Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN NERVOUS SYSTEM-ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human
nervous system
s associated proteins and to the use of these sequences in the diagnosis,
treatment, and prevention of
neurological. inflammatory, and cell proliferative disorders, including
cancer.
BACKGROUND OF THE INVENTION
The human nervous system, which regulates all bodily functions. is composed of
the central
nervous system (CNS), consisting of the brain and spinal cord, and the
peripheral nervous system
(PNS), consisting of afferent neural pathways for conducting nerve impulses
from sensory organs to the
CNS, and efferent neural pathways for conducting motor impulses from the CNS
to effector organs.
The PNS can be further divided into the somatic nervous system, which
regulates voluntary motor
activity such as for skeletal muscle, and the autonomic nervous system, which
regulates involuntary
motor activity for internal organs such as the heart, lungs, and viscera.
There are two subdivisions of
the autonomic nervous system: the sympathetic nervous system and the
parasympathetic nervous
system.
A nerve cell (neuron) contains tour regions, the cell body, axon, dendrites,
and axon terminal.
The cell body contains the nucleus and other organelles. The dendrites are
processes which extend
outward from the cell body and receive signals from sense organs or from the
axons of other neurons.
These signals are converted to electrical impulses and transmitted to the cell
body. The axon, whose
size can range from one millimeter to more than one meter, is a single process
that conducts the nerve
impulse away from the cell body. Cytoskeletal fibers, including microtubules
and neurofilaments, run
the length of the axon and function in transporting proteins, membrane
vesicles, and other
macromolecules from the cell body along the axon to the axon terminal. Some
axons are surrounded by
a myelin sheath made up of membranes from either an oligodendrocyte cell (CNS)
or a Schwann cell
(PNS). Myelinated axons conduct electrical impulses faster than unmyelinated
ones of the same
diameter. The axon terminal is at the tip of the axon away from the cell body.
(See Lodish, H. et al.
(1986) Molecular Cell Bioloey Scientific American Books New York NY> pp. 715-
719.)
Contact from one neuron to another occurs at a specialized site called the
synapse. At this site.
the axon terminal from one neuron (the presynaptic cell) sends a signal to
another neuron (the
postsynaptic cell). Synapses may be connected either electrically or
chemically. An electrical synapse
consists of gap junctions connecting the two neurons, allowing electrical
impulses to pass directly from
the presynaptic to the postsynaptic cell. In a chemical synapse, the axon
terminal of the presynaptic cell
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contains membrane vesicles containing a particular neurotransmitter molecule.
A change in electrical
potential at the nerve terminal resulting from the electrical impulse triggers
the release of the
neurotransmitter from the synaptic vesicle by exocytosis. The neurotransmitter
rapidly diffuses
across the synaptic cleft separating the presynaptic nerve cell from the
postsynaptic cell. The
neurotransmitter then binds receptors and opens transmitter-gated ion channels
located in the plasma
membrane of the postsynaptic cell, provoking a change in the cell's electrical
potential. This change
in membrane potential of the postsynaptic cell may serve either to excite or
inhibit further transmission
of the nerve impulse.
Neurotransmitters comprise a diverse group of more than 30 small molecules
which include
acetylcholine, monoamines such as serotonin, dopamine, epinephrine,
norepinephrine, and histamine,
and amino acids such as gamma-aminobutyric acid (GABA), glutamate, and
aspartate, and
neuropepddes such as endorphins and enkephalins (McCance, K.L. and Huether,
S.E. (1994)
PATHOPHYSIOLOGY, The Biologic Basis for Disease in Adults and Children, 2nd
edition, Mosby,
St. Louis, MO, pp 403-404). Many of these molecules have more than one
function and the effects
may be excitatory, e.g. to depolarize the postsynaptic cell plasma membrane
and stimulate nerve
impulse transmission, or inhibitory, e.g. to hyperpolarize the plasma membrane
and inhibit nerve
impulse transmission.
Neurotransmitters and their receptors are targets of pharmacological agents
aimed at
controlling neurological function. For example GABA is the major inhibitory
neurotransmitter in the
CNS, and GABA receptors are the principal target of sedatives such as
benzodiazepines and
barbiturates which act by enhancing GABA-mediated effects (Katzung, B.G.
(1995) Basic and Clinical
Pharmacoloev> 6th edition, Appleton & Lange, Norwalk, CT, pp. 338-339).
Aberrant activity of
neurotransmitters and their receptors is involved in various neurological
conditions, including
Alzheimer's disease, myasthenia gravis, stroke, epilepsy, and Parkinson's
disease. (See Planells-Cases,
R. et al. (1993) Proc. Natl. Acad. Sci. USA 90:5057-5061.)
Each of over a trillion neurons in adult humans connects with over a thousand
target cells
(Tessier-Lavigne, M. et al. (1996) Science 274:1123-1133). These neuronal
connections form during
embryonic development. Each differentiating neuron sends out an axon tipped at
the leading edge by a
growth cone. Aided by molecular guidance cues, the growth cone migrates
through the embryonic
environment to its synaptic target.
Axon growth is guided in part by contact-mediated mechanisms involving cell
surface and
extracellular matrix (ECM) molecules. Many ECM molecules, including
ribronectin, vitronectin,
members of the laminin. tenascin, collagen, and thrombospondin families, and a
variety of
proteoglycans, can act either as promoters or inhibitors of neurite outgrowth
and extension (Tessier-
3~ Lavigne et al., supra). Receptors for ECM molecules include integrins,
immunoglobulin superfamily
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members, and proteoglycans. ECM molecules and their receptors have also been
implicated in the
adhesion, maintenance, and differentiation of neurons (Reichardt, L.F. et al.
( 1991 ) Ann. Rev.
Neurosci. 14:5 31-571 ).
The adrenal medulla is the central portion of the adrenal gland and is
functionally related to the
sympathetic nervous system. The neurotransmitters epinephrine and
norepinephrine are secreted by the
adrenal medullae into the blood stream which causes a systemic response. A
cDNA encoding a 50 kD
protein has been isolated from the adrenal medulla. The possible function of
this protein has not been
determined (NCBI Entrez Protein query, 8483843 on 20 July 1999).
Another nervous system-associated protein is 4F5. The gene encoding the 4F5
protein has been
identified as a candidate modifying gene for spinal muscular atrophy (SMA), a
recessive disorder.
SMA is characterized by the loss of lower motor neurons in the spinal cord.
The age of onset and
severity of the disease allows it to be classified into three types. All three
types of SMA have been
mapped to chromosome Sql3. The genetic basis for the phenotypic variability of
SMA is unclear
(Scharf, J.M. et al. (1998) Nat. Genet. 20:83-86).
To understand the biology of specific neuron types, cell-type specific
molecules are being
identified. One group selected cDNA clones by comparing libraries of normal
mouse cerebellar cDNA
and cerebellar cDNA from Purkinje cell degeneration (pcd) mice. One clone
identified to correspond
with mRNA present in Purkinje neurons encodes a protein of 99 amino acids.
PCDS. PCDS's
expression is restricted to the cerebellum and the eye. The gene encoding PCDS
was localized to mouse
chromosome 8 (Nordquist, D.T. et al. (1988) J. Neurosci. 8(12):4780-4789).
The discovery of new human nervous system-associated proteins and the
polynucleotides
encoding them satisfies a need in the art by providing new compositions which
are useful in the
diagnosis, prevention, and treatment of neurological, inflammatory, and cell
proliferative disorders,
including cancer.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, human nervous system-associated
proteins,
referred to collectively as "NSPRT" and individually as "NSPRT-1," "NSPRT-2,''
"NSPRT-3," and
"NSPRT-4." 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:l-4, b) a naturally occurring amino acid sequence
having at least 9090
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:l-4, c) a
biologically active fragment of an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-4, and d) an immunogenic fragment of an amino acid sequence selected from
the group consisting
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of SEQ ID NO:1-4. In one alternative, the invention provides an isolated
polypeptide comprising the
amino acid sequence of SEQ ID NO:1-=I.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising an
amino acid sequence selected from the group consisting of a) an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-4, 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:1-4, c) a
biologically active fragment of an amino acid sequence selected from the group
consisting of SEQ ID
NO:l-4, and d) an immunogenic fragment of an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-4. In one alternative, the polynucleotide encodes a polypeptide
selected from the
group consisting of SEQ ID NO:1-4. In another alternative, the polynucleotide
is selected from the
group consisting of SEQ ID NO:S-8.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypepdde comprising
an amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
1~ consisting of SEQ ID NO:1-4, 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:l-4, c) a
biologically active fragment of an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-4, and d) an immunogenic fragment of an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-4. 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:1-4, 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:1-4, c) a
biologically active fragment of an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-4, and d) an immunogenic fragment of an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-4. 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: l -4, b) a naturally
occurring amino acid
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sequence having at least 90% sequence identity to an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-4, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-4, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-4.
The invention further provides an isolated polynucleotide comprising a
polynucleotide sequence
selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID NO:S-8, b) a naturally occurring polynucleotide sequence having at
least 70% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID NO:S-8, 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 NO:S-8, b) a naturally occurring polynucleotide sequence having at
least 70% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID NO:S-8, 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
tiom the group consisting of
SEQ ID NO:S-8, b) a naturally occurring polynucleotide sequence having at
least 70% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID NO:S-8, 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 polymerise 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.
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The invention further provides a pharmaceutical composition comprising an
effective amount
of a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:l-4, 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:I-4, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-4, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-4, and a
pharmaceutically acceptable
excipient. In one embodiment, the pharmaceutical composition comprises an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-4. The invention
additionally provides a method of
treating a disease or condition associated with decreased expression of
functional NSPRT, comprising
administering to a patient in need of such treatment the pharmaceutical
composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide comprising an amino acid sequence selected from the
group consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO:1-4, 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:1-4, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-4, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-4. The method
comprises a)
exposing a sample comprising the polypeptide to a compound, and b) detecting
agonist activity in the
sample. In one alternative, the invention provides a pharmaceutical
composition comprising an agonist
compound identified by the method and a pharmaceutically acceptable excipient.
In another
alternative, the invention provides a method of treating a disease or
condition associated with
decreased expression of functional NSPRT, comprising administering to a
patient in need of such
treatment the pharmaceutical composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide comprising an amino acid sequence selected from
the group consisting
of a) an amino acid sequence selected from the group consisting of SEQ ID NO:l-
4, 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:1-4, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:l-4, and d) an
immunogenic fragment
of an amino acid sequence selected from the group consisting of SEQ ID NO:I-4.
The method
comprises a) exposing a sample comprising the polypeptide to a compound, and
b) detecting
antagonist activity in the sample. In one alternative, the invention provides
a pharmaceutical
composition comprising an antagonist compound identified by the method and a
pharmaceutically
acceptable excipient. In another alternative, the invention provides a method
of treating a disease or
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condition associated with overexpression of functional NSPRT, comprising
administering to a patient
in need of such treatment the pharmaceutical composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-4, 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:1-4, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-4, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-4. 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)
IS an amino acid sequence selected from the group consisting of SEQ ID NO:l-4,
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:1-4, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-4, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-4. 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 NO:S-8, the method
comprising a) exposing a
sample comprising the target polynucleotide to a compound, and b) detecting
altered expression of
the target polynucleotide.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs),
clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments
used to assemble full-
length sequences encoding NSPRT.
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Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of NSPRT.
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
encodine NSPRT 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
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.
DEFINIT10NS
"NSPRT" refers to the amino acid sequences of substantially purit7ed NSPRT
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
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NSPRT. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of NSPRT either by
directly interacting with
NSPRT or by acting on components of the biological pathway in which NSPRT
participates.
An "allelic variant" is an alternative form of the gene encoding NSPRT.
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 NSPRT include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as NSPRT or a
polypeptide with at least one functional characteristic of NSPRT. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding NSPRT, and improper or unexpected hybridization to
allelic variants, with
a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding NSPRT.
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 NSPRT.
Deliberate amino acid substitutions may be made on the basis of similarity in
polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues, as long as the
biological or immunological activity of NSPRT 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 polymerise chain reaction (PCR)
technologies well known
in the art.
3~ The term "antagonist'' refers to a molecule which inhibits or attenuates
the biological activity of
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NSPRT. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of NSPRT either by
directly interacting with NSPRT or by acting on components of the biological
pathway in which
NSPRT participates.
The term "antibody'' refers to intact immunoglobulin molecules as well as to
fragments thereof,
such as Fab, F(ab'),, and Fv fragments, which are capable of binding an
epitopic determinant.
Antibodies that bind NSPRT 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; 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 ~-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 NSPRT, or
of any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
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"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 NSPRT or fragments
of NSPRT 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 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
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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 polvpeptide
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
poly~peptide. Chemical
moditlcations 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 NSPRT or the polynucleotide encoding NSPRT
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer. antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
2~ 15, 16, 20, 25, 30, 40. 50. 60, 75, 100, 150, 250 or at least 500
contiguous nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 259c 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 NO:~-8 comprises a region of unique polynucleotide
sequence that
specifically identities SEQ ID NO:~-8, for example, as distinct from any other
sequence in the
genome from which the fragment was obtained. .~ fragment of SEQ ID NO:S-8 is
useful. for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID NO:~-8 from related polynucleotide sequences. The precise length of a
fragment of SEQ ID
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NO:S-8 and the region of SEQ ID NO:S-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 fragment of SEQ ID NO:1-4 is encoded by a fragment of SEQ ID NO:S-8. A
fragment of
SEQ ID NO:1-4 comprises a region of unique amino acid sequence that
specifically identifies SEQ ID
NO:1-4. For example, a fragment of SEQ ID NO:1-4 is useful as an immunogenic
peptide for the
development of antibodies that specifically recognize SEQ ID NO:1-4. The
precise length of a
fragment of SEQ ID NO:1-4 and the region of SEQ ID NO:1-4 to which the
fragment corresponds are
routinely determinable by one of ordinary skill in the art based on the
intended purpose for the
fragment.
A "full-length" polynucleotide sequence is one containing at least a
translation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full-
length" polynucleotide sequence encodes a "full-length" polypeptide sequence.
"Homology'' refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity' and "% identity," as applied to polynucleotide
sequences, refer to
the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps in
the sequences being compared in order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence
alignment program. This program is part of the LASERGENE software package, a
suite of molecular
biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in
Higgins, D.G.
and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992)
CABIOS 8:189-191.
For pairwise alignments of polynucleotide sequences, the default parameters
are set as follows:
Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted"
residue weight table is
selected as the default. Percent identity is reported by CLUSTAL V as the
"percent similarity" between
aligned polynucleotide sequences.
Alternatively. a suite of commonly used and freely available sequence
comparison algorithms is
provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment Search
Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410). which
is available fiom 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
3~ polynucleotide sequences from a variety of databases. Also available is a
tool called "BLAST 2
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Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http:/lwww.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: I
Penalh~ for mismatch: -2
Open Gap: ~ and Extension Gap: 2 penalties
Gap x drop-off.' ~0
Expect: 10
Word Site: II
Filter: on
1S Percent identity may be measured over the length of an entire defined
sequence, for example, as
defined by a particular SEQ ID number, or may be measured over a shorter
length, for example, over
the length of a fragment taken from a larger, defined sequence, for instance,
a fragment of at least 20, at
least 30, at least 40, at least 50, at least 70, at least 100, or at least 200
contiguous nucleotides. Such
lengths are exemplary only, and it is understood that any fragment length
supported by the sequences
shown herein, in the tables, figures, or Sequence Listing, may be used to
describe a length over which
percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes in
a nucleic acid sequence can be made using this degeneracy to produce multiple
nucleic acid sequences
that all encode substantially the same protein.
The phrases "percent identity' and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above. generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default parameters
of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e
sequence alignment
program (described and referenced above). For pairwise alignments of
polypeptide sequences using
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CLUSTAL V, the default parameters are set as follows: Ktuple=I, gap penalty=3,
window=~, 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: I penalties
Gap x drop-off' S0
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
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
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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 19c (w/v)
SDS, and about 100 ~tg/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (T,~ for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 500 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 0.190.
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 35-5090 v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex'' refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A hybridization
complex may be formed in solution (e.g., Cat 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 affeca cellular
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and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of NSPRT
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
NSPRT 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 NSPRT. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological.
functional, or immunological properties of NSPRT.
The phrases "nucleic acid'' and ''nucleic acid sequence' refer to a
nucleotide, oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and. where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid'' (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an NSPRT 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 NSPRT.
"Probe" refers to nucleic acid sequences encoding NSPRT, 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
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complementary base-pairing. The primer may then be extended along the target
DNA strand by a DNA
polymerase enzyme. Primer pairs can be used for amplification (and
identification) of a nucleic acid
sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least I S contiguous
S 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,
S0, 60, 70, 80, 90, 100,
or at least 1S0 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 CloninE: A Laboratory Manual,
2°d ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et a1.,1987, Current
Protocols in Molecular
Biology, 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
1S can be derived from a known sequence, for example, by using computer
programs intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas South
West Medical Center, Dallas TX) is capable of choosing specific primers fiom
megabase sequences
and is thus useful for designing primers on a genome-wide scope. The Primer3
primer selection
program (available to the public from the Whitehead Institute/MIT Center for
Genome Research.
Cambridge MA) allows the user to input a "mispriming library," in which
sequences to avoid as primer
binding sites are user-specified. Primer3 is useful, in particular, for the
selection of oligonucleotides for
microarrays. (The source axle 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
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polynucleotide fragments. The oligonucleotides and polynucleotide fragments
identified by any of the
above selection methods are useful in hybridization technologies, for example,
as PCR or sequencing
primers, microarray elements, or specific probes to identify fully or
partially complementary
polynucleotides in a sample of nucleic acids. Methods of oligonucleotide
selection are not limited to
those described above.
A "recombinant nucleic acid'' is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, su ra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for
example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A ''regulatory element'' refers to a nucleic acid sequence usually derived
from untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions (UTRs).
Regulatory elements interact with host or viral proteins which control
transcription, translation, or RNA
stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes:
fluorescent,
chemiluminescent, or chromogenic agents: substrates: cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding NSPRT, or fragments thereof, or NSPRT 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
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synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recognized by the
binding molecule. For
example, if an antibody is specific for epitope "A,'' the presence of a
polypeptide comprising the epitope
A, or the presence of free unlabeled A, in a reaction containing free labeled
A and the antibody will
reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution' refers to the replacement of one or more amino acid residues
or nucleotides by
different amino acid residues or nucleotides, respectively.
''Substrate'' refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" refers to the collective pattern of gene expression by a
particular cell type
or tissue under given conditions at a given time.
''Transformation" describes a process by which exogenous DNA is introduced
into a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods well
known in the art, and may rely on any known method for the insertion of
foreign nucleic acid sequences
into a prokaryotic or eukaryotic host cell. The method for transformation is
selected based on the type
of host cell being transformed and may include, but is not limited to,
bacteriophage or viral infection,
electroporation, heat shock, lipofection, and particle bombardment. The term
"transformed" cells
includes stably transformed cells in which the inserted DNA is capable of
replication either as an
autonomously replicating plasmid or as part of the host chromosome, as well as
transiently transformed
cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation. such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria.
3i cyanobacteria, fungi, plants, and animals. The isolated DNA of the present
invention can be
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introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook et al. ( 1989),
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-07-1999)
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 nervous system-
asscx:iated proteins
(NSPRT), the polynucleotides encoding NSPRT, and the use of these compositions
for the diagnosis.
treatment, or prevention of neurological, inflammatory, and cell proliferative
disorders, including
cancer.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
NSPRT. 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 NSPRT were identified. and colunm 4 shows the cDNA
libraries from
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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
NSPRT and are useful as fragments in hybridization technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; column 6
shows homologous sequences as identified by BLAST analysis; and column 7 shows
analytical methods
and in some cases, searchable databases to which the analytical methods were
applied. The methods of
column 7 were used to characterize each polypeptide through sequence homology
and protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding NSPRT. The first column of Table
3 lists the nucleotide
SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1.
These fragments are
useful, for example, in hybridization or amplification technologies to
identify SEQ ID N0:5-8 and to
distinguish between SEQ ID N0:5-8 and related polynucleotide sequences. The
polypeptides encoded
by these fragments are useful, for example, as immunogenic peptides. Column 3
lists tissue categories
which express NSPRT as a fraction of total tissues expressing NSPRT. Column 4
lists diseases,
disorders, or conditions associated with those tissues expressing NSPRT as a
fraction of total tissues
expressing NSPRT. Column 5 lists the vectors used to subclone each cDNA
library. Of particular
interest is the expression of SEQ ID N0:5 exclusively in nervous tissue (100%)
and the expression of
SEQ ID N0:7 predominately in nervous tissue (78.690).
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
from which cDNA clones encoding NSPRT were isolated. Column 1 references the
nucleotide SEQ ID
NOs, column 2 shows the cDNA libraries from which these clones were isolated,
and column 3 shows
the tissue origins and other descriptive information relevant to the cDNA
libraries in column 2.
'The invention also encompasses NSPRT variants. A preferred NSPRT variant is
one which
has at least about 8090, or alternatively at least about 90%, or even at least
about 95'70 amino acid
sequence identity to the NSPRT amino acid sequence, and which contains at
least one functional or
structural characteristic of NSPRT.
The invention also encompasses polynucleotides which encode NSPRT. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected from
the group consisting of SEQ ID N0:5-8, which encodes NSPRT. The polynucleotide
sequences of
SEQ ID NO:S-8, as presented in the Sequence Listing, embrace the equivalent
RNA sequences, wherein
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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
NSPRT. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at least
about 85 %, or even at least about 95 % polynucleotide sequence identity to
the polynucleotide sequence
encoding NSPRT. A particular aspect of the invention encompasses a variant of
a polynucleotide
sequence comprising a sequence selected from the group consisting of SEQ ID
N0:5-8 which has at
least about 70%, or alternatively at least about 85%, or even at least about
95% polynucleotide
sequence identity to a nucleic acid sequence selected from the group
consisting of SEQ ID N0:5-8.
Any one of the polynucleotide variants described above can encode an amino
acid sequence which
contains at least one functional or structural characteristic of NSPRT.
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 NSPRT, 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 NSPRT, and all such variations are to be considered as being
specifically disclosed.
Although nucleotide sequences which encode NSPRT and its variants are
generally capable of
hybridizing to the nucleotide sequence of the naturally occurring NSPRT under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding NSPRT 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 NSPRT 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 NSPRT
and
NSPRT 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 NSPRT or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
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hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:5-8 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,
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
Biosystetns). 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 Biology, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
(1995) Molecular
BioloeY and Biotechnology, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding NSPRT 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 perfornung 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. 19:3055-30601.
Additionally. one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
=1
CA 02375571 2002-O1-21
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Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intronlexon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 Primer Analysis software
(National Biosciences,
Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides
in length, to have a
GC content of about 50% or more, and to anneal to the template at temperatures
of about 68°C to
72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T) library
does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence into 5'
non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze the
size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, 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 NSPRT may be cloned in recombinant DNA molecules that direct expression
of NSPRT, 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 NSPRT.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter NSPRT-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction sites,
alter glycosylation patterns, change colon preference, produce splice
variants, and so forth.
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
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
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 NSPRT, such as its biological or
enzymatic activity or its ability
to bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
IS In another embodiment, sequences encoding NSPRT may be synthesized, in
whole or in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively,
NSPRT 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
NSPRT, 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 NSPRT, the nucleotide sequences
encoding NSPRT 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
3~ encoding NSPRT. Such elements may vary in their strength and specificity.
Specific initiation signals
26
CA 02375571 2002-O1-21
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may also be used to achieve more efficient translation of sequences encoding
NSPRT. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding NSPRT 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 NSPRT and appropriate transcriptional
and translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
CloninE, 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 Biology, 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 NSPRT. 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;
Brogue, R. et al.
(1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105; The
McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York
NY, pp.
191-196: Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-
3659; and Harrington,
J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from
retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al., (1993)
Proc. Natl. Acad. Sci.
USA 90(13):6340-6344: Buller, R.M. et al. (1985) Nature 317(6040):813-815:
McGregor, D.P. et al.
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(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.)
The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding NSPRT. For
example, routine cloning,
subcloning, and propagation of polynucleotide sequences encoding NSPRT 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 NSPRT 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 NSPRT are needed, e.g. for the
production of antibodies,
vectors which direct high level expression of NSPRT may be used. For example,
vectors containing the
strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of NSPRT. 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
oastoris. 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, su ra;
Bitter, supra; and Scorer, supra.)
Plant systems may also be used for expression of NSPRT. Transcription of
sequences
encoding NSPRT 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, su ra; 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 Technology
(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 NSPRT
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses NSPRT 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
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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
NSPRT in cell lines is preferred. For example, sequences encoding NSPRT can be
transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include,
but are not limited to, the herpes simplex virus thymidine kinase and adenine
phosphoribosylVansferase
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; ~zeo 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 13-glucuronide, or luciferase and its
substrate luciferin may be used.
These markers can be used not only to identify transformants, but also to
quantify the amount of
transient or stable protein expression attributable to a specific vector
system. (See, e.g., Rhodes, C.A.
(1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be conf
rmed. For example, if the
sequence encoding NSPRT is inserted within a marker gene sequence, transformed
cells containing
sequences encoding NSPRT can be identified by the absence of marker gene
function. Alternatively, a
29
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WO 01/07470 PCT/US00/19837
marker gene can be placed in tandem with a sequence encoding NSPRT 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 NSPRT
and that express
NSPRT 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 NSPRT
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 NSPRT is preferred, but
a competitive binding
assay may be employed. These and other assays are well known in the art. (See,
e.g., Hampton, R. et
al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN,
Sect. IV; Coligan, J.E.
et al. ( 1997) Current Protocols in ImmunoloQV, 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
NSPRT include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, the sequences encoding NSPRT, 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 NSPRT 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 NSPRT may be designed to contain signal sequences
which direct
CA 02375571 2002-O1-21
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secretion of NSPRT 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 NSPRT 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 NSPRT protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of NSPRT
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 NSPRT encoding sequence and the heterologous protein
sequence, so that NSPRT
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 NSPRT 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. ~'S-methionine.
NSPRT of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to NSPRT. At least one and up to a plurality of test
compounds may be
31
CA 02375571 2002-O1-21
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screened for specific binding to NSPRT. 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
NSPRT, e.g., a ligand or fragment thereof, a natural substrate, a structural
or functional mimetic, or a
natural binding partner. (See, Coligan, J.E. et al. (1991) Current Protocols
in Immunoloey 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which NSPRT
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 NSPRT,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing NSPRT or cell membrane fractions which contain NSPRT
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either NSPRT or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
NSPRT, either in
solution or affixed to a solid support, and detecting the binding of NSPRT 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.
NSPRT of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of NSPRT. Such compounds may include agonists,
antagonists, or partial
or inverse agonists. In one embodiment, an assay is performed under conditions
permissive for
NSPRT activity, wherein NSPRT is combined with at least one test compound, and
the activity of
NSPRT in the presence of a test compound is compared with the activity of
NSPRT in the absence of
the test compound. A change in the activity of NSPRT in the presence of the
test compound is
indicative of a compound that modulates the activity of NSPRT. Alternatively,
a test compound is
combined with an in vitro or cell-free system comprising NSPRT under
conditions suitable for
NSPRT activity, and the assay is performed. In either of these assays, a test
compound which
modulates the activity of NSPRT 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 NSPRT 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
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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 (March, J.D. (1996) Clin. Invest. 97:1999-2002: Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding NSPRT 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 NSPRT 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 NSPRT 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 NSPRT, e. g., by secreting NSPRT
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 NSPRT and human nervous system-associated proteins. In
addition, the
expression of NSPRT is closely associated with cancerous, proliferating,
inflamed, nervous,
reproductive, hematopoietic/immune, urologic, cardiovascular, and
gastrointestinal tissue. Therefore,
NSPRT appears to play a role in neurological, inflammatory, and cell
proliferative disorders, including
cancer. In the treatment of disorders associated with increased NSPRT
expression or activity, it is
3~ desirable to decrease the expression or activity of NSPRT. In the treatment
of disorders associated
33
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with decreased NSPRT expression or activity, it is desirable to increase the
expression or activity of
NSPRT.
Therefore, in one embodiment, NSPRT 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 NSPRT. Examples of such disorders include, but are not limited to,
a neurological
disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral
neoplasms, Alzheimer's
disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease
and other extrapyra.midal
disorders, amyotrophic lateral sclerosis and other motor neuron disorders,
progressive neural muscular
atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and
other demyelinating diseases,
bacterial and viral meningitis, brain abscess, subdural empyema, epidural
abscess, suppurative
intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous
system disease, prion
diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-
Scheinker syndrome,
fatal familial insomnia, nutritional and metabolic diseases of the nervous
system, neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central nervous system,
cerebral palsy,
neuroskeletal disorders, autonomic nervous system disorders, cranial nerve
disorders, spinal cord
diseases, muscular dystrophy and other neuromuscular disorders, peripheral
nervous system disorders,
dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic
myopathies, myasthenia
gravis, periodic paralysis, mental disorders including mood, anxiety, and
schizophrenic disorders,
seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic
neuropathy, tardive
dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's
disorder, progressive
supranuclear palsy, corticobasal degeneration, and familial frontotemporal
dementia; an inflammatory
disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease,
adult respiratory
distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
polyendocrinopathy-
candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact
dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
episodic lymphopenia
with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic
gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroidids,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis. scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
3~ fungal, parasitic, protozoal, and helminthic infections, and trauma; and a
cell proliferative disorder
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WO 01/07470 PCT/US00/19837
such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria,
polycythemia vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers of
the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall
bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas,
parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus .
In another embodiment, a vector capable of expressing NSPRT 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 NSPRT including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
NSPRT 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
NSPRT including, but not
limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of NSPRT
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or activity
of NSPRT including, but not limited to, those listed above.
In a further embodiment, an antagonist of NSPRT may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of NSPRT.
Examples of such
disorders include, but are not limited to, those neurological, inflammatory,
and cell proliferative
disorders, including cancer, described above. In one aspect, an antibody which
specifically binds
NSPRT may be used directly as an antagonist or indirectly as a targeting or
delivery mechanism for
bringing a pharmaceutical agent to cells or tiSSlles Which express NSPRT.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding NSPRT may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of NSPRT including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made by
one of ordinary skill in the art, according to conventional pharmaceutical
principles. The combination
of therapeutic agents may act synergistically to effect the treatment or
prevention of the various
disorders described above. Using this approach, one may be able to achieve
therapeutic efficacy with
lower dosages of each agent, thus reducing the potential for adverse side
effects.
An antagonist of NSPRT may be produced using methods which are generally known
in the art.
3~ In particular, purified NSPRT may be used to produce antibodies or to
screen libraries of
3~
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
pharmaceutical agents to identify those which specifically bind NSPRT.
Antibodies to NSPRT 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 NSPRT 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 Corynebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
NSPRT 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
NSPRT 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 NSPRT 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
NSPRT-specific single
chain antibodies. Antibodies with related specificity. but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g., Burton,
D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
36
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
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 NSPRT may also be
generated.
For example, such fragments include, but are not limited to, F(ab), 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
NSPRT and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering NSPRT 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 NSPRT. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of NSPRT-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
NSPRT epitopes, represents the average affinity, or avidity, of the antibodies
for NSPRT. The Ka
determined for a preparation of monoclonal antibodies, which are monospecific
for a particular NSPRT
epitope, represents a true measure of affinity. High-affinity antibody
preparations with Ka ranging from
about 109 to 101' L/mole are preferred for use in immunoassays in which the
NSPRT-antibody complex
must withstand rigorous manipulations. Low-affinity antibody preparations with
I~ ranging from
about 106 to 10' L/mole are preferred for use in immunopurification and
similar procedures which
ultimately require dissociation of NSPRT, preferably in active form, from the
antibody (Catty, D.
(1988) Antibodies. Volume I: A Practical Approach, IRL Press, Washington DC;
Liddell, J.E. and A.
Cryer (1991) A Practical Guide to Monoclonal Antibodies. John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
the quality and suitability of such preparations for certain downstream
applications. For example, a
polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably ~-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of NSPRT-antibody
37
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
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, su ra, and
Coligan et al., supra.)
In another embodiment of the invention, the polynucleotides encoding NSPRT, 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 NSPRT.
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 NSPRT.
(See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Moms, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding NSPRT may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-linked
inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe
combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and Somia, N. (1997) Nature
389:239-242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
3~ against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore. D. (1988)
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CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
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 NSPRT expression or regulation causes
disease, the expression of
NSPRT 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
NSPRT are treated by constructing mammalian expression vectors encoding NSPRT
and introducing
these vectors by mechanical means into NSPRT-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).
I S Expression vectors that may be effective for the expression of NSPRT
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). NSPRT 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 encodine NSPRT from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham. F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of these
standardized mammalian transfection protocols.
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In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to NSPRT expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding NSPRT under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in an
appropriate vector producing cell line (VPCL) that expresses an envelope gene
with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses a
method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ T-cells), and the
return of transduced cells to a patient are procedures well known to persons
skilled in the art of gene
therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et
al. (1997) Blood 89:2259-2267: Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
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 NSPRT to cells which have one or more genetic
abnormalities with respect to
the expression of NSPRT. 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 NSPRT to target cells which have one or more genetic
abnormalities with
respect to the expression of NSPRT. The use of herpes simplex virus (HSV)-
based vectors may be
especially valuable for introducing NSPRT to cells of the central nervous
system. for which HSV has a
CA 02375571 2002-O1-21
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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 I-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 uansfection of
multiple plasmids
containing different segments of the large herpesvirus genomes, the growth and
propagation of
hezpesvirus, 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-suanded RNA virus)
vector is used to
deliver polynucleotides encoding NSPRT to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based on
the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotech. 9:464-
469). During alphavirus
RNA replication, a subgenomic RNA is generated that normally encodes the viral
capsid proteins. This
subgenomic RNA replicates to higher levels than the full-length genomic RNA,
resulting in the
overproduction of capsid proteins relative to the viral proteins with
enzymatic activity (e.g., protease
and polymerase). Similarly, inserting the coding sequence for NSPRT into the
alphavirus genome in
place of the capsid-coding region results in the production of a large number
of NSPRT-coding RNAs
and the synthesis of high levels of NSPRT 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
NSPRT into a variety of cell types. The specific transduction of a subset of
cells in a population may
require the sorting of cells prior to transduction. The methods of
manipulating infectious cDNA clones
of alphaviruses, performing alphavirus cDNA and RNA transfections, and
performing alphavirus
infections. are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site. e. g.,
between about positions -10
-I1
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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 Immunoloeic 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 NSPRT.
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 NSPRT. Such DNA sequences may be incorporated into a wide variety of
vectors with
suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA
constructs that
synthesize complementary RNA, constitutively or inducibly, can be introduced
into cell lines, cells, or
tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include. but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine, and
wybutosine, as well as acetyl-. methyl-, thio-, and similarly modified forms
of adenine, cytidine,
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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 NSPRT.
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
NSPRT expression or activity, a compound which specifically inhibits
expression of the
polynucleotide encoding NSPRT may be therapeutically useful, and in the
treament of disorders
associated with decreased NSPRT expression or activity, a compound which
specifically promotes
expression of the polynucleotide encoding NSPRT 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 NSPRT 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
NSPRT 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 NSPRT. The amount of hybridization may be
quantified, thus
forming the basis for a comparison of the expression of the polynucleotide
both with and without
exposure to one or more test compounds. Detection of a change in the
expression of a polynucleotide
exposed to a test compound indicates that the test compound is effective in
altering the expression of
the polynucleotide. A screen for a compound effective in altering expression
of a specific
polynucleotide can be carried out, for example, using a Schizosaccharomyces
pombe gene expression
system (Atkins, D. et al. (1999) U.S. Patent No. 5.932,435; Arndt, G.M. et al.
(2000) Nucleic Acids
Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al.
(2000) Biochem. Biophys.
Res. Commun. 268:8-13). A particular embodiment of the present invention
involves screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
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WO 01/07470 PCT/US00/19837
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691 ).
Many methods for introducing vectors into cells or tissues are available and
equally suitable for
use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells taken
from the patient and clonally propagated for autologous transplant back into
that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15 :462-466. )
Any of the therapeutic methods described above may be applied to any subject
in need of such
therapy, including, for example, mammals such as humans, dogs, cats, cows,
horses, rabbits. and
monkeys.
An additional embodiment of the invention relates to the administration of a
pharmaceutical
composition which generally comprises an active ingredient formulated with a
pharmaceutically
acceptable excipient. Excipients may include, for example, sugars, starches,
celluloses, gums. and
proteins. Various formulations are commonly known and are thoroughly discussed
in the latest edition
of ReminQton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
pharmaceutical
compositions may consist of NSPRT, antibodies to NSPRT, and mimetics,
agonists, antagonists, or
inhibitors of NSPRT.
The pharmaceutical compositions utilized in this invention may be administered
by any number
of routes including, but not limited to, oral, intravenous, intramuscular,
infra-arterial. intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal, enteral,
topical, sublingual, or rectal means.
Pharmaceutical compositions for pulmonary administration may be prepared in
liquid or dry
powder form. These compositions are generally aerosolized immediately prior to
inhalation by the
patient. In the case of small molecules (e.g. traditional low molecular weight
organic drugs). aerosol
delivery of fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g. larger
peptides and proteins), recent developments in the field of pulmonary delivery
via the alveolar region of
the lung have enabled the practical delivery of drugs such as insulin to blood
circulation (see. e.g.,
Patton, J.S. et al., U.S. Patent No. x.997.848). Pulmonary delivery has the
advantage of administration
without needle injection, and obviates the need for potentially toxic
penetration enhancers.
Pharmaceutical compositions suitable for use in the invention include
compositions wherein the
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 pharmaceutical compositions may be prepared for direct
intracellular
delivery of macromolecules comprising NSPRT or fragments thereof. For example,
liposome
preparations containing a cell-impermeable macromolecule may promote cell
fusion and intracellular
delivery of the macromolecule. Alternatively, NSPRT 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 NSPRT
or fragments thereof, antibodies of NSPRT, and agonists, antagonists or
inhibitors of NSPRT, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDso (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDso/EDso ratio.
Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained from cell
culture assays and animal
studies are used to formulate a range of dosage for human use. The dosage
contained in such
compositions is preferably within a range of circulating concentrations that
includes the ED;o with little
or no toxicity. The dosage varies within this range depending upon the dosage
form employed, the
sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the subject
requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the active
moiety or to maintain the desired effect. Factors which may be taken into
account include the severity
of the disease state, the general health of the subject, the age, weight, and
gender of the subject, time
and frequency of administration, drug combination(s), reaction sensitivities,
and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3 to 4 days,
every week, or
biweekly depending on the half-life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~ g to 100,000 fig, 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.
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Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind NSPRT may be used
for the
diagnosis of disorders characterized by expression of NSPRT, or in assays to
monitor patients being
treated with NSPRT or agonists, antagonists, or inhibitors of NSPRT.
Antibodies useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for NSPRT include methods which utilize the antibody and a label to detect
NSPRT 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 NSPRT, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
NSPRT expression. Normal
or standard values for NSPRT expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibody to NSPRT under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of NSPRT
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 NSPRT 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 NSPRT may
be correlated with
2~ disease. The diagnostic assay may be used to determine absence, presence,
and excess expression of
NSPRT, and to monitor regulation of NSPRT levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding NSPRT or closely related
molecules may be used to
identify nucleic acid sequences which encode NSPRT. 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 NSPRT, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 5090
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sequence identity to any of the NSPRT encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
NO:S-8 or from
genomic sequences including promoters, enhancers, and introns of the NSPRT
gene.
Means for producing specific hybridization probes for DNAs encoding NSPRT
include the
cloning of polynucleotide sequences encoding NSPRT or NSPRT 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
polymerises and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a variety
of reporter groups, for example, by radionuclides such as 32P or 3'S, or by
enzymatic labels, such as
alkaline phosphatase coupled to the probe via avidin/biotin coupling systems,
and the like.
Polynucleotide sequences encoding NSPRT may be used for the diagnosis of
disorders
associated with expression of NSPRT. Examples of such disorders include, but
are not limited to, a
neurological disorder such as epilepsy, ischemic cerebrovascular disease,
stroke, cerebral neoplasms,
Alzheimer's disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and other
extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron
disorders, progressive
neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple
sclerosis and other
demyelinating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis,
viral central nervous system
disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-Straussler-Scheinker
syndrome, fatal familial insomnia, nutritional and metabolic diseases of the
nervous system,
neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal
syndrome, mental retardation and other developmental disorders of the central
nervous system, cerebral
palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial
nerve disorders, spinal cord
diseases, muscular dystrophy and other neuromuscular disorders, peripheral
nervous system disorders,
?5 dermatomyositis and polymyositis, inherited, metabolic, endocrine, and
toxic myopathies, myasthenia
gravis, periodic paralysis, mental disorders including mood, anxiety, and
schizophrenic disorders,
seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic
neuropathy, tardive
dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's
disorder. progressive
supranuclear palsy, corticobasal degeneration, and familial frontotemporal
dementia: an inflammatory
disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease.
adult respiratory
distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
polyendocrinopathy-
candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact
dermatitis. Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
episodic lymphopenia
with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic
gastritis,
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glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections, and trauma; and a
cell proliferative disorder
such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria,
polycythemia vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers of
the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall
bladder, ganglia.
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas,
parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus . The
polynucleotide sequences
IS encoding NSPRT 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 NSPRT
expression. Such
qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding NSPRT may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding NSPRT 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
NSPRT 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
NSPRT, 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 NSPRT, 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
3~ polynucleotide is used. Standard values obtained in this manner may be
compared with values obtained
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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 NSPRT
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
NSPRT, or a fragment of a polynucleotide complementary to the polynucleotide
encoding NSPRT, and
will be employed under optimized conditions for identification of a specific
gene or condition.
Oligomers may also be employed under less stringent conditions for detection
or quantification of
closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding NSPRT may be used to detect single nucleotide polvmorphisms (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 NSPRT 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
lluorescently 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-
49
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
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 NSPRT include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C. et
al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple
samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described in Seilhamer, J.J. et al.,
"Comparative Gene Transcript
Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference. The
microarray may also be
used to identify genetic variants, mutations, and polymorphisms. This
information may be used to
determine gene function, to understand the genetic basis of a disorder, to
diagnose a disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic profile.
In another embodiment, antibodies specific for NSPRT, or NSPRT or fragments
thereof may
be used as elements on a microarray. The microarray may be used to monitor or
measure protem-
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
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CA 02375571 2002-O1-21
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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
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.
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.
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CA 02375571 2002-O1-21
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USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116:
Shalon, D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are well
known and thoroughly described in DNA Microarravs: 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 NSPRT
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a multi-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial Pl
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop genetic
linkage maps, for example, which correlate the inheritance of a disease state
with the inheritance of a
particular chromosome region or restriction fragment length polymorphism
(RFLP). (See. 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
NSPRT on a physical
map and a specific disorder, or a predisposition to a specific disorder, may
help define the region of
DNA associated with that disorder and thus may further positional cloning
efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse, may
reveal associated markers even if the exact chromosomal locus is not known.
This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely localized
by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia
to l 1q22-23, any sequences
mapping to that area may represent associated or regulatory genes for further
investigation. (See. e.~T.,
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.,
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CA 02375571 2002-O1-21
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among normal, carrier, or affected individuals.
In another embodiment of the invention, NSPRT, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between NSPRT 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 NSPRT,
or fragments thereof,
and washed. Bound NSPRT is then detected by methods well known in the art.
Purified NSPRT 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 NSPRT specifically compete with a test compound
for binding NSPRT.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with NSPRT.
In additional embodiments, the nucleotide sequences which encode NSPRT 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.
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 LT.S. Ser. No. 60/144,994, 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
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were homogenized and lysed in guanidinium isothiocyanate, while others were
homogenized and lysed
in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life
Technologies), a monophasic
solution of phenol and guanidine isothiocyanate. The resulting lysates were
centrifuged over CsCl
cushions or extracted with chloroform. RNA was precipitated from the lysates
with either isopropanol
or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, su~a, 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-
BlueMRF, or SOLR
from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo excision
using the UNIZAP rvector 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 amplilled from host cell lysates using direct
link PCR in a
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WO 01/07470 PCT/US00/19837
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in 384-
well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (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 Biosysten~s) in conjunction with standard ABI
protocols and base calling
software; or other sequence analysis systems known in the art. Reading frames
within the cDNA
sequences were identified using standard methods (reviewed in Ausubel, 1997,
su ra, unit 7.7). Some
of the cDNA sequences were selected for extension using the techniques
disclosed in Example V.
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
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CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
programing, and dinucleotide nearest neighbor analysis. The sequences were
then queried against a
selection of public databases such as the GenBank primate, rodent, mammalian,
vertebrate, and
eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation
using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled
into full
length polynucleotide sequences using programs based on Phred, Phrap, and
Consed, and were screened
for open reading frames using programs based on GeneMark, BLAST, and FASTA.
The full length
polynucleotide sequences were translated to derive the corresponding full
length amino acid sequences,
and these full length sequences were subsequently analyzed by querying against
databases such as the
GenBank databases (described above), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM,
Prosite,
and Hidden Markov Model (HMM)-based protein family databases such as PFAM. HMM
is a
probabilistic approach which analyzes consensus primary structures of gene
families. (See, e.g.,
Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide and
amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
NO:S-8. 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,
supra, ch. 4 and 16. )
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomicsl. 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
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every mismatch. Two sequences may share more than one HSP (separated by gaps).
If there is more
than one HSP, then the pair with the highest BLAST score is used to calculate
the product score. The
product score represents a balance between fractional overlap and quality in a
BLAST alignment. For
example, a product score of 100 is produced only for 100% identity over the
entire length of the shorter
of the two sequences being compared. A product score of 70 is produced either
by 100% identity and
70% overlap at one end, or by 88% identity and 100% overlap at the other. A
product score of 50 is
produced either by 100% identity and 50% overlap at one end, or 79% identity
and 100% overlap.
The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding NSPRT 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
acxoss all categories.
Percentage values of tissue-specific and disease- or condition-specific
expression are reported in Table
3.
V. Extension of NSPRT Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID NO:S-8 were produced by
extension of an
appropriate fragment of the full length molecule using oligonucleotide primers
designed from this
fragment. One primer was synthesized to initiate 5' extension of the known
fragment, and the other
primer, to initiate 3' extension of the known fragment. The initial primers
were designed using OLIGO
4.06 software (National Biosciences), or another appropriate program, to be
about 22 to 30 nucleotides
in length, to have a GC content of about 50% or more, and to anneal to the
target sequence at
temperatures of about 68 °C to about 72°C. Any stretch of
nucleotides which would result in hairpin
structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one extension
was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc. ). The reaction
mix contained DNA template. 200 nmol of each primer, reaction buffer
containing Mg'+, (NH.,),SO.~.
and ~-mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerise (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 9~°C. 3 min; Step 2: 94°C, 1~ sec:
Step 3: 60°C, 1 min: Step 4: 68°C,
2 min: Step ~: Steps 2. 3, and 4 repeated 20 times; Step 6: 68°C, 5
nun: Step 7: storage at 4°C. In the
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CA 02375571 2002-O1-21
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alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, IS 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 q1
PICOGREEN
quantitation reagent (0.25~Io (v1v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 q1 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 ~I aliquot of the reaction mixture was
analyzed by electrophoresis
on a 1 % agarose mini-gel to determine which reactions were successful in
extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%) agarose
IS gels, fragments were excised, and agar digested with Agar ACE (Promega).
Extended clones were
relegated using T4 lipase (New England Biolabs, Beverly MA) into pUC 18 vector
(Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site overhangs,
and transfected into competent E. coli cells. Transformed cells were selected
on antibiotic-containing
media, and individual colonies were picked and cultured overnight at
37°C in 384-well plates in LB/2x
carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following parameters:
Step 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 2090
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 NO:S-8 are used to
obtain 5'
regulatory sequences using the procedure above, along with oligonucleotides
designed for such
extension, and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID NO:S-8 are employed to screen cDNAs,
genomic
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CA 02375571 2002-O1-21
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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 ~cCi of
[~y-3~P) 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 Rh Pst I, Xba I, or
Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7'7o 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.
VII. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra), mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), supra). Suggested
substrates include silicon. silica, glass slides, glass chips, and silicon
wafers. Alternatively, a procedure
analogous to a dot or slot blot may also be used to arrange and link elements
to the surface of a
substrate using thermal, UV, chemical, or mechanical bonding procedures. A
typical array may be
produced using available methods and machines well known to those of ordinary
skill in the art and may
contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470:
Shalom D. et al. (1996) Genome Res. 6:639-645: Marshall, A. and J. Hodgson
(1998) Nat. Biotechnol.
16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The array
elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
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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/E.il oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/~.M RNase inhibitor, 500 NM dATP, 500 pNI dGTP, 500
LiM dTTP, 40 pM
dCTP, 40 ~rNI 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
viuo transcription
from non-coding yeast genomic DNA. After incubation at 37 °C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium
hydroxide and
incubated for 20 minutes at 85 °C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 p1 5X SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
fig. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0. I % 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
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
Patent No. 5,807,522, incorporated herein by reference. 1 Etl of the array
element DNA, at an average
concentration of 100 ng/E~l, 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).
Microatrays 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 1M of sample mixture consisting of 0.2 pg
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65 °C for ~ minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm' coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of
140 ~.~1 of SX SSC in a corner of the chamber. The chamber containing the
arrays is incubated for
about 6.5 hours at 60°C. The arrays are washed for 10 min at 45
°C in a first wash buffer (1X SSC,
0.1 % SDS), three times for 10 minutes each at 45 °C in a second wash
buffer (0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater 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 56~ nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per tluorophore 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
3~ the array contains a complementary DNA sequence, allowing the intensity of
the signal at that
61
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
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
Iluorophore, 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).
VIII. Complementary Polynucleotides
Sequences complementary to the NSPRT-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring NSPRT. Although
use of oligonucleotides
comprising from about 15 to 30 base pairs is described, essentially the same
procedure is used with
smaller or with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of NSPRT. 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
2~ designed to prevent ribosomal binding to the NSPRT-encoding transcript.
IX. Expression of NSPRT
Expression and purification of NSPRT is achieved using bacterial or virus-
based expression
systems. For expression of NSPRT 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 (tnc) 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 NSPRT upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of NSPRT in eukaryotic cells is
achieved by infecting insect
3~ or mammalian cell lines with recombinant Autoeraphica californica nuclear
polyhedrosis virus
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CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding NSPRT 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 ~odoptera fruEiperda (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, NSPRT 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 Schistosomajaponicum, 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 NSPRT 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 NSPRT obtained by these methods can be used directly
in the assays shown in
Examples X and XIV.
X. Demonstration of NSPRT Activity
NSPRT, or biologically active fragments thereof, are labeled with''5I Bolton-
Hunter reagent.
(See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules
previously arrayed in the
wells of a multi-well plate are incubated with the labeled NSPRT, washed, and
any wells with labeled
NSPRT complex are assayed. Data obtained using different concentrations of
NSPRT are used to
calculate values for the number, affinity, and association of NSPRT with the
candidate molecules.
XI. Functional Assays
NSPRT function is assessed by expressing the sequences encoding NSPRT 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 elecaroporation. I-2 ~g of an additional plasmid containing
sequences encoding a
63
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
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 Cytometrv, Oxford, New York NY.
The influence of NSPRT on gene expression can be assessed using highly
purified populations
of cells transfected with sequences encoding NSPRT 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 NSPRT and other genes of interest can be analyzed by northern
analysis or
microarray techniques.
XII. Production of NSPRT Specific Antibodies
NSPRT substantially purified using polyacrylamide gel electrophoresis (PAGE;
see. e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the NSPRT 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, su ra, ch. 11.1
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431 A
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 inc,~rease
64
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
immunogenicity. (See, e.g., Ausubel, 1995, su ra.) Rabbits are immunized with
the oligopeptide-KLH
complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-NSPRT
activity by, for example, binding the peptide or NSPRT to a substrate,
blocking with 1 % BSA, reacting
with rabbit antisera, washing, and reacting with radio-iodinated goat anti-
rabbit IgG.
XIII. Purification of Naturally Occurring NSPRT Using Specific Antibodies
Naturally occurring or recombinant NSPRT is substantially purified by
immunoafYinity
chromatography using antibodies specific for NSPRT. An immunoaffinity column
is constructed by
covalently coupling anti-NSPRT 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 NSPRT are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of NSPRT (e. g.
> high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/NSPRT 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 NSPRT is collected.
XIV. Identification of Molecules Which Interact with NSPRT
Molecules interacting with NSPRT 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).
NSPRT may also be used in the PATHCALLING process (CuraGen Corp., New Haven
CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S. Patent
No. 6,057,101 ).
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.
6~
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
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CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
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CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
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SUBSTITUTE SHEET (RULE 26)
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
TANG, Y. Tom
YUE, Henry
LU, Dyung Aina M.
YANG, Junming
REDDY, Roopa
AZIMZAI, Yalda
<120> HUMAN NERVOUS SYSTEM-ASSOCIATED PROTEINS
<130> PF-0724 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/144,994
<151> 1999-07-22
<160> 8
<170> PERL Program
<210> 1
<211> 168
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_teature
<223> Incyte ID No:1741504CD1
<400> 1
Met Gly His Ser Gln Gly Pro Met Gly Ile Ala Arg Ala Leu Trp
1 5 10 15
Val Leu Trp Lys Gln Glu Arg G1y Pro Trp Lys Gln Pro Arg Gln
20 25 30
Leu Gly Phe Thr Gln Arg Gly Pro Lys Ser Gln Ser Leu Pro Phe
35 40 45
Ser Gln Asn Thr Asp Ile Phe Ala Ser Gly His Arg Ala Thr Gly
50 55 60
Ala Leu Ser Ser Lys Met Arg Phe Leu Lys Pro Gly Ile Asp Trp
65 70 75
Ser Pro Lys Asn Arg Cys Trp Asp Gly Glu Trp Gly Phe Ile Trp
80 85 90
Val Ser Val Lys Gln Gly Gly Leu Asp Lys Ser Gly Trp Ala Thr
95 100 105
Trp Tyr Pro His Thr Arg Thr His Thr Gly Ala Asn Pro Leu Gln
110 115 120
Leu Asn Lys Gln Arg Asn Ser Val Trp Lys Gly Pro Ser Cys Leu
125 130 135
Leu Lys Ser Leu Arg Pro Cys His Thr Ser Pro Arg His Cys His
140 145 150
His Ser Gly His His Cys Thr Val Gln Gln Val Arg Arg Pro Arg
155 160 165
Ser Tyr Ser
<210> 2
<211> 447
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_teature
<223> Incyte ID No:1831392CD1
1/J
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
<400> 2
Met Arg Ser Val Ser Tyr Val Gln Arg Val Ala Leu Glu Phe Ser
1 5 10 15
Gly Ser Leu Phe Pro His Ala Ile Cys Leu Gly Asp Val Asp Asn
20 25 30
Asp Thr Leu Asn Glu Leu Val Val Gly Asp Thr Ser Gly Lys Val
35 40 45
Ser Val Tyr Lys Asn Asp Asp Ser Arg Pro Trp Leu Thr Cys Ser
50 55 60
Cys Gln Gly Met Leu Thr Cys Val Gly Val Gly Asp Val Cys Asn
65 70 75
Lys Gly Lys Asn Leu Leu Val Ala Val Ser Ala Glu Gly Trp Phe
80 85 90
His Leu Phe Asp Leu Thr Pro Ala Lys Val Leu Asp Ala Ser Gly
95 100 105
His His Glu Thr Leu Ile Gly Glu Glu Gln Arg Pro Val Phe Lys
110 115 120
Gln His Ile Pro Ala Asn Thr Lys Val Met Leu Ile Ser Asp Ile
125 130 135
Asp Gly Asp Gly Cys Arg Glu Leu Val Val Gly Tyr Thr Asp Arg
140 145 150
Val Val Arg Ala Phe Arg Trp Glu Glu Leu Gly Glu Gly Pro Glu
155 160 165
His Leu Thr Gly G1n Leu Val Ser Leu Lys Lys Trp Met Leu Glu
170 175 180
Gly Gln Val Asp Ser Leu Ser Val Thr Leu Gly Pro Leu Gly Leu
185 190 195
Pro Glu Leu Met Val Ser Gln Pro Gly Cys Ala Tyr Ala Ile Leu
200 205 210
Leu Cys Thr Trp Lys Lys Asp Thr Gly Ser Pro Pro Ala Ser Glu
215 220 225
Gly Pro Thr Asp G1y Ser Arg Glu Thr Pro Ala Ala Arg Asp Val
230 235 240
Val Leu His Gln Thr Ser Gly Arg Ile His Asn Lys Asn Val Ser
245 250 255
Thr His Leu Ile Gly Asn Ile Lys Gln Gly His Gly Thr Glu Ser
260 265 270
Ser Gly Ser Gly Leu Phe Ala Leu Cys Thr Leu Asp Gly Thr Leu
275 280 285
Lys Leu Met Glu Glu Met Glu Glu Ala Asp Lys Leu Leu Trp Ser
290 295 300
Val Gln Val Asp His Gln Leu Phe Ala Leu Glu Lys Leu Asp Val
305 310 315
Thr Gly Asn Gly His Glu Glu Val Val Ala Cys Ala Trp Asp Gly
320 325 330
Gln Thr Tyr Ile Ile Asp His Asn Arg Thr Val Val Arg Phe Gln
335 340 345
Val Asp Glu Asn Ile Arg Ala Phe Cys Ala Gly Leu Tyr Ala Cys
350 355 360
Lys Glu Gly Arg Asn Ser Pro Cys Leu Val Tyr Val Thr Phe Asn
365 370 375
Gln Lys Ile Tyr Val Tyr Trp Glu Val Gln Leu Glu Arg Met Glu
380 385 390
Ser Thr Asn Leu Val Lys Leu Leu Glu Thr Lys Pro Glu Tyr His
395 400 405
Ser Leu Leu Gln Glu Leu Gly Val Asp Pro Asp Asp Leu Pro Val
410 415 420
Thr Arg Ala Leu Leu His Gln Thr Leu Tyr His Pro Asp Gln Pro
425 430 435
Pro Gln Cys Ala Pro Ser Ser Leu Gln Asp Pro Thr
440 445
<210> 3
<211> 208
<212> PRT
<213> Homo sapiens
<220>
2/S
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
<221> misc_feature
<223> Incyte ID No:3853885CD1
<400> 3
Met Gly Ser Cys Ser Gly Arg Cys Ala Leu Val Val Leu Cys Ala
1 5 10 15
Phe Gln Leu Val Ala Ala Leu Glu Arg Gln Val Phe Asp Phe Leu
20 25 30
Gly Tyr Gln Trp Ala Pro Ile Leu Ala Asn Phe Val His Ile Ile
35 40 45
Ile Val Ile Leu Gly Leu Phe Gly Thr Ile Gln Tyr Arg Leu Arg
50 55 60
Tyr Val Met Val Tyr Thr Leu Trp Ala Ala Val Trp Val Thr Trp
65 70 75
Asn Val Phe Ile Ile Cys Phe Tyr Leu Glu Val Gly Gly Leu Leu
80 85 90
Gln Asp Ser Glu Leu Leu Thr Phe Ser Leu Ser Arg His Arg Ser
95 100 105
Trp Trp Arg Glu Arg Trp Pro Gly Cys Leu His Glu Glu Val Pro
110 115 120
Ala Val G1y Leu Gly Ala Pro His Gly Gln Ala Leu Val Ser Gly
125 130 135
Ala Gly Cys Ala Leu Glu Pro Ser Tyr Val Glu Ala Leu His Ser
140 145 150
G1V_ Leu Gln Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly Cys
155 160 165
Gln Val Val Ser Val Phe Thr Glu Glu Glu Asp Ser Phe Asp Phe
170 175 180
Ile Gly Gly Phe Asp Pro Phe Pro Leu Tyr His Val Asn Glu Lys
185 190 195
Pro Ser Ser Leu Leu Ser Lys Gln Val Tyr Leu Pro Ala
200 205
<210> 4
<211> 154
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No:5059410CD1
<400> 4
Met Met Asp Gln Glu Glu Lys Thr Glu Glu Gly Ser Gly Pro Cys
1 5 10 15
Ala Glu Ala Gly Ser Pro Asp Gln Glu Gly Phe Phe Asn Leu Leu
20 25 30
Ser His Val Gln Gly Asp Arg Met Glu Gly Gln Arg Cys Ser Leu
35 40 45
Gln Ala Gly Pro Gly Gln Thr Thr Lys Ser Gln Ser Asp Pro Thr
50 55 60
Pro Glu Met Asp Ser Leu Met Asp Met Leu Ala Ser Thr Gln Gly
65 70 75
Arg Arg Met Asp Asp Gln Arg Val Thr Val Ser Ser Leu Pro Gly
80 85 90
Phe Gln Pro Val Gly Ser Lys Asp Gly Ala Gln Lys Arg Ala Trp
95 100 105
Thr Leu Ser Pro Gln Pro Leu Leu Asn Pro Gln Asp Pro Thr Ala
110 115 120
Leu Gly Phe Arg Arg Asn Ser Ser Pro Gln Pro Pro Thr Gln Ala
125 130 135
Pro Leu Gly Ala Glu Ala Ser Trp Val Ser Leu Gly Leu G1n Lys
140 145 150
Leu Val Gly Gly
<210> 5
<211> 1315
<212> DNA
3/5
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No:1741504CB1
<400> 5
acagaacccc ttgtaggctg gaggcaagat tgaatgtggg agaaaatcgg agagaagcga 60
taggagttag aacatctgga tgtgtctgca gcctgctgtc agcccaattg ggccaggggg 120
tcccaaagac gcatattctc accccacctc tacctgcttc ctgatcacat cccagtcacc 180
agcggcagct tcctggatag tgagggagaa caactgcaag ttgagagagg cagaggggtg 240
gaagggacct gaagctggcc tggagaaaag cataggccca ggagagcctg ccctgggaca 300
gcgcctgtct cccacacagc agcactggcc cagcaaggac ctcctccctt ggccctggcc 360
acatcccact cctgcccttt cataagcccc ctggggaaag cactccagtc ttctctgttc 420
caggctgggc agatagggtc ctatggggca cagccagggt cctatgggca tagccagggc 480
cctatgggtc ctctggaagc aagaaagggg gccatggaag cagcccagac agctggggtt 540
cactcagaga ggacccaagt cccagtccct tcctttcagt caaaacacgg atatctttgc 600
ctcaggtcac agggccactg gggccctgtc atcaaagatg agattcctga agcctggcat 660
tgactggtcc cctaagaaca gatgttggga tggagaatgg ggattcattt gggtttcagt 720
aaaacagggg ggtctggaca agagcgggtg ggctacttgg tatccacaca cacgcactca 780
cacaggagcc aacccattgc agctgaacaa gcagagaaac tcagtctgga aaggcccctc 840
ctgcctgctg aagtcactga gaccctgcca cacctctcct cgccactgtc accactcagg 900
gcaccactgt acagtgcaac aagtcaggag acctaggtcc tactcctgac acttgctaat 960
tagctctatg actctgggca aatcgcatat ctgggcctca gtttcctcat ctgtaaaaat 1020
gacagcaaac tcgtaatgct caataaatgt ttaaataaca actgaaaaga aagaaaccaa 1080
gtcaggcgac aaggagcgta gaacagacca aacgaggcgg ccgccgaagg agacggaagc 1140
caggtgtggg cgaggagtaa gaagaggggg cgcgcagccc gaaataaggg ttgcaggacc 1200
agcgaccgag agatagatat acagagagcc ggagcgaaga gcacgcgagc acacagcctc 1260
cgctccagcc gaagagaggg cagctaacaa gaagaaacgc agatgaccat aacat 1315
<210> 6
<211> 2500
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No:1831392CB1
<400> 6
ttttttttta caaagaaggt ataactttaa taaccctatt tacagataag aaaaccaaga 60
ctcagaagtt aaatggaaag gtgaggtctc tgaactaatc cagactctcc catgaggttc 120
ctttggcaag tcctgggctt ctgtcctcat ctgcaaaatc gaaagcattc ctgaggtttc 180
ttccagctct gcatcatcag agttgggaag cccgcctcca agcccggaag acccgcctcc 240
tctgtaaggt taaaggagga agaggcagag attggaggtt caggcagtga cgtaacttgc 300
tgccttaggt ggccttccgc tctggcggct gtcgcgacgg gggttcaggg aatatttact 360
gggcctctcc gctccctctg ctcttggagg tgccatgagg tcagttagct acgtgcagcg 420
cgtggcgctg gagttcagcg ggagcctctt cccgcacgca atctgcctcg gagacgttga 480
taacgatacg ttaaatgaac tggtggtggg agacaccagc gggaaggtgt ctgtgtataa 540
aaatgatgac agtcggccat ggctcacctg ttcctgccag ggaatgctga cttgcgttgg 600
ggttggagac gtgtgtaata aaggaaagaa cctgttggtg gcagtgagtg ctgaaggctg 660
gtttcatttg tttgacctga cacctgccaa ggtgttggat gcttctgggc accacgagac 720
actaatcgga gaggagcagc gtccagtctt caagcagcac atccctgcca acaccaaggt 780
catgctgatc agcgacatcg atggagatgg gtgtcgtgag ctggtggtgg gctacacaga 840
ccgtgtggtg cgagctttcc gctgggagga gctaggtgag ggtcctgaac atctgacagg 900
gcagctggtg tccctcaaga aatggatgct ggagggtcag gtggacagcc tctcagtgac 960
tctggggcca ctgggtcttc ctgaactgat ggtgtctcag ccaggttgtg cgtatgcaat 1020
tctactgtgt acctggaaaa aggacactgg gtcccctcct gcctctgaag ggcccacgga 1080
tggtagtagg gagaccccag ctgcccgaga cgtggtgctg caccagacat ctggccgtat 1140
ccacaacaag aatgtctcca ctcacctaat tggcaacatc aaacaaggcc acggcactga 1200
gagtagtggc tctggcctct ttgccctgtg caccctggat gggacactga agctcatgga 1260
agaaatggaa gaagcagaca agctgctgtg gtcagtgcag gtggatcacc agctctttgc 1320
cctggagaaa ctggatgtca ccggcaacgg gcatgaggag gtagttgcat gcgcctggga 1380
tggacagaca tatatcattg atcacaaccg caccgtcgtc cgcttccaag tggatgaaaa 1440
tatccgtgcc ttctgtgcag gcctgtacgc ctgcaaagag ggccgcaaca gcccctgcct 1500
cgtatatgtc actttcaacc agaagatcta tgtgtactgg gaggtgcagc tggagcggat 1560
ggagtctacc aatctggtga aactgctgga gaccaagccg gagtaccaca gcctgctgca 1620
4/5
CA 02375571 2002-O1-21
WO 01/07470 PCT/US00/19837
ggagctgggc gtggatcctg acgacctccc tgtgactcgt gccctgcttc accaaacgct 1680
ctaccatcca gaccagccac cacagtgtgc tccctcaagc ctccaggatc ccacctagct 1740
gtacttgcct catagctggt gaaggattct tctgaacccc caccctaccc cctaaaggta 1800
tctgtggtat tggcaggata gggaatatgc attacagaaa tgcaggattt gactctgggc 1860
atgaaagatg gcagcagccc tagggtgacc gtgaactata gacctcgcag tcttttcggt 1920
gaaagaagag acaagttgac cctctgccca tttccttatg gacctcaccc atcatgccag 1980
cagggtcata ggaccctggc cttgttccaa atcatctggg acatgaccca ctccccactg 2040
tcactgtgtt gaaaacagag acttgtttgt gtggccccaa cacccataag gaaaccaggc 2100
tttaggccca ggggagcagt ggaggtaagg gctccacccc atcttaagct ctgtcttccg 2160
tggcacaatt ccaagttctt gacgttagta attgttaaag gaatggcaaa ctgttttgtt 2220
ttgaaggatc tttctacagt ctggtcttac ccatgttcct agcaaccctg agatgatttt 2280
cttccattta ccaaagcagc cgggtcagtg ctttctcacg ttgccgtatt cttcaggtat 2340
tagtcagctt cagaagccct gctcccattt ttccacccac ccattccccc ataaaacagc 2400
ttattgtctc caagacaata gacatttaaa atgtgatgcg ggtttatgat ccagaccaca 2460
atcagaatta tatcttgggt catttaaaaa aaaaaaaaaa 2500
<210> 7
<211> 900
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No:3853885CB1
<400> 7
ccgagcgcgg ggcaccgggg gcctcctgta taggcgggca ccatgggctc ctgctccggc 60
cgctgcgcgc tcgtcgtcct ctgcgctttt cagctggtcg ccgccctgga gaggcaggtg 120
tttgacttcc tgggctacca gtgggcgccc atcctggcca actttgtcca catcatcatc 180
gtcatcctgg gactcttcgg caccatccag taccggctgc gctacgtcat ggtgtacacg 240
ctgtgggcag ccgtctgggt cacctggaac gtcttcatca tctgcttcta cctggaagtc 300
ggtggcctct tacaggacag cgagctactg accttcagcc tctcccggca tcgctcctgg 360
tggcgtgagc gctggccagg ctgtctgcat gaggaggtgc cagcagtggg cctcggggcc 420
ccccatggcc aggccctggt gtcaggtgct ggctgtgccc tggagcccag ctatgtggag 480
gccctacaca gtggcctgca gatcctgatc gcgcttctgg gctttgtctg tggctgccag 540
gtggtcagcg tgtttacgga ggaagaggac agctttgatt tcattggtgg atttgatcca 600
tttcctctct accatgtcaa tgaaaagcca tccagtctct tgtccaagca ggtgtacttg 660
cctgcgtaag tgaggaaaca gctgatcctg ctcctgtggc ctccagcctc agcgaccgac 720
cagtgacaat gacaggagct cccaggcctt gggacgcgcc cccacccagc accccccagg 780
cggccggcag cacctgccct gggttctaag tactggacac cagccagggc ggcagggcag 840
tgccacggct ggctgcagcg tcaagagagt ttgtaatttc ctttctctta aaaaaaaaaa 900
<210> 8
<211> 499
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No:5059410CB1
<400> 8
gggacatgat ggatcaggag gagaagacgg aggaaggctc aggcccctgt gccgaggcgg 60
gctccccaga ccaggagggc ttcttcaatc tgctgagcca cgtgcagggc gaccggatgg 120
agggacagcg ctgttcactg caagccgggc cgggccagac caccaagagc cagagcgacc 180
ccacccccga gatggacagc ctcatggaca tgctggccag tacccagggc cgccgcatgg 240
atgaccaacg tgtgacagtc agcagcctgc ccggcttcca gcccgtgggg tccaaggacg 300
gagcacagaa acgagcttgg accctcagtc cccaacccct gcttaaccct caggacccga 360
ccgctctcgg ctttcgtcgg aacagcagcc cccagccccc gacacaagcc cccttagggg 420
ctgaggcatc ctgggtctca ctcgggctcc aaaaactcgt aggaggatag acgcttgaat 480
gggtagcaag aaaaaataa 499
5/5
