Note: Descriptions are shown in the official language in which they were submitted.
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AMINOACYL TRNA SYNTHETASES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of aminoacyl
tRNA
synthetases and to the use of these sequences in the diagnosis, treatment, and
prevention of cell
proliferative and autoimmune/inflammatory disorders, and in the assessment of
the effects of
exogenous compounds on the expression of nucleic acid and amino acid sequences
of aminoacyl
tRNA synthetases.
l0 BACKGROUND OF THE INVENTION
Correct translation of the genetic code depends upon each amino acid forming a
linkage with
the appropriate transfer RNA (tRNA). The aminoacyl-tRNA synthetases (aaRSs)
are essential
proteins found in all living organisms. The aaRSs are responsible for the
activation and correct
attachment of an amino acid with its cognate tRNA, as the first step in
protein biosynthesis.
Prokaryotic organisms have at least twenty different types of aaRSs, one for
each different amino
acid, while eukaryotes usually have two aaRSs, a cytosolic form and a
mitochondrial form, for each
different amino acid. The 20 aaRS enzymes can be divided into two structural
classes. Class I
enzymes add amino acids to the 2' hydroxyl at the 3' end of tRNAs while Class
II enzymes add amino
acids to the 3' hydroxyl at the 3' end of tRNAs. Each class is characterized
by a distinctive topology
of the catalytic domain. Class I enzymes contain a catalytic domain based on
the nucleotide-binding
'Rossman fold'. In particular, a consensus tetrapeptide motif is highly
conserved (Prosite Document
PDOC00161, Aminoacyl-transfer RNA synthetases class-I signature). Class I
enzymes are specific
for arginine, cysteine, glutamic acid, glutamine, isoleucine, leucine,
methionine, tyrosine, tryptophan,
and valine. Class II enzymes contain a central catalytic domain, which
consists of a seven-stranded
antiparallel 13-sheet domain, as well as N- and C- terminal regulatory
domains. Class II enzymes are
separated into two groups based on the heterodimeric or homodimeric structure
of the enzyme; the
latter group is further subdivided by the structure of the N- and C-terminal
regulatory domains
(Hartlein, M. and Cusack, S. (1995) J. Mol. Evol. 40:519-530). Class II
enzymes are specific for
alanine, asparagine, aspartic acid, glycine, histidine, lysine, phenylalanine,
proline, serine, and
threonine.
Certain aaRSs also have editing functions. IleRS, for example, can misactivate
valine to form
Val-tRNA"e, but this product is cleared by a hydrolytic activity that destroys
the mischarged product.
This editing activity is located within a second catalytic site found in the
connective polypeptide 1
region (CP1), a long insertion sequence within the Rossman fold domain of
Class I enzymes
(Schimmel, P. et al. (1998) FASEB J. 12:1599-1609). AaRSs also play a role in
tRNA processing. It
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has been shown that mature tRNAs are charged with their respective amino acids
in the nucleus
before export to the cytoplasm, and charging may serve as a quality control
mechanism to insure the
tRNAs are functional (Martinis, S.A. et al. (1999) EMBO J. 18:4591-4596).
In addition to their function in protein synthesis, specific aminoacyl tRNA
synthetases also
play roles in cellular fidelity, RNA splicing, RNA trafficking, apoptosis, and
transcriptional and
translational regulation. For example, human tyrosyl-tRNA synthetase can be
proteolytically cleaved
into two fragments with distinct cytokine activities. The carboxy-terminal
domain exhibits monocyte
and leukocyte chemotaxis activity as well as stimulating production of
myeloperoxidase, tumor
necrosis factor-a, and tissue factor. The N-terminal domain binds to the
interleukin-8 type A receptor
and functions as an interleukin-8-like cytokine. Human tyrosyl-tRNA synthetase
is secreted from
apoptotic tumor cells and may accelerate apoptosis (Wakasugi, K., and
Schimmel, P. (1999) Science
284:147-151). Mitochondrial Neurospora crassa TyrRS and S. cerevisiae LeuRS
are essential factors
for certain group I intron splicing activities, and human mitochondrial LeuRS
can substitute for the
yeast LeuRS in a yeast null strain. Certain bacterial aaRSs are involved in
regulating their own
transcription or translation (Martinis, su ra). Several aaRSs are able to
synthesize diadenosine
oligophosphates, a class of signalling molecules with roles in cell
proliferation, differentiation, and
apoptosis (Kisselev, L.L et al. (1998) FEBS Lett. 427:157-163; Vartanian, A.
et al. (1999) FEBS Lett.
456:175-180).
Autoantibodies against aminoacyl-tRNAs are generated by patients with
autoimmune
diseases such as rheumatic arthritis, dermatomyositis and polymyositis, and
correlate strongly with
complicating interstitial lung disease (ILD) (Freist, W. et al. (1999) Biol.
Chem. 380:623-646; Freist,
W. et al. (1996) Biol. Chem. Hoppe Seyler 377:343-356). These antibodies
appear to be generated in
response to viral infection, and coxsackie virus has been used to induce
experimental viral myositis in
animals.
Comparison of aaRS structures between humans and pathogens has been useful in
the design
of novel antibiotics (Schimmel, supra). Genetically engineered aaRSs have been
utilized to allow
site-specific incorporation of unnatural amino acids into proteins in vivo
(Liu, D.R. et al. (1997) Proc.
Natl. Acad. Sci. USA 94:10092-10097).
The discovery of new aminoacyl tRNA synthetases, 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 cell proliferative and autoimmune/inflammatory
disorders, and in the
assessment of the effects of exogenous compounds on the expression of nucleic
acid and amino acid
sequences of aminoacyl tRNA synthetases.
SUMMARY OF THE INVENTION
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The invention features purified polypeptides, aminoacyl tRNA synthetases,
referred to
collectively as "ATRS" and individually as "ATRS-1," "ATRS-2," "ATRS-3," "ATRS-
4," "ATRS-
5," "ATRS-6." In one aspect, the invention provides an isolated polypeptide
selected from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-6, b) a polypeptide comprising a naturally occurring amino acid
sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO:1-6, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-6, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-6. In one
alternative, the invention
provides an isolated polypeptide comprising the amino acid sequence of SEQ )D
NO:l-6.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected
from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-6, b) a polypeptide comprising a naturally
occurring amino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-6, c) a biologically active fragment of a polypeptide having an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-6, and d) an immunogenic fragment of
a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-6. In one
alternative, the polynucleotide encodes a polypeptide selected from the group
consisting of SEQ ID
NO:1-6. In another alternative, the polynucleotide is selected from the group
consisting of SEQ ID
N0:7-12.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-6, b) a polypeptide comprising a naturally occurring amino acid
sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ )D NO:1-6, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO: l-6, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO: l-6. 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 selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-6, b) a polypeptide comprising a naturally occurring amino acid
sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO:1-6, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
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consisting of SEQ ID NO:1-6, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO: l-6. 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 selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ ID NO: l-6, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-6, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-6, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-6.
The invention further provides an isolated polynucleotide selected from the
group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ ID N0:7-12, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ ID N0:7-
12, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
In one alternative, the
polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
selected from the group
consisting of a) a polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:7-12, b) a polynucleotide comprising a naturally
occurring polynucleotide
sequence at least 90% identical to a polynucleotide sequence selected from the
group consisting of
SEQ ID N0:7-12, c) a polynucleotide complementary to the polynucleotide of a),
d) a polynucleotide
complementary to the polynucleotide of 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 selected from
the group consisting
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of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ ID N0:7-12, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ ID N0:7-
12, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of 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 composition comprising an effective amount of
a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ ID N0:1-6, b) a polypeptide
comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-6, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ >D
N0:1-6, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:l-6, and a pharmaceutically acceptable excipient. In
one embodiment, the
composition comprises an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
6. The invention additionally provides a method of treating a disease or
condition associated with
decreased expression of functional ATRS, comprising administering to a patient
in need of such
treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO: l-6, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-6, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
N0:1-6, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID N0:1-6. 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 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 ATRS, comprising
administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
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amino acid sequence selected from the group consisting of SEQ ID NO:1-6, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ ID NO:l-6, c) a
biologically active fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-6,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID N0:1-6. 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 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
ATRS, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-6, b) a polypeptide
comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO: l-6, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:l-6, and d) an
immunogenic fragment of a polypeptide having an amino acid,sequence selected
from the group
consisting of SEQ ID NO:1-6. 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
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:l-6, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-6, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-6, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:l-6. 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
polypeptide 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
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altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected from the group consisting of SEQ ID N0:7-12, the method
comprising a) exposing
a sample comprising the target polynucleotide to a compound, and b) detecting
altered expression of
the target polynucleotide. ,
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:7-12, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:7-12,
iii) a polynucleotide
having a sequence complementary to i), iv) a polynucleotide complementary to
the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions
whereby a specific
hybridization complex is formed between said probe and a target polynucleotide
in the biological
sample, said target polynucleotide selected from the group consisting of i) a
polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:7-12, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:7-12,
iii) a polynucleotide
complementary to the polynucleotide of i), iv) a polynucleotide complementary
to the polynucleotide
of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target
polynucleotide comprises a
fragment of a polynucleotide sequence selected from the group consisting of i)-
v) above; c)
quantifying the amount of hybridization complex; and d) comparing the amount
of hybridization
complex in the treated biological sample with the amount of hybridization
complex in an untreated
biological sample, wherein a difference in the amount of hybridization complex
in the treated
biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability score for the match
between each
polypeptide and its GenBank homolog is also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
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Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 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
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to 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
"ATRS" refers to the amino acid sequences of substantially purified ATRS
obtained from any
species, particularly a mammalian species, including bovine, ovine, porcine,
murine, 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
ATRS. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
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compound or composition which modulates the activity of ATRS either by
directly interacting with
ATRS or by acting on components of the biological pathway in which ATRS
participates.
An "allelic variant" is an alternative form of the gene encoding ATRS. 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 ATRS include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as ATRS or a
polypeptide with at least one functional characteristic of ATRS. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding ATRS, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
ATRS. 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 ATRS. 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 ATRS 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 ATRS. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
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molecules, or any other compound or composition which modulates the activity
of ATRS either by
directly interacting with ATRS or by acting on components of the biological
pathway in which ATRS
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')Z, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind ATRS polypeptides 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 (KL,H). 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
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 ATRS, 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,
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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 ATRS or fragments of
ATRS 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., NaCI), 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
(Applied
Biosystems, Foster City CA) in the 5' andlor the 3' direction, and
resequenced, or which has been
assembled from 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.
"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
His 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
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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 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 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.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
A "fragment" is a unique portion of ATRS or the polynucleotide encoding ATRS
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°Io) 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 N0:7-12 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:7-12, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID NO:7-12 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:7-12 from related polynucleotide sequences. The precise length of a
fragment of SEQ
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ID N0:7-12 and the region of SEQ ID N0:7-12 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:l-6 is encoded by a fragment of SEQ ID N0:7-12. A
fragment of
SEQ ID NO:1-6 comprises a region of unique amino acid sequence that
specifically identifies SEQ
ID NO:1-6. For example, a fragment of SEQ ID NO:l-6 is useful as an
immunogenic peptide for the
development of antibodies that specifically recognize SEQ ID NO:1-6. The
precise length of a
fragment of SEQ ID NO:1-6 and the region of SEQ ID NO:1-6 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" polynucleotide 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" polynucleotide 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 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 WI). 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 (NCBI) 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:l/www.ncbi.nlm.nih.govlBLAST/. 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
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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: S and Extension Gap: 2 penalties
Gap x drop-off. 50
Expect: 10
Word Size: Il
Filter: ora
Percent identity may be measured over the length of an entire defined
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 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: I~tuple=l, gap
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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.
Alternatively 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 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 1l azzd Extensiozz Gap: 1 petzalties
Gap x drop-off. SO
Expect: 10
Word Size: 3
Filter: ozz
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, 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 compleriientarity.
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
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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 (Tm) 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 Cloning: A Laboratory Manual, 2°a 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, 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 hybridizations. 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 ATRS
which is
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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 ATRS 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 ATRS. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of ATRS.
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
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 ATRS 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 ATRS.
"Probe" refers to nucleic acid sequences encoding ATRS, 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
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DNA strand by a DNA polymerise enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerise 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 Laboratory Manual,
2°d ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, 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
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
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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, supra. 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 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 ATRS,
nucleic acids encoding ATRS, or fragments thereof 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
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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 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
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into such organisms are widely known and provided in references such as
Sambrook et al. (1989),
supra.
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
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% 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 will generally 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.
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 91%, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
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 aminoacyl tRNA
synthetases (ATRS),
the polynucleotides encoding ATRS, and the use of these compositions for the
diagnosis, treatment,
or prevention of cell proliferative and autoimmune/inflammatory disorders. The
tRNA synthetase of
the present invention catalyzes a number of important biological reactions
including, but not limited
to, the activation and correct attachment of an amino acid to its cognate
tRNA. In certain contexts,
tRNA synthetases can load amino acids onto tRNAs other than their cognates,
with varying kinetic
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efficiency.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ ID NO:) and
an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID)
as shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the
invention. Column 3
shows the GenBank identification number (Genbank ID NO:) of the nearest
GenBank homolog.
Column 4 shows the probability score for the match between each polypeptide
and its GenBank
homolog. Column 5 shows the annotation of the GenBank homolog along with
relevant citations
where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and
2 show the polypeptide sequence identification number (SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention. Column
3 shows the number of amino acid residues in each polypeptide. Column 4 shows
potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the
MOTIFS program of the GCG sequence analysis software package (Genetics
Computer Group,
Madison WI). Column 6 shows amino acid residues comprising signature
sequences, domains, and
motifs. Column 7 shows analytical methods for protein structure/function
analysis and in some cases,
searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are aminoacyl tRNA
synthetases. For example,
SEQ ID NO:l is 43% identical from amino acids 44 to 548 to Schizosaccharomyces
pombe
mitochondrial methionyl tRNA synthetase (GenBank ID g2388946) as determined by
the Basic Local
Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is
S.le-102, which
indicates the probability of obtaining the observed polypeptide sequence
alignment by chance. SEQ
ID NO:1 also contains a tRNA synthetase class I domain as determined by
searching for statistically
significant matches in the hidden Markov model (HMM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from BLIMPS analyses provide further
corroborative evidence
that SEQ ID NO:1 is a methionyl tRNA synthetase. In an alternative example,
SEQ ID N0:2 is 64%
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identical to human threonyl-tRNA synthetase (GenBank >D g1464742) as
determined by the Basic
Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability
score is 5.4e-288,
which indicates the probability of obtaining the observed polypeptide sequence
alignment by chance.
SEQ ID N0:2 also contains conserved signature sequences for class II tRNA
synthetases, as
determined by searching for statistically significant matches in the hidden
Markov model (HMM)-
based PFAM database of conserved protein family domains. (See Table 3.) The
presence of these
motifs is confirmed by BLIMPS and MOTIFS analyses, providing further
corroborative evidence that
SEQ ID N0:2 is a class II aminoacyl tRNA synthetase. In an alternative
example, SEQ ID N0:3
shares significant amino acid identity with A- uc~ifex aeolicus alanyl-tRNA
synthetase (GenBank ID
g2983727) as determined by the Basic Local Alignment Search Tool (BLAST). (See
Table 2.) The
BLAST probability score is 6.9e-20, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ ID N0:3 also contains a tRNA
synthetases class II
(A) domain as determined by searching for statistically significant matches in
the hidden Markov
model (HMM)-based PFAM database of conserved protein family domains. (See
Table 3.) Data from
BLIMPS analysis provides further corroborative evidence that SEQ ID N0:3 is an
alanyl-tRNA
synthetase. In an alternative example, SEQ ID N0:4 is 52% identical to human
threonyl-tRNA
synthetase (GenBank ID g1464742) as determined by the Basic Local Alignment
Search Tool
(BLAST). (See Table 2.) The BLAST probability score is 7.4e-205, which
indicates the probability
of obtaining the observed polypeptide sequence alignment by chance. SEQ ID
N0:4 also contains a
tRNA synthetase class II (G, H, P, S, and T) domain as determined by searching
for statistically
significant matches in the hidden Markov model (HMM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses provide
further
corroborative evidence that SEQ ID N0:4 is a threonyl-tRNA synthetase. In an
alternative example,
SEQ ID NO:S is 46% identical to Aduifex aeolicus proline-tRNA synthetase
(GenBank ID g2983039)
as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
The BLAST
probability score is 2.2e-78, which indicates the probability of obtaining the
observed polypeptide
sequence alignment by chance. SEQ ID N0:5 also contains a prolyl-tRNA
synthetase signature as
determined by searching for statistically significant matches in the PRINTS
database of conserved
protein family domains. (See Table 3.) Data from BLIMPS, and MOTIFS analyses
provide further
corroborative evidence that SEQ ID N0:5 is a prolyl-tRNA synthetase. In an
alternative example,
SEQ ID N0:6 is 40% identical to a portion of Mesorhizobium loti alanyl-tRNA
synthetase (GenBank
ID g14021340) and is 34% identical to a portion of Pyrococcus abyssi alanyl-
tRNA synthetase
(GenBank ID g5457541) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 9.0e-44 and 4.1e-26 respectively,
which indicates the
probability of obtaining the observed polypeptide sequence alignment by
chance. SEQ ID N0:6 also
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WO 02/04611 PCT/USO1/20723
contains an alanyl-tRNA synthetase domain as determined by searching for
statistically significant
matches in the hidden Markov model (HMM)-based PFAM database of conserved
protein family
domains (See Table 3), providing further corroborative evidence that SEQ ID
N0:6 is an alanyl-
tRNA synthetase. The algorithms and parameters for the analysis of SEQ ID NO:1-
6 are described in
Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Columns 1 and 2 list the
polynucleotide sequence
identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte
polynucleotide
consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide
of the invention.
Column 3 shows the length of each polynucleotide sequence in basepairs. Column
4 lists fragments
of the polynucleotide sequences which are useful, for example, in
hybridization or amplification
technologies that identify SEQ ID N0:7-12 or that distinguish between SEQ ID
N0:7-12 and related
polynucleotide sequences. Column 5 shows identification numbers corresponding
to cDNA
sequences, coding sequences (exons) predicted from genomic DNA, and/or
sequence assemblages
comprised of both cDNA and genomic DNA. These sequences were used to assemble
the full length
polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the
nucleotide start (5')
and stop (3') positions of the cDNA andlor genomic sequences in column 5
relative to their respective
full length sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
3393851H1 is the
identification number of an Incyte cDNA sequence, and LUNGNOT28 is the cDNA
library from
which it is derived. Incyte cDNAs for which cDNA libraries are not indicated
were derived from
pooled cDNA libraries (e.g., 70531465V1). Alternatively, the identification
numbers in column 5
may refer to GenBank cDNAs or ESTs which contributed to the assembly of the
full length
polynucleotide sequences. In addition, the identification numbers in column 5
may identify
sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UI~)
database (i.e., those
sequences including the designation "ENST"). Alternatively, the identification
numbers in column 5
may be derived from the NCBI RefSeq Nucleotide Sequence Records Database
(i.e., those sequences
including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence
Records (i.e., those
sequences including the designation "NP"). Alternatively, the identification
numbers in column 5
may refer to assemblages of both cDNA and Genscan-predicted exons brought
together by an "exon
stitching" algorithm. For example, FL XXXXXX N, NZ YYYYY N3 N4 represents a
"stitched"
sequence in which XXXXXX is the identification number of the cluster of
sequences to which the
algorithm was applied, and YYYYY is the number of the prediction generated by
the algorithm, and
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N,,~,3..., if present, represent specific exons that may have been manually
edited during analysis (See
Example V). Alternatively, the identification numbers in column 5 may refer to
assemblages of
exons brought together by an "exon-stretching" algorithm. For example,
FLX~~~XXX_gAAAAA_gBBBBB_1 N is the identification number of a "stretched"
sequence, with
XXXXXX being the Incyte project identification number, gA~4A~9A being the
GenBank identification
number of the human genomic sequence to which the "exon-stretching" algorithm
was applied,
gBBBBB being the GenBank identification number or NCBI RefSeq identification
number of the
nearest GenBank protein homolog, and N referring to specific exons (See
Example V). In instances
where a RefSeq sequence was used as a protein homolog for the "exon-
stretching" algorithm, a
RefSeq identifier (denoted by "NM," "NP," or "NT") may be used in place of the
GenBank identifier
(i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V)
Prefix Type of analysis and/or examples of programs
GNN, GFG, Exon prediction from genomic sequences using,
ENST for example,
GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UI~).
GBI Hand-edited analysis of genomic sequences.
, FL Stitched or stretched genomic sequences
(see Example V).
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in
column 5 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant
Incyte cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library is
the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences which
were used to assemble and confirm the above polynucleotide sequences. The
tissues and vectors
which were used to construct the cDNA libraries shown in Table 5 are described
in Table 6.
The invention also encompasses ATRS variants. A preferred ATRS 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 ATRS amino acid sequence, and which contains at least
one functional or
structural characteristic of ATRS.
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
The invention also encompasses polynucleotides which encode ATRS. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:7-12, which encodes ATRS. The
polynucleotide sequences
of SEQ ID N0:7-12, 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
ATRS. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at
least about 85%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding ATRS. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:7-
12 which has at least about 70%, or alternatively at least about 85%, or even
at least about 95%
polynucleotide sequence identity to a nucleic acid sequence selected from the
group consisting of
SEQ ID N0:7-12. Any one of the polynucleotide variants described above can
encode an amino acid
sequence which contains at least one functional or structural characteristic
of ATRS.
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 ATRS, some
bearing minimal
similarity to the polynucleotide 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 accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring ATRS, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode ATRS and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring ATRS under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding ATRS 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 eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding ATRS 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 ATRS
and
ATRS derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the
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WO 02/04611 PCT/USO1/20723
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 ATRS 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
N0:7-12 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 polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Applied
Biosystems), thermostable T7 polymerise (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerises 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 NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
sequencing system (Applied 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 irx the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biolo~y and Biotechnology, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding ATRS 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 unknown
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 legations may be used to insert an engineered double-stranded
sequence into a region
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of unknown sequence before performing PCR. Other methods which may be used to
retrieve
unknown 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 5' 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, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
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 ATRS may be cloned in recombinant DNA molecules that direct
expression of ATRS,
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 ATRS.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter ATRS-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 colon preference, produce splice
variants, and so forth.
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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 ATRS, such as its biological or enzymatic
activity omits 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,a 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 ATRS 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, ATRS 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., Creighton, T. (1984) Proteins, Structures and Molecular Pro eu
rties, 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 (Applied Biosystems).
Additionally, the
amino acid sequence of ATRS, 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, su ra, pp. 28-53.)
In order to express a biologically active ATRS, the nucleotide sequences
encoding ATRS 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
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WO 02/04611 PCT/USO1/20723
inducible promoters, and 5' and 3' untranslated regions in. the vector and in
polynucleotide sequences
encoding ATRS. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
ATRS. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the I~ozak
sequence. In cases where
sequences encoding ATRS 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 ATRS 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 ATRS. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasxnid, or cosmid DNA expression vectors;
yeast transformed with
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, su ~~ra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; 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; The McGraw Hill Yearbook of Science and Technoloay (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; Butler, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
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(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 polynucleotide sequences encoding ATRS. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding ATRS 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 ATRS 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 ATRS- are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of ATRS may be used.
For example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of ATRS. 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; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and
Scorer, C.A. et al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of ATRS. Transcription of
sequences
encoding ATRS maybe driven by 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, G. et al. (1984) EMBO J. 3:1671-
1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J, et al. (1991) Results Probl. Cell
Differ. 17:85-105.)
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 ATRS
may be ligated into
an adenovirus transcription/txanslation 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 ATRS in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
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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 ATRS in cell lines is preferred. For example, sequences encoding ATRS can
be transformed into
cell lines using expression vectors which may contain viral origins of
replication andlor 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
a1. (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,
dlZfr confers resistance to
methotrexate; r2eo confers 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 liisD, 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),13 glucuronidase and its substrate 13-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 ATRS is inserted within a marker gene sequence, transformed
cells containing
32
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sequences encoding ATRS can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding ATRS 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 ATRS
and that express
ATRS 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 ATRS 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 ATRS 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 Immunolo~y, 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 ATRS
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding ATRS, 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 polymerase
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 ATRS 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
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containing polynucleotides which encode ATRS may be designed to contain signal
sequences which
direct secretion of ATRS 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 ATRS 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 ATRS protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of ATRS 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 these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the ATRS encoding sequence and the
heterologous protein
sequence, so that ATRS may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel (1995, su ra, 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 ATRS 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.
ATRS of the present invention or fragments thereof may be used to screen for
compounds
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that specifically bind to ATRS. At least one and up to a plurality of test
compounds may be screened
for specific binding to ATRS. 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
ATRS, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which ATRS
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 ATRS,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or
E. coli. Cells expressing ATRS or cell membrane fractions which contain ATRS
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either ATRS 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
ATRS, either in
solution or affixed to a solid support, and detecting the binding of ATRS 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.
ATRS of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of ATRS. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for ATRS
activity, wherein ATRS is combined with at least one test compound, and the
activity of ATRS in the
presence of a test compound is compared with the activity of ATRS in the
absence of the test
compound. A change in the activity of ATRS in the presence of the test
compound is indicative of a
compound that modulates the activity of ATRS. Alternatively, a test compound
is combined with an
in vitro or cell-free system comprising ATRS under conditions suitable for
ATRS activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of ATRS
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 ATRS or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem
CA 02413810 2003-O1-06
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(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 Number 5,175,383 and U.S.
Patent Number
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 ATRS 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 ATRS 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 ATRS 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
treated with potential pharmaceutical agents to obtain information on
treatment of a human disease.
Alternatively, a mammal inbred to overexpress ATRS, e.g., by secreting ATRS 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 ATRS and aminoacyl tRNA synthetases. In addition, the
expression of ATRS is
closely associated with neurological, fetal brain, pancreatic, and transformed
kidney epithelial tissues
and bone marrow neuroblastoma tumor cells. Therefore, ATRS appears to play a
role in cell
proliferative and autoimmune/inflammatory disorders. In the treatment of
disorders associated with
increased ATRS expression or activity, it is desirable to decrease the
expression or activity of ATRS.
In the treatment of disorders associated with decreased ATRS expression or
activity, it is desirable to
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increase the expression or activity of ATRS.
Therefore, in one embodiment, ATRS 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 ATRS. Examples of such disorders include, but are not limited to,
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; and 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
polyendocrinopathy-
candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, 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
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.
In another embodiment, a vector capable of expressing ATRS 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 ATRS including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
ATRS 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 ATRS including,
but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of ATRS
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of ATRS including, but not limited to, those listed above.
In a further embodiment, an antagonist of ATRS may be administered to a
subject to treat or
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CA 02413810 2003-O1-06
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prevent a disorder associated with increased expression or activity of ATRS.
Examples of such
disorders include, but are not limited to, those cell proliferative and
autoimmunelinflammatory
disorders described above. In one aspect, an antibody which specifically binds
ATRS 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 ATRS.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding ATRS may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of ATRS including, but not limited to, those
described above.
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 ATRS may be produced using methods which are generally known
in the
art. In particular, purified ATRS may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind ATRS.
Antibodies to ATRS 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 ATRS 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-Gueriri) and Corynebacterium auap rvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
ATRS 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 ATRS amino acids may be fused with those of another protein, such as I~LH,
and antibodies to the
chimeric molecule may be produced.
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Monoclonal antibodies to ATRS 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
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
ATRS-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., Qrlandi, 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 ATRS 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
ATRS and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering ATRS 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 ATRS. Affinity
is expressed as an
association constant, Ka, which is defined as the molar concentration of ATRS-
antibody complex
39
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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 ATRS epitopes, represents the average affinity, or
avidity, of the antibodies for
ATRS. The Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular ATRS epitope, represents a true measure of affinity. High-affinity
antibody preparations
with Ka ranging from about 109 to 10'2 L/mole are preferred for use in
immunoassays in which the
ATRS-antibody complex must withstand rigorous manipulations. Low-affinity
antibody preparations
with Ka ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of ATRS, 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 ATRS-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, s- upra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding ATRS, or
any
fragment or complement thereof, may be used fox 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 ATRS. 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 ATRS. (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, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
CA 02413810 2003-O1-06
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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 Morris, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding ATRS may be
used for
somatic or gernnline 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),
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 N. Somia (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 falcinarum and
Trypanosoma cruzi). In the
case where a genetic deficiency in ATRS expression or regulation causes
disease, the expression of
ATRS 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
ATRS are treated by constructing mammalian expression vectors encoding ATRS
and introducing
these vectors by mechanical means into ATRS-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 ATRS 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). ATRS may be expressed
using (i) a
41
CA 02413810 2003-O1-06
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constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40
virus, thymidine kinase (TK), or (3-actin genes), (ii) an inducible promoter
(e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA
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 Blau, H.M. supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding ATRS from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
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 ATRS expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding ATRS 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. USA 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;
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Ranga, U, et al. (1998) Proc. Natl. Acad. Sci. USA 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 ATRS to cells which have one or more genetic
abnormalities with respect
to the expression of ATRS. 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
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding ATRS to target cells which have one or more genetic
abnormalities with
respect to the expression of ATRS. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing ATRS 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 ATRS 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 I~.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
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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
ATRS into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
ATRS-coding RNAs and the synthesis of high levels of ATRS 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 ATRS into a variety of cell types. The specific
transduction of a subset of
cells in a population may 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 ig c 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 ATRS.
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
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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 ATRS. 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 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 ATRS. 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 ATRS
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding ATRS may be therapeutically useful, and in the treatment of disorders
associated with
decreased ATRS expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding ATRS 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
CA 02413810 2003-O1-06
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library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding ATRS 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
ATRS 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 ATRS. 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 Schizosacchaxomyces pombe gene
expression system
(Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000)
Nucleic Acids 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.l5:462-466.)
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
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
Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
compositions may
consist of ATRS, antibodies to ATRS, and mimetics, agonists, antagonists, or
inhibitors of ATRS.
~ The compositions utilized in this invention may be administered by any
number of routes
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including, but not limited to, oral, intravenous, intramuscular, intra-
arterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
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.
Compositions suitable for use in the invention include compositions wherein
the 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 compositions may be prepared for direct intracellular
delivery of
macromolecules comprising ATRS or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of
the macromolecule. Alternatively, ATRS 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 ATRS
or fragments thereof, antibodies of ATRS, and agonists, antagonists or
inhibitors of ATRS, 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°10 of the population) statistics. The dose ratio of toxic
to therapeutic effects is the
therapeutic index, which can be expressed as the LDSOlEDSO ratio. 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
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CA 02413810 2003-O1-06
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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 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 ,ug to 100,000 beg, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
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 ATRS may be used for
the
diagnosis of disorders characterized by expression of ATRS, or in assays to
monitor patients being
treated with ATRS or agonists, antagonists, or inhibitors of ATRS. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for ATRS include methods which utilize the antibody and a label to detect ATRS
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 ATRS, including ELISAs, RIAs, and FACS,
are known
.in the art and provide a basis for diagnosing altered or abnormal levels of
ATRS expression. Normal
or standard values for ATRS expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibodies to ATRS under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of ATRS
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 ATRS may
be used for
diagnostic purposes. The polynucleotides,which may be used include
oligonucleotide sequences,
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complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of ATRS
may be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
ATRS, and to monitor regulation of ATRS levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding ATRS or closely related
molecules may be used
to identify nucleic acid sequences which encode ATRS. 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 ATRS, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the ATRS encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:7-12 or from
genomic sequences including promoters, enhancers, and introns of the ATRS
gene.
Means for producing specific hybridization probes for DNAs encoding ATRS
include the
cloning of polynucleotide sequences encoding ATRS or ATRS 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 355,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding ATRS may be used for the diagnosis of
disorders
associated with expression of ATRS. Examples of such disorders include, but
are not limited to, 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; and 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
polyendocrinopathy-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. The
polynucleotide sequences encoding ATRS 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 ATRS
expression. Such qualitative or quantitative methods are well known in the
art.
In a particular aspect, the nucleotide sequences encoding ATRS may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding ATRS 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 ATRS 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
ATRS, 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 ATRS, 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 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
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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 ATRS
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 ATRS, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
ATRS, 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 ATRS may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic
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,
oligonucleotide primers derived from the polynucleotide sequences encoding
ATRS 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), axe 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 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 ATRS include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
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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 ox 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 below. 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, ATRS, fragments of ATRS, or antibodies specific for
ATRS 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.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines,
biopsies, or other biological samples. The transcript image may thus reflect
gene expression in vivo,
as in the case of a tissue or biopsy sample, or in vitro, as in the case of a
cell line.
Transcript images which profile the expression of the polynucleotides of the
present
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invention may also be used in conjunction with in vitro model systems and
preclinical evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share
those toxic properties. These fingerprints or signatures are most useful and
refined when they contain
expression information from a large number of genes and gene families.
Ideally, a genome-wide
measurement of expression provides the highest quality signature. Even genes
whose expression is
not altered by any tested compounds are important as well, as the levels of
expression of these genes
are used to normalize the rest of the expression data. The normalization
procedure is useful for
comparison of expression data after treatment with different compounds. While
the assignment of
gene function to elements of a toxicant signature aids in interpretation of
toxicity mechanisms,
knowledge of gene function is not necessary for the statistical matching of
signatures which leads to
prediction of toxicity. (See, for example, Press Release 00-02 from the
National Institute of
Environmental Health Sciences, released February 29, 2000, available at
http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a
proteome can be subjected individually to further analysis. Proteome
expression patterns, or profiles,
are analyzed by quantifying the number of expressed proteins and their
relative abundance under
given conditions and at a given time. A profile of a cell's proteome may thus
be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample are
separated by isoelectric focusing in the first dimension, and then according
to molecular weight by
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sodium dodecyl sulfate slab gel electrophoresis in the second dimension
(Steiner and Anderson,
supra). The proteins are visualized in the gel as discrete and uniquely
positioned spots, typically by
staining the gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical
density of each protein spot is generally proportional to the level of the
protein in the sample. The
optical densities of equivalently positioned protein spots from different
samples, for example, from
biological samples either treated or untreated with a test compound or
therapeutic agent, are
compared to identify any changes in protein spot density related to the
treatment. The proteins in the
spots are partially sequenced using, for example, standard methods employing
chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein in a spot
may be determined by
comparing its partial sequence, preferably of at least 5 contiguous amino acid
residues, to the
polypeptide sequences of the present invention. In some cases, further
sequence data may be
obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for ATRS
to quantify the
levels of ATRS expression. In one embodiment, the antibodies are used as
elements on a microarray,
and protein expression levels are quantified by exposing the microarray to the
sample and detecting
the levels of protein bound to each array element (Lueking, A. et al. (1999)
Anal. Biochem. 270:103-
111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a
variety of methods known in the art, for example, by reacting the proteins in
the sample with a thiol-
or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at each
array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to
rapid degradation of mRNA, so proteomic profiling may be more reliable and
informative in such
cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological
sample. A difference in the amount of protein between the two samples is
indicative of a toxic
response to the test compound in the treated sample. Individual proteins are
identified by sequencing
the amino acid residues of the individual proteins and comparing these partial
sequences to the
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polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are
incubated with antibodies specific to the polypeptides of the present
invention. The amount of
protein recognized by the antibodies is quantified. The amount of protein in
the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount of
protein between the two samples is indicative of a toxic response to the test
compound in the treated
sample.
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;
Shalom 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 ATRS
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
of a multi-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 Pl
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, for example, 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
(OM1M) World Wide Web site. Correlation between the location of the gene
encoding ATRS 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.
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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, ATRS, 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 ATRS 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
synthesized on a solid substrate. The test compounds are reacted with ATRS, or
fragments thereof,
and washed. Bound ATRS is then detected by methods well known in the art.
Purified ATRS 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 ATRS specifically compete with a test compound
for binding ATRS.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with ATRS.
In additional embodiments, the nucleotide sequences which encode ATRS 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 embodiments are,
therefore, to be construed as merely illustrative, and not limitative of the
remainder of the disclosure
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in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below,
including U.S. Ser. No. 60/216,748, U.S. Ser. No. 60/219,019, U.S. Ser. No.
60/223,058, U.S. Ser.
No. 60/234,693, and U.S. Ser. No. 60/239,797, are hereby expressly
incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database
(Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. 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
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), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBI~-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo Alto
CA), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli cells
including XL1-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DHlOB, or
ElectroMAX
DHlOB from Life Technologies.
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II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP 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 FLUOROSKAN 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 (Applied 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 (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied 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, su ra, unit 7.7). Some of the cDNA sequences were selected for extension
using the techniques
disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof 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 hidden Markov model (HMM)-based protein
family
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databases such as PFAM. (HMM is a probabilistic approach which analyzes
consensus primary
structures of gene families. See, for example, Eddy, S.R. (1996) Curr. Opin.
Struct. Biol. 6:361-365.)
The queries were performed using programs based on BLAST, FASTA, BLIMPS, and
HMMER. The
Incyte cDNA sequences were assembled to produce full length polynucleotide
sequences.
Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched
sequences, or
Genscan-predicted coding sequences (see Examples IV and V) were used to extend
lilcyte cDNA
assemblages to full length. Assembly was performed using programs based on
Phred, Phrap, and
Consed, and cDNA assemblages 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 polypeptide sequences. Alternatively, a
polypeptide of the invention
may begin at any of the methionine residues of the full length translated
polypeptide. Full length
polypeptide sequences were subsequently analyzed by querying against databases
such as the
GenBank protein databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM,
Prosite,
and hidden Markov model (HMM)-based protein family databases such as PFAM.
Full length
polynucleotide sequences are also analyzed using MACDNASIS PRO software
(Hitachi Software
Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide
and polypeptide sequence alignments are generated using 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.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and
threshold parameters. The first column of Table 7 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 or the lower
the probability value,
the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide
and polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ
ID N0:7-12. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization
and amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative aminoacyl tRNA synthetases were initially identified by running the
Genscan gene
identification program against public genomic sequence databases (e.g., gbpri
and gbhtg). Genscan is
a general-purpose gene identification program which analyzes genomic DNA
sequences from a
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variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-
94, and Burge, C. and
S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program
concatenates predicted exons to
form an assembled cDNA sequence extending from a methionine to a stop codon.
The output of
Genscan is a FASTA database of polynucleotide and polypeptide sequences. The
maximum range of
sequence for Genscan to analyze at once was set to 30 kb. To determine which
of these Genscan
predicted cDNA sequences encode aminoacyl tRNA synthetases, the encoded
polypeptides were
analyzed by querying against PFAM models for aminoacyl tRNA synthetases.
Potential aminoacyl
tRNA synthetases were also identified by homology to Incyte cDNA sequences
that had been
annotated as aminoacyl tRNA synthetases. These selected Genscan-predicted
sequences were then
compared by BLAST analysis to the genpept and gbpri public databases. Where
necessary, the
Genscan-predicted sequences were then edited by comparison to the top BLAST
hit from genpept to
correct errors in the sequence predicted by Genscan, such as extra or omitted
exons. BLAST analysis
was also used to find any Incyte cDNA or public cDNA coverage of the Genscan-
predicted
sequences, thus providing evidence for transcription. When Incyte cDNA
coverage was available,
this information was used to correct or confirm the Genscan predicted
sequence. Full length
polynucleotide sequences were obtained by assembling Genscan-predicted coding
sequences with
Incyte cDNA sequences andlor public cDNA sequences using the assembly process
described in
Example III. Alternatively, full length polynucleotide sequences were derived
entirely from edited or
unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
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genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for
homologous genomic sequences from the public human genome databases. Partial
DNA sequences
were therefore "stretched" or extended by the addition of homologous genomic
sequences. The
resultant stretched sequences were examined to determine whether it contained
a complete gene.
VI. Chromosomal Mapping of ATRS Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:7-12 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 N0:7-12 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). 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 Genethon 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.
Map locations are represented by ranges, or intervals, of human chromosomes.
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. Human
genome maps and
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other resources available to the public, such as the NCBI "GeneMap' 99" World
Wide Web site
(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
VII. 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 is
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.
Alternatively, polynucleotide sequences encoding ATRS are analyzed with
respect to the
tissue sources from which they were derived. For example, some full length
sequences are
assembled, at least in part, With overlapping Incyte cDNA sequences (see
Example III). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following organ/tissue categories: cardiovascular
system; connective tissue;
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digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female;
genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal
system; nervous
system; pancreas; respiratory system; sense organs; skin; stomatognathic
system; unclassified/mixed;
or urinary tract. The number of libraries in each category is counted and
divided by the total number
of libraries across all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
by the total number of libraries across all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding ATRS. cDNA sequences and cDNA
library/tissue
information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto
CA).
VIII. Extension of ATRS Encoding Polynucleotides
Full length polynucleotide sequences were also 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 was
synthesized 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,
and 2-mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerise (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 ~1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 p1 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
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(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ,u1 to 10 ~1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose 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 relegation 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 relegated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerise (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 polymerise
(Amersham Pharmacia Biotech) and Pfu DNA polymerise (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 (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5'regulatory sequences using the above procedure along with
oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:7-12 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 ,uCi of
[y 3zP~ 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 dextrin bead column (Amersham Pharmacia
Biotech).
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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.
X. 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, supra.),
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),
supra). 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
polynucleotides in the
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-(dT) cellulose method. Each poly(A)+
RNA sample is
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reverse transcribed using MMLV reverse-transcriptase, 0.05 pgl~.tl oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/~1 RNase inhibitor, 500 l.~M dATP, 500 E,iM
dGTP, 500 ~.iM dTTP, 40
i.~M dCTP, 40 NM 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
NY) and
resuspended in 14 ~l 5X 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
pg. 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 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 N1 of the array
element DNA, at an average
concentration of 100 ng/~1, 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 microarrays 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.
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Hybridization
Hybridization reactions contain 9 p1 of sample mixture consisting of 0.2 pg
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 p1 of 5X 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 NJ) 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
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
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(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).
XI. Complementary Polynucleotides
Sequences complementary to the ATRS-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring ATRS. 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 ATRS. 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
ATRS-encoding
transcript.
XII. Expression of ATRS
Expression and purification of ATRS is achieved using bacterial or virus-based
expression
systems. For expression of ATRS 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 TS 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 ATRS upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of ATRS in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Auto raphica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding ATRS 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 SRodoptera frugiperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
68
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
Infection of the latter requires additional genetic 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, ATRS 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 iaponicum, 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
ATRS 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 ATRS obtained by these methods can be used
directly in the assays
shown in Examples XVI, XVII, and XVITI, where applicable.
XIII. Functional Assays
ATRS function is assessed by expressing the sequences encoding ATRS 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 (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA),
both of which
contain the cytomegalovirus promoter. 5-10 ,ug 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 ,ug 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
69
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow Cytometry_, Oxford, New York NY.
The influence of ATRS on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding ATRS 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
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
NY). mRNA can be purified from the cells using methods well known by those of
skill in the art.
Expression of mRNA encoding ATRS and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XIV. Production of ATRS Specific Antibodies
ATRS 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 ATRS 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 (Applied Biosystems) using FMOC chemistry and coupled to
I~LH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccininnide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-I~LH complex in complete Freund's adjuvant. Resulting antisera
are tested for
antipeptide and anti-ATRS activity by, for example, binding the peptide or
ATRS to a substrate,
blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat
anti-rabbit IgG.
XV. Purification of Naturally Occurring ATRS Using Specific Antibodies
Naturally occurring or recombinant ATRS is substantially purified by
immunoaffinity
chromatography using antibodies specific for ATRS. An immunoaffinity column is
constructed by
covalently coupling anti-ATRS 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 ATRS are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of ATRS (e.g.,
high ionic strength
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/ATRS 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 ATRS is collected.
XVI. Identification of Molecules Which Interact with ATRS
ATRS, 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
previously arrayed in the wells of a mufti-well plate are incubated with the
labeled ATRS, washed,
and any wells with labeled ATRS complex are assayed. Data obtained using
different concentrations
of ATRS are used to calculate values for the number, affinity, and association
of ATRS with the
candidate molecules.
Alternatively, molecules interacting with ATRS 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).
ATRS 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. 6,057,101).
XVII. Demonstration of ATRS Activity
tRNA synthetase activity is measured as the aminoacylation of a substrate tRNA
in the
presence of ['4C]-labeled amino acid. ATRS is incubated with ['4C]-labeled
amino acid and the
appropriate cognate tRNA (for example, ['~C]alanine and tRNAa'a; ['øC]proline
and tRNAp'° ) in a
buffered solution. '4C-labeled product is separated from free ['4C]amino acid
by chromatography,
and the incorporated'4C is quantified by scintillation counter. The amount of
'4C-labeled product
detected is proportional to the activity of ATRS in this assay.
Alternatively, tRNA synthetase activity is measured as the aminoacylation of a
substrate
tRNA in the presence of [35S]methionine. ATRS is incubated with tRNAme' and
[35S]methionine in a
buffered solution. 35S-labeled product is separated from free [35S]methionine
by chromatography, and
the incorporated 35S is quantified by scintillation counter. The amount of 35S
detected is proportional
to the activity of ATRS in this assay.
XVIII. Identification of ATRS Agonists and Antagonists
Agonists or antagonists of ATRS activation or inhibition may be tested using
the assay
described in section XVII. Agonists cause an increase in ATRS activity and
antagonists cause a
decrease in ATRS activity.
Various modifications and variations of the described methods and systems of
the invention
71
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
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.
72
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
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CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
<110> INCYTE GENOMICS, INC.
LEE, Ernestine A.
TANG, Y. Tom
LU, Dyung Aina M.
TRIBOULEY, Catherine M.
GANDHI, Ameena R.
LU, Yan
BAUGHN, Mariah R.
WARREN, Bridget A.
THORNTON, Michael
YUE, Henry
HILLMAN, Jennifer L.
PATTERSON, Chandra
ELLIOTT, Vicki S.
THANGAVELU, Kavitha
RAMKUMAR, Jayalaxini
WALIA, Narinder K.
YAO, Monique G.
GANDHI, Ameena R.
<120> AMINOACYL TRNA SYNTHETASES
<130> PI-0153 PCT
<140> To Be Assigned
<141> Herewith
<150> 60!216,748; 60J219,019; 60/223,058; 60!234,693; 60/239,797
<151> 2000-07-07; 2000-07-18; 2000-08-04; 2000-09-21; 2000-10-11
<160> 12
<170> PERL Program
<210> 1
<211> 593
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3393851CD1
<400> 1
Met Leu Arg Thr Ser Val Leu Arg Leu Leu Gly Arg Thr Gly Ala
1 5 10 15
Ser Arg Leu Ser Leu Leu Glu Asp Phe Gly Pro Arg Tyr Tyr Ser
20 25 30
Ser Gly Ser Leu Ser Ala Gly Asp Asp Ala Cys Asp Val Arg Ala
35 40 . 45
Tyr Phe Thr Thr Pro Ile Phe Tyr Val Asn Ala Ala Pro His Ile
50 55 60
Gly His Leu Tyr Ser Ala Leu Leu Ala Asp Ala Leu Cys Arg His
65 70 75
Arg Arg Leu Arg Gly Pro Ser Thr Ala Ala Thr Arg Phe Ser Thr
80 85 90
Gly Thr Asp Glu His Gly Leu Lys Ile Gln Gln Ala Ala Ala Thr
95 100 105
Ala Gly Leu Ala Pro Thr Glu Leu Cys Asp Arg Val Ser Glu Gln
110 115 120
Phe Gln Gln Leu Phe Gln Glu Ala Gly Ile Ser Cys Thr Asp Phe
125 130 135
Ile Arg Thr Thr Glu Ala Arg His Arg Val Ala Val Gln His Phe
1/13
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
140 145 150
Trp Gly Val Leu Lys Ser Arg Gly Leu Leu Tyr Lys Gly Val Tyr
155 160 165
Glu Gly Trp Tyr Cys Ala Ser Asp Glu Cys Phe Leu Pro Glu Ala
170 175 180
Lys Val Thr G1n Gln Pro Gly Pro Ser Gly Asp Ser Phe Pro Val
185 190 195
Ser Leu Glu Ser Gly His Pro Val Ser Trp Thr Lys Glu Glu Asn
200 205 210
Tyr Ile Phe Arg Leu Ser Gln Phe Arg Lys Pro Leu Gln Arg Trp
215 220 225
Leu Arg Gly Asn Pro Gln Ala Ile Thr Pro Glu Pro Phe His His
230 235 240
Val Val Leu Gln Trp Leu Asp Glu Glu Leu Pro Asp Leu Ser Val
245 250 255
Ser Arg Arg Ser Ser His Leu His Trp Gly Ile Pro Val Pro Gly
260 265 270
Asp Asp Ser Gln Thr Ile Tyr Val Trp Leu Asp Ala Leu Val Asn
275 280 285
Tyr Leu Thr Val Ile Gly Tyr Pro Asn Ala Glu Phe Lys Ser Trp
290 295 300
Trp Pro Ala Thr Ser His Ile Ile Gly Lys Asp Ile Leu Lys Phe
305 310 315
His Ala Ile Tyr Trp Pro Ala Phe Leu Leu Gly Ala Gly Met Ser
320 325 330
Pro Pro Gln Arg Ile Cys Val His Ser His Trp Thr Val Cys Gly
335 340 345
Gln Lys Met Ser Lys Ser Leu Gly Asn Val Val Asp Pro Arg Thr
350 355 360
Cys Leu Asn Arg Tyr Thr Val Asp Gly Phe Arg Tyr Phe Leu Leu
365 370 375
Arg Gln Gly Val Pro Asn Trp Asp Cys Asp Tyr Tyr Asp Glu Lys
380 385 390
Val Val Lys Leu Leu Asn Ser Glu Leu Ala Asp Ala Leu Gly Gly
395 400 405
Leu Leu Asn Arg Cys Thr Ala Lys Arg Ile Asn Pro Ser Glu Thr
410 415 420
Tyr Pro Ala Phe Cys Thr Thr Cys Phe Pro Ser Glu Pro Gly Leu
425 430 435
Val Gly Pro Ser Val Arg Ala Gln Ala Glu Asp Tyr Ala Leu Val
440 445 450
Ser Ala Val Ala Thr Leu Pro Lys Gln Val Ala Asp His Tyr Asp
455 460 465
Asn Phe Arg Ile Tyr Lys Ala Leu Glu Ala Val Ser Ser Cys Val
470 475 480
Arg Gln Thr Asn Gly Phe Val Gln Arg His Ala Pro Trp Lys Leu
485 490 495
Asn Trp G1u Ser Pro Val Asp Ala Pro Trp Leu Gly Thr Val Leu
500 505 510
His Val Ala Leu Glu Cys Leu Arg Val Phe Gly Thr Leu Leu Gln
515 520 525
Pro Val Thr Pro Ser Leu Ala Asp Lys Leu Leu Ser Arg Leu Gly
530 535 540
Val Ser Ala Ser Glu Arg Ser Leu Gly Glu Leu Tyr Phe Leu Pro
545 550 555
Arg Phe Tyr Gly His Pro Cys Pro Phe Glu Gly Arg Arg Leu Gly
560 565 570
Pro Glu Thr Gly Leu Leu Phe Pro Arg Leu Asp Gln Ser Arg Thr
575 580 585
Trp Leu Val Lys Ala His Arg Thr
590
<210> 2
2113
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
<211> 802
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475756CD1
<400> 2
Met Ala Ala Glu Ala Leu Ala Ala Glu Ala Val Ala Ser Arg Leu
1 5 10 15
Glu Arg Gln Glu Glu Asp Ile Arg Trp Leu Trp Ser Glu Val Glu
20 25 30
Arg Leu Arg Asp G1u Gln Leu Asn Ser Pro Tyr Ser Cys Gln Ala
35 40 45
Glu Gly Pro Cys Leu Thr Arg Glu Val Ala Gln Leu Arg Ala Glu
50 55 60
Asn Cys Asp Leu Arg His Arg Leu Cys Ser Leu Arg Leu Cys Leu
65 70 75
Ala Glu Glu Arg Ser Arg Gln Ala Thr Leu Glu Ser Ala Glu Leu
80 85 90
Glu Ala Ala Gln Glu Ala Gly Ala Gln Pro Pro Pro Ser Gln Ser
95 100 105
Gln Asp Lys Asp Met Lys Lys Lys.Lys Met Lys Glu Ser Glu Ala
110 115 120
Asp Ser Glu Val Lys His Gln Pro Ile Phe Ile Lys Glu Arg Leu
125 130 135
Lys Leu Phe Glu Ile Leu Lys Lys Asp His Gln Leu Leu Leu Ala
140 145 150
Ile Tyr Gly Lys Lys Gly Asp Thr Ser Asn Ile Ile Thr Val Arg
155 160 165
Val Ala Asp Gly Gln Thr Val Gln Gly Glu Val Trp Lys Thr Thr
170 175 180
Pro Tyr Gln Val Ala Ala Glu Ile Ser Gln Glu Leu Ala Glu Ser
185 190 195
Thr Val Ile Ala Lys Val Asn Gly Glu Leu Trp Asp Leu Asp Arg
200 205 210
Pro Leu Glu Gly Asp Ser Ser Leu Glu Leu Leu Thr Phe Asp Asn
215 220 ' 225
Glu Glu Ala Gln Ala Val Tyr Trp His Ser Ser Ala His Ile Leu
230 235 240
Gly Glu Ala Met Glu Leu Tyr Tyr Gly Gly His Leu Cys Tyr Gly
245 250 255
Pro Pro Ile Glu Asn Gly Phe Tyr Tyr Asp Met Phe Ile Glu Asp
260 265 270
Arg Ala Val Ser Ser Thr Glu Leu Ser Ala Leu Glu Asn Ile Cys
275 280 285
Lys Ala I1e Ile Lys Glu Lys Gln Pro Phe Glu Arg Leu Glu Val
290 295 300
Ser.Lys Glu Ile Leu Leu Glu Met Phe Lys Tyr Asn Lys Phe Lys
305 310 315
Cys Arg Ile Leu Asn Glu Lys Val Asn Thr Ala Thr Thr Thr Val
320 325 330
Tyr Arg Cys Gly Pro Leu Ile Asp Leu Cys Lys Gly Pro His Val
335 340 345
Arg His Thr Gly Lys Ile Lys Thr Ile Lys Ile Phe Lys Asn Ser
350 355 360
Ser Thr Tyr Trp Glu Gly Asn Pro Glu Met Glu Thr Leu Gln Arg
365 370 375
Ile Tyr Gly Ile Ser Phe Pro~Asp Asn Lys Met Met Arg Asp Trp
380 385 390
Glu Lys Phe Gln Glu Glu Ala Lys Asn Arg Asp His Arg Lys Ile
395 400 405
3/ 13
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
Gly Lys Glu Gln Glu Leu Phe Phe Phe His Asp Leu Ser Pro Gly
410 415 420
Ser Cys Phe Phe Leu Pro Arg Gly Ala Phe Ile Tyr Asn Thr Leu
425 430 435
Thr Asp Phe Ile Arg Glu Glu Tyr His Lys Arg Asp Phe Thr Glu
440 445 450
Val Leu Ser Pro Asn Met Tyr Asn Ser Lys Leu Trp Glu Ala Ser
455 460 465
Gly His Trp Gln His Tyr Ser Glu Asn Met Phe Thr Phe Glu Ile
470 475 480
Glu Lys Asp Thr Phe Ala Leu Lys Pro Met Asn Cys Pro Gly His
485 490 495
Cys Leu Met Phe Ala His Arg Pro Arg Ser Trp Arg Glu Met Pro
500 505 510
Tle Arg Phe Ala Asp Phe Gly Val Leu His Arg Asn Glu Leu Ser
515 520 525
Gly Thr Leu Ser Gly Leu Thr Arg Val Arg Arg Phe Gln Gln Asp
530 535 540
Asp Ala His Ile Phe Cys Thr Val Glu Gln Ile Glu Glu Glu Ile
545 550 555
Lys Gly Cys,Leu Gln Phe Leu Gln Ser Val Tyr Ser Thr Phe Gly
560 565 570
Phe Ser Phe Gln Leu Asn Leu Ser Thr Arg Pro Glu Asn Phe Leu
575 580 585
Gly Glu Ile Glu Met Trp Asn Glu Ala Glu Lys Gln Leu Gln Asn
590 595 600
Ser Leu Met Asp Phe Gly Glu Pro Trp Lys Met Asn Pro Gly Asp
605 610 615
Gly Ala Phe Tyr Gly Pro Lys Ile Asp Ile Lys Ile Lys Asp A1a
620 625 630
Ile Gly Arg Tyr His Gln Cys Ala Thr Ile Gln Leu Asp Phe Gln
635 640 645
Leu Pro Ile Arg Phe Asn Leu Thr Tyr Val Ser Lys Asp Gly Asp
650 655 660
Asp Lys Lys Arg Pro Val Ile Ile His Arg Ala Ile Leu Gly Ser
665 670 675
Val Glu Arg Met I1e Ala Ile Leu Ser Glu Asn Tyr Gly Gly Lys
680 685 690
Trp Pro Phe Trp Leu Ser Pro Arg Gln Val Met Val Ile Pro Val
695 700 705
Gly Pro Thr Cys Glu Lys Tyr Ala Leu Gln Val Ser Ser Glu Phe
710 715 720
Phe Glu Glu Gly Phe Met Ala Asp Val Asp Leu Asp His Ser Cys
725 730 735
Thr Leu Asn Lys Lys Ile Arg Asn Ala Gln Leu Ala Gln Tyr Asn
740 745 750
Phe Ile Leu Val Val Gly Glu Lys Glu Lys Ile Asp Asn Ala Val
755 760. 765
Asn Val Arg Thr Arg Asp Asn Lys Ile His Gly Glu Ile Leu Val
770 775 780
Thr Ser Ala Ile Asp Lys Leu Lys Asn Leu Arg Lys Thr Arg Thr
785 790 795
Leu Asn Ala Glu Glu Ala Phe
800
<210> 3
<211> 422
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1554103CD1
4/ 13
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
<400> 3
Met Pro Ile Pro Leu Thr Pro Pro Ala Gly Met Ala Phe Trp Cys
1 5 10 15
Gln Arg Asp Ser Tyr Ala Arg Glu Phe Thr Thr Thr Val Val Ser
20 25 30
Cys Cys Pro Ala Glu Leu Gln Thr Glu Gly Ser Asn Gly Lys Lys
35 40 45
Glu Val Leu Ser Gly Phe Gln Val Val Leu Glu Asp Thr Val Leu
50 55 60
Phe Pro Glu Gly Gly Gly Gln Pro Asp Asp Arg Gly Thr Ile Asn
65 70 75
Asp Ile Ser Val Leu Arg Val Thr Arg Arg Gly Glu Gln Ala Asp
80 85 90
His Phe Thr Gln Thr Pro Leu Asp Pro Gly Ser Gln Val Leu Val
95 100 105
Arg Val Asp Trp Glu Arg Arg Phe Asp His Met Gln Gln His Ser
110 115 120
Gly Gln His Leu Ile Thr Ala Val Ala Asp His Leu Phe Lys Leu
125 130 135
Lys Thr Thr Ser Trp Glu Leu Gly Arg Phe Arg Ser Ala Ile Glu
140 145 150
Leu Asp Thr Pro Ser Met Thr Ala Glu Gln Val Ala Ala Ile Glu
155 160 165
Gln Ser Val Asn Glu Lys Ile Arg Asp Arg Leu Pro Val Asn Val
170 175 180
Arg Glu Leu Ser Leu Asp Asp Pro Glu Val Glu Gln Val Ser Gly
185 190 195
Arg Gly Leu Pro Asp Asp His Ala Gly Pro Ile Arg Val Val Asn
200 205 210
Ile Glu Gly Val Asp Ser Asn Met Cys Cys Gly Thr His Val Ser
215 220 225
N
Asn Leu Ser Asp Leu Gln Val Ile Lys Ile Leu Gly Thr Glu Lys
230 235 240
Gly Lys Lys Asn Arg Thr Asn Leu Ile Phe Leu Ser Gly Asn Arg
245 250 255
Val Leu Lys Trp Met Glu Arg Ser His Gly Thr Glu Lys Ala Leu
260 265 270
Thr Ala Leu Leu Lys Cys Gly Ala Glu Asp His Va1 Glu Ala Va1
275 280 285
Lys Lys Leu Gln Asn Ser Thr Lys Ile Leu Gln Lys Asn Asn Leu
290 295 300
Asn Leu Leu Arg Asp Leu Ala Val His Ile Ala His Ser Leu Arg
305 310 315
Asn Ser Pro Asp Trp Gly Gly Val Val Ile Leu His Arg Lys Glu
320 325 330
Gly Asp Ser Glu Phe Met Asn Ile Ile Ala Asn Glu Ile Gly Ser
335 340 345
Glu Glu Thr Leu Leu Phe Leu Thr Val Gly Asp Glu Lys Gly Gly
350 355 360
Gly Leu Phe Leu Leu Ala Gly Pro Pro Ala Ser Val Glu Thr Leu
365 370 375
Gly Pro Arg Val Ala Glu Val Leu Glu Gly Lys Gly Ala Gly Lys
380 385 390
Lys Gly Arg Phe Gln Gly Lys Ala Thr Lys Met Ser Arg Arg Met
395 400 405
Glu Ala Gln Ala Leu Leu Gln Asp Tyr Ile Ser Thr Gln Ser Ala
410 415 420
Lys Glu
<210> 4
<211> 718
<212> PRT
5/ 13
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2184108CD1
<400> 4
Met Ala Leu Tyr Gln Arg Trp Arg Cys Leu Arg Leu Gln Gly Leu
1 5 10 15
Gln Ala Cys Arg Leu His Thr Ala Val Val Ser Thr Pro Pro Arg
20 25 30
Trp Leu Ala Glu Arg Leu Gly Leu Phe Glu Glu Leu Trp Ala Ala
35 40 45
Gln. Val Lys Arg Leu Ala Ser Met Ala Gln Lys Glu Pro Arg Thr
50 55 60
Ile Lys Ile Ser Leu Pro Gly Gly Gln Lys Ile Asp Ala Val Ala
65 70 75
Trp Asn Thr Thr Pro Tyr Gln Leu Ala Arg Gln Ile Ser Ser Thr
80 85 90
Leu Ala Asp Thr Ala Val Ala Ala Gln Val Asn Gly Glu Pro Tyr
95 100 105
Asp Leu Glu Arg Pro Leu Glu Thr Asp Ser Asp Leu Arg Phe Leu
110 115 120
Thr Phe Asp Ser Pro Glu Gly Lys Ala Val Phe Trp His Ser Ser
125 130 135
Thr His Val Leu Gly Ala Ala Ala Glu Gln Phe Leu Gly Ala Val
140 145 150
Leu Cys Arg Gly Pro Ser Thr Glu Tyr Gly Phe Tyr His Asp Phe
155 160 165
Phe Leu Gly Lys Glu Arg Thr Ile Arg Gly Ser Glu Leu Pro Val
170 175 180
Leu Glu Arg Ile Cys Gln Glu Leu Thr Ala Ala Ala Arg Pro Phe
185 190 195
Arg Arg Leu Glu Ala Ser Arg Asp Gln Leu Arg Gln Leu Phe Lys
200 205 210
Asp Asn Pro Phe Lys Leu His Leu Ile Glu Glu Lys Val Thr Gly
215 220 225
Pro Thr Ala Thr Val Tyr Gly Cys Gly Thr Leu Val Asp Leu Cys
230 235 240
Gln Gly Pro His Leu Arg His Thr Gly Gln Ile Gly Gly Leu Lys
245 250 255
Leu Leu Ser Asn Ser Ser Ser Leu Trp Arg Ser Ser Gly A1a Pro
260 265 270
Glu Thr Leu Gln Arg Val Ser Gly Ile Ser Phe Pro Thr Thr Glu
275 280 285
Leu Leu Arg Val Trp Glu Ala Trp Arg Glu Glu Ala G1u Leu Arg
290 295 300
Asp His Arg Arg Ile Gly Lys Glu Gln Glu Leu Phe Phe Phe His
305 310 315
Glu Leu Ser Pro Gly Ser Cys Phe Phe Leu Pro Arg Gly Thr Arg
320 325 330
Val Tyr Asn Ala Leu Val Ala Phe Ile Arg Ala Glu Tyr Ala His
335 340 345
Arg Gly Phe Ser Glu Val Lys Thr Pro Thr Leu Phe Ser Thr Lys
350 355 360
Leu Trp Glu Gln Ser Gly His Trp Glu His Tyr Gln Glu Asp Met
365 370 375
Phe Ala Val Gln Pro Pro Gly Ser Asp Arg Pro Pro Ser Ser Gln
380 385 390
Ser Asp Asp Ser Thr Arg His Ile Thr Asp Thr Leu Ala Leu Lys
395 400 405
Pro Met Asn Cys Pro Ala His Cys Leu Met Phe Ala His Arg Pro
410 415 420
6/13
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
Arg Ser Trp Arg Glu Leu Pro Leu Arg Leu Ala Asp Phe Gly Ala
425 430 435
Leu His Arg Ala Glu Ala Ser Gly Gly Leu Gly Gly Leu Thr Arg
440 445 450
Leu Arg Cys Phe Gln Gln Asp Asp Ala His Ile Phe Cys Thr Thr
455 460 465
Asp Gln Leu Glu Ala Glu Ile Gln Ser Cys Leu Asp Phe Leu Arg
470 475 480
Ser Val Tyr Ala Val Leu Gly Phe Ser Phe Arg Leu Ala Leu Ser
485 490 495
Thr Arg Pro Ser Gly Phe Leu Gly Asp Pro Cys Leu Trp Asp Gln
500 505 510
Ala Glu Gln Val Leu Lys Gln Ala Leu Lys Glu Phe G1y Glu Pro
515 520 525
Trp Asp Leu Asn Ser Gly Asp Gly Ala Phe Tyr Gly Pro Lys Ile
530 535 540
Asp Val His Leu His Asp Ala Leu Gly Arg Pro His Gln Cys Gly
545 550 555
Thr Ile Gln Leu Asp Phe Gln Leu Pro Leu Arg Phe Asp Leu Gln
560 565 570
Tyr Lys Gly Gln Ala Gly Ala Leu Glu Arg Pro Val Leu Ile His
575 580 585
Arg Ala Val Leu Gly Ser Val Glu Arg Leu Leu Gly Val Leu Ala
590 595 600
Glu Ser Cys Gly Gly Lys Trp Pro Leu Trp Leu Ser Pro Phe Gln
605 610 615
Val Val Val Ile Pro Val Gly Ser Glu Gln Glu Glu Tyr Ala Lys
620 625 630
Glu Ala Gln Gln Ser Leu Arg Ala Ala Gly Leu Val Ser Asp Leu
635 640 645
Asp Ala Asp Ser Gly Leu Thr Leu Ser Arg Arg Ile Arg Arg Ala
650 655 660
Gln Leu Ala His Tyr Asn Phe Gln Phe Val Val Gly Gln Lys Glu
665 670 675
Gln Ser Lys Arg Thr Val Asn Ile Arg Thr Arg Asp Asn Arg Arg
680 685 690
Leu Gly Glu Trp Asp Leu Pro Glu Ala Val Gln Arg Leu Val Glu
695 700 705
Leu Gln Asn Thr Arg Val Pro Asn Ala G1u Glu Ile Phe
710 715
<210> 5
<211> 475
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2115996CD1
<400> 5
Met Glu Gly Leu Leu Thr Arg Cys Arg Ala Leu Pro Ala Leu Ala
1 5 10 15
Thr Cys Ser Arg Gln Leu Ser Gly Tyr Val Pro Cys Arg Phe His
20 25 30
His Cys Ala Pro Arg Arg Gly Arg Arg Leu Leu Leu Ser Arg Val
35 40 45
Phe Gln Pro Gln Asn Leu Arg Glu Asp Arg Val Leu Ser Leu Gln
50 55 60
Asp Lys Ser Asp Asp Leu Thr Cys Lys Ser Gln Arg Leu Met Leu
65 70 75
Gln Val Gly Leu Ile Tyr Pro Ala Ser Pro Gly Cys Tyr His Leu
80 85 90
7113
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
Leu Pro Tyr Thr Val Arg Ala Met Glu Lys Leu Val Arg Val Ile
95 100 105
Asp Gln Glu Met Gln Ala Ile Gly Gly Gln Lys Val Asn Met Pro
110 115 120
Ser Leu Ser Pro Ala Glu Leu Trp Gln Ala Thr Asn Arg Trp Asp
125 130 135
Leu Met Gly Lys Glu Leu Leu Arg Leu Arg Asp Arg His Gly Lys
140 145 150
Glu Tyr Cys Leu Gly Pro Thr His Glu Glu Ala Ile Thr Ala Leu
155 160 165
Ile Ala Ser Gln Lys Lys Leu Ser Tyr Lys Gln Leu Pro Phe Leu
170 175 180
Leu Tyr Gln Val Thr Arg Lys Phe Arg Asp Glu Pro Arg Pro Arg
185 190 195
Phe Gly Leu Leu Arg Gly Arg Glu Phe Tyr Met Lys Asp Met Tyr
200 205 210
Thr Phe Asp Ser Ser Pro Glu Ala Ala Gln Gln Thr Tyr Ser Leu
215 220 225
Val Cys Asp Ala Tyr Cys Ser Leu Phe Asn Lys Leu Gly Leu Pro
230 235 240
Phe Val Lys Val Gln Ala Asp Val Gly Thr Ile Gly Gly Thr Val
245 250 255
Ser His Glu Phe Gln Leu Pro Val Asp Ile Gly Glu Asp Arg Leu
260 265 270
Ala Ile Cys Pro Arg Cys Ser Phe Ser Ala Asn Met Glu Thr Leu
275 280 285
Asp Leu Ser Gln Met Asn Cys Pro Ala Cys Gln Gly Pro Leu Thr
290 295 300
Lys Thr Lys Gly Ile Glu Val Gly His Thr Phe Tyr Leu Gly Thr
305 310 315
Lys Tyr Ser Ser Ile Phe Asn Ala Gln Phe Thr Asn Val Cys G1y
320 325 330
Lys Pro Thr Leu Ala Glu Met Gly Cys Tyr Gly Leu Gly Val Thr
335 340 345
Arg Ile Leu Ala Ala Ala 21e Glu Val Leu Ser Thr Glu Asp Cys
350 355 360
Val Arg Trp Pro Ser Leu Leu Ala Pro Tyr Gln Ala Cys Leu Ile
365 370 375
Pro Pro Lys Lys Gly Ser Lys Glu Gln Ala Ala Ser Glu Leu Ile
380 385 390
Gly Gln Leu Tyr Asp His Ile Thr Glu Ala Val Pro Gln Leu His
395 400 405
Gly Glu Val Leu Leu Asp Asp Arg Thr His Leu Thr Ile Gly Asn
410 415 420
Arg Leu Lys Asp Ala Asn Lys Phe Gly Tyr Pro Phe Val Ile Ile
425 430 435
Ala Gly Lys Arg Ala Leu Glu Asp Pro Ala His Phe Glu Val Trp
440 445 450
Cys Gln Asn Thr Gly Glu Val Ala Phe Leu Thr Lys Asp Gly Val
455 460 465
Met Asp Leu Leu Thr Pro Val Gln Thr Val
470 475
<210> 6
<211> 244
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6971530CD1
<400> 6
8/13
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
Met Asn Met Ser Val His Thr Met Glu Thr Leu Ala Leu Phe Asp
1 ' 5 10 15
Ser Ala Pro Tyr Gln Asn Ala Phe Asn Ala Arg Va1 Ile Ala Val
20 25 30
Ser Glu His Gly Ile Ala Leu Glu His Thr Leu Phe,Tyr Pro Thr
35 40 45
Gly Gly Gly Gln Pro Gly Asp Thr Gly His Phe Thr Leu Ala Asp
50 55 60
Gly Thr Arg Val Asp Ile Thr Gly Thr Val Arg Asp Ala Glu Ala
65 70 75
Arg Ser Ile Ile Trp His Gln Val Glu Asn Cys Pro Glu Pro Leu
80 85 90
Val Ala Gly Val Gln Val Glu Ala Asn Leu Asp Trp Glu Arg Arg
95 100 105
Tyr Gln His Met Lys Met His Thr Cys Leu His Leu Leu Cys Ser
110 115 120
Leu Ile Asp Ala Pro Val Thr Gly Cys Ser Ile Ser Ala Asp Lys
125 130 135
Gly Arg Leu Asp Phe Asp Leu Pro Glu Met Thr Leu Asp Lys Asp
140 145 150
Ser Ile Thr Arg Asp.Leu Asn Ala Leu Ile Ala Gln Ala His Glu
155 160 165
Val Lys Thr Leu Ser Met Pro Ala Thr Glu Tyr Ala Thr Leu Leu
170 175 180
Gln Ile Thr Arg Thr Gln Ala Val Ala Pro Pro Val Ile Gln Gly
185 190 195
Ser Val Arg Val Ile Glu Ile Gly Gly Ile Asp Ile Gln Pro Cys
200 205 210
Gly Gly Thr His Val Ala Asn Thr Glu Glu Ile Gly Arg Va1 Phe
215 220 225
Cys Glu Lys Ile Glu Lys Lys Ser Lys His Asn Arg Arg Val Ile
230 235 240
Leu Arg Phe Glu
<210> 7
<211> 1959
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3393851CB1
<400> 7
tacggcacca tgctgcgaac gtccgtcctc cgcctgctag gacgcacggg ggctagtagg 60
ctgtctctcc tggaggactt cggcccacgc tactacagtt cgggctccct cagtgccggc 120
gatgatgctt gtgatgtgcg cgcctacttc actacaccca ttttctacgt gaacgcggcg 180
ccgcacatcg ggcacctgta ctcggcacta ctggcggacg ccctatgccg ccaccgtcgc 240
ctccgaggtc ccagcacggc cgccacgcga ttctccactg gtaccgacga gcacgggctg 300
aagattcagc aggcagcagc taccgcgggc ctggccccga ccgagctgtg cgaccgagtc 360
tctgagcagt tccagcagct tttccaggag gccggtatct cctgcacaga tttcatccgc 420
accacggagg cccggcaccg ggtggctgtg cagcacttct ggggggtgct taagtcccgc 480
ggtctgctct acaagggcgt ctatgaaggt tggtattgcg cttccgacga gtgcttcctg 540
cctgaggcca aggtcaccca gcagccgggc ccatcggggg attcgtttcc tgtatctctc 600
gagagcgggc atccagtctc ctggaccaag gaagaaaact acattttcag gctttcccag 660
ttccggaagc cactccagcg gtggctgcgg ggcaaccctc aggcgatcac ccccgaacca 720
tttcatcacg tagttcttca gtggctggac gaggagctgc ccgacctgtc cgtgtctcgc 780
agaagtagcc acttgcactg gggcattccg gtgcccgggg atgattcgca gaccatctat 840
gtatggctgg atgccctggt caactacctc actgtaattg gctacccaaa tgctgagttc 900
aaatcttggt ggccggccac ctctcatatc ataggtaagg acattctcaa attccatgcc 960
atctattggc ctgccttcct gttaggggcc ggcatgagcc cgccacagcg catctgtgtc 1020
cattcccact ggacagtctg tggccaaaag atgtccaaga gcttgggcaa cgtggtggat 1080
9/13
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
cctaggactt gccttaaccg ctataccgtg gatggcttcc gctactttct ccttcggcag 1140
ggcgtcccca actgggactg tgactactat gatgaaaagg tggttaagtt gctgaactcc 1200
gagctggcag atgccttggg aggtctcttg aaccgatgca ctgccaaaag aataaatcct 1260
tctgagacct acccagcctt ctgcactacc tgcttcccta gtgagccagg gttggtgggg 1320
ccgtcagttc gtgctcaggc agaggattat gctctggtga gcgcagtggc cactttgcca 1380
aagcaggtag cagaccacta tgataacttt cggatatata aggctctgga ggccgtgtcc 1440
agctgtgtcc ggcaaactaa tggttttgtc caaaggcatg caccatggaa gctgaactgg 1500
gagagcccag tggatgctcc ctggctgggt actgtgcttc atgtggcctt ggaatgtttg 1560
cgagtctttg ggactttgct gcagcctgtc accccaagcc tagctgacaa gctgctgtct 1620
aggctggggg tctctgcctc agagaggagt cttggagagc tctatttctt gcctcgattc 1680
tatggacatc catgcccttt tgaagggagg aggctgggac ctgaaactgg gcttttgttt 1740
ccaagactag accagtccag gacttggctg gtgaaagccc accggaccta gaaactcagt 1800
tcttaccggc ttgtggtaaa aaagcaaatg tgttatcttt ttatttttta ttttcaggaa 1860
agttatactt gtattttctt aagtgtggaa tcaaatgagc acataagctg tgtcctgtga 1920
aaagaggttt gtagcctttc agtgcctgct cctattcat 1959
<210> 8
<211> 3101
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475756CB1
<400> 8
cgcgggcacc ccgctcggcg tcggtgcctg agggaggccg cgatggcggc cgaggccctg 60
gcggcggagg ccgtggcgtc gcgcctggag cggcaggagg aggacatccg ctggctgtgg 120
tcggaggtcg agcgcctgag ggacgagcag ctgaactcgc cctacagctg ccaggcggag 180
gggccgtgcc tcacgcggga ggtggcgcag ctccgggccg agaactgcga cctgcgccac 240
cgcctgtgca gcctgcggct gtgcctcgcc gaggagcgga gccgccaggc cacgctggag 300
agcgcggagc tagaggcggc gcaggaggcc ggcgcacagc ctcctcctag tcaaagccaa 360
gacaaggaca tgaaaaagaa gaaaatgaag gaaagcgagg ctgacagcga ggtgaagcat 420
caaccaattt tcataaaaga aagattgaag ctttttgaaa tactgaagaa agaccatcag 480
ctcttacttg ccatttatgg aaaaaagggg gatacaagca acatcatcac agtaagagtg 540
gctgatgggc aaacagtgca aggggaagtc tggaaaacaa cgccttacca agtggctgct 600
gaaattagtc aggaactggc tgaaagcacg gtaatagcca aagtcaatgg tgaactgtgg 660
gacctggacc gcccattgga aggggactct tctctagagc tgcttacatt tgataatgag 720
gaagctcaag ctgtgtactg gcactccagt gctcacattc ttggggaggc catggagctt 780
tactatggag gccacctgtg ctacggtccg cccattgaaa atggatttta ttatgacatg 840
ttcattgaag acagagcagt gtccagcaca gaattgtcag ccctggagaa tatatgtaaa 900
gccatcataa aagaaaagca accttttgaa agactagaag tcagcaagga aatcctcctg 960
gaaatgttta agtacaataa atttaaatgc cgcattctga atgagaaagt taacactgca 1020
actaccaccg tgtacaggtg cggtccatta attgaccttt gcaaaggtcc acatgtaaga 1080
cacactggaa aaattaaaac catcaaaatt tttaagaatt cctcaacata ttgggagggc 1140
aatccggaaa tggaaacatt gcagaggatc tatggaatat cctttcctga taacaagatg 1200
atgagagact gggaaaagtt ccaagaggaa gcaaagaacc gagatcacag gaagatcggg 1260
aaggaacaag aacttttctt tttccacgat ttgagtcctg gaagctgttt tttccttccc 1320
agaggagcct tcatttataa tacgcttaca gatttcatac gagaggaata tcacaaacgg 1380
gacttcacgg aggtgctctc tcccaatatg tacaacagta aactctggga agcctcaggc 1440
cactggcagc attacagcga gaacatgttt acctttgaga ttgaaaagga cacttttgcc 1500
ctcaaaccca tgaattgtcc agggcactgt ctaatgtttg cccatcgtcc acgatcttgg 1560
agggaaatgc ctattagatt tgctgatttt ggagttctgc atagaaatga actgtcgggg 1620
actctcagtg gcttgaccag agtgaggcgc ttccagcagg acgatgctca cattttttgc 1680
acagtggagc agattgaaga agaaataaag gggtgtttgc agtttttgca atctgtttac 1740
tcaacatttg gcttctcctt tcaattaaac ctgtcaacaa ggccggaaaa cttcctagga 1800
gagattgaga tgtggaatga ggctgagaag caactgcaga acagcttgat ggactttgga 1860
gaaccgtgga aaatgaaccc aggagatgga gcattttatg gccctaaaat tgacataaaa 1920
atcaaggatg ctattggcag ataccatcaa tgtgctacaa ttcagctgga cttccaactg 2980
cctattagat ttaatctcac atatgttagt aaggatgggg atgataagaa gagacctgtg 2040
atcattcatc gagccatttt gggatcagtg gaaagaatga tagccattct ttcagaaaac 2100
tatggcggaa aatggccttt ctggctatct cctcgtcagg tgatggtcat ccctgtgggg 2160
ccaacttgtg aaaaatatgc acttcaggta tccagtgaat tttttgaaga aggatttatg 2220
10113
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
gctgacgttg acttggatca cagttgtaca ctaaataaga aaatacgaaa tgcacagctg 2280
gctcagtata attttatttt ggtggttgga gaaaaggaaa agatagataa tgctgtaaac 2340
gtgcgaacaa gagacaacaa aattcatgga gagattttag taacttctgc cattgataaa 2400
ctgaagaatc tcaggaagac acggacactc aatgctgagg aggccttttg aagtccttcc 2460
ctgatatttg cttctgtgta actttgtttt gacccttaaa aatgtatttt tcttaacatg 2520
ttagtacttc tacgactttg gagccactga tgggtccact catggcctca gctgagaaag 2580
gagacgatga acgtgtagct gacatgcacg aagtttaatt tactcatgtC cacgggggac 2640
gtttagaggg cacgtgggaa attttccagc aatcaatgcc ttgagaaact taaatgggga 2700
aatattattc atcgagaaag tgaaacaaaa cactaggaaa tgattatgaa atgttagtga 2760
ttttcaaaag atgggcttca aataaaagtc tgcagagttt tttttaaata ggagggaaaa 2820
tcttattttc tagtatgtct caggtatttt tatgacttct actaaaattc acactgaaac 2880
tttatcttct aaactggaat cattacttaa ttttaactaa ccaacaacca caaaagcagc 2940
agctactact aaatattgga ttactgacaa aggaattcag ttttgtggaa tctggtgttt 3000
gcactatagg ttaagagttg ccatttaaat gtttcttatt cataattagg ttttgttccg 3060
ctttagaaaa aaataaattc ccaaatgaat tgcaaaaaaa a 3101
<210> 9
<211> 1345
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1554103CB1
<400> 9
atgccaatac ctttaactcc acctgcaggt atggcgttct ggtgtcagcg tgacagttat 60
gcccgagagt tcaccaccac cgtggtctct tgctgtcccg cggagctgca gactgaaggg 120
agcaacggca agaaagaagt gctgagcggt ttccaagtgg tgctggaaga cacagtgctt 180
ttccctgagg gcgggggaca gcctgatgac cgtggtacaa tcaatgacat ctctgtgctg 240
agagtgactc gccgtgggga acaggctgat catttcaccc agacacccct ggatccagga 300
agccaggttc tggtccgggt agattgggag cggaggtttg accacatgca gcagcattca 360
gggcagcatc tcatcacggc agttgctgac catctattta agctgaagac aacatcatgg 420
gagttaggga gatttcggag tgcgattgag ctggacaccc cctctatgac tgcagagcaa 480
gtagctgcca ttgagcagag cgtcaatgaa aaaatcagag atcggctgcc tgtgaatgtc 540
cgagaactga gcctggatga tcctgaggtg gagcaggtga gtggccgggg tttgcctgat 600
gatcatgctg ggcccattcg ggttgttaac atcgagggcg ttgattccaa catgtgctgt 660
gggacccatg tgagcaatct cagtgacctt caggtcatta agattctggg cactgagaag 720
gggaaaaaga acagaaccaa cctgatattt ctgtctggga accgggtgct gaagtggatg 780
gagagaagtc atggaactga aaaagcactg actgctctgc ttaagtgtgg agcagaggat 840
catgtggaag cagtgaaaaa gctccagaac tccaccaaga tcctgcagaa gaataacctg 900
aatctgctca gagacctggc tgtgcacatt gcccatagcc tcaggaacag tccagactgg 960
ggaggtgtgg tcatattaca caggaaggag ggtgattcag agttcatgaa tatcattgcc 1020
aatgagattg ggtcagagga gaccctcctg ttcttaactg tgggcgatga gaaaggtggt 1080
ggactcttct tactggcagg gccacctgcg tctgtggaga ccctggggcc cagggtggct 1140
gaggtcctgg aaggcaaagg agcagggaag aaaggccgtt ttcagggcaa ggccaccaag 1200
atgagccggc ggatggaggc gcaggcgctt ctccaggact acatcagcac gcagagtgct 1260
aaggagtgag ggcttagggc actcacctcc tgtttccaca ggaatctttt ggtcaataaa 1320
atagattgac tcagaaaaaa aaaaa 1345
<210> 10
<211> 2518
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2184108CB1
<400> 10
agtgtgctgg aaagggcact ggtgtgaagg aacatggccc tgtatcagag gtggcggtgt 60
ctccggctcc aaggtttaca ggcttgcagg ctacacacgg cagttgtgtc gacccctcca 120
cgctggttgg cagagcggct tggccttttt gaggagctgt gggctgctca ggtaaagaga 180
11/13
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
ttagcaagca tggcacagaa ggaaccccgg actattaaga tatcacttcc tggaggccag 240
aaaattgatg ctgtggcatg gaacacaacc ccctaccaac tagcccggca gatcagttca 300
acactggcag atactgcagt ggctgctcaa gtgaatggag aaccttatga tctggagcgg 360
cccttggaga cagattctga cctcagattt ctgacattcg attccccaga ggggaaagca 420
gtgttctggc actccagcac ccatgtcctg ggggcagcag ctgaacaatt cctaggtgct 480
gttctctgca gaggtccaag tacagaatat ggcttttacc atgatttctt cctgggaaag 540
gagaggacaa tccggggctc agagctgcct gttttggagc ggatttgcca ggaacttaca 600
gctgctgctc gacccttccg gaggctagag gcttcacggg atcagcttcg ccagttgttc 660
aaggataacc cctttaagct tcacttgatt gaggagaaag tgacaggtcc aacagcaaca 720
gtatatgggt gtggcacatt ggttgacctt tgccagggcc cccaccttcg gcatactgga 780
cagattggag gactgaagct gctatcgaac tcatcatcct tatggaggtc ttcaggggcc 840
ccagagacac tgcagagagt gtcagggatt tccttcccca caacagaatt gctgagggtc 900
tgggaagcat ggagggagga agcagaattg cgggaccacc ggcgcattgg gaaggaacag 960
gagctcttct tcttccatga actgagccct gggagctgct tcttcctgcc acgagggaca 1020
agggtgtata atgcactagt ggcgtttatc agggctgagt atgcccatcg tggtttctcc 1080
gaggtgaaaa ctcccacact gttttctacg aagctctggg aacagtcagg gcactgggag 1140
cattatcagg aagacatgtt tgccgtgcag cccccaggct ctgacaggcc tcccagctcc 1200
cagagtgacg attctaccag gcatatcaca gatacactcg ccctcaagcc tatgaactgc 1260
cctgcacact gcctgatgtt cgcccaccgg cccagatcct ggcgggaact gcccctgcga 1320
ctagctgact ttggggctct acaccgggcc gaagcctctg gtggtctggg gggactgacc 1380
cgactgcggt gcttccagca ggatgacgct cacatcttct gtacaacaga tcagctggaa 1440
gcagagatcc aaagctgtct tgatttcctc CgttCCgtCt atgccgttct tggcttctcc 1500
ttccgcctgg cactgtccac ccggccatct ggcttcctgg gggacccttg cctttgggac 1560
Caggccgaac aggtccttaa acaggccctg aaggaatttg gagaaccctg ggacctcaac 1620
tctggagatg gtgccttcta tggacctaag attgacgtgc acctccacga tgccctgggc 1680
cggccacatc agtgtgggac aattcagctt gacttccaac tgcccctgag atttgacctc 1740
cagtataagg ggcaggcggg tgccctggag cgtccagtcc tcattcaccg agcagtgctc 1800
ggttctgtgg aaagactgtt gggagtgctg gcagaaagct gcggggggaa atggccactg 1860
tggctgtccc cgttccaggt ggtggtcatc cctgtgggga gtgagcaaga ggaatacgcc 1920
aaagaggcac agcagagcct gcgggctgca ggactggtca gtgacctgga tgcagactct 1980
ggactgaccc tcagccggag aatccgccgg gcccagcttg cccactacaa ttttcagttt 2040
gtggttggcc agaaagagca aagtaagaga acagtgaaca ttcggactcg agataatcgt 2100
cgccttgggg agtgggactt gcctgaggct gtgcagcgac tggtggagct acagaacacg 2160
agggtcccaa atgccgaaga aattttctga gcctttgtac atagatgagg caaaaacctg 2220
cgagtgccat cagcctccct cacatgggag accccaaccc agctgacaat gtggagcccc 228.0
cagaacttca gaactgtgtg gaggcacatg tctgctctcc tgaaaagaga cttggtttgg 23.40
ggaccccaca aaaggaggga agctgtagct gtttggatgt gaggagaatg aaactacaaa 2400
aaaaaataaa ttgggccagg cgcagtggct catgcctgta atcecagcac tctgggaggc 2460
tgaggcggac ggatcatgag gtcaggagat caagaccacc ctggtaacac ggtgaacg 2518
<210> 11
<211> 2372
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2115996CB1
<400> 11
cccacgcgtc cggcgcgctg gcccggcacg gcggtggtct tgcgggaggc gtgggctggg 60
attgcggtgc ctgtgcttcc cggtgccagg gtgtcatgga agggctgctg acaagatgca 120
gagcattgcc cgccctggcc acctgcagcc gccagctctc tgggtatgtt ccttgcaggt 180
ttcaccactg tgccccaaga agagggcggc gcctgctgct gtctcgtgtg ttccagccac 240
agaaccttcg ggaagaccgg gtgctctccc tgcaggacaa atctgatgac ctgacctgta 300
agagccagcg gctgatgctg caggtgggcc tgatctaccc agcaagcccc ggctgttacc 360
acctcctgcc atataccgtc cgtgccatgg agaagctcgt gcgagtgata gaccaggaga 420
tgcaggccat cgggggccag aaagtcaaca tgcccagcct cagcccggca gagctctggc 480
aagccaccaa ccggtgggac ttgatgggca aagagctgct aagacttaga gacaggcatg 540
gcaaggaata ctgcttagga ccaactcacg aggaagccat tacggcctta attgcctccc 600
agaagaaact gtcctacaag cagcttccct tcctgctgta ccaagtgaca aggaagtttc 660
gggatgagcc caggccccgc tttggtcttc tccgtggccg agagttttac atgaaggata 720
tgtacacctt tgactcctcc ccagaggctg cccagcagac ctacagcctg gtgtgtgatg 780
12/13
CA 02413810 2003-O1-06
WO 02/04611 PCT/USO1/20723
cctactgcag cctgttcaac aagctagggc tgccatttgt caaggtccag gccgatgtgg 840
gcaccatcgg gggcacagtg tctcatgagt tccagctccc agtggatatt ggagaggacc 900
ggcttgccat ctgtccccgc tgcagcttct cagccaacat ggagacacta gacttgtcac 960
aaatgaactg ccctgcttgc cagggcccat tgactaaaac caaaggcatt gaggtggggc 1020
acacatttta cctgggtacc aagtactcat ccattttcaa tgcccagttt accaatgtct 1080
gtggcaaacc aaccctggct gaaatggggt gctatggctt gggtgtgaca cggatcttgg 1140
ctgctgccat tgaagtcctc tctacagaag actgtgtccg ctggcccagc ctactggccc 1200
cttaccaagc ctgcctcatc ccccctaaga agggcagtaa ggagcaggcg gcctccgagc 1260
tcatagggca gctgtacgac cacatcacag aggcagtgcc tcagcttcac ggggaggtgc 1320
tcctggacga caggacccat ctgaccatcg gaaacagact gaaagatgcc aacaagtttg 1380
gctacccctt tgtgataatc gctggcaaga gggccctgga ggaccctgca cattttgagg 1440
tttggtgtca gaacactggt gaggtggcct tcctcaccaa agatggagtc atggatttac 1500
tgaccccagt gcagactgtc taaatgcccc cagcccaccc ctgcccccat ttgcagcctt 1560
ggtgttcgtt ctaacactgc attttcctac acccctttcc tggactgctc tctccagaaa 1620
cagcacagct catggagggt gagatcatgt tagggaaatc aatttttatc tagtcatttg 1680
ttcagattat ttgtatttaa agtcattaac ttgatccctt tctccagact ggccatgccg 1740
ccatatacct ttccttttgt attccattgt ggaagcttct aggaggctga gaagcaggag 1800
atCCaagctc cagtcctggg tgttaccctg aggggggaag tcacttccct tctctcagct 1860
gagtttttca cgtgtaagat gagaatggct gatggctgag agcccaccaa ccttccccac 1920
cccgtatcag tttgaccacg ggccttttgc tacattctat ccagagagca ggcctgccag 1980
cccctctcca ctgagaagtt gggctgagag tatggggaaa agaatcaaga gacctgactg 2040
ccgccaactc actagtgact tagatacatc ccctccccct gctggggcct cagtttcccc 2100
atctgtaact tgagaaaata gaactagatc tgtaaagtct ggaactagat ctgtaacgtc 2160
cttataggtg tcactagggg gttccatgag aggtgtgtga caggcagtct gattcctctc 2220
attctccata gtctgtttcc tggaaagtcg atgtaattaa ctgatggccc aaaaactacc 2280
tcaagagacc tggccctgtt aagacggctt aaccactgag atcccgttct attgatttaa 2340
taaagtcaaa ctattggaaa aaaaaaaaaa as 2372
<210> 12
<211> 1042
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6971530CB1
<400> 12
caatatctgc aatttcgcct gcatcgcgtc gaaacggccg accacctgcc gcgcctgcca 60
ttgaacctca aagtctgcgg cccggaattc gaaatcactt tgctctcccc ccctggcttg 120
agcgcaatgg tgcgccactt gctgcaagcc agcccactga tcgtcggagg atgaacatgt 180
ccgtgcacac catggaaacc ctggcgctgt ttgacagcgc gccctaccag aacgcattca 240
acgcccgggt gattgccgtc agcgagcacg gcatcgccct ggagcacacg ctgttctacc 300
caaccggcgg cgggcaaccg ggtgacactg gccacttcac cctggcggat ggcacgcgcg 360
tcgacatcac cggtaccgtg cgcgacgccg aagcgcgctc gatcatctgg catcaggtgg 420
aaaattgccc cgagccattg gttgccgggg tgcaggtgga ggccaacctc gattgggaac 480
ggcgctacca gcacatgaag atgcacacct gcctgcacct gctgtgctcg ctcatcgatg 540
cccctgtgac cggatgcagc atcagtgccg acaagggccg cctggatttc gacctgccgg 600
aaatgacgct ggacaaggac agcatcaccc gcgacctcaa cgcgctgatc gcccaggccc 660
atgaggtcaa gaccctctcg atgcccgcca cggagtacgc caccctgctg cagatcaccc 720
gcacccaggc ggtcgcgccg ccggtgatcc agggctcggt acgcgtgatt gaaattggcg 780
ggatcgatat ccaaccgtgc ggcggcaccc atgtggccaa caccgaggaa atcggccggg 840
tgttctgcga aaaaatcgag aagaagagca aacacaaccg ccgcgtgatc ctgcggtttg 900
aataacagct gagcggctgg ggtgctcgcg ccccagtctc agttcgtaca ggaaactgac 960
agcaaaattg acgcgcttgt aacattcaga aacgtacaat tcctccagca tcttcttgat 1020
gcccctgact catgaacagc ac 1042
13/13
