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ISOMERASE PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of isomerases
and to the use
of these sequences in the diagnosis, treatment, and prevention of immune and
cell proliferation
disorders including cancer.
BACKGROUND OF THE INVENTION
Proteins may be modified after translation through structural rearrangements
and/or by the
addition of phosphate, sugar, prenyl, fatty acid, and other chemical groups.
Cells contain a number
of specialized molecules that assist in the formation of protein structure.
Enzymes involved in post
translational modification include kinases, phosphatases,
glycosyltransferases, and prenyltransferases.
These molecules facilitate the folding of newly synthesized proteins, prevent
aggregation and
improper glycosylation, and remove denatured protein. These modifications are
often required for
proper protein activity. The conformation of proteins may also be modified
after translation for
example, by the introduction and rearrangement of disulfide bonds
(rearrangement catalyzed by
protein disulfide isomerases), the isomerization of proline side chains by
prolyl isomerase, and by
interactions with molecular chaperone proteins.
Numerous essential biochemical reactions involve the isomerization of a
substrate. Enzymes
which catalyze such reactions are known as isomerases. Isomerases are a class
of enzymes that
catalyze geometric or structural changes within a molecule to form a single
product. A number of
isomerases have been described catalyzing steps in a wide variety of
biochemical pathways including
protein folding, phototransduction, and various anabolic and catabolic
pathways (e.g., glycolysis), in
organisms ranging from bacteria to humans. Within the class of isomerases are
racemases and
epimerases, cis-trans-isomerases, intramolecular oxidoreductases and
intramolecular transferases
(mutases).
Racemases and Epimerases
Racemases are a subset of isomerases that catalyze inversion of a molecules
configuration
around the asymmetric carbon atom in a substrate having a single center of
asymmetry, thereby
interconverting two racemers. Epimerases are another subset of isomerases that
catalyze inversion of
configuration around an asymmetric carbon atom in a substrate with more than
one center of
symmetry, thereby interconverting two epimers. Racemases and epimerases can
act on amino acids
and derivatives, hydroxy acids and derivatives, as well as carbohydrates and
derivatives. The
interconversion of UDP-galactose and UDP-glucose is catalyzed by UDP-galactose-
4=epimerase.
Proper regulation and function of this epimerase is essential to the synthesis
of glycoproteins and
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glycolipids. Elevated blood galactose levels have been correlated with UDP-
galactose-4'-epimerase
deficiency in screening programs of infants (Gitzelmann, R. (1972) Helv.
Paediat. Acta 27:125-130).
Pe~tidyl prolyl cis-traps isomerases
Correct folding of newly synthesized proteins is assisted by molecular
chaperones and folding
catalysts, two unrelated groups of helper molecules. Chaperones suppress non-
productive side
reactions by stoichiometric binding to folding intermediates, whereas folding
enzymes catalyze some
of the multiple folding steps that enable proteins to attain their final
functional configurations (Kern,
G. et al. (1994) FEBS Lett. 348: 145-148). One class of folding enzymes, the
peptidyl prolyl cis-
trans isomerases (PPIases), isomerize certain proline imidic bonds in what is
considered to be a rate
limiting step in protein maturation and export. PPIases catalyze the cis to
traps isomerization of
certain proline imidic bonds in proteins. There are three sequence-unrelated
families of PPIases, the
cyclophilins, the FK506 binding proteins, and the newly characterized parvulin
family (Rahfeld, J.U.
et al. (1994) FEBS Lett. 352: 180-184).
The cyclophilins (CyP), were originally identified as major receptors for the
immunosuppressive drug cyclosporin A (CsA) an inhibitor of T-cell activation
(Handschumacher,
R.E. et al. (1984) Science 226: 544-547; Harding, M.W. et al. (1986) J. Biol.
Chem. 261: 8547-8555).
Thus, the peptidyl-prolyl isomerase activity of CyP may be part of the
signaling pathway that leads to
T-cell activation. Subsequent work demonstrated that CyP's isomerase activity
is essential for correct
protein folding and/or protein trafficking, and may also be involved in
assembly/disassembly of
protein complexes and regulation of protein activity. For example, in
Drosophila, the CyP NinaA is
required for correct localization of rhodopsins, while a mammalian CyP (Cyp40)
is part of the
Hsp90/Hsp70 complex that binds steroid receptors. The mammalian CyP (CypA) has
been shown to
bind the gag protein from human immunodeficiency virus 1 (HIV-1), an
interaction that can be
inhibited by cyclosporin. Since cyclosporin has potent anti-HIV-1 activity,
CypA may play an
essential function in HIV-1 replication. Finally, Cyp40 has been shown to bind
and inactivate the
transcription factor c-Myb, an effect that is reversed by cyclosporin. This
effect implicates CyPs in
the regulation of transcription, transformation, and differentiation (Bergsma,
D.J. et al (1991) J. Biol.
Chem. 266:23204 - 23214; Hunter, T. (1998) Cell 92: 141-143; and Leverson,
J.D. and Ness, S.A.
( 1998) Mol. Cell. 1:203-211 ).
Protein Disulfide Isomerases
One of the major rate limiting steps in protein folding is the thiol:disulfide
exchange that is
necessary for correct protein assembly. Although incubation of reduced,
unfolded proteins in buffers
with defined ratios of oxidized and reduced thiols can lead to native
conformation, the rate of folding
is slow and the attainment of native conformation decreases proportionately to
the size and number of
cysteines in the protein. Certain cellular compartments such as the
endoplasmic reticulum of
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eukaryotes and the periplasmic space of prokaryotes are maintained in a more
oxidized state than the
surrounding cytosol. Correct disulfide formation can occur in these
compartments but at a rate that is
insufficient for normal cell processes and not adequate for synthesizing
secreted proteins. The
protein disulfide isomerases, thioredoxins and glutaredoxins are able to
catalyze the formation of
disulfide bonds and regulate the redox environment in cells to enable the
necessary thiol:disulfide
exchanges (Loferer, H. (1995) J. Biol Chem. 270:26178-26183).
Each of these proteins have somewhat different functions but all belong to a
group of
disulfide-containing redox proteins that contain a conserved active-site
sequence and are ubiquitously
distributed in eukaryotes and prokaryotes. Protein disulfide isomerases are
found in the endoplasmic
reticulum of eukaryotes and in the periplasmic space of prokaryotes. They
function by exchanging
their own disulfide for a thiol in a folding peptide chain. In contrast, the
reduced thioredoxins and
glutaredoxins are generally found in the cytoplasm and function by directly
reducing disulfides in the
substrate proteins.
These catalytic molecules not only facilitate disulfide formation but also
regulate and
participate in a wide variety of physiological processes. The thioredoxin
system serves, for example,
as a hydrogen donor for ribonucleotide reductase and as a regulator of enzymes
by redox control. It
also modulates the activity of transcription factors such as NF-KB, AP-1, and
steroid receptors. More
recently, several cytokines or secreted cytokine-like factors such as adult T-
cell leukemia-derived
factor, 3B6-interleukin-1, T-hybridoma-derived (MP-6) B cell stimulatory
factor, and early pregnancy
factor have been reported to be identical to thioredoxin (Holmgren, A. (1985)
Annu. Rev. Biochem.
54:237-271, Abate, C. et al., (1990) Science 249:1157-1161, Tagaya, Y. et al.
(1989) EMBO J.
8:757-764, Wakasugi, H. (1987) Proc. Natl. Acad. Sci. 84:804-808, Rosen, A. et
al. (1995) Int.
Immunol. 7:625-633). Thioredoxin has also been shown to have many
extracellular activities
including a role as a regulator of cell growth and a mediator in the immune
system (Miranda-Vizuete,
A. et al. (1996) J. Biol. Chem. 271:19099-19103, Yamauchi, A. et al (1992)
Mol. Immunol. 29:263-
270).
Intramolecular oxidoreductases
Oxidoreductases can be isomerases as well. Oxidoreductases catalyze the
reversible transfer
of electrons from a substrate that becomes oxidized to a substrate that
becomes reduced. This class of
enzymes includes dehydrogenases, hydroxylases, oxidases, oxygenases,
peroxidases, and reductases.
Proper maintenance of oxidoreductase levels is physiologically important. The
pentose phosphate
pathway for example, utilizes enzymes which are responsible for generating the
reducing agent
NADPH, while at the same time oxidizing glucose-6-phosphate to ribose-5-
phosphate. NADPH
serves as the fuel for reactions undergoing reductive biosynthesis. Ribose-5-
phosphate and its
derivatives become part of critical biological molecules such as ATP, Coenzyme
A, NAD+, FAD,
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RNA, and DNA. The pentose phosphate pathway has both oxidative and non-
oxidative branches.
The oxidative branch steps, which are catalyzed by the enzymes glucose-6-
phosphate dehydrogenase,
lactonase, and 6-phosphogluconate dehydrogenase, convert glucose-6-phosphate
and NADP+ to
ribulose-6-phosphate and NADPH. The non-oxidative branch steps, which are
catalyzed by the
enzymes phosphopentose isomerase, phosphopentose epimerase, transketolase, and
transaldolase,
allow the interconversion of three-, four-, five-, six-, and seven-carbon
sugars.
Transferases
Another subgroup of isomerases are the transferases (or mutases). Transferases
transfer a
chemical group from one compound (the donor) to another compound (the
acceptor). The types of
groups transferred by these enzymes include acyl groups, amino groups,
phosphate groups
(phosphotransferases or phosphomutases), and others. The transferase carnitine
palmitoyltransferase
is an important component of fatty acid metabolism. Genetically-linked
deficiencies in this
transferase can lead to myopathy (Scriver C.R. et. al. (1995) The Metabolic
and Molecular Basis of
Inherited Disease, McGraw-Hill New York NY pp.1501-1533).
Isomerases are critical components of cellular biochemistry with roles in
metabolic energy
production including glycolysis, as well as other diverse enzymatic processes
(Stryer, L. (1995)
Biochemistry W.H. Freeman and Co. New York, NY pp.483-507).
The discovery of new isomerases 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
immune and cell proliferation disorders including cancer.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, isomerases, referred to
collectively as "ISOM"
and individually as "ISOM-1," "ISOM-2," "ISOM-3," "ISOM-4," "ISOM-5," "ISOM-
6," "ISOM-7,"
and "ISOM-8." In one aspect, the invention provides an isolated polypeptide
comprising an amino
acid sequence selected from the group consisting of a) an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-8, b) a naturally occurring amino acid
sequence having at least
90% sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-8, c) a biologically active fragment of an amino acid sequence selected
from the group
consisting of SEQ )D NO: l-8, and d) an immunogenic fragment of an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-8. In one alternative, the invention
provides an isolated
polypeptide comprising the amino acid sequence of SEQ )D NO:1-8.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising
an amino acid sequence selected from the group consisting of a) an amino acid
sequence selected
from the group consisting of SEQ )D NO:l-8, b) a naturally occurring amino
acid sequence having at
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least 90% sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-8, c) a biologically active fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-8, and d) an immunogenic fragment of an amino acid
sequence selected
from the group consisting of SEQ )D NO:1-8. In one alternative, the
polynucleotide encodes a
polypeptide selected from the group consisting of SEQ ID NO:1-8. In another
alternative, the
polynucleotide is selected from the group consisting of SEQ ID N0:9-16.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising
an amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-8, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ )D NO:1-8, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-8, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-8. In one alternative, the invention provides a cell
transformed with the
recombinant polynucleotide. In another alternative, the invention provides a
transgenic organism
comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising an
amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-8, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-8, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-8, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-8. The method comprises a) culturing a cell under
conditions suitable for
expression of the polypeptide, wherein said cell is transformed with a
recombinant polynucleotide
comprising a promoter sequence operably linked to a polynucleotide encoding
the polypeptide, and b)
recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-8, b) a
naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-8, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-8, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-8.
The invention further provides an isolated polynucleotide comprising a
polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
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consisting of SEQ >D N0:9-16, b) a naturally occurring polynucleotide sequence
having at least 90%
sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID N0:9
16, c) a polynucleotide sequence complementary to a), d) a polynucleotide
sequence complementary
to b), and e) an RNA equivalent of a)-d). In one alternative, the
polynucleotide comprises at least 60
contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
comprising a polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:9-16, b) a naturally occurring polynucleotide sequence
having at least 90%
sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID N0:9-
16, c) a polynucleotide sequence complementary to a), d) a polynucleotide
sequence complementary
to b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing
the sample with a
probe comprising at least 20 contiguous nucleotides comprising a sequence
complementary to said
target polynucleotide in the sample, and which probe specifically hybridizes
to said target
polynucleotide, under conditions whereby a hybridization complex is formed
between said probe and
said target polynucleotide or fragments thereof, and b) detecting the presence
or absence of said
hybridization complex, and optionally, if present, the amount thereof. In one
alternative, the probe
comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide comprising a
polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ >D N0:9-16, b) a naturally occurring polynucleotide sequence
having at least 90%
sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID N0:9-
16, c) a polynucleotide sequence complementary to a), d) a polynucleotide
sequence complementary
to b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying
said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting
the presence or absence of said amplified target polynucleotide or fragment
thereof, and, optionally, if
present, the amount thereof.
The invention further provides a composition comprising an effective amount of
a
polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ >D NO:1-8, b) a
naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-8, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ >D NO:1-8, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-8, and a
pharmaceutically
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acceptable excipient. In one embodiment, the composition comprises an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-8. The invention
additionally provides a method
of treating a disease or condition associated with decreased expression of
functional ISOM,
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 comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ 1D NO:1-8, b)
a naturally
occurring amino acid sequence having at least 90°lo sequence identity
to an amino acid sequence
selected from the group consisting of SEQ ID NO:1-8, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-8, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-8. 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 ISOM, 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 comprising an amino acid sequence selected from
the group consisting
of a) an amino acid sequence selected from the group consisting of SEQ >D NO:1-
8, b) a naturally
occurring amino acid sequence having at least 90°lo sequence identity
to an amino acid sequence
selected from the group consisting of SEQ ID NO:1-8, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-8, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-8. 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 ISOM, 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 comprising an amino acid sequence selected from the group
consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO:1-8, b) a
naturally occurring
amino acid sequence having at least 90% sequence identity to an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-8, c) a biologically active fragment of an
amino acid sequence
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selected from the group consisting of SEQ ID NO:1-8, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-8. 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 comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-8, b)
a naturally
occurring amino acid sequence having at least 90°lo sequence identity
to an amino acid sequence
selected from the group consisting of SEQ ID NO:1-8, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-8, and d) an
imrnunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-8. 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
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected from the group consisting of SEQ ID N0:9-16, 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 comprising a polynucleotide
sequence selected from the
group consisting of i) a polynucleotide sequence selected from the group
consisting of SEQ ID N0:9-
16, ii) a naturally occurring polynucleotide sequence having at least 90%
sequence identity to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:9-16,
iii) a polynucleotide
sequence complementary to i), iv) a polynucleotide sequence complementary to
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 comprising a polynucleotide sequence selected from the group
consisting of i) a
polynucleotide sequence selected from the group consisting of SEQ ID N0:9-16,
ii) a naturally
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occurring polynucleotide sequence having at least 90% sequence identity to a
polynucleotide
sequence selected from the group consisting of SEQ ID N0:9-16, iii) a
polynucleotide sequence
complementary to i), iv) a polynucleotide sequence complementary to 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 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs),
clone identification numbers (clone 117s), cDNA libraries, and cDNA fragments
used to assemble full-
length sequences encoding ISOM.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of ISOM.
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-
specific
expression patterns of each nucleic acid sequence as determined by northern
analysis; diseases,
disorders, or conditions associated with these tissues; and the vector into
which each cDNA was
cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA clones
encoding ISOM were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
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
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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
"ISOM" refers to the amino acid sequences of substantially purified ISOM
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
ISOM. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of ISOM either by
directly interacting with
ISOM or by acting on components of the biological pathway in which ISOM
participates.
An "allelic variant" is an alternative form of the gene encoding ISOM. 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 ISOM include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as ISOM or a
polypeptide with at least one functional characteristic of ISOM. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding ISOM, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
ISOM. 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 ISOM. 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
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residues, as long as the biological or immunological activity of ISOM 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 ISOM. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of ISOM either by
directly interacting with ISOM or by acting on components of the biological
pathway in which ISOM
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 ISOM 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 (KLH). The coupled peptide is then used to
immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
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;
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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 ISOM, or of
any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide
or amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding ISOM or fragments of
ISOM 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
(PE Biosystems,
Foster City CA) in the 5' and/or 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
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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
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide sequence can include, for example,
replacement of
hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative
polynucleotide encodes a
polypeptide which retains at least one biological or immunological function of
the natural molecule.
A derivative polypeptide is one modified by glycosylation, pegylation, or any
similar process that
retains at least one biological or immunological function of the polypeptide
from which it was
derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
A "fragment" is a unique portion of ISOM or the polynucleotide encoding ISOM
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
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used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 16, 20, 25, 30, 40, S0, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide)
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:9-16 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:9-16, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:9-16 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:9-16 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:9-16 and the region of SEQ ID N0:9-16 to which the fragment corresponds
are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID NO:1-8 is encoded by a fragment of SEQ ID N0:9-16. A
fragment of
SEQ ID NO:1-8 comprises a region of unique amino acid sequence that
specifically identifies SEQ
ID NO:l-8. For example, a fragment of SEQ ID NO:1-8 is useful as an
immunogenic peptide for the
development of antibodies that specifically recognize SEQ ID NO:1-8. The
precise length of a
fragment of SEQ ID NO:1-8 and the region of SEQ ID NO:1-8 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
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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://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off:' SO
Expect: l0
Word Size: 11
Filter: on
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
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similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the charge
and hydrophobicity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
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 (Apr-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Cap x drop-off :~ SO
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, 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
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chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1 % (w/v) SDS, and about 100 pg/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"d ed., vol. 1-3, Cold Spring
Harbor Press,
Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present
invention include wash conditions of 68°C in the presence of about 0.2
x SSC and about 0.1% SDS,
for 1 hour. Alternatively, temperatures of about 65°C, 60°C,
55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present
at about 0.1 %.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents
include, for instance, sheared and denatured salmon sperm DNA at about 100-200
pg/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.
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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 ISOM
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment
of ISOM 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 ISOM. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of ISOM.
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
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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 ISOM 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 ISOM.
"Probe" refers to nucleic acid sequences encoding ISOM, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA 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. ( I 987) 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
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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/MTT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may also be obtained from their respective sources and modified to
meet the user's specific
needs.) The PrimeGen program (available to the public from the UK Human Genome
Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, 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
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other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding ISOM, or fragments thereof, or ISOM itself, may comprise a
bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a cell; a cell;
genomic DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90°lo 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
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replication either as an autonomously replicating plasmid or as part of the
host chromosome, as well
as transiently transformed cells which express the inserted DNA or RNA for
limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants, and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook, J. 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
95% or at least 98% or
greater sequence identity over a certain defined length. A variant may be
described as, for example,
an "allelic" (as defined above), "splice," "species," or "polymorphic"
variant. A splice variant may
have significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternative splicing of exons during mRNA processing.
The corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides generally will have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
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-
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1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least
98% or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human isomerases (ISOM), the
polynucleotides encoding ISOM, and the use of these compositions for the
diagnosis, treatment, or
prevention of immune and cell proliferation disorders including cancer.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
ISOM. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of
the polypeptide
and nucleotide sequences, respectively. Column 3 shows the clone IDs of the
Incyte clones in which
nucleic acids encoding each ISOM were identified, and column 4 shows the cDNA
libraries from
which these clones were isolated. Column 5 shows Incyte clones and their
corresponding cDNA
libraries. Clones for which cDNA libraries are not indicated were derived from
pooled cDNA
libraries. The Incyte clones in column 5 were used to assemble the consensus
nucleotide sequence of
each ISOM and are useful as fragments in hybridization technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the invention:
column 1 references the SEQ >D NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; column 6
shows homologous sequences as identified by BLAST analysis; and column 7 shows
analytical
methods and in some cases, searchable databases to which the analytical
methods were applied. The
methods of column 7 were used to characterize each polypeptide through
sequence homology and
protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding ISOM. The first column of Table
3 lists the
nucleotide SEQ 117 NOs. Column 2 lists fragments of the nucleotide sequences
of column 1. These
fragments are useful, for example, in hybridization or amplification
technologies to identify SEQ ID
N0:9-16 and to distinguish between SEQ ID N0:9-16 and related polynucleotide
sequences. The
polypeptides encoded by these fragments are useful, for example, as
immunogenic peptides. Column
3 lists tissue categories which express ISOM as a fraction of total tissues
expressing ISOM. Column
4 lists diseases, disorders, or conditions associated with those tissues
expressing ISOM as a fraction
of total tissues expressing ISOM. Column 5 lists the vectors used to subclone
each cDNA library.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
from which cDNA clones encoding ISOM were isolated. Column 1 references the
nucleotide SEQ )D
NOs, column 2 shows the cDNA libraries from which these clones were isolated,
and column 3 shows
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the tissue origins and other descriptive information relevant to the cDNA
libraries in column 2.
SEQ ID N0:13 maps to chromosome 2 within the interval from 188.2 to 201.7. SEQ
ID
N0:16 maps to chromosome 16 within the interval from 19.70 to 33.30.
centiMorgans. The interval
on chromosome 16 from 19.70 to 21.80 centiMorgans also contains the gene and
ESTs encoding the
protein stannin. The presence of stannin in a cell renders it sensitive to the
effects of the drug
trimethyltin (TMT) which induces neuronal damage in the brain of humans
(Krady, J.K. et al. (1990)
Brain Res. Molec. Brain Res. 7:287-297 and Toggas, S.M. et al. (1992) Molec.
Pharm. 42:44-56).
Stannin has also been shown to expressed in atherosclerotic lesions when
activated by tumor necrosis
factor-alpha (Online Mendelian Inheritance in Man (OMIM) *603032 Stannin; SNN;
Horrevoets,
A.J. et al. (1999) Blood 93:3418-3431). The interval on chromosome 16 from
27.00 to 34.60
centiMorgans also contains the gene encoding multidrug resistance-associated
protein (MRP) as
mapped in a small cell lung carcinoma cell line NCI-H69. MRP has sequence
similarity to an ATP-
binding cassette superfamily of transport systems which include the genes
encoding the
transmembrane transport protein P-glycoprotein (MDR1) and the cystic fibrosis
transmembrane
conductance regulator (CFTR) (OMIM *158343 Multidrug Resistance-Associated
Protein 1; MRP1;
Cole, S.P.C. et al. (1992) Science 258:1650-1654).
The invention also encompasses ISOM variants. A preferred ISOM 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 ISOM amino acid sequence, and which contains at least
one functional or
structural characteristic of ISOM.
The invention also encompasses polynucleotides which encode ISOM. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:9-16, which encodes ISOM. The
polynucleotide sequences
of SEQ >D N0:9-16, 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
ISOM. In
particular, such a variant polynucleotide sequence will have at least about
80%, or alternatively at
least about 90%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding ISOM. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:9-
16 which has at least about 80%, or alternatively at least about 90%, or even
at least about 95%
polynucleotide sequence identity to a nucleic acid sequence selected from the
group consisting of
SEQ ff~ N0:9-16. Any one of the polynucleotide variants described above can
encode an amino acid
sequence which contains at least one functional or structural characteristic
of ISOM.
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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 ISOM, 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 ISOM, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode ISOM and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring ISOM under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding ISOM 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 ISOM 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 ISOM
and
ISOM derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell
systems using reagents well known in the art. Moreover, synthetic chemistry
may be used to
introduce mutations into a sequence encoding ISOM 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
N0:9-16 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
(PE
Biosystems, Foster City CA), 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,
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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 (PE Biosystems). Sequencing is then carried out
using either the
ABI 373 or 377 DNA sequencing system (PE Biosystems), the MEGABACE 1000 DNA
sequencing
system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art.
The resulting
sequences are analyzed using a variety of algorithms which are well known in
the art. (See, e.g.,
Ausubel, F.M. (1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons,
New York NY, unit
7.7; Meyers, R.A. (1995) Molecular Biology and BiotechnoloQV, Wiley VCH, New
York NY, pp.
856-853.)
The nucleic acid sequences encoding ISOM 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 ligations may be used to insert an engineered double-stranded
sequence into a region
of unknown sequence before performing PCR. Other methods which may be used to
retrieve
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.
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Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
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 ISOM may be cloned in recombinant DNA molecules that direct
expression of ISOM,
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 ISOM.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter ISOM-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of ISOM, such as its biological or enzymatic
activity or its ability
to bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
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maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding ISOM 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; Horn, T. et al. ( 1980) Nucleic Acids Symp. Ser. 7:225-
232.) Alternatively,
ISOM 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 Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated
synthesis may be achieved
using the ABI 431A peptide synthesizer (PE Biosystems). Additionally, the
amino acid sequence of
ISOM, or any part thereof, may be altered during direct synthesis and/or
combined with sequences
from other proteins, or any part thereof, to produce a variant polypeptide or
a polypeptide having a
sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier ( 1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active ISOM, the nucleotide sequences
encoding ISOM or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in
a suitable host. These elements include regulatory sequences, such as
enhancers, constitutive and
inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotide sequences
encoding ISOM. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
ISOM. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding ISOM 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 ISOM and appropriate transcriptional and
translational control
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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 ISOM. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, 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, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Bitter, G.A. et al. (1987) Methods
Enzymol. 153:516-544;
Scorer, C.A. et al. (1994) Bio/Technology 12:181-184; Engelhard, E.K. et al.
(1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-
1945; Takamatsu,
N. (1987) EMBO J. 6:307-311; Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;
Broglie, R. et al.
(1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105; The
McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York
NY, pp.
191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-
3659; and Harrington,
J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from
retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. ( 1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (
1993) Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Butler, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.)
The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding ISOM. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding ISOM 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 ISOM 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 ISOM are needed, e.g. for the
production of
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antibodies, vectors which direct high level expression of ISOM may be used.
For example, vectors
containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of ISOM. 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, supra; and Scorer, supra.)
Plant systems may also be used for expression of ISOM. Transcription of
sequences
encoding ISOM may be driven viral promoters, e.g., the 35S and 19S promoters
of CaMV used alone
or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311 ). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, supra; Brogue, supra; and Winter,
supra.) These
constructs can be introduced into plant cells by direct DNA transformation or
pathogen-mediated
transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y (
1992) McGraw
Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding ISOM
may be ligated into
an adenovirns transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses ISOM in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of ISOM in cell lines is preferred. For example, sequences encoding ISOM can
be transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media
before being switched to selective media. The purpose of the selectable marker
is to confer resistance
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to a selective agent, and its presence allows growth and recovery of cells
which successfully express
the introduced sequences. Resistant clones of stably transformed cells may be
propagated using
tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk- and apr cells, respectively.
(See, e.g., Wigler, M. et
al. ( 1977) Cell 11:223-232; Lowy, I. et al. ( 1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech), f3 glucuronidase and its substrate f3-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 ISOM is inserted within a marker gene sequence, transformed
cells containing
sequences encoding ISOM can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding ISOM 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 ISOM
and that express
ISOM 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 ISOM 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 ISOM is
preferred, but a
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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 ISOM
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding ISOM, 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 ISOM may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors
containing polynucleotides which encode ISOM may be designed to contain signal
sequences which
direct secretion of ISOM 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 ISOM 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 ISOM protein
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containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of ISOM 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 ISOM encoding sequence and the
heterologous protein
sequence, so that ISOM may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel ( 1995, supra, ch. 10).
A variety of commercially available kits may also be used to facilitate
expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled ISOM 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.
ISOM of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to ISOM. At least one and up to a plurality of test
compounds may be screened
for specific binding to ISOM. 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
ISOM, 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 ISOM
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 ISOM,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Droso~hila, or
E. coli. Cells expressing ISOM or cell membrane fractions which contain ISOM
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either ISOM or the
compound is analyzed.
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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
ISOM, either in
solution or affixed to a solid support, and detecting the binding of ISOM 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.
ISOM of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of ISOM. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for ISOM
activity, wherein ISOM is combined with at least one test compound, and the
activity of ISOM in the
presence of a test compound is compared with the activity of ISOM in the
absence of the test
compound. A change in the activity of ISOM in the presence of the test
compound is indicative of a
compound that modulates the activity of ISOM. Alternatively, a test compound
is combined with an
in vitro or cell-free system comprising ISOM under conditions suitable for
ISOM activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of ISOM
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 ISOM or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem
(ES) cells. Such techniques are well known in the art and are useful for the
generation of animal
models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent
No. 5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi,
M.R. (1989) Science 244:1288-1292). The vector integrates into the
corresponding region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
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Polynucleotides encoding ISOM 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 ISOM 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 ISOM 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 ISOM, e.g., by secreting ISOM 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 ISOM and isomerases. In addition, the expression of ISOM is
closely associated
with reproductive, hematopoietic/immune, and gastrointestinal tissues.
Therefore, ISOM appears to
play a role in immune and cell proliferation disorders including cancer. In
the treatment of disorders
associated with increased ISOM expression or activity, it is desirable to
decrease the expression or
activity of ISOM. In the treatment of disorders associated with decreased ISOM
expression or
activity, it is desirable to increase the expression or activity of ISOM.
Therefore, in one embodiment, ISOM 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 ISOM. Examples of such disorders include, but are not limited to,
an immune disorder
such as inflammation, actinic keratosis, acquired immunodeficiency syndrome
(AIDS), Addison's
disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia,
arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune thyroiditis,
bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's
disease, atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis,
erythema nodosum, atrophic
gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's
thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis,
hypereosinophilia, irritable bowel
syndrome, episodic lymphopenia with lymphocytotoxins, mixed connective tissue
disease (MCTD),
multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
myelofibrosis,
osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis,
psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
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lupus erythematosus, systemic sclerosis, primary thrombocythemia,
thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of cancer,
hemodialysis, and
extracorporeal circulation, trauma, and hematopoietic cancer including
lymphoma, leukemia, and
myeloma; and a cell proliferative disorder such as actinic keratosis,
arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD),
myelofibrosis, paroxysmal
nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and cancers
including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma,
and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow,
brain, breast, cervix,
gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,
muscle, ovary, pancreas,
parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus.
In another embodiment, a vector capable of expressing ISOM 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 ISOM including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
ISOM 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 ISOM including,
but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of ISOM
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of ISOM including, but not limited to, those listed above.
In a further embodiment, an antagonist of ISOM may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of ISOM.
Examples of such
disorders include, but are not limited to, those immune and cell proliferation
disorders including
cancer described above. In one aspect, an antibody which specifically binds
ISOM 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 ISOM.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding ISOM may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of ISOM 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
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efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of ISOM may be produced using methods which are generally known
in the
art. In particular, purified ISOM may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind ISOM.
Antibodies to ISOM 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 ISOM or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among
adjuvants used in
humans, BCG (bacilli Calmette-Guerin) and Cor~ebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
ISOM 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 ISOM amino acids may be fused with those of another protein, such as KLH,
and antibodies to the
chimeric molecule may be produced.
Monoclonal antibodies to ISOM 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
ISOM-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.,
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Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for ISOM may also be
generated.
For example, such fragments include, but are not limited to, F(ab~z 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
ISOM and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering ISOM 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 ISOM. Affinity
is expressed as an
association constant, Ka, which is defined as the molar concentration of ISOM-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium conditions.
The Ka determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple ISOM epitopes, represents the average affinity, or
avidity, of the antibodies for
ISOM. The Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular ISOM epitope, represents a true measure of affinity. High-affinity
antibody preparations
with K3 ranging from about 109 to 10'2 L/mole are preferred for use in
immunoassays in which the
ISOM-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 ISOM, 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
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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 ISOM-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, supra, and Coligan et al., supra.)
In another embodiment of the invention, the polynucleotides encoding ISOM, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, modifications
of gene expression can be achieved by designing complementary sequences or
antisense molecules
(DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory
regions of the gene
encoding ISOM. 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 ISOM. (See, e.g., Agrawal, S., ed. (1996) Antisense Theraveutics,
Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, su ra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding ISOM may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SC>D)-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)
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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 (HN)
(Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA. 93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trypanosoma cruzi). In the
case where a genetic deficiency in ISOM expression or regulation causes
disease, the expression of
ISOM 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
ISOM are treated by constructing mammalian expression vectors encoding ISOM
and introducing
these vectors by mechanical means into ISOM-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 ISOM 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). ISOM may be
expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV),
Rous sarcoma virus
(RSV), SV40 virus, thymidine kinase (TK), or ~i-actin genes), (ii) an
inducible promoter (e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. 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 H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding ISOM 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.
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(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 ISOM expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding ISOM under the control of an independent promoter oi~
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;
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 ISOM to cells which have one or more genetic
abnormalities with respect
to the expression of ISOM. 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 ISOM to target cells which have one or more genetic
abnormalities with
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respect to the expression of ISOM. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing ISOM 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 ISOM to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full-length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
ISOM into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
ISOM-coding RNAs and the synthesis of high levels of ISOM 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 ISOM 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
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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 i~Yc 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 ISOM.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding ISOM. 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,
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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 ISOM. 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 ISOM
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding ISOM may be therapeutically useful, and in the treament of disorders
associated with
decreased ISOM expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding ISOM may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding ISOM 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
ISOM 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 ISOM. 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 earned out, for example, using a Schizosaccharom~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.
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Commun. 268:8-13). A particular embodiment of the present invention involves
screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition
which generally comprises an active ingredient formulated with a
pharmaceutically acceptable
excipient. Excipients may include, for example, sugars, starches, celluloses,
gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the
latest edition of
ReminQton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
compositions may
consist of ISOM, antibodies to ISOM, and mimetics, agonists, antagonists, or
inhibitors of ISOM.
The compositions utilized in this invention may be administered by any number
of routes
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.
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Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising ISOM or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusiow
intracellular delivery of
the macromolecule. Alternatively, ISOM 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 ISOM
or fragments thereof, antibodies of ISOM, and agonists, antagonists or
inhibitors of ISOM, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDso ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting compositions may be administered every 3 to 4
days, every week,
or biweekly depending on the half-life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 ,ug, 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
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inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind ISOM may be used for
the
diagnosis of disorders characterized by expression of ISOM, or in assays to
monitor patients being
treated with ISOM or agonists, antagonists, or inhibitors of ISOM. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for ISOM include methods which utilize the antibody and a label to detect ISOM
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 ISOM, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
ISOM expression. Normal
or standard values for ISOM expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibody to ISOM under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of ISOM
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 ISOM may
be used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
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 ISOM
may be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
ISOM, and to monitor regulation of ISOM levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding ISOM or closely related
molecules may be used to
identify nucleic acid sequences which encode ISOM. 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 ISOM, 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 ISOM encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:9-16 or from
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genomic sequences including promoters, enhancers, and introns of the ISOM
gene.
Means for producing specific hybridization probes for DNAs encoding ISOM
include the
cloning of polynucleotide sequences encoding ISOM or ISOM 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 ISOM may be used for the diagnosis of
disorders
associated with expression of ISOM. Examples of such disorders include, but
are not limited to, an
immune disorder such as inflammation, actinic keratosis, acquired
immunodeficiency syndrome
(AIDS), Addison's disease, adult respiratory distress syndrome, allergies,
ankylosing spondylitis,
amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune
hemolytic anemia,
autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis,
contact dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria,
hepatitis, hypereosinophilia,
irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed
connective tissue
disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation,
myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera,
polymyositis, psoriasis,
Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome,
systemic anaphylaxis,
systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia,
thrombocytopenic
purpura, ulcerative colitis, uveitis, Werner syndrome, complications of
cancer, hemodialysis, and
extracorporeal circulation, trauma, and hematopoietic cancer including
lymphoma, leukemia, and
myeloma; and a cell proliferative disorder such as actinic keratosis,
arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD),
myelofibrosis, paroxysmal
nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and cancers
including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma,
and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow,
brain, breast, cervix,
gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,
muscle, ovary, pancreas,
parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus . The
polynucleotide sequences encoding ISOM 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 ISOM
expression. Such qualitative or quantitative methods are well known in the
art.
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In a particular aspect, the nucleotide sequences encoding ISOM may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding ISOM 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 ISOM 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
ISOM, 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 ISOM, 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
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 ISOM
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 ISOM, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
ISOM, and will be employed under optimized conditions for identification of a
specific gene or
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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 ISOM 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
ISOM are used to
amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived,
for example,
from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause
differences in the secondary and tertiary structures of PCR products in single-
stranded form, and
these differences are detectable using gel electrophoresis in non-denaturing
gels. In fSCCP, the
oligonucleotide primers are fluorescently labeled, which allows detection of
the amplimers in high-
throughput equipment such as DNA sequencing machines. Additionally, sequence
database analysis
methods, termed in silico SNP (isSNP), are capable of identifying
polymorphisms by comparing the
sequence of individual overlapping DNA fragments which assemble into a common
consensus
sequence. These computer-based methods filter out sequence variations due to
laboratory preparation
of DNA and sequencing errors using statistical models and automated analyses
of DNA sequence
chromatograms. In the 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 ISOM include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives
rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described in Seilhamer, J.J. et al.,
"Comparative Gene Transcript
Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference. The
microarray may also be
used to identify genetic variants, mutations, and polymorphisms. This
information may be used to
determine gene function, to understand the genetic basis of a disorder, to
diagnose a disorder, to
monitor progression/regression of disease as a function of gene expression,
and to develop and
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monitor the activities of therapeutic agents in the treatment of disease. In
particular, this information
may be used to develop a pharmacogenomic profile of a patient in order to
select the most appropriate
and effective treatment regimen for that patient. For example, therapeutic
agents which are highly
effective and display the fewest side effects may be selected for a patient
based on his/her
pharmacogenomic profile.
In another embodiment, antibodies specific for ISOM, or ISOM or fragments
thereof may be
used as elements on a microarray. The microarray may be used to monitor or
measure protein-protein
interactions, drug-target interactions, and gene expression profiles, as
described above.
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
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
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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
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.
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A proteomic profile may also be generated using antibodies specific for ISOM
to quantify the
levels of ISOM 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
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;
Shalon, D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
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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 ISOM
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 mufti-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. ( 1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
(See, e.g., Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA
83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. ( 1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding ISOM on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to 11 q22-23,
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, ISOM, its catalytic or immunogenic
fragments, or
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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 ISOM 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 ISOM, or
fragments thereof,
and washed. Bound ISOM is then detected by methods well known in the art.
Purified ISOM 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 ISOM specifically compete with a test compound
for binding ISOM. In
IS this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with ISOM.
In additional embodiments, the nucleotide sequences which encode ISOM may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below, in
particular U.S. Ser. No.60/149,388, are hereby expressly incorporated by
reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some
tissues were homogenized and lysed in guanidinium isothiocyanate, while others
were homogenized
and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL
(Life Technologies), a
monophasic solution of phenol and guanidine isothiocyanate. The resulting
lysates were centrifuged
over CsCI cushions or extracted with chloroform. RNA was precipitated from the
lysates with either
isopropanol or sodium acetate and ethanol, or by other routine methods.
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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), or pINCY plasmid (Incyte Genomics, Palo Alto CA).
Recombinant
plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-
BIueMRF, or
SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DHl OB from Life
Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the 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).
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III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (PE Biosystems) thermal cycler or the PTC-200
thermal cycler (MJ
Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or
the MICROLAB
2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were
prepared using reagents
provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such
as the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
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 (PE Biosystems) in conjunction with standard ABI
protocols and base
calling software; or other sequence analysis systems known in the art. Reading
frames within the
cDNA sequences were identified using standard methods (reviewed in Ausubel,
1997, supra, unit
7.7). Some of the cDNA sequences were selected for extension using the
techniques disclosed in
Example VI.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilled in the
art. Table 5 summarizes the tools, programs, and algorithms used and provides
applicable
descriptions, references, and threshold parameters. The first column of Table
5 shows the tools,
programs, and algorithms used, the second column provides brief descriptions
thereof, the third
column presents appropriate references, all of which are incorporated by
reference herein in their
entirety, and the fourth column presents, where applicable, the scores,
probability values, and other
parameters used to evaluate the strength of a match between two sequences (the
higher the score, the
greater the homology between two sequences). Sequences were analyzed using
MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software
(DNASTAR). Polynucleotide and polypeptide sequence alignments were generated
using the default
parameters specified by the clustal algorithm as incorporated into the
MEGALIGN multisequence
alignment program (DNASTAR), which also calculates the percent identity
between aligned
sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
were then queried
against a selection of public databases such as the GenBank primate, rodent,
mammalian, vertebrate,
and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences
were assembled
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into full length polynucleotide sequences using programs based on Phred,
Phrap, and Consed, and
were screened for open reading frames using programs based on GeneMark, BLAST,
and FASTA.
The full length polynucleotide sequences were translated to derive the
corresponding full length
amino acid sequences, and these full length sequences were subsequently
analyzed by querying
against databases such as the GenBank databases (described above), SwissProt,
BLOCKS, PRINTS,
DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family
databases such
as PFAM. HMM is a probabilistic approach which analyzes consensus primary
structures of gene
families. (See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide
and amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ )D
N0:9-16. Fragments from about 20 to about 4000 nucleotides which are useful in
hybridization and
amplification technologies were described in The Invention section above.
IV. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel,
1995, s-upra, 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
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either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding ISOM occurred. Analysis involved the categorization of
cDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease/condition categories included cancer,
inflammation, trauma,
cell proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
sequence of interest was counted and divided by the total number of libraries
across all categories.
Percentage values of tissue-specific and disease- or condition-specific
expression are reported in
Table 3.
V. Chromosomal Mapping of ISOM Encoding Polynucleotides
The cDNA sequences which were used to assemble SEQ ID N0:9-16 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 N0:9-16 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 5). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and 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.
The genetic map locations of SEQ ID N0:13 and SEQ ID N0:16 are described in
The
Invention as 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. Diseases associated with the
public and Incyte
sequences located within the indicated intervals are also reported in the
Invention where applicable.
VI. Extension of ISOM Encoding Polynucleotides
The full length nucleic acid sequences of SEQ 1D N0:9-16 were produced by
extension of an
appropriate fragment of the full length molecule using oligonucleotide primers
designed from this
fragment. One primer was synthesized to initiate 5' extension of the known
fragment, and the other
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primer, to initiate 3' extension of the known fragment. The initial primers
were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mgz+, (NH4)ZSOa,
and ~i-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step S: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 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
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~1 to 10 ~l aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose mini-gel to determine which reactions were
successful in extending
the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to 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 polymerase (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37°C in
384-well plates in LB/2x carb liquid media.
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The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
In like manner, the polynucleotide sequences of SEQ ID N0:9-16 are used to
obtain 5'
regulatory sequences using the procedure above, along with oligonucleotides
designed for such
extension, and an appropriate genomic library.
VII. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:9-16 are employed to screen cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of-the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~Ci of
[y 32P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VIII. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, 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),
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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
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/pl oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/E.~l RNase inhibitor, 500 E.~M dATP, 500 ECM
dGTP, 500 ECM dTTP, 40
l.~M dCTP, 40 l.iM 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 O.SM 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 p1 SX SSC/0.2% SDS.
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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 p1 of the array
element DNA, at an average
concentration of 100 ng/pl, 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.
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 SX SSC in a corner of the chamber. The chamber
containing the arrays is
incubated for about 6.5 hours at 60 °C. The arrays are washed for 10
min at 45 °C in a first wash
buffer (1X SSC, 0.1% SDS), three times for 10 minutes each at 45 °C in
a second wash buffer (0.1X
SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
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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
I S the array contains a complementary DNA sequence, allowing the intensity of
the signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are
differentially expressed, the calibration is done by labeling samples of the
calibrating cDNA with the
two fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each
spot is centered in each element of the grid. The fluorescence signal within
each element is then
integrated to obtain a numerical value corresponding to the average intensity
of the signal. The
software used for signal analysis is the GEMTOOLS gene expression analysis
program (Incyte).
IX. Complementary Polynucleotides
Sequences complementary to the ISOM-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring ISOM. Although
use of oligonucleotides
comprising from about 15 to 30 base pairs is described, essentially the same
procedure is used with
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smaller or with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of ISOM. 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 ISOM-encoding transcript.
X. Expression of ISOM
Expression and purification of ISOM is achieved using bacterial or virus-based
expression
systems. For expression of ISOM 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 ISOM upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of ISOM in eukaryotic cells is
achieved by infecting insect
or mammalian cell lines with recombinant Autogrraphica californica nuclear
polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding ISOM 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 Spodontera frugiperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
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, ISOM 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 i_aponicum, 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
ISOM 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 ISOM obtained by these methods can be used
directly in the assays
shown in Examples XI and XV.
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XI. Demonstration of ISOM Activity
ISOM activity is demonstrated through a variety of specific enzyme assays,
some of which
are outlined below.
Peptidyl Prolyl cis-traps Isomerase Activity
ISOM-6 peptidyl prolyl cis-traps isomerase activity can be assayed as
described (Rahfeld,
J.U. et al. (1994) FEBS Lett. 352:180-184). The assay is performed at
10°C in 35 mM HEPES
buffer, pH 7.8, containing chymotrypsin (0.5 mg/ml) and ISOM-6 at a variety of
concentrations. In
this assay, the substrate is a peptide containing four hydrophobic residues.
The peptide contains a
succinate group at the N-terminus and a nitroanilide group at the C-terminus.
The substrate is in
equilibrium with respect to the prolyl bond, with 80-95% in traps and 5-20% in
cis conformation. An
aliquot (2 p1) of the substrate dissolved in dimethyl sulfoxide (10 mg/ml) is
added to the reaction
mixture described above. Only the cis isomer of the substrate is a substrate
for cleavage by
chymotrypsin. Thus, as the substrate is isomerized by ISOM-6, the product is
cleaved by
chymotrypsin to produce 4-nitroanilide, which is detected by its absorbance at
390 nm. 4-
Nitroanilide appears in a time-dependent and an ISOM-6 concentration-dependent
manner.
Alternatively, peptidyl prolyl cis-traps isomerase activity of ISOM-6 can be
assayed using a
chromogenic peptide in a coupled assay with chymotrypsin (Fischer, G. et al.
(1984) Biomed.
Biochim. Acta 43:1101-1111 ).
Thioredoxin Activity
ISOM thioredoxin activity is assayed as described (Luthman, M. (1982)
Biochemistry
21:6628-6633). Thioredoxins catalyze the formation of disulfide bonds and
regulate the redox
environment in cells to enable the necessary thiol:disulfide exchanges. One
way to measure the
thiol:disulfide exchange is by measuring the reduction of insulin in a mixture
containing O.1M
potassium phosphate, pH 7.0, 2 mM EDTA, 0.16 pM insulin, 0.33 mM DTT, and 0.48
mM NADPH.
Different concentrations of ISOM are added to the mixture, and the reaction
rate is followed by
monitoring the oxidation of NADPH at 340 nM.
Transferase Activity
ISOM transferase activity is measured through a methyl transferase assay in
which the
transfer of radiolabeled methyl groups between a donor substrate and an
acceptor substrate is
measured (Bokar, J.A. et al. (1994) J. Biol. Chem. 269:17697-17704). Reaction
mixtures (50 p1 final
volume) contain 15 mM HEPES, pH 7.9, 1.5 mM MgClz, 10 mM dithiothreitol, 3%
polyvinylalcohol,
donor substrate (1.5 pCi [methyl-3H]AdoMet (0.375 pM AdoMet, DuPont-NEN), 0.6
pg ISOM, and
acceptor substrate (0.4 pg [ASS]RNA or 6-mercaptopurine (6-MP) to 1 mM final
concentration).
Reaction mixtures are incubated at 30°C for 30 minutes, then
65°C for 5 minutes. The products are
separated by chromatography or electrophoresis and the level of methyl
transferase activity is
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determined by quantification of methyl-3H-RNA or methyl-3H-6-MP recovery.
XII. Functional Assays
ISOM function is assessed by expressing the sequences encoding ISOM at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid
(Invitrogen), both of which
contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ~cg of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
The influence of ISOM on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding ISOM 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 ISOM and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XIII. Production of ISOM Specific Antibodies
ISOM 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.
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Alternatively, the ISOM 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. 1 l.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with
the oligopeptide-
KLH complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-
ISOM activity by, for example, binding the peptide or ISOM to a substrate,
blocking with 1% BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
XIV. Purification of Naturally Occurring ISOM Using Specific Antibodies
Naturally occurring or recombinant ISOM is substantially purified by
immunoaffinity
chromatography using antibodies specific for ISOM. An immunoaffinity column is
constructed by
covalently coupling anti-ISOM 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 ISOM are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of ISOM (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/ISOM 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 ISOM is collected.
XV. Identification of Molecules Which Interact with ISOM
ISOM, 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 multi-well plate are incubated with the
labeled ISOM, washed,
and any wells with labeled ISOM complex are assayed. Data obtained using
different concentrations
of ISOM are used to calculate values for the number, affinity, and association
of ISOM with the
candidate molecules.
Alternatively, molecules interacting with ISOM 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).
ISOM 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
68
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the
invention. Although the invention has been described in connection with
certain embodiments, it
should be understood that the invention as claimed should not be unduly
limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying
out the invention
which are obvious to those skilled in molecular biology or related fields are
intended to be within the
scope of the following claims.
69
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
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CA 02382019 2002-02-14
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SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
BANDMAN, Olga
LU, Dyung Aina M.
YUE, Henry
TRAN, Bao
HILLMAN, Jennifer L.
BAUGHN, Mariah R.
LAL, Preeti
TANG, Y. Tom
<120> ISOMERASE PROTEINS
<130> PF-0730 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/149,388
<151> 1999-08-17
<160> 16
<170> PERL Program
<210> 1
<211> 542
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 011886CD1
<400> 1
Met Asp Leu Gly Ala Ile Thr Lys Tyr Ser Ala Leu His Ala Lys
1 5 10 15
Pro Asn Gly Leu Ile Leu Gln Tyr Gly Thr Ala Gly Phe Arg Thr
20 25 30
Lys Ala Glu His Leu Asp His Val Met Phe Arg Met Gly Leu Leu
35 40 45
Ala Val Leu Arg Ser Lys Gln Thr Lys Ser Thr Ile Gly Val Met
50 55 60
Val Thr Ala Ser His Asn Pro Glu Glu Asp Asn Gly Val Lys Leu
65 70 75
Val Asp Pro Leu Gly Glu Met Leu Ala Pro Ser Trp Glu Glu His
80 85 90
Ala Thr Cys Leu Ala Asn Ala Glu Glu Gln Asp Met Gln Arg Val
95 100 105
Leu Ile Asp Ile Ser Glu Lys Glu Ala Val Asn Leu Gln Gln Asp
110 115 120
Ala Phe Val Val Ile Gly Arg Asp Thr Arg Pro Ser Ser Glu Lys
125 130 135
Leu Ser Gln Ser Val Ile Asp Gly Val Thr Val Leu Gly Gly Gln
140 145 150
Phe His Asp Tyr Gly Leu Leu Thr Thr Pro Gln Leu His Tyr Met
155 160 165
Val Tyr Cys Arg Asn Thr Gly Gly Arg Tyr Gly Lys Ala Thr Ile
170 175 180
Glu Gly Tyr Tyr Gln Lys Leu Ser Lys Ala Phe Val Glu Leu Thr
1/16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
185 190 195
Lys Gln Ala Ser Cys Ser Gly Asp Glu Tyr Arg Ser Leu Lys Val
200 205 210
Asp Cys Ala Asn Gly Ile Gly Ala Leu Lys Leu Arg Glu Met Glu
215 220 225
His Tyr Phe Ser Gln Gly Leu Ser Val Gln Leu Phe Asn Asp Gly
230 235 240
Ser Lys Gly Lys Leu Asn His Leu Cys Gly Ala Asp Phe Val Lys
245 250 255
Ser His Gln Lys Pro Pro Gln Gly Met Glu Ile Lys Ser Asn Glu
260 265 270
Arg Cys Cys Ser Phe Asp Gly Asp Ala Asp Arg Ile Val Tyr Tyr
275 280 285
Tyr His Asp Ala Asp Gly His Phe His Leu Ile Asp Gly Asp Lys
290 295 300
Ile Ala Thr Leu Ile Ser Ser Phe Leu Lys Glu Leu Leu Val Glu
305 310 315
Ile Gly Glu Ser Leu Asn Ile Gly Val Val Gln Thr Ala Tyr Ala
320 325 330
Asn Gly Ser Ser Thr Arg Tyr Leu Glu Glu Val Met Lys Val Pro
335 340 345
Val Tyr Cys Thr Lys Thr Gly Val Lys His Leu His His Lys Ala
350 355 360
Gln Glu Phe Asp Ile Gly Val Tyr Phe Glu Ala Asn Gly His Gly
365 370 375
Thr Ala Leu Phe Ser Thr Ala Val Glu Met Lys Ile Lys Gln Ser
380 385 390
Ala Glu Gln Leu Glu Asp Lys Lys Arg Lys Ala Ala Lys Met Leu
395 400 405
Glu Asn Ile Ile Asp Leu Phe Asn Gln Ala Ala Gly Asp Ala Ile
410 415 420
Ser Asp Met Leu Val Ile Glu Ala Ile Leu Ala Leu Lys Gly Leu
425 430 435
Thr Val Gln Gln Trp Asp Ala Leu Tyr Thr Asp Leu Pro Asn Arg
440 445 450
Gln Leu Lys Val Gln Val Ala Asp Arg Arg Val Ile Ser Thr Thr
455 460 465
Asp Ala Glu Arg Gln Ala Val Thr Pro Pro Gly Leu Gln Glu Ala
470 475 480
Ile Asn Asp Leu Val Lys Lys Tyr Lys Leu Ser Arg Ala Phe Val
485 490 495
Arg Pro Ser Gly Thr Glu Asp Val Val Arg Val Tyr Ala Glu Ala
500 505 510
Asp Ser Gln Glu Ser Ala Asp His Leu Ala His Glu Val Ser Leu
515 520 525
Ala Val Phe Gln Leu Ala Gly Gly Ile Gly Glu Arg Pro Gln Pro
530 535 540
Gly Phe
<210> 2
<211> 311
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1863189CD1
<400> 2
Met Gln Arg Pro Gly Pro Phe Ser Thr Leu Tyr Gly Arg Val Leu
2/ 16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
1 5 10 15
Ala Pro Leu Pro Gly Arg Ala Gly Gly Ala Ala Ser Gly Gly Gly
20 25 30
Gly Asn Ser Trp Asp Leu Pro Gly Ser His Val Arg Leu Pro Gly
35 40 45
Arg Ala Gln Ser Gly Thr Arg Gly Gly Ala Gly Asn Thr Ser Thr
50 55 60
Ser Cys Gly Asp Ser Asn Ser Ile Cys Pro Ala Pro Ser Thr Met
65 70 75
Ser Lys Ala Glu Glu Ala Lys Lys Leu Ala Gly Arg Ala Ala Val
80 85 90
Glu Asn His Val Arg Asn Asn Gln Val Leu Gly Ile Gly Ser Gly
95 100 105
Ser Thr Ile Val His Ala Val Gln Arg Ile Ala Glu Arg Val Lys
110 115 120
Gln Glu Asn Leu Asn Leu Val Cys Ile Pro Thr Ser Phe Gln Ala
125 130 135
Arg Gln Leu Ile Leu Gln Tyr Gly Leu Thr Leu Ser Asp Leu Asp
140 145 150
Arg His Pro Glu Ile Asp Leu Ala Ile Asp Gly Ala Asp Glu Val
155 160 165
Asp Ala Asp Leu Asn Leu Ile Lys Gly Gly Gly Gly Cys Leu Thr
170 175 180
Gln Glu Lys Ile Val Ala Gly Tyr Ala Ser Arg Phe Ile Val Ile
185 190 195
Ala Asp Phe Arg Lys Asp Ser Lys Asn Leu Gly Asp Gln Trp His
200 205 210
Lys Gly Ile Pro Ile Glu Val Ile Pro Met Ala Tyr Val Pro Val
215 220 225
Ser Arg Ala Val Ser Gln Lys Phe Gly Gly Val Val Glu Leu Arg
230 235 240
Met Ala Val Asn Lys Ala Gly Pro Val Val Thr Asp Asn Gly Asn
245 250 255
Phe Ile Leu Asp Trp Lys Phe Asp Arg Val His Lys Trp Ser Glu
260 265 270
Val Asn Thr Ala Ile Lys Met Ile Pro Gly Val Val Asp Thr Gly
275 280 285
Leu Phe Ile Asn Met Ala Glu Arg Val Tyr Phe Gly Met Gln Asp
290 295 300
Gly Ser Val Asn Met Arg Glu Lys Pro Phe Cys
305 310
<210> 3
<211> 273
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2088868CD1
<400> 3
Met Glu Ala Ala Pro Ser Arg Phe Met Phe Leu Leu Phe Leu Leu
1 5 10 15
Thr Cys Glu Leu Ala Ala Glu Val Ala Ala Glu Val Glu Lys Ser
20 25 30
Ser Asp Gly Pro Gly Ala Ala Gln Glu Pro Thr Trp Leu Thr Asp
35 40 45
Val Pro Ala Ala Met Glu Phe Ile Ala Ala Thr Glu Val Ala Val
50 55 60
3/16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
Ile Gly Phe Phe Gln Asp Leu Glu Ile Pro Ala Val Pro Ile Leu
65 70 75
His Ser Met Val Gln Lys Phe Pro Gly Val Ser Phe Gly Ile Ser
80 85 90
Thr Asp Ser Glu Val Leu Thr His Tyr Asn Ile Thr Gly Asn Thr
95 100 105
Ile Cys Leu Phe Arg Leu Val Asp Asn Glu Gln Leu Asn Leu Glu
110 115 120
Asp Glu Asp Ile Glu Ser Ile Asp Ala Thr Lys Leu Ser Arg Phe
125 130 135
Ile Glu Ile Asn Ser Leu His Met Val Thr Glu Tyr Asn Pro Val
140 145 150
Thr Val Ile Gly Leu Phe Asn Ser Val Ile Gln Ile His Leu Leu
155 160 165
Leu Ile Met Asn Lys Ala Ser Pro Glu Tyr Glu Glu Asn Met His
170 175 180
Arg Tyr Gln Lys Ala Ala Lys Leu Phe Gln Gly Lys Ile Leu Phe
185 190 195
Ile Leu Val Asp Ser Gly Met Lys Glu Asn Gly Lys Val Ile Ser
200 205 210
Phe Phe Lys Leu Lys Glu Ser Gln Leu Pro Ala Leu Ala Ile Tyr
215 220 225
Gln Thr Leu Asp Asp Glu Trp Asp Thr Leu Pro Thr Ala Glu Val
230 235 240
Ser Val Glu His Val Gln Asn Phe Cys Asp Gly Phe Leu Ser Gly
245 250 255
Lys Leu Leu Lys Glu Asn Arg Glu Ser Glu Gly Lys Thr Pro Lys
260 265 270
Val Glu Leu
<210> 4
<211> 228
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2481256CD1
<400> 4
Met Ala Ser Gly Cys Lys Ile Gly Pro Ser Ile Leu Asn Ser Asp
1 5 10 15
Leu Ala Asn Leu Gly Ala Glu Cys Leu Arg Met Leu Asp Ser Gly
20 25 30
Ala Asp Tyr Leu His Leu Asp Val Met Asp Gly His Phe Val Pro
35 40 45
Asn Ile Thr Phe Gly His Pro Val Val Glu Ser Leu Arg Lys Gln
50 55 60
Leu Gly Gln Asp Pro Phe Phe Asp Met His Met Met Val Ser Lys
65 70 75
Pro Glu Gln Trp Val Lys Pro Met Ala Val Ala Gly Ala Asn Gln
80 85 90
Tyr Thr Phe His Leu Glu Ala Thr Glu Asn Pro Gly Ala Leu Ile
95 100 105
Lys Asp Ile Arg Glu Asn Gly Met Lys Val Gly Leu Ala Ile Lys
110 115 120
Pro Gly Thr Ser Val Glu Tyr Leu Ala Pro Trp Ala Asn Gln Ile
125 130 135
Asp Met Ala Leu Val Met Thr Val Glu Pro Gly Phe Gly Gly Gln
140 145 150
4/16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
Lys Phe Met Glu Asp Met Met Pro Lys Val His Trp Leu Arg Thr
155 160 165
Gln Phe Pro Ser Leu Asp Ile Glu Val Asp Gly Gly Val Gly Pro
170 175 180
Asp Thr Val His Lys Cys Ala Glu Ala Gly Ala Asn Met Ile Val
185 190 195
Ser Gly Ser Ala Ile Met Arg Ser Glu Asp Pro Arg Ser Val Ile
200 205 210
Asn Leu Leu Arg Asn Val Cys Ser Glu Ala Ala Gln Lys Arg Ser
215 220 225
Leu Asp Arg
<210> 5
<211> 793
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2505257CD1
<400> 5
Met Gly Val Trp Leu Asn Lys Asp Asp Asp Ile Arg Asp Leu Lys
1 5 10 15
Arg Ile Ile Leu Cys Phe Leu Ile Val Tyr Met Ala Ile Leu Val
20 25 30
Gly Thr Asp Gln Asp Phe Tyr Ser Leu Leu Gly Val Ser Lys Thr
35 40 45
Ala Ser Ser Arg Glu Ile Arg Gln Ala Phe Lys Lys Leu Ala Leu
50 55 60
Lys Leu His Pro Asp Lys Asn Pro Asn Asn Pro Asn Ala His Gly
65 70 75
Asn Phe Leu Lys Ile Asn Arg Ala Tyr Glu Val Leu Lys Asp Glu
80 g5 90
Asp Leu Arg Lys Lys Tyr Asp Lys Tyr Gly Glu Lys Gly Leu Glu
95 100 105
Asp Asn Gln Gly Gly Gln Tyr Glu Ser Trp Asn Tyr Tyr Arg Tyr
110 115 120
Asp Phe Gly Ile Tyr Asp Asp Asp Pro Glu Ile Ile Thr Leu Glu
125 130 135
Arg Arg Glu Phe Asp Ala Ala Val Asn Ser Gly Glu Leu Trp Phe
140 145 150
Val Asn Phe Tyr Ser Pro Gly Cys Ser His Cys His Asp Leu Ala
155 160 165
Pro Thr Trp Arg Asp Phe Ala Lys Glu Val Asp Gly Leu Leu Arg
170 175 180
Ile Gly Ala Val Asn Cys Gly Asp Asp Arg Met Leu Cys Arg Met
185 190 195
Lys Gly Val Asn Ser Tyr Pro Ser Leu Phe Ile Phe Arg Ser Gly
200 205 210
Met Ala Pro Val Lys Tyr His Gly Asp Arg Ser Lys Glu Ser Leu
215 220 225
Val Ser Phe Ala Met Gln His Val Arg Ser Thr Val Thr Glu Leu
230 235 240
Trp Thr Gly Asn Phe Val Asn Ser Ile Gln Thr Ala Phe Ala Ala
245 250 255
Gly Ile Gly Trp Leu Ile Thr Phe Cys Ser Lys Gly Gly Asp Cys
260 265 270
Leu Thr Ser Gln Thr Arg Leu Arg Leu Ser Gly Met Leu Asp Gly
275 280 285
5/16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
Leu Val Asn Val Gly Trp Met Asp Cys Ala Thr Gln Asp Asn Leu
290 295 300
Cys Lys Ser Leu Asp Ile Thr Thr Ser Thr Thr Ala Tyr Phe Pro
305 310 315
Pro Gly Ala Thr Leu Asn Asn Lys Glu Lys Asn Ser Ile Leu Phe
320 325 330
Leu Asn Ser Leu Asp Ala Lys Glu Ile Tyr Leu Glu Val Ile His
335 340 345
Asn Leu Pro Asp Phe Glu Leu Leu Ser Ala Asn Thr Leu Glu Asp
350 355 360
Arg Leu Ala His His Arg Trp Leu Leu Phe Phe His Phe Gly Lys
365 370 375
Asn Glu Asn Ser Asn Asp Pro Glu Leu Lys Lys Leu Lys Thr Leu
380 385 390
Leu Lys Asn Asp His Ile Gln Val Gly Arg Phe Asp Cys Ser Ser
395 400 405
Ala Pro Asp Ile Cys Ser Asn Leu Tyr Val Phe Gln Pro Ser Leu
410 415 420
Ala Val Phe Lys Gly Gln Gly Thr Lys Glu Tyr Glu Ile His His
425 430 435
Gly Lys Lys Ile Leu Tyr Asp Ile Leu Ala Phe Ala Lys Glu Ser
440 445 450
Val Asn Ser His Val Thr Thr Leu Gly Pro Gln Asn Phe Pro Ala
455 460 465
Asn Asp Lys Glu Pro Trp Leu Val Asp Phe Phe Ala Pro Trp Cys
470 475 480
Pro Pro Cys Arg Ala Leu Leu Pro Glu Leu Arg Arg Ala Ser Asn
485 490 495
Leu Leu Tyr Gly Gln Leu Lys Phe Gly Thr Leu Asp Cys Thr Val
500 505 510
His Glu Gly Leu Cys Asn Met Tyr Asn Ile Gln Ala Tyr Pro Thr
515 520 525
Thr Val Val Phe Asn Gln Ser Asn Ile His Glu Tyr Glu Gly His
530 535 540
His Ser Ala Glu Gln Ile Leu Glu Phe Ile Glu Asp Leu Met Asn
545 550 555
Pro Ser Val Val Ser Leu Thr Pro Thr Thr Phe Asn Glu Leu Val
560 565 570
Thr Gln Arg Lys His Asn Glu Val Trp Met Val Asp Phe Tyr Ser
575 580 585
Pro Trp Cys His Pro Cys Gln Val Leu Met Pro Glu Trp Lys Arg
590 595 600
Met Ala Arg Thr Leu Thr Gly Leu Ile Asn Val Gly Ser Ile Asp
605 610 615
Cys Gln Gln Tyr His Ser Phe Cys Ala Gln Glu Asn Val Gln Arg
620 625 630
Tyr Pro Glu Ile Arg Phe Phe Pro Pro Lys Ser Asn Lys Ala Tyr
635 640 645
Gln Tyr His Ser Tyr Asn Gly Trp Asn Arg Asp Ala Tyr Ser Leu
650 655 660
Arg Ile Trp Gly Leu Gly Phe Leu Pro Gln Val Ser Thr Asp Leu
665 670 675
Thr Pro Gln Thr Phe Ser Glu Lys Val Leu Gln Gly Lys Asn His
680 685 690
Trp Val Ile Asp Phe Tyr Ala Pro Trp Cys Gly Pro Cys Gln Asn
695 700 705
Phe Ala Pro Glu Phe Glu Leu Leu Ala Arg Met Ile Lys Gly Lys
710 715 720
Val Lys Ala Gly Lys Val Asp Cys Gln Ala Tyr Ala Gln Thr Cys
725 730 735
Gln Lys Ala Gly Ile Arg Ala Tyr Pro Thr Val Lys Phe Tyr Phe
740 745 750
Tyr Glu Arg Ala Lys Arg Asn Phe Gln Glu Glu Gln Ile Asn Thr
6/ 16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
755 760 765
Arg Asp Ala Lys Ala Ile Ala Ala Leu Ile Ser Glu Lys Leu Glu
770 775 780
Thr Leu Arg Asn Gln Gly Lys Arg Asn Lys Asp Glu Leu
785 790
<210> 6
<211> 492
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3325534CD1
<400> 6
Met Ala Val Leu Leu Glu Thr Thr Leu Gly Asp Val Val Ile Asp
1 5 10 15
Leu Tyr Thr Glu Glu Arg Pro Arg Ala Cys Leu Asn Phe Leu Lys
20 25 30
Leu Cys Lys Ile Lys Tyr Tyr Asn Tyr Cys Leu Ile His Asn Val
35 40 45
Gln Arg Asp Phe Ile Ile Gln Thr Gly Asp Pro Thr Gly Thr Gly
50 55 60
Arg Gly Gly Glu Ser Ile Phe Gly Gln Leu Tyr Gly Asp Gln Ala
65 70 75
Ser Phe Phe Glu Ala Glu Lys Val Pro Arg Ile Lys His Lys Lys
80 85 90
Lys Gly Thr Val Ser Met Val Asn Asn Gly Ser Asp Gln His Gly
95 100 105
Ser Gln Phe Leu Ile Thr Thr Gly Glu Asn Leu Asp Tyr Leu Asp
110 115 120
Gly Val His Thr Val Phe Gly Glu Val Thr Glu Gly Met Asp Ile
125 130 135
Ile Lys Lys Ile Asn Glu Thr Phe Val Asp Lys Asp Phe Val Pro
140 145 150
Tyr Gln Asp Ile Arg Ile Asn His Thr Val Ile Leu Asp Asp Pro
155 160 165
Phe Asp Asp Pro Pro Asp Leu Leu Ile Pro Asp Arg Ser Pro Glu
170 175 180
Pro Thr Arg Glu Gln Leu Asp Ser Gly Arg Ile Gly Ala Asp Glu
185 190 195
Glu ale Asp Asp Phe Lys Gly Arg Ser Ala Glu Glu Val Glu Glu
200 205 210
Ile Lys Ala Glu Lys Glu Ala Lys Thr Gln Ala Ile Leu Leu Glu
215 220 225
Met Val Gly Asp Leu Pro Asp Ala Asp Ile Lys Pro Pro Glu Asn
230 235 240
Val Leu Phe Val Cys Lys Leu Asn Pro Val Thr Thr Asp Glu Asp
245 250 255
Leu Glu Ile Ile Phe Ser Arg Phe Gly Pro Ile Arg Ser Cys Glu
260 265 270
Val Ile Arg Asp Trp Lys Thr Gly Glu Ser Leu Cys Tyr Ala Phe
275 280 285
Ile Glu Phe Glu Lys Glu Glu Asp Cys Glu Lys Ala Phe Phe Lys
290 295 300
Met Asp Asn Val Leu Ile Asp Asp Arg Arg Ile His Val Asp Phe
305 310 315
Ser Gln Ser Val Ala Lys Val Lys Trp Lys Gly Lys Gly Gly Lys
320 325 330
7/ 16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
Tyr Thr Lys Ser Asp Phe Lys Glu Tyr Glu Lys Glu Gln Asp Lys
335 340 345
Pro Pro Asn Leu Val Leu Lys Asp Lys Val Lys Pro Lys Gln Asp
350 355 360
Thr Lys Tyr Asp Leu Ile Leu Asp Glu Gln Ala Glu Asp Ser Lys
365 370 375
Ser Ser His Ser His Thr Ser Lys Lys His Lys Lys Lys Thr His
380 385 390
His Cys Ser Glu Glu Lys Glu Asp Glu Asp Tyr Met Pro Ile Lys
395 400 405
Asn Thr Asn Gln Asp Ile Tyr Arg Glu Met Gly Phe Gly His Tyr
410 415 420
Glu Glu Glu Glu Ser Cys Trp Glu Lys Gln Lys Ser Glu Lys Arg
425 430 435
Asp Arg Thr Gln Asn Arg Ser Arg Ser Arg Ser Arg Glu Arg Asp
440 445 450
Gly His Tyr Ser Asn Ser His Lys Ser Lys Tyr Gln Thr Asp Leu
455 460 465
Tyr Glu Arg Glu Arg Ser Lys Lys Arg Asp Arg Ser Arg Ser Pro
470 475 480
Lys Lys Ser Lys Asp Lys Glu Lys Ser Lys Tyr Arg
485 490
<210> 7
<211> 160
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3817050CD1
<400> 7
Met Val Ile Pro Thr Val Pro Phe Asn Ile Thr Ile Asn Ser Lys
1 5 10 15
Pro Leu Gly His Ile Ser Phe Gln Leu Phe Ala Asp Lys Phe Pro
20 25 30
Lys Thr Gly Glu Asn Phe His Thr Leu Asn Asn Lys Asp Lys Gly
35 40 45
Phe Gly Ser Cys Phe His Arg Ile Ile Pro Glu Phe Ile Cys Gln
50 55 60
Gly Asp Asp Phe Thr Pro His Asn Gly Ile Gly Gly Lys Ser Ile
65 70 75
Tyr Gly Asp Lys Phe Asp Asp Lys Asn Phe Ile Val Lys His Thr
80 85 90
Gly Leu Gly Ile Leu Ser Met Ala Asn Ala Ala Pro Lys Thr Asn
95 100 105
Glu Ser Gln Phe Phe Ile Cys Thr Ala Met Ala Lys Trp Trp Asp
110 115 120
Gly Lys His Val Ile Phe Gly Arg Val Lys Glu Gly Met Asn Ile
125 130 135
Val Glu Ala Met Glu Cys Phe Gly Ser Arg Asn Gly Lys Thr Ser
140 145 150
Lys Ile Ala Ile Ala Asn Cys Arg Gln Leu
155 160
<210> 8
8/16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
<211> 744
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5324378CD1
<400> 8
Met Gln Lys Thr Glu Thr Leu Leu Leu Phe Ser Cys Asn Ile Ser
1 5 10 15
Val Ser Ser Glu Pro Gly Val Leu Gly Tyr Phe Glu Phe Ser Gly
20 25 30
Ser Pro Gln Pro Pro Gly Tyr Leu Thr Phe Phe Thr Ser Ala Leu
35 40 45
His Ser Leu Lys Lys Asp Tyr Leu Gly Thr Val Arg Phe Gly Val
50 55 60
Ile Thr Asn Lys His Leu Ala Lys Leu Val Ser Leu Val His Ser
65 70 75
Gly Ser Val Tyr Leu His Arg His Phe Asn Thr Ser Leu Val Phe
80 85 90
Pro Arg Glu Val Leu Asn Tyr Thr Ala Glu Asn Ile Cys Lys Trp
95 100 105
Ala Leu Glu Asn Gln Glu Thr Leu Phe Arg Trp Leu Arg Pro His
110 115 120
Gly Gly Lys Ser Leu Leu Leu Asn Asn Glu Leu Lys Lys Gly Pro
125 130 135
Ala Leu Phe Leu Phe Ile Pro Phe Asn Pro Leu Ala Glu Ser His
140 145 150
Pro Leu Ile Asp Glu Ile Thr Glu Val Ala Leu Glu Tyr Asn Asn
155 160 165
Cys His Gly Asp Gln Val Val Glu Arg Leu Leu Gln His Leu Arg
170 175 180
Arg Val Asp Ala Pro Val Leu Glu Ser Leu Ala Leu Glu Val Pro
185 190 195
Ala Gln Leu Pro Asp Pro Pro Thr Ile Thr Ala Ser Pro Cys Cys
200 205 210
Asn Thr Val Val Leu Pro Gln Trp His Ser Phe Ser Arg Thr His
215 220 225
Asn Val Cys Glu Leu Cys Val Asn Gln Thr Ser Gly Gly Met Lys
230 235 240
Pro Ser Ser Val Ser Val Pro Gln Cys Ser Phe Phe Glu Met Ala
245 250 255
Ala Ala Leu Asp Ser Phe Tyr Leu Lys Glu Gln Thr Phe Tyr His
260 265 270
Val Ala Ser Asp Ser Ile Glu Cys Ser Asn Phe Leu Thr Ser Tyr
275 280 285
Ser Pro Phe Ser Tyr Tyr Thr Ala Cys Cys Arg Thr Ile Ser Arg
290 295 300
Gly Val Ser Gly Phe Ile Asp Ser Glu Gln Gly Val Phe Glu Ala
305 310 315
Pro Thr Val Ala Phe Ser Ser Leu Glu Lys Lys Cys Glu Val Asp
320 325 330
Ala Pro Ser Ser Val Pro His Ile Glu Glu Asn Arg Tyr Leu Phe
335 340 345
Pro Glu Val Asp Met Thr Ser Thr Asn Phe Thr Gly Leu Ser Cys
350 355 360
Arg Thr Asn Lys Thr Leu Asn Ile Tyr Leu Leu Asp Ser Asn Leu
365 370 375
Phe Trp Leu Tyr Ala Glu Arg Leu Gly Ala Pro Ser Ser Thr Gln
380 385 390
Val Lys Glu Phe Ala Ala Ile Val Asp Val Lys Glu Glu Ser His
395 400 405
9/ 16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
Tyr Ile Leu Asp Pro Lys Gln Ala Leu Met Lys Leu Thr Leu Glu
410 415 420
Ser Phe Ile Gln Asn Phe Ser Val Leu Tyr Ser Pro Leu Lys Arg
425 430 435
His Leu Ile Gly Ser Gly Ser Ala Gln Phe Pro Ser Gln His Leu
440 445 450
Ile Thr Glu Val Thr Thr Asp Thr Phe Trp Glu Val Val Leu Gln
455 460 465
Lys Gln Asp Val Leu Leu Leu Tyr Tyr Ala Pro Trp Cys Gly Phe
470 475 480
Cys Pro Ser Leu Asn His Ile Phe Ile Gln Leu Ala Arg Asn Leu
485 490 495
Pro Met Asp Thr Phe Thr Val Ala Arg Ile Asp Val Ser Gln Asn
500 505 510
Asp Leu Pro Trp Glu Phe Met Val Asp Arg Leu Pro Thr Val Leu
515 520 525
Phe Phe Pro Cys Asn Arg Lys Asp Leu Ser Val Lys Tyr Pro Glu
530 535 540
Asp Val Pro Ile Thr Leu Pro Asn Leu Leu Arg Phe Ile Leu His
545 550 555
His Ser Asp Pro Ala Ser Ser Pro Gln Asn Val Ala Asn Ser Pro
560 565 570
Thr Lys Glu Cys Leu Gln Ser Glu Ala Val Leu Gln Arg Gly His
575 580 585
Ile Ser His Leu Glu Arg Glu Ile Gln Lys Leu Arg Ala Glu Ile
590 595 600
Ser Ser Leu Gln Arg Ala Gln Val Gln Val Glu Ser Gln Leu Ser
605 610 615
Ser Ala Arg Arg Asp Glu His Arg Leu Arg Gln Gln Gln Arg Ala
620 625 630
Leu Glu Glu Gln His Ser Leu Leu His Ala His Ser Glu Gln Leu
635 640 645
Gln Ala Leu Tyr Glu Gln Lys Thr Arg Glu Leu Gln Glu Leu Ala
650 655 660
Arg Lys Leu Gln Glu Leu Ala Asp Ala Ser Glu Asn Leu Leu Thr
665 670 675
Glu Asn Thr Trp Leu Lys Ile Leu Val Ala Thr Met Glu Arg Lys
680 685 690
Leu Glu Gly Arg Asp Gly Ala Glu Ser Leu Ala Ala Gln Arg Glu
695 700 705
Val His Pro Lys Gln Pro Glu Pro Ser Ala Thr Pro Gln Leu Pro
710 715 720
Gly Ser Ser Pro Pro Pro Ala Asn Val Ser Ala Thr Leu Val Ser
725 730 735
Glu Arg Asn Lys Glu Asn Arg Thr Asp
740
<210> 9
<211> 2015
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 011886CB1
<400> 9
agacgttgtt gcttgggcgc ttctccgctg cgtgtaggtg aagggggctt cctgaccgag 60
acatggattt aggtgctatt acaaaatact cagcattaca cgccaagccc aatggactga 120
tccttcaata cgggactgct ggatttcgaa cgaaggcaga acatcttgat catgtcatgt 180
10/16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
ttcgcatggg attattagct gtcctgaggt caaaacagac aaaatccact ataggagtca 240
tggtaacagc gtcccacaat cctgaggaag acaatggtgt aaaattggtt gatcctttgg 300
gtgaaatgtt ggcaccatcc tgggaggaac atgccacctg tttagcaaat gctgaggaac 360
aagatatgca gagagtgctt attgacatca gcgagaaaga agctgtgaat ctgcaacaag 420
atgcctttgt agttattggt agagatacca ggcccagcag tgagaaactt tcacaatctg 480
taatagatgg tgtgactgtt ctaggaggtc aattccatga ttatggcttg ttaacaacac 540
cccagctgca ctacatggtg tattgtcgaa acacgggtgg ccgatatgga aaggcaacta 600
tagaaggtta ctaccagaaa ctctctaagg cttttgtgga actcaccaaa caggcttctt 660
gcagtggaga tgaatacaga tcacttaagg ttgactgtgc aaatggcata ggggccctga 720
agctaaggga aatggaacac tacttctcac agggcctgtc agttcagctg tttaatgatg 780
ggtccaaggg caaactcaat catttatgtg gagctgactt tgtgaaaagt catcagaaac 840
ctccacaggg aatggaaatt aagtccaatg aaagatgctg ttcttttgat ggagatgcag 900
acagaattgt ttattactac catgatgcag atggccactt tcatctcata gatggagaca 960
agatagcaac gttaattagc agtttcctta aagagctcct ggtggagatt ggagaaagtt 1020
tgaatattgg tgttgtacaa actgcatatg caaatggaag ttcaacacgg tatcttgaag 1080
aagttatgaa ggtacctgtc tattgcacta agactggtgt aaaacatttg caccacaagg 1140
ctcaagagtt tgacattgga gtttattttg aagcaaatgg gcatggcact gcactgttta 1200
gtacagctgt tgaaatgaag ataaaacaat cagcagaaca actggaagat aagaaaagaa 1260
aagctgctaa gatgcttgaa aacattattg acttgtttaa ccaggcagct ggtgatgcta 1320
tttctgacat gctggtgatt gaagcaatct tggctctgaa gggcttgact gtacaacagt 1380
gggatgctct ctatacagat cttccaaaca gacaacttaa agttcaggtt gcagacagga 1440
gagttattag cactaccgat gctgaaagac aagcagttac acccccagga ttacaggagg 1500
caatcaatga cctggtgaag aagtacaagc tttctcgagc ttttgtccgg ccctctggta 1560
cagaagatgt cgtccgagta tatgcagaag cagactcaca agaaagtgca gatcaccttg 1620
cacatgaagt gagcttggca gtatttcagc tggctggagg aattggagaa aggccccaac 1680
caggtttctg aagataattt tcatattcct gagaaactgg actttttaca agtctttaca 1740
aaactgtcaa taataatggc agtactaaga gatttataat cataatgttt acaatgcagc 1800
ctactggatt gtctctagat ctgtttttct taaacactaa cagaataatt ctttataaat 1860
aggtaagcct tacacttgtt aaagaaattt acctctaatt tcagtctcac taatgtaaaa 1920
tactgggact taagtataca attcagtcac taactgtaca gttttatgtg gggaacaatt 1980
catgcaggct actggaaaat taaatcttat tacca 2015
<210> 10
<211> 1823
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1863189CB1
<400> 10
cggctcgagc cggagcgagg cgtcgggatg cagcgccccg ggcccttcag caccctctac 60
gggcgggtct tggccccgct gcccgggagg gccgggggcg cggcctccgg cggaggaggg 120
aacagctggg acctcccggg ttcccacgtg cggctgccgg ggcgtgcaca gtctgggacc 180
cgtggcggtg ctggcaacac aagcaccagc tgcggggact ccaacagcat ctgcccggcc 240
ccctccacga tgtccaaggc cgaggaggcc aagaagctgg cgggccgcgc ggctgtggag 300
aaccacgtga ggaataacca agtgctggga attggaagtg gttctacaat tgtccatgct 360
gtgcagcgaa tagctgaaag ggtgaagcaa gagaatctga acctcgtctg tattcccact 420
tccttccagg cccgccagct catcctgcag tatggcttga ccctcagtga tctggatcga 480
cacccagaga tcgaccttgc catcgatggt gctgatgaag tagatgctga tctcaatctc 540
atcaagggtg gcggaggctg cctgacccag gagaagattg tggctggcta tgctagtcgc 600
ttcatcgtga tcgctgattt caggaaagat tcgaagaatc tcggggatca gtggcacaag 660
ggaatcccca tcgaggtcat cccaatggcc tatgtcccag tgagccgagc tgtgagccag 720
aagtttgggg gcgtggttga acttcgaatg gctgtcaaca aggctggtcc tgtggtgaca 780
gataatggga attttatctt ggactggaag tttgaccggg tacacaaatg gagtgaagtg 840
aatacagcta tcaaaatgat cccaggtgtg gtggacacag gcctattcat caacatggct 900
gagagagtct actttgggat gcaggatggc tcagtgaaca tgagggagaa gcctttctgt 960
tgaccctgca aggagcagag tgtgttcacc ttgagtctcc agcccacagc caaggtggac 1020
gtacctctcc aggagccttt gccttaatgt atctctgcct ggacaacttg tggtgggggg 1080
tggggggaag agtgggaggg ggagttaaat ccagtcttat gaagtattgt tattaaatgt 1140
ctttttaaaa agagaaatat aaacatatat ttttactatt aaaatattca gttttttaaa 1200
11/16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
tgaagtagaa cttgagttca tgttttatat gaaatattta ccaaaaaaaa aaaatgaggt 1260
aaactgtatt taaaaccttt gacttgagtc tgctggtaaa gcttctgaat attgagtttg 1320
ctgagaaata aaaatcaaaa cttctttaag ctggtaaagt gaggggccca ccagcagtga 1380
tctcctgatg ccttactgga aactttgttt acttgtctgc taccctctga tttgttttta 1440
gttagttttt attgtgagca cacatagtac ctagttacat cttaagatca ggtttataaa 1500
actgtggagt ggagcggtat ggtatggaat gacttggaat gtaagctgtc agggagaaaa 1560
tgttgttaca cttttgctaa gatctggggg tttcttcata ttcctgctgt tggaagcagt 1620
tgaccagaaa tgcttgccag tactgccaaa gcactgctgt gaaatgtgaa gtactttgtt 1680
tttttatttt taatgatttt ctttttgtta ttaatatttt tctctgttcc tttgttatta 1740
cttgcatggt ttggcgtcag aagtccttac ctctttatat tgtttgcagg tttaaataaa 1800
acagtgtggt gccaaaaaaa aaa 1823
<210> 11
<211> 1526
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2088868CB1
<400> 11
gcaggagcag gagagggaca atggaagctg ccccgtccag gttcatgttc ctcttatttc 60
tcctcacgtg tgagctggct gcagaagttg ctgcagaagt tgagaaatcc tcagatggtc 120
ctggtgctgc ccaggaaccc acgtggctca cagatgtccc agctgccatg gaattcattg 180
ctgccactga ggtggctgtc ataggcttct tccaggattt agaaatacca gcagtgccca 240
tactccatag catggtgcaa aaattcccag gcgtgtcatt tgggatcagc actgattctg 300
aggttctgac acactacaac atcactggga acaccatctg cctctttcgc ctggtagaca 360
atgaacaact gaatttagag gacgaagaca ttgaaagcat tgatgccacc aaattgagcc 420
gtttcattga gatcaacagc ctccacatgg tgacagagta caaccctgtg actgtgattg 480
ggttattcaa cagcgtaatt cagattcatc tcctcctgat aatgaacaag gcctccccag 540
agtatgaaga gaacatgcac agataccaga aggcagccaa gctcttccag gggaagattc 600
tctttattct ggtggacagt ggtatgaaag aaaatgggaa ggtgatatca tttttcaaac 660
taaaggagtc tcaactgcca gctttggcaa tttaccagac tctagatgac gagtgggata 720
cactgcccac agcagaagtt tccgtagagc atgtgcaaaa cttttgtgat ggattcctaa 780
gtggaaaatt gttgaaagaa aatcgtgaat cagaaggaaa gactccaaag gtggaactct 840
gacttctcct tggaactaca tatggccaag tatctacttt atgcaaagta aaaaggcaca 900
actcaaatct cagagacact aaacaacagg atcactaggc ctgccaacca cacacacacg 960
cacgtgcaca cacgcacgca cgcgtgcaca cacacacgcg cacacacaca cacacacaca 1020
cagagcttca tttcctgtct taaaatctcg ttttctcttc ttccttcttt taaatttcat 1080
atcctcactc cctatccaat ttccttctta tcgtgcattc atactctgta agcccatctg 1140
taacacacct agatcaaggc tttaagagac tcactgtgat gcctctatga aagagaggca 1200
ttcctagaga aagattgttc caatttgtca tttaatatca agtttgtata ctgcacatga 1260
cttacacaca acatagttcc tgctctttta aggttaccta agggttgaaa ctctaccttc 1320
tttcataagc acatgtccgt ctctgactca ggatcaaaaa ccaaaggatg gttttaaaca 1380
cctttgtgaa attgtctttt tgccagaagt taaaggctgt ctccaagtcc ctgaactcag 1440
cagaaataga ccatgtgaaa actccatgct tggttagcat ctccaactcc ctatgtaaat 1500
caacaacctg cataataaat aacaga 1526
<210> 12
<211> 1205
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2481256CB1
<400> 12
gcggtatggc gtcgggctgc aagattggcc cgtccatcct caacagcgac ctggccaatt 60
taggggccga gtgcctccgg atgctagact ctggggccga ttatctgcac ctggacgtaa 120
12/16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
tggacgggca ttttgttccc aacatcacct ttggtcaccc tgtggtagaa agccttcgaa 180
agcagctagg ccaggaccct ttctttgaca tgcacatgat ggtgtccaag ccagaacagt 240
gggtaaagcc aatggctgta gcaggagcca atcagtacac ctttcatctc gaggctactg 300
agaacccagg ggctttgatt aaagacattc gggagaatgg gatgaaggtt ggccttgcca 360
tcaaaccagg aacctcagtt gagtatttgg caccatgggc taatcagata gatatggcct 420
tggttatgac agtggaaccg gggtttggag ggcagaaatt catggaagat atgatgccaa 480
aggttcactg gttgaggacc cagttcccat ctttggatat agaggtcgat ggtggagtag 540
gtcctgacac tgtccataaa tgtgcagagg caggagctaa catgattgtg tctggcagtg 600
ctattatgag gagtgaagac cccagatctg tgatcaatct attaagaaat gtttgctcag 660
aagctgctca gaaacgttct cttgatcggt gaaaccataa ggagcccagt gttcctgttc 720
atgaaatctc ccttttactg gaaaacagga atattgacta ccaaatcaca atgcaattga 780
agccgtactg cttttttgag cagttattca ttccagtgat taaaactgat tgtgcagaat 840
attctaagag gtcagaaatt ggtgtgtata actacatttt tagtgatgca atttattgat 900
tagtgagtaa gatactgttt ttattgagag atttgatttt tataaagtaa aaatacggct 960
gcattagggt tacaaacaga aaagtgtctt aatgtctaag gagggcatat tagctacact 1020
acaaaaacaa attttgtctg tacttctgaa aagaattttg ttgtttctca gctgttttcc 1080
aaaagcaaag gaagtcttta tggttttttt ctatttcatg ttattgtgat ttgtttataa 1140
gtttgggtgg ggtgcatacc atattcttgg ttcttaaaat ctatcacttt tcaccttata 1200
cttga 1205
<210> 13
<211> 4796
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2505257CB1
<400> 13
gccccggctc gccgtggaga ccggcgcgtg aggcacctac cggtaccggc cgcgcgctgg 60
tagtcgccgg tgtggctgca cctcaccaat cccgtgcgcc gcggctgggc cgtcggagag 120
tgcgtgtgct tctctcctgc acgcggtgct tgggctcggc caggcggggt ccgccgccag 180
ggtttgagga tgggggagta gctacaggaa gcgaccccgc gatggcaagg tatatttttg 240
tggaatgaaa aggaagtatt agaaatgagc tgaagaccat tcacagatta atatttttgg 300
ggacagattt gtgatgcttg attcaccctt gaagtaatgt agacagaagt tctcaaattt 360
gcatattaca tcaactggaa ccagcagtga atcttaatgt tcacttaaat cagaacttgc 420
ataagagaga gaatgggagt ctggttaaat aaagatgacg atatcagaga cttgaaaagg 480
atcattctct gttttctgat agtgtatatg gccattttag tgggcacaga tcaggatttt 540
tacagtttac ttggagtgtc caaaactgca agcagtagag aaataagaca agctttcaag 600
aaattggcat tgaagttaca tcctgataaa aacccgaata acccaaatgc acatggcaat 660
tttttaaaaa taaatagagc atatgaagta ctcaaagatg aagatctacg gaaaaagtat 720
gacaaatatg gagaaaaggg acttgaggat aatcaaggtg gccagtatga aagctggaac 780
tattatcgtt atgattttgg tatttatgat gatgatcctg aaatcataac attggaaaga 840
agagaatttg atgctgctgt taattctgga gaactgtggt ttgtaaattt ttactcccca 900
ggctgttcac actgccatga tttagctccc acatggagag actttgctaa agaagtggat 960
gggttacttc gaattggagc tgttaactgt ggtgatgata gaatgctttg ccgaatgaaa 1020
ggagtcaaca gctatcccag tctcttcatt tttcggtctg gaatggcccc agtgaaatat 1080
catggagaca gatcaaagga gagtttagtg agttttgcaa tgcagcatgt tagaagtaca 1140
gtgacagaac tttggacagg aaattttgtc aactccatac aaactgcttt tgctgctggt 1200
attggctggc tgatcacttt ttgttcaaaa ggaggagatt gtttgacttc acagacacga 1260
ctcaggctta gtggcatgtt ggatggtctt gttaatgtag gatggatgga ctgtgccacc 1320
caggataacc tttgtaaaag cttagatatt acaacaagta ctactgctta ttttcctcct 1380
ggagccactt taaataacaa agagaaaaac agtattttgt ttctcaactc attggatgct 1440
aaagaaatat atttggaagt aatacataat cttccagatt ttgaactact ttcggcaaac 1500
acactagagg atcgtttggc tcatcatcgg tggctgttat tttttcattt tggaaaaaat 1560
gaaaattcaa atgatcctga gctgaaaaaa ctaaaaactc tacttaaaaa tgatcatatt 1620
caagttggca ggtttgactg ttcctctgca ccagacatct gtagtaatct gtatgttttt 1680
cagccgtctc tagcagtatt taaaggacaa ggaaccaaag aatatgaaat tcatcatgga 1740
aagaagattc tatatgatat acttgccttt gccaaagaaa gtgtgaattc tcatgttacc 1800
acgcttggac ctcaaaattt tcctgccaat gacaaagaac catggcttgt tgatttcttt 1860
gccccctggt gtccaccatg tcgagcttta ctaccagagt tacgaagagc atcaaatctt 1920
13/16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
ctttatggtc agcttaagtt tggtacacta gattgtacag ttcatgaggg actctgtaac 1980
atgtataaca ttcaggctta tccaacaaca gtggtattca accagtccaa cattcatgag 2040
tatgaaggac atcactctgc tgaacaaatc ttggagttca tagaggatct tatgaatcct 2100
tcagtggtct cccttacacc caccaccttc aacgaactag ttacacaaag aaaacacaac 2160
gaagtctgga tggttgattt ctattctccg tggtgtcatc cttgccaagt cttaatgcca 2220
gaatggaaaa gaatggcccg gacattaact ggactgatca acgtgggcag tatagattgc 2280
caacagtatc attctttttg tgcccaggaa aacgttcaaa gataccctga gataagattt 2340
tttcccccaa aatcaaataa agcttatcag tatcacagtt acaatggttg gaatagggat 2400
gcttattccc tgagaatctg gggtctagga tttttacctc aagtatccac agatctaaca 2460
cctcagactt tcagtgaaaa agttctacaa gggaaaaatc attgggtgat tgatttctat 2520
gctccttggt gtggaccttg ccagaatttt gctccagaat ttgagctctt ggctaggatg 2580
attaaaggaa aagtgaaagc tggaaaagta gactgtcagg cttatgctca gacatgccag 2640
aaagctggga tcagggccta tccaactgtt aagttttatt tctacgaaag agcaaagaga 2700
aattttcaag aagagcagat aaataccaga gatgcaaaag caatcgctgc cttaataagt 2760
gaaaaattgg aaactctccg aaatcaaggc aagaggaata aggatgaact ttgataatgt 2820
tgaagatgaa gaaaaagttt aaaagaaatt ctgacagatg acatcagaag acacctattt 2880
agaatgttac atttatgatg ggaatgaatg aacattatct tagacttgca gttgtactgc 2940
cagaattatc tacagcactg gtgtaaaaga agggtctgca aactttttct gtaaagggcc 3000
ggtttataaa tattttagac tttgcaggct ataatatatg gttcacacat gagaacaaga 3060
atagagtcat catgtattct ttgttatttg cttttaacaa cctttaaaaa atattaaaac 3120
gattcttagc tcagagccat acaaaagtag gctggattca gtccatggac catagattgc 3180
tgtccccctc gacggactta taatgtttca ggtggctggc ttgaacatga gtctgctgtg 3240
ctatctacat aaatgtctaa gttgtataaa gtccactttc ccttcacgtt ttttggctga 3300
cctgaaaaga ggtaacttag tttttggtca cttgttctcc taaaaatgct atccctaacc 3360
atatatttat atttcgtttt aaaaacaccc atgatgtggc acagtaaaca aaccctgtta 3420
tgctgtatta ttatgaggag attcttcatt gttttctttc cttctcaaag gttgaaaaaa 3480
tgcttttaat ttttcacagc cgagaaacag tgcagcagta tatgtgcaca cagtaagtac 3540
acaaatttga gcaacagtaa gtgcacaaat tctgtagttt gctgtatcat ccaggaaaac 3600
ctgagggaaa aaaattatag caattaactg ggcattgtag agtatcctaa atatgttatc 3660
aagtatttag agttctatat tttaaagata tatgtgttca tgtattttct gaaattgctt 3720
tcatagaaat tttcccactg atagttgatt tttgaggcat ctaatattta catatttgcc 3780
ttctgaactt tgttttgacc tgtatccttt atttacattg ggtttttctt tcgtagtttt 3840
ggtttttcac tcctgtccag tctatttatt attcaaatag gaaaaattac tttacaggtt 3900
gttttactgt agcttataat gatactgtag ttattccagt tactagttta ctgtcagagg 3960
gctgcctttt tcagataaat attgacataa taactgaagt tatttttata agaaaatcaa 4020
gtatataaat ctaggaaagg gatcttctag tttctgtgtt gtttagactc aaagaatcac 4080
aaatttgtca gtaacatgta gttgtttagt tataattcag agtgtacaga atggtaaaaa 4140
ttccaatcag tcaaaagagg tcaatgaatt aaaaggcttg caactttttt caaaaacctg 4200
ttagaatatg ctttattgtg ttttgaggag ttttcctttt tttcttttca atatcacttt 4260
atcctccagt atttcctcat aagggttatt atagccataa ttaatgttaa aatagacttt 4320
gttcttcata ttctcccatc tttttcgcta ctatatactc tgtctggatt ctgctgtatg 4380
cctgttggca tatatggaac agtcaccact tgtcacactt aacaccagct ttttgaatta 4440
tgatcagtaa tggcaagagc ctttcattct cgaatgttta aagcctagga gttctacaaa 4500
attggcttct ttctacaaga atcccaaaat ggaatgccta aagaagtctt acttgggtaa 4560
atacttacta aaatatactg gttatgtgca tatcaccaca ctggacactg aggagtgttc 4620
aaaaggaatc taagacatgg tccccatctt ccaactgtct gtaattcact gttttgtcat 4680
tgagctcata aggtacttac attactacct ataaatgttt cctgtacttg ttagttgttg 4740
agaaacattt taggcagtaa ataaaatagt aaatattatg tgtcctataa tttgac 4796
<210> 14
<211> 1680
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3325534CB1
<400> 14
acacccccct cctcccgggg tttgtagcgg aggaggagcg ggcgccatgg cggttctact 60
ggagaccact ttaggcgacg tcgtcatcga cttgtacacc gaagaacggc cgcgtgcctg 120
cttgaatttc ttgaaactgt gcaaaataaa atattacaat tattgcctta ttcacaatgt 180
14/16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
acagagggat tttatcatac aaactggcga tcctacaggg actggccgtg gaggagagtc 240
tatctttggc caactgtatg gtgatcaagc aagctttttt gaggcagaaa aagtcccaag 300
aattaagcac aagaagaaag gcacagtgtc catggtgaat aatggcagtg atcaacatgg 360
atctcagttt cttatcacca caggagaaaa tctagattat cttgatggtg tccatacggt 420
gtttggtgag gtgacagaag gcatggacat aattaagaaa attaatgaga cctttgttga 480
caaggacttt gtaccatatc aggatatcag gataaatcat acggtgattt tagatgatcc 540
atttgatgac cctcctgatt tattaatccc tgatcgatca ccagaaccta caagggaaca 600
attagatagt ggtcgaatag gagcagatga agaaattgat gatttcaaag gaagatcagc 660
tgaggaagta gaagaaataa aggcagaaaa agaggctaaa actcaggcta tacttttgga 720
gatggtggga gacctacctg atgcagatat taaacctcca gaaaatgtac tgtttgtgtg 780
taaattgaac ccagtgacca cagatgagga tctggaaata atattctcta gatttgggcc 840
aataagaagt tgtgaagtta tccgagactg gaagacagga gagtccctct gttacgcttt 900
tattgaattt gaaaaggaag aagattgtga gaaagcattc ttcaaaatgg acaatgtgct 960
tatagatgac agaagaatac atgtggattt tagccagtcg gttgcaaagg ttaaatggaa 1020
aggaaaaggt gggaaataca ccaagagtga tttcaaggag tatgaaaaag aacaggataa 1080
accacctaat ttggttctga aagataaagt aaagcccaaa caggatacaa aatacgatct 1140
tatattagat gagcaggccg aagactcaaa atcaagtcac tcacacacaa gtaaaaaaca 1200
caagaagaaa acccatcact gttctgaaga gaaagaagat gaggactaca tgccaatcaa 1260
aaatactaat caggatatct atagagaaat ggggtttggt cactatgaag aagaagaaag 1320
ctgttgggag aaacaaaaga gtgaaaagag agaccgaact cagaaccgaa gtcgtagccg 1380
atctcgagag agggatggcc attatagtaa tagtcataaa tcaaaatacc aaacagatct 1440
ttatgaaaga gaaaggagta aaaagagaga ccgaagcaga agtccaaaga agtccaaaga 1500
taaagaaaaa tctaagtata gatgaaagat gaagaggcag aattgagagg ctaacatatt 1560
tactcttgtc taacttaaga gtgccaggaa agcagatgct tagattttgt gtcaaagctt 1620
gttatttttt tcatactagg attatggtct ttagattaat actgattata tagagcacgc 1680
<210> 15
<211> 1403
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3817050CB1
<400> 15
cctgtgcact gttggtggga atataaaatg atgcagctgg ctttgcagac actgctgtcc 60
cccaacaccc cctgtcacta ggccatggtc atcccgactg tgcccttcaa catcaccatc 120
aacagcaagc ccttaggaca catctccttt cagctatttg cagacaaatt tccaaagaca 180
ggagaaaact ttcacactct gaacaataaa gacaaaggat ttggttcctg ctttcacaga 240
attattccgg agtttatatg ccagggtgat gacttcacac cccataatgg cattggtggc 300
aagtccatct acggggataa atttgatgat aagaacttta ttgtgaagca tacaggtctt 360
ggcatcttgt ccatggcaaa tgctgcaccc aaaacaaatg agtcccagtt tttcatctgc 420
actgccatgg ccaaatggtg ggatggcaag catgtgatct ttggcagggt gaaagagggc 480
atgaatattg tggaagccat ggaatgcttt gggtccagga atggcaagac aagcaagatc 540
gccattgcca actgcagaca actctgataa atttgacttg tgttttatct taaccaccag 600
acctttcctt ttgtagctca ggagagcacc gttccacccc attcgctcac aatatcctat 660
aatatttgtg ctctcactgc agttctttga gttctatatt ttcattatgt ccctccacgt 720
atagctggat tgcagagtta agtttatgat tatgaaataa aaactaacaa aaaaaaatga 780
tgcagccact atggaaaaca gtatcacagt ttctcaaata attaaacatt gaattactat 840
atgattcagc agttccactc ctggatatat atccaaaaga attgaaagca gaattccaaa 900
gaaatatttg cacatccatg ttcatagcta taccattcac agtagccaag aggtggaagc 960
catctgtgtg cccatccaca gatgaatgga taaacaaaat atgggatata cacactatga 1020
atacagcctt aaaaaggaag gaaattccaa cacatgctac aacatggagg aatcttgagg 1080
aattaacggt aagtgaaata agccagtcac aaaaaggcca atactgaatg attccactta 1140
tgtgaggtat ctagagtagt catattcata gagacagaaa atagaatgat tgttgccagc 1200
aactgggagg aagggggtgt gaaaagttgt ttaatggata ttgagtttgt tttcccagac 1260
gaagaagttc tgaaggttgg ttacatgatg tgaatatact aaacactact gaactgtgta 1320
cttagaatgg ttaagataaa ttttatgcgt tttcactaca ataacaagta gaacagtaga 1380
acagatgatt agtcacagca gaa 1403
15/16
CA 02382019 2002-02-14
WO 01/12790 PCT/US00/22518
<210> 16
<211> 2665
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5324378CB1
<400> 16
ccgtggcgct cggctgcgcg ctgctcctcg ccctcaagtt cacctgcagt cgagcaaaag 60
atgtgataat accagcaaag ccacctgtca gctttttctc cttgaggtct ccagtccttg 120
acctcttcca ggggcagctg gattatgcag agtacgttcg acgggattca gaggtggtac 180
tgctcttctt ctatgcccct tggtgtggac agtccatcgc tgccagggca gaaattgagc 240
aagcagcaag tcggctttca gatcaggtgt tgtttgtggc aattaactgt tggtggaacc 300
aggggaaatg cagaaaacag aaacacttct tttattttcc tgtaatatat ctgtatcatc 360
ggagcctgga gtactcgggt actttgagtt cagtggctca ccccagcctc ctggttattt 420
gaccttcttc acctcagcat tacattcatt aaagaaagat tacctaggaa cagtacgatt 480
tggggttatc acaaataaac atcttgcgaa actggtatcc ttagtacact ctggaagtgt 540
gtatttacat agacatttca acacatcact tgtcttcccc agggaggtcc tgaactacac 600
agctgagaac atctgtaagt gggccttaga aaaccaggag acgctctttc ggtggctgcg 660
gccacacgga ggcaagagtc tcctgctgaa taacgagctg aagaaaggac cagcgctgtt 720
tctgttcata ccttttaatc ccctggccga aagtcatcct ttaatagacg agatcaccga 780
agtggccttg gagtacaaca actgtcatgg ggaccaggtg gtggagcgtc tccttcagca 840
cctgcggcgg gtggatgctc cagtgctgga gtccctggcc ctggaagtgc cggcacagct 900
gccagacccg ccaacgatca cagcgtcccc ctgctgcaac actgtggtgc tgccccagtg 960
gcactccttc tccaggaccc acaacgtctg tgaactctgt gtcaaccaga cctccggggg 1020
catgaagccg agctcggtca gcgtgccaca gtgcagcttt tttgaaatgg cagcagctct 1080
ggattctttc tacctcaagg agcagacctt ttatcatgtg gcatcagaca gcatagaatg 1140
cagcaatttt ttaacttcct atagcccctt cagctactac actgcatgtt gcaggaccat 1200
aagcaggggt gtgtcaggct tcatcgactc tgaacaaggt gtctttgaag cccctactgt 1260
tgcattttct tcccttgaga agaaatgtga ggttgatgcc ccaagctccg ttcctcacat 1320
tgaggagaac aggtatctct ttccagaagt ggacatgact agcacaaact tcacaggcct 1380
gagctgcaga accaacaaga ctctcaacat ctaccttttg gattcaaatt tgttttggtt 1440
atatgcagag agactgggtg ctccgagctc cactcaggtg aaagaatttg cggcaattgt 1500
tgacgtgaaa gaagaatctc attacatctt ggatccaaag caagcactga tgaagctcac 1560
cctagagtct tttattcaaa acttcagcgt tctctatagt cccttgaaaa ggcatctcat 1620
tggaagtggc tctgcccagt tcccgtctca gcatttaatc actgaagtga caactgatac 1680
cttttgggaa gtagtccttc aaaaacagga cgttctcctg ctctattacg ctccgtggtg 1740
cggcttctgt ccatccctca atcacatctt catccagcta gctcggaacc tgcccatgga 1800
cacattcact gtggcaagga ttgacgtgtc tcagaatgac cttccttggg aatttatggt 1860
cgatcgtctt cctactgtct tgttttttcc ctgcaacaga aaggacctaa gtgtgaaata 1920
ccccgaagac gtccccatca cccttccaaa cctgttgagg ttcattttgc atcactcaga 1980
ccctgcttcc agcccccaga atgtggctaa ctctcctacc aaggagtgtc ttcagagcga 2040
ggcagtctta cagcgggggc acatctccca cttggagaga gagatccaga aactgagagc 2100
agaaataagc agcctccagc gagcacaagt gcaggtggag tcccagctct ccagtgcccg 2160
cagagatgag caccggctgc ggcagcagca gcgggccctg gaagagcagc acagcctgct 2220
ccacgcacac agtgagcagc tgcaggccct ctatgagcag aagacacgtg agctgcagga 2280
gctggcccgc aagctgcagg agctggccga tgcctcagaa aacctcctta ccgagaacac 2340
gtggctcaag atcctggtgg cgaccatgga gaggaaactg gagggcaggg atggagctga 2400
aagcctggcg gcccagagag aggtccaccc caagcagcct gagccctcag ccacccccca 2460
gctccctggc agctcccctc cacctgccaa tgtcagcgcc acactggtgt ctgaaaggaa 2520
taaggagaac aggacagact aactttttaa atgatatgaa gaaatcagag gtgaaaattg 2580
tacattggga atatatttat gcaaatttta ttgaaattta ttgtaaataa agattttctc 2640
agtggtctag aaaaaaaaaa aaaaa 2665
16/16
