa catggataca gatgatgaaa ggcaatacca 1440
agattttctt gaagatcttg aagaagatga ggcaattcga aaaaatgtca acatttacag 1500
agattcagcc atccctgtgg aaagtgacac cgatgatgaa ggagcacctc gaattagtct 1560
ggctgagatg cttgaagacc ttcatatttc ccaagatgcc actggtgaag aaggtgcatc 1620
aatgctgaca taatgagatg ttgtagactg tttccataca tgggcttaag aagttggaca 1680
gagttacctt aagtgtctct actatctttg cctccagatt tcaagaggag aaatttagtt 1740
ttaaacctga ataaacatgt ttgttttcag tgctcactca aaccactaaa acagatggat 1800
agctttgagg ttttagataa ggaaagatta tggagaatgt agttgttatt gatttttggc 1860
aattttacat ttggaatttt atcactgtgc ttttttatat gaggcactgt agtattttca 1920
catagtatag tactctggat gtaaaagctc aaaaattgtg attccttgaa cgttcactaa 1980
atcttcaagc aaaaacacat ttttacatta tttttacgtt gattatttta gtgaaagacc 2040
atatgaagaa gcatttttaa tattaacttg ttacatactt tgatccactt tacatcattt 2100
ttatgttgtt gaggtaggga aattagggtt cagtttatca ctggacattc aggaggcaag 2160
tcaatctttt ttatttcctt ataaaattaa ctcttcaaaa gctgttaaac agagagttat 2220
cttaattttt attgcagtag gaggaaatat atttaaaata tttgtagatt tatagcaaat 2280
agagactcgt tatttaa 2297
<210> 15
<211> 2144
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 618791
<400> 15
gacctgcgct ggaggcttca tctttgccgc cgctgccgtc gccttcctgg gattggagtc 60
tcgagctttc ttcgttcgtt cgtcggcggg ttcgcgccct tctcgcgcct cggggctgcg 120
15/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
aggctgggga aggggttgga gggggctgtt gatcgccgcg tttaagttgc gctcggggcg 180
gccatgtcgg ccggcgaggt cgagcgccta gtgtcggagc tgagcggcgg gaccggaggg 240
gatgaggagg aagagtggct ctatggcggc ccatgggacg tgcatgtgca cagtgatttg 300
gcaaaggacc tagatgaaaa tgaagttgaa aggccagaag aagaaaatgc cagtgctaat 360
cctccatctg gaattgaaga tgaaactgct gaaaatggtg taccaaaacc gaaagtgact 420
gagaccgaag atgatagtga tagtgacagc gatgatgatg aagatgatgt tcatgtcact 480
ataggagaca ttaaaacggg agcaccacag tatgggagtt atggtacagc acctgtaaat 540
cttaacatca agacaggggg aagagtttat ggaactacag ggacaaaagt caaaggagta 600
gaccttgatg cacctggaag cattaatgga gttccactct tagaggtaga tttggattct 660
tttgaagata aaccatggcg taaacctggt gctgatcttt ctgattattt taattatggg 720
tttaatgaag atacctggaa agcttactgt gaaaaacaaa agaggatacg aatgggactt 780
gaagttatac cagtaacctc tactacaaat aaaattacgg ccgaagactg tactatggaa 840
gttacaccag gtgcagagat ccaagatggc agattcaatc tttttaaggt acagcaggga 900
agaactggaa actcagagaa agaaactgcc cttccatcta caaaagctga gtttacttct 960
cctccttctt tgttcaagac tgggcttcca ccgagcagaa acagcacttc ttctcagtct 1020
cagacaagta ctgcctccag aaaagccaat tcaagcgttg ggaagtggca ggatcgatat 1080
gggagggccg aatcacctga tctaaggaga ttacctgggg caattgatgt tatcggtcag 1140
actataacta tcagccgagt agaaggcagg cgacgggcaa atgagaacag caacatacag 1200
gtcctttctg aaagatctgc tactgaagta gacaacaatt ttagcaaacc acctccgttt 1260
ttccctccag gagctcctcc cactcacctt ccacctcctc catttcttcc acctcctccg 1320
actgtcagca ctgctccacc tctgattcca ccaccgggtt ttcctcctcc accaggcgct 1380
ccacctccat ctcttatacc aacaatagaa agtggacatt cctctggtta tgatagtcgt 1440
tctgcacgtg catttccata tggcaatgtt gcctttcccc atcttcctgg ttctgctcct 1500
tcgtggccta gtcttgtgga caccagcaag cagtgggact attatgccag aagagagaaa 1560
gaccgagata gagagagaga cagagacaga gagcgagacc gtgatcggga cagagaaaga 1620
gaacgcacca gagagagaga gagggagcgt gatcacagtc ctacaccaag tgttttcaac 1680
agcgatgaag aacgatacag atacagggaa tatgcagaaa gaggttatga gcgtcacaga 1740
gcaagtcgag aaaaagaaga acgacataga gaaagacgac acagggagaa agaggaaacc 1800
agacataagt cttctcgaag taatagtaga cgtcgccatg aaagtgaaga aggagatagt 1860
cacaggagac acaaacacaa aaaatctaaa agaagcaaag aaggaaaaga agcgggcagt 1920
gagcctgccc ctgaacagga gagcaccgaa gctacacctg cagaataggc atggttttgg 1980
ccttttgtgt atattagtac cagaagtaga tactataaat cttgttattt ttctggataa 2040
tgtttaagaa atttacctta aatcttgttc tgtttgttag tatgaaaagt taactttttt 2100
tccaaaataa aagagtgaat ttttcatgtt aagttaaaaa aaaa 2144
<210> 16
<211> 1343
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1240366
<400> 16
cggacgcgtg ggttggaatt ctcggatccc gagagatgtc agagacacag tactcgagcc 60
ttacgcagac ccttattatg actatgaaat tgagcggttt tggcgtggcg gacagtatga 120
gaatttcagg gtgcagtata cagaaacaga gccgtatcat aattaccgac cgccaagctg 180
agccaccaaa gaaggaggct gccaccacgg ggccgcaggt gaagagagca gatgagtgga 240
aggacccttg gcgccgatcc aagtctccca agaagaaact cggggtgtcg gtctccccga 300
gccgggctcg aaggcgtcgg aaaacatcag cctcgtcagc ctctgcctct aattcctcca 360
ggtcgtcttc gcggtcatcg tcctactctg gctccggctc ctcccggtcg cgatcccggt 420
cttcatccta cagctcctac tccagccgct cttccagaca cagctcgttc tcaggaagcc 480
ggtccaggtc ccggtccttc tcttcgtccc cgtccccgtc cccaacacct tccccacata 540
gaccttccat cagaaccaag ggagagccgg ccccgccgcc cgggaaagca ggagagaagt 600
cagtgaagaa gccggccccg cctccagccc caccacaggc caccaaaacc actgctcctg 660
tccccgagcc caccaagcca ggagaccctc gggaagccag gaggaaggag cggccagcca 720
ggaccccccc caggaggcgg acgctaagcg gcagcggcag tggcagtggt agcagctata 780
gtggttccag ctcccgatcc aggtccctga gcgtgagcag cgtctcctca gtgtccagtg 840
ctacgtcgag cagcagctct gcacacagcg tggactcgga ggacatgtac gcagacctgg 900
16/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
ctagccccgt gtcctcagcc agctctcggt ccccggcccc agcccagacc aggaaggaga 960
aaggaaaatc taagaaagaa gacggtgtta aagaggaaaa gcggaaaagg gattcgtcca 1020
cacaaccacc caaatctgca aaacctccag caggggggaa gtcctcccag cagccctcga 1080
caccccagca ggcacccccc gggcagcccc agcagggcac atttgtggcc cacaaggaga 1140
tcaagttgac actgttgaat aaggcggctg ataaaggaag caggaagcgc tatgaaccat 1200
cagacaagga caggcagagc cctcctccag ccaagcggcc caacacatcc ccagaccgag 1260
gttctcggga ccgatagtca ggtgggagac tgggctcccc gaagccagag cggcagcaag 1320
cttattccct ttagtgaggg gac
1343
<210> 17
<211> 1346
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1295773
<400> 17
atctaggacc ttgacagcac tgagtatcaa ggcaaaagaa tgcatgtgca gttgtccaca 60
agccggcttc ggactgcccc tggtatggga gaccagagtg gctgctatcg gtgtgggaaa 120
gaagggcact ggtccaaaga gtgcccagta gatcgtacgg gtcgtgtggc agactttact 180
gagcagtata atgaacaata tggagcagtt cgaacacctt acaccatggg ctacggggaa 240
tccatgtatt acaacgatgc atatggagca ctcgactact ataagcgata ccgggtccgc 300
tcttatgagg cagtagcagc ggcggcagcg gcttctgcat acaactacgc agagcagacc 360
atgtcccatc tgcctcaagt ccaaagcaca actgtgacca gccacctcaa ctctacttct 420
gttgatccct atgacagaca cctattgcca aactctggcg ctgctgccac ttcagctgct 480
atggctgctg ctgcagccac cacttcctcc tactatggaa gggacaggag cccactgcgt 540
cgtgctgcag ccatgctccc cacagttgga gagggctacg gttatgggcc agagagtgaa 600
ttatctcagg cttccgcagc tacacggaat tctctgtatg acatggcccg gtatgaacgg 660
gagcagtatg tggaccgagc ccggtactca gccttttaaa aactggaggt aggataattg 720
cggactgaac cctcgggctg cggtcatata tgagaacttg ctccgcgcgg tcccctttgc 780
cgggatgttt ccattgcttc atgtttcagt aaacaaaagg agtttgtgac caactatgtt 840
ttctttctta attcttctaa gttgactttt ctttcctcct gaaactagtc tctgtagcct 900
ttcactctgt tccttatatt ctcagcctct gagcagccct aggtaaggat tatgctggca 960
tccccttttt cctgtgcagt ggaacccctc ttatcttgct ttccctagga gttgaatcct 1020
tctccctgcc tacctgcagc atctcctttc cctttaaaat gaccatgtag tggcaagcag 1080
ccttttactc ttctgttagc tctggactct taacacttaa gttactcttc tgaaattgct 1140
aggaccattg ggggttttgt tgttttgttt gttttttatg tccgacctgt gatcgtggta 1200
cagcattagc tgaaatttac ccttgtttta ctccactcct ccctttttta aaaaaatttt 1260
ttgacaaata aatgtttcta acacttaaaa aaaaaaatga agaataaaca aagaaaaaat 1320
ccaagtacat aacagaaaaa aaaaaa 1346
<210> 18
<211> 2720
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 1858421
<400> 18
gtgaggagtg cggaggggcg cgaggtttca agatggcggt agctgagggg ttgaccgaga 60
gacccagttg aaggccttta cgaagtgaaa gaggccggga atcgccccct acccgcttct 120
cgtagtcctg ggagcacagc agaagtgttt ttcttttttt aatgaacaag taaaccatac 180
aaattgtcaa catgggacgg agatctacat catccaccaa gagtggaaaa tttatgaacc 240
ccacagacca agcccgaaag gaagcccgga agagagaatt aaagaagaac aaaaaacagc 300
gcatgatggt tcgagctgca gttttaaaga tgaaggatcc aaaacagata atccgagaca 360
17/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
tggagaaatt ggatgaaatg gagtttaacc cagtgcaaca gccacaatta aatgagaaag 420
tactgaaaga caagcgtaaa aagctgcgtg aaacctttga acgtattcta cgactctatg 480
aaaaagagaa tccagatatt tacaaagaat tgagaaagct agaagtagaa tatgaacaga 540
agagggctca acttagccaa tattttgatg ctgtcaagaa tgctcagcat gtggaagtgg 600
agagtattcc tttgccagat atgccacatg ctccttccaa cattttgatc caggacattc 660
cacttcctgg tgcccagcca ccctctatcc taaagaaaac ctcagcctat ggacctccaa 720
ctcgggcagt ttctatcctt cctcttcttg gacatggtgt tccacgtttg ccccctggca 780
gaaaacctcc tggccctccc cctggtccac ctcctcctca agtcgtgcag atgtatggcc 840
gtaaagtggg ttttgcccta gatcttcccc ctcgtaggcg agatgaagac atgttatata 900
gtcctgaact tgcccagcga ggtcatgatg atgatgtttc tagcaccagt gaagatgatg 960
gctatcctga ggacatggat caagataagc atgatgacag tactgatgac agtgacaccg 1020
acaaatcaga tggagaaagt gacggggatg aatttgtgca ccgtgataat ggtgagagag 1080
acaacaatga agaaaagaag tcaggtctga gtgtacggtt tgcagatatg cctggaaaat 1140
caaggaagaa aaagaagaac atgaaggaac tgactcctct tcaagccatg atgcttcgta 1200
tggcaggtca agaaatccct gaggagggac gggaagtaga ggaattttca gaggacgatg 1260
atgaagatga ttctgatgac tctgaagcag aaaagcaatc acaaaagcag cataaagagg 1320
aatcccattc tgatggcaca tccactgctt cttcacagca gcaggctccg ccgcagtctg 1380
ttcctccttc tcagatacaa gcacctccca tgccaggacc accacctctt ggaccaccac 1440
ctgctccacc attacggcct cctgggccac ctacaggcct tcctcctggt ccacctccag 1500
gagctcctcc attcctgaga ccacctggaa tgccaggact ccgagggccc ttaccccgac 1560
ttttacctcc aggaccacca ccaggccgac cccctggccc tcccccaggt ccacctccag 1620
gtctgcctcc tggtccccct cctcgtggac ccccaccaag gctacctccc cctgcacctc 1680
caggtattcc tccacctcgt cctggcatga tgcgcccacc tttggtgcct ccccttggac 1740
ctgccccccc tgggctgttc ccaccagctc ccttgccaaa ccctggggtt ttaagtgccc 1800
cacccaactt gattcagcga cccaaggcgg atgatacaag tgcagccacc attgagaaga 1860
aagccacagc aaccatcagt gccaagccac agatcactaa tcccaaggca gagattactc 1920
gatttgtgcc cactgcactg agagtacgtc gggagaataa aggggctact gctgctcccc 1980
aaagaaagtc agaggatgat tctgctgtgc ctcttgccaa agcagcaccc aaatctggtc 2040
cttctgttcc tgtctcagta caaactaagg atgatgtcta tgaggctttc atgaaagaga 2100
tggaagggct actgtgacag cttttgatgc cagaaaaggc ttctgttcac aacagtggcc 2160
catggagaaa gaggctctta ttaaacttag atgaaagagc tgcttccatt gtcagggtat 2220
tttctaattt cagttcaagg aatatcctaa aatttagcct tgttcagaat ttactgcaca 2280
taaaaaaggg tatttcatcc agaatagatc agttattgaa gcagtgctgc taacatccat 2340
tccctttcat accaccattt tcaccctgtt tcttcccctc ctccagttct ttggaaattt 2400
gtgatcgggg gatcttagtt gcttatttgt tttgactctt gtgtgctgtg ggcactggag 2460
tagagatttc tggagaaaaa aaaacagttt atttcatctt gccttttgtg tttgagttat 2520
ttttaatatt ttcctgtaaa tattttgtaa tattttactt gtaatgaaat ggatcacaat 2580
gtcatttcct aatacaaggc aggatatgtg ggaagaatat gtacaattat ttgattaaaa 2640
ttatttccca ctgacctaaa ctttcagtga tttgtgggaa aaataaataa atgttctaca 2700
ccaagaaaaa aaaaaaaaaa 2720
<210> 19
<211> 676
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2152431
<400> 19
gggaccagcg cgggtgcgca gacgaaaggc gctctttgcc agctgaaagt tcccacggaa 60
aaactaccat ctcccctgcc caccatggca gacgaaattg atttcactac tggagatgcc 120
ggggcttcca gcacttaccc tatgcagtgc tcggccttgc gcaaaaacgg cttcgtggtg 180
ctcaaaggac gaccatgcaa aatagtggag atgtcaactt ccaaaactgg aaagcatggt 240
catgccaagg ttcaccttgt tggaattgat attttcacgg gcaaaaaata tgaagatatt 300
tgtccttcta ctcacaacat ggatgttcca aatattaaga gaaatgatta tcaactgata 360
tgcattcaag atggttacct ttccctgctg acagaaactg gtgaagttcg tgaggatctt 420
aaactgccag aaggtgaact aggcaaagaa atagagggaa aatacaatgc aggtgaagat 480
18/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
gtacaggtgt ctgtcatgtg tgcaatgagt gaagaatatg ctgtagccat aaaaccctgc 540
aaataaacgg aaacatcagg catgaacact gtttatgtct gaatcaactg cagatctaat 600
ttggttctaa gttgtcacca aagctatagc cttcataagc aacctcattt ctttttttaa 660
ttgttttcag attgtg 676
<210> 20
<211> 909
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 2641494
<400> 20
acggaaactg actggggtca attcaagtca tgcaggctgt gagaaacgcg gggtcgcggt 60
tcctgcggtc ctggacttgg ccccagacag ccggcagggt cgtggccaga acgccggccg 120
ggaccatctg cacaggcgct cgacagctcc aagacgctgc ggccaagcag aaagttgaac 180
agaacgcggc tcccagccac accaagttca gcatttaccc tcccattcca ggagaggaga 240
gctctctgag gtgggcagga aagaaatttg aggagatccc aattgcacac attaaagcat 300
cccacaacaa cacacagatc caggtagtct ctgctagtaa tgagcccctt gcctttgctt 360
cctgtggcac agagggattt cggaatgcca agaagggcac aggcatcgca gcacagacag 420
caggcatagc cgcagcggcg agagctaaac aaaagggcgt gatccacatc cgagttgtgg 480
tgaaaggcct ggggccagga cgcttgtctg ccatgcacgg actgatcatg ggcggcctgg 540
aagtgatctc aatcacagac aacaccccaa tcccacacaa cggctgccgc cccaggaagg 600
ctcggaagct gtgatgggaa ggaggcctgc acttggacct gacctcaagc ctcagctcca 660
gtgggacctt gtaaaatgct ccctgtcaga gctctccaga atatgcttgt tggagatcct 720
tcaggcagta agggagagtt ttgcctcctt acacagtggc ctttgcttgc acctccagct 780
ggagatgggt gtgccccaga agtaagcttt gcatctctta caagagggga gctacagggg 840
cagccgtggc ctaggcccaa actctgctct gagaaaataa atatctgtac cacctgtcaa 900
aaaaaaaaa gpg
<210> 21
<211> 2405
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 3803409
<400> 21
cttcaagacc tggaatgtat ccgcctccag ggtcgtatag acctaccccc tcctatgggc 60
aaaccaccag gttcaattgt aagaccctct gctccaccag caagatcatc tgttcctgtg 120
accaggccac ctgtcccaat accaccacct ccacctcctc cacctctacc tcctcctcct 180
ccagtgataa agccacaaac ttcagctgta gaacaggaac gatgggatga agattctttc 240
tatgggctct gggatacaaa tgatgaacaa ggactgaatt cagaatttaa gtcagaaact 300
gcagcaattc catctgctcc agtattacca cccccacctg ttcactcttc cattccccct 360
cctggcccag tgcctatggg tatgccacca atgtccaagc caccaccagt acaacagact 420
gttgattatg gccatggccg agatatatcc actaataaag ttgaacagat accttatgga 480
gaaagaataa ctctacgccc agatccacta cctgaaagat caacttttga gacagagcat 540
gcaggccaac gtgatcgtta tgatagagaa agagatcgtg agccttattt tgatcgtcaa 600
agtaatgtca tagcagatca tcgagatttt aaaagggatc gtgagacaca tagagatcga 660
gaccgggatc gtggtgttat tgactatgac cgggatcgat ttgacagaga acgccgaccc 720
cgagatgata gagctcagtc atatcgagac aaaaaagacc attcctcatc cagaagaggg 780
ggttttgata ggccatccta tgaccggaag tctgaccgac cagtctatga aggaccatcc 840
atgtttggag gagaacgaag gacttatcct gaggagcgaa tgcctctgcc agctccttca 900
ctgagccacc agccacctcc agctccacga gtcgagaaga agcctgaatc aaagaatgtg 960
gacgatattt tgaaaccacc gggccgggag agcagacctg agagaattgt tgttataatg 1020
19/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
agaggattac ctggcagtgg aaagacacat gttgcaaaac ttattcgaga taaggaggta 1080
gaatttggag gacctgcacc cagagttcta agcctggatg attacttcat cactgaagtg 1140
gaaaaagaag aaaaagatcc agattctgga aagaaagtga aaaagaaggt aatggaatat 1200
gaatatgaag ctgagatgga ggagacttac cgcaccagca tgttcaaaac tttcaaaaag 1260
actctggatg atggcttttt tcccttcatc atcctggatg ccatcaatga cagagttagg 1320
cattttgacc agttttggag tgcagcaaaa accaagggat ttgaggtata tttggctgaa 1380
atgagtgcag ataaccagac ttgtggcaag agaaatattc atggaagaaa gcttaaagaa 1440
ataaataaga tggctgatca ctgggaaact gcacctcgtc acatgatgcg tctagatatt 1500
cgttctttgc tgcaagatgc tgctattgaa gaggtagaga tggaagattt tgatgcaaat 1560
atcgaagaac agaaagaaga aaagaaagat gcagaggaag aggaaagcga actgggttac 1620
attccgaaaa gcaaatggga gatggacaca tctgaggcaa agctagacaa gttggatggc 1680
ttgaggactg gtactaaaag gaaacgtgac tgggaggcca ttgccagcag aatggaggat 1740
tatcttcagc tccccgatga ttatgatact cgtgcttctg agcctgggaa gaagagggtc 1800
agatgggcag acctggaaga gaagaaggat gcagatagga aaagggccat aggttttgtg 1860
gtcggacaga ctgattggga gaagatcaca gatgaaagtg gtcacctggc tgaaaaagcc 1920
ctcaatcgaa ccaaatatat atgagactta gtttttgaac ggagtcatta ttcctctaag 1980
gtggttcgct ttgaggtggt ctgaagccaa ggcctcgcgg agcttctttg tgtgtcacct 2040
tgcttccacg tttcagttct tgttttgttt ctactgcttt agtttttttt aaagttctcc 2100
agtgtcccca agaggtatta gaatcttgct gtacccaagc aagacgttaa tttttctttt 2160
aactgttttg gggagggagg gagtgatagc ttaactgctg aagccaggcg ggggtctgct 2220
ggaggattcc aacagagagt atttcctcca ctgtacaatg tcacagacta tctctatcat 2280
cattgctttg tggctgtttc tgttttttac tgtatgtaac tggtagctga ttgtactagg 2340
attaaaaaca ataaactttc atgataaagc cgatgagatt catgggctat acagaaaaaa 2400
aaaaa 2405
<210> 22
<211> 1754
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 3979009
<400> 22
cgggtttgtc gggctgaaat gtggcgggtc tcggaaggtt ccgacctcag taaagagagc 60
taacgtgtat tcttcttttt cttagatgct gagatgaatc gtcacctgtg tgtttggctt 120
tttagacatc catctcttaa tggttacctc cagtgtcaca tccagctcca ttctcatcaa 180
tttagacaga tacatcttga tacaaggctg caagttttta gacaaaacag gaattgcatt 240
cttcatctgt taagtaagaa ttggtccagg agatattgcc atcaagacac caagatgctc 300
tggaagcata aagcactaca gaaatatatg gagaacctga gtaaggagta ccaaacactt 360
gagcaatgtc tgcagcatat ccctgtgaat gaagaaaacc gaaggtcctt gaacagaagg 420
catgctgagt tggcacctct tgcagccatt taccaagaaa ttcaggagac tgaacaagca 480
attgaagaat tagaatcaat gtgtaaaagc ctaaataaac aagatgaaaa gcagttacaa 540
gaacttgcac tggaagaaag gcaaaccatt gatcaaaaaa tcaacatgtt gtacaatgag 600
cttttccaga gccttgtgcc aaaggagaaa tatgacaaaa atgatgttat tttagaggtg 660
acagctggaa ggactactgg aggtgacatc tgccaacaat ttacccgaga aatatttgac 720
atgtaccaga attattcgtg ctataaacac tggcaatttg aacttctgaa ttatacacca 780
gcagattatg gtggactaca tcatgcagcc gcccgaattt ccggtgacgg tgtctataag 840
catttgaagt atgagggtgg gattcaccga gttcagcgca tccccgaggt gggcctgtcc 900
tcaaggatgc agcgcattca cacaggaacg atgtcggtta ttgtccttcc tcagccagat 960
gaggtggatg tgaaattgga ccccaaggat ttgcgaatag atacatttcg agccaaagga 1020
gcaggagggc agcatgttaa taaaactgat agtgccgtca gacttgtcca catccccaca 1080
gggctagtag tagaatgcca acaagaaaga tcacagataa aaaataaaga aatagccttt 1140
cgtgtgttga gagctagact ctaccagcag attattgaga aagacaagcg tcagcaacaa 1200
agtgctagaa aactgcaggt gggaacaaga gcccagtcag agcgaattcg gacatataat 1260
ttcacccagg atagagtcag tgaccacagg atagcatatg aagttcgtga tattaaggaa 1320
tttttatgtg gtgggaaggg cctggatcag ctaattcaga gactgcttca atcagcagat 1380
gaagaagcca ttgctgaact tttggatgaa caccttaaat cagcaaaata aatactaact 1440
tattattatt tatgattata taaatgaaat ggacctatat caagaggcag actgaagctt 1500
20/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
ggaaatcatt atgaatattt gtaaattaca gctttaagaa cacattacac ataaatatat 1560
gttttgtaat taatcgaagt cacatttcct gacctaagaa tttattttag gtttcctgta 1620
aagtacaatc caactcatca agtagaaaat aagcatgcat cattgaaaag ggaaagtatt 1680
gagaattgat tgtgtcattt aggacaagtc acttgttctc ttaaaatgcc ttttttcccc 1740
agccatctat gaat 1754
<210> 23
<211> 1221
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 3992058
<400> 23
ccgcgctccc gggtggcaag atggtggcgc gcaggaggaa gtgcgccgcg cgggacccgg 60
aggaccgtat ccccagccca ctgggctacg cagctattcc aatcaagttc tctgaaaagc 120
aacaggcttc tcactacctc tatgtgagag cacacggcgt tcgacaaggc accaagtcca 180
cctggcctca gaagaggact ctttttgtcc tcaatgtgcc cccatactgc acagaggaga 240
gcctgtcccg cctcctgtcc acctgtggcc tcgtccagtc tatagagttg caggagaagc 300
cggacctggc tgagagccca aaggagtcaa ggtcgaagtt ttttcatccc aagccagttc 360
cgggtttcca ggtagcctac gtggtgttcc agaagccaag tggggtgtca gcggccttgg 420
ccctgaaggg ccccctgctg gtgtccacag agagccaccc tgtgaagagt ggcattcaca 480
agtggatcag tgactacgca gactctgtgc ccgaccctga ggccctgagg gtggaagtgg 540
acacgttcat ggaggcatat gaccagaaga tcgctgagga agaagctaag gccaaggagg 600
aggagggggt ccctgacgag gagggctggg tgaaggtgac ccgccggggc cggcggcctg 660
tgctcccccg gactgaggca gccagcttgc gggtgctgga gagggagaga cggaagcgca 720
gccgaaaaga gctgctcaac ttctacgcct ggcagcatcg agagagcaag atggagcatc 780
tagcgcagct gcgcaagaag ttcgaggagg acaagcagag gatcgagctg ctgcgggccc 840
agcgcaaatt ccgaccgtac tgagctgtga gagccgcagt gaatggctgg aggtgcaggg 900
ccaggaggag gcgaggcagg gcctgcagcg gtctctgaga ggccgagctc tggccaacgg 960
gccccaggtt gaaggccacc gcgtccaaca gccccatcag agtccacaca ggccaggagg 1020
gaaggaccag gccacccctc gggtcttgtg cttcagcagt cctggggacc caggcgtgcc 1080
gagaggagga cttgtccttc ctgcttcttg cctccacacc ctcctctcca ggaccctgga 1140
tgaatccgtt ctgtgcttcc ttttccctca atgcaaaagc ccttgctggc aacgaaaaag 1200
cctcaaaagc aaaaaaaaaa a 1221
<210> 24
<211> 628
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 4011179
<400> 24
ggaaacccta gccgccatat ctcacgatcc actcgagcac caagccgagg gaaggtgagg 60
agcgatggcg cgcggcgtgg tgtcggcgaa gggcggcgcg gtcgcgggca agaagaaggg 120
gtcggtttcc ttcacgatcg actgcaccaa gccagtggag gacaagatca tggaggtcgc 180
ctcgctcgag aagttcctgc aggagcgcat caaggtcgcc ggcggcaagg ctggcaacct 240
cggcgactcc gtcaccatct ctcgcgagaa gaccaaggtc accgtcacct ctgacggacc 300
cttctccaag aggtacctga agtacttgac caagaagtac ttgaagaagc acaacgtgcg 360
ggattggcta cgcgtggttg ctgccaacaa ggaccgaaac gtctatgagc tccgctactt 420
caacattgct gagaacgagg gcgaggaaga agattagatt gcactacgct tatattttag 480
tattgaactc gttgcatttt gatacctgta cccgtagttt cgcaaatgtc ccatgttatg 540
gtgtggtatg gttaatttga agaatcctta tgtactgaat ctctgcaaaa agctatgttg 600
tggacagaag tgtaacgtgc cagatttt 628
21/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
<210> 25
<211> 1500
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 5425219
<400> 25
gtctgctaac gtagtccctc agtgcgcatc cggacgtagg aggtggaggt tgtggaattc 60
gccgttcgaa agcagggact aaaagcccca cttcgtctta cgttccgaaa ggaaggcgtc 120
tgttgagcct ttctctcagt cgtgagggag gcgtcgacgg cgtgcggaag tcctgagttg 180
aggcttgcgg gatcctttcc ggagaaagcg caggctaaag ccgcaggtga agatgtccaa 240
ctacgtgaac gacatgtggc cgggctcgcc gcaggagaag gattcgccct cgacctcgcg 300
gtcgggcggg tccagccggc tgtcgtcgcg gtctaggagc cgctcttttt ccagaagctc 360
tcggtcccat tcccgcgtct cgagccggtt ttcgtccagg agtcggagga gcaagtccag 420
gtcccgttcc cgaaggcgcc accagcggaa gtacaggcgc tactcgcggt catactcgcg 480
gagccggtcg cgatcccgca gccgccgtta ccgagagagg cgctacgggt tcaccaggag 540
atactaccgg tctccttcgc ggtaccggtc ccggtcccgt agcaggtcgc gctctcgggg 600
aaggtcgtac tgcggaaggg cgtacgcgat cgcgcgggga cagcgctact acggctttgg 660
tcgcacagtg tacccggagg agcacagcag atggagggac agatccagga cgaggtcgcg 720
gagcagaacc ccctttcgct taagtgaaaa agatcgaatg gagctgttag aaatagcaaa 780
aaccaatgca gcgaaagctc taggaacaac caacattgac ttgccagcta gtctcagaac 840
tgttccttca gccaaagaaa caagccgtgg aataggtgta tcaagtaatg gtgcaaagcc 900
tgaaaaatca tgaatgtggt ctgcagacat tgatgaagaa aatctgttgc tgtcggaaaa 960
ggtaacagaa gatggaactc gaaatcccaa tgaaaaacct acccagcaaa gaagcatagc 1020
ttttagctct aataattctg tagcaaagcc aatacaaaaa tcagctaaag ctgccacaga 1080
agaggcatct tcaagatcac caaaaataga tcagaaaaaa agtccatatg gactgtggat 1140
acctatctaa aagaagaaaa ctgatggcta agtttgcatg aaaactgcac tttattgcaa 1200
gttagtgttt ctagcattat cccatccctt tgagccattc aggggtactt gtgcatttaa 1260
aaaccaacac aaaaagatgt aaatacttaa cactcaaata ttaacatttt aggtttctct 1320
tgcagatatg agagatagca cagatggacc aaaggttatg cacaggtggg agtcttttgt 1380
atatagttgt aaatattgtc ttggttatgt aaaaatgaaa ttttttagac acagtaattg 1440
aactgtattc ctgttttgta tatttaataa atttcttgtt ttcattctta aaaaaaaaaa 1500
<210> 26
<211> 1143
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte Clone No: 5522684
<400> 26
gaccacgtgg cctccgagca gctcagggcg cccttgaaag ttcttggatc tgcgggttat 60
ggccggtccc ttgcagggcg gtggggcccg ggccctggac ctactccggg gcctgccgcg 120
tgtgagcctg gccaacttaa agccgaatcc cggctccaag aaaccggaga gaagaccaag 180
aggtcggaga agaggtagaa aatgtggcag aggccataaa ggagaaaggc aaagaggaac 240
ccggccccgc ttgggctttg agggaggcca gactccattt tacatccgaa tcccaaaata 300
cgggtttaac gaaggacata gtttcagacg ccagtataag cctttgagtc tcaatagact 360
gcagtatctt attgatttgg gtcgtgttga tcctagtcaa cctattgact taacccagct 420
tgtcaatggg agaggtgtga ccatccagcc acttaaaagg gattatggtg tccagctggt 480
tgaggagggt gctgacacct ttacggcaaa agttaatatt gaagtacagt tggcttcaga 540
actagctatt gctgccattg aaaaaaatgg tggtgttgtt actacagcct tctatgatcc 600
aagaagtctg gacattgtat gcaaacctgt tccattcttt cttcgtggac aacccattcc 660
aaaaagaatg cttccaccag aagaactggt accatattac actgatgcaa agaaccgtgg 720
gtacctggcg gatcctgcca aatttcctga agcacgactt gaactcgcca ggaagtatgg 780
22/23
CA 02375407 2001-11-28
WO 00/78952 PCT/US00/16644
ttatatctta cctgatatca ctaaagatga actcttcaaa atgctctgta ctaggaagga 840
tccaaggcag attttctttg gtcttgctcc aggatgggtg gtgaatatgg ccgataagaa 900
aatcctaaaa cctacagatg aaaatctcct taagtattat acctcatgaa ttcccgtcca 960
aggaagcaga gttgttaaag agtactggaa taggggctga aggatctata ttcccttatt 1020
gcattttcct tatgtataat tttccagatg gtgatgttac ttttcagtgt actcatatgt 1080
ctcattttca tctaaaatta aatggcagga aacaaggact gcatagagaa aaaaaaaaaa 1140
aaa
1143
23/23
