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SECRETION AND TRAFFICKING MOLECULES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of secretion
and trafficking
molecules and to the use of these sequences in the diagnosis, treatment, and
prevention of vesicle
trafficking, transport, neurological, autoimmune/inflammatory, and cell
proliferative disorders, and in
the assessment of the effects of exogenous compounds on the expression of
nucleic acid and amino
acid sequences of secretion and trafficking molecules.
BACKGROUND OF THE INVENTION
Eukaryotic cells are bound by a lipid bilayer membrane and subdivided into
functionally
distinct, membrane-bound compartments. The membranes maintain the essential
differences between
the cytosol, the extracellular environment, and the lumenal space of each
intracellular organelle.
Eukaryotic ,proteins including integral membrane proteins, secreted proteins,
and proteins destined for
the lumen of organelles are synthesized within the endoplasmic reticulum (ER),
delivered to the Golgi
complex for post-translational processing and sorting, and then transported to
specific intracellular and
extracellular destinations. Material is internalized from the extracellular
environment by endocytosis, a
process essential for transmission of neuronal, metabolic, and proliferative
signals; uptake of many
essential nutrients; and defense against invading organisms. This
intracellular and extracellular
movement of protein molecules is termed vesicle trafficking. Trafficking is
accomplished by the
packaging of protein molecules into specialized vesicles which bud from the
donor organelle
membrane and fuse to the target membrane (Rothman, J.E and Wieland, F.T.
(1996) Science
272:227-234).
The transport of proteins across the ER membrane involves a process that is
similar in
bacteria, yeast, and mammals (Gorlich, D. et al. (1992) Cell 71: 489-503). In
mammalian systems,
transport is initiated by the action of a cytoplasmic signal recognition
particle (SRP) which recognizes
a signal sequence on a growing, nascent polypeptide and binds the polypeptide
and its ribosome
complex to the ER membrane through an SRP receptor located on the ER membrane.
The signal
peptide is cleaved and the ribosome complex, together with the attached
polypeptide, becomes,
membrane bound. The polypeptide is subsequently translocated across the ER
membrane and into a
vesicle (Blobel, G. and B. Dobberstein (1975) J. Cell Biol. 67:852-862).
Proteins implicated in the translocation of polypeptides across the ER
membrane in yeast
include SEC6lp, SEC62p, and SEC63p. Mutations in the genes encoding these
proteins lead to
defects in the translocation process. SEC61 may be of particular importance
since certain mutations
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in the gene for this protein inhibit the translocation of many proteins
(Gorlich, supra).
Mammalian homologs of yeast SEC61 (mSEC61) have been identified in dog and rat
(Gorlich,
supra). Mammalian SEC61 is also structurally similar to SECYp, the bacterial
cytoplasmic membrane
translocation protein. mSEC61 is found in tight association with membrane-
bound ribosomes. This
association is induced by membrane-targeting of nascent polypeptide chains and
is weakened by
dissociation of the ribosomes into their constituent subunits. mSEC61 is
postulated to be a component
of a putative protein-conducting channel, located in the ER membrane, to which
nascent polypeptides
are transferred following the completion of translation by ribosomes (Gorlich,
supra).
Several steps in the transit of material along the secretory and endocytic
pathways require the
i0 formation of transport vesicles. Specifically, vesicles form at the
transitional endoplasmic reticulum
(tER), the rim of Golgi cisternae, the face of the Trans-Golgi Network (TGN),
the plasma membrane
(PM), and tubular extensions of the endosomes. Vesicle formation occurs when a
region of
membrane buds off from the donor organelle. The membrane-bound vesicle
contains proteins to be
transported and is surrounded by a proteinaceous coat, the components of which
are recruited from
the cytosol. The initial budding and coating processes are controlled by a
cytosolic ras-like
GTP-binding protein, ADP-ribosylating factor (Arf), and adapter proteins (AP).
Cytosolic GTP-bound
Arf is also incorporated into the vesicle as it forms. Different isoforms of
both Arf and AP are
involved at different sites of budding. For example, Arfs 1, 3, and 5 are
required for Golgi budding,
Arf4 for endosomal budding, and Arf6 for plasma membrane budding. Two
different classes of coat
protein have also been identified. Clathrin coats form on vesicles derived
from the TGN and PM,
whereas coatomer (COP) coats form on vesicles derived from the ER and Golgi
(Mellman, I. (1996)
Annu. Rev. Cell Dev. Biol. 12:575-625).
In clathrin-based vesicle formation, APs bring vesicle cargo and coat proteins
together at the
surface of the budding membrane. APs are heterotetrameric complexes composed
of two large
chains: one chain comprised of an a, 'y, 8, or ~ chain with a (3 chain, a
medium chain (~,), and a small
chain (6). Clathrin binds to APs via the carboxy-terminal appendage domain of
the ~i-adaptin subunit
(Le Bourgne, R. and Hoflack, B. (1998) C~rr. Opin. Cell. Biol. 10:499-503). AP-
1 functions in protein
sorting from the TGN and endosomes to compartments of the endosomal/lysosomal
system. AP-2
functions in clathrin-mediated endocytosis at the plasma membrane, while AP-3
is associated with
endosomes and/or the TGN and recruits integral membrane proteins for transport
to lysosomes and
lysosome-related organelles. The recently isolated AP-4 complex localizes to
the TGN or a
neighboring compartment and may play a role in sorting events thought to take
place in post-Golgi
compartments (Dell'Angelica, E. C. et al. (1999) J. Biol. Chem. 274:7278-
7285). Cytosolic GTP-
bound Arf is also incorporated into the vesicle as it forms. Another GTP-
binding protein, dynamin,
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forms a ring complex around the neck of the forming vesicle and provides the
mechanochemical force
required to release the vesicle from the donor membrane. The coated vesicle
complex is then
transported through the cytosol. During the transport process, Arf bound GTP
is hydrolyzed to GDP
and the coat dissociates from the transport vesicle (West, M.A. et al. (1997)
J. Cell Biol. 138:1239-
1254).
Coatomer (COP) coats, a second class of coat proteins, form on vesicles
derived from the
ER and Golgi. COP coats can further be classified as COPI, involved in
retrograde traffic through the
Golgi and from the Golgi to the ER, and COPII, involved in anterograde traffic
from the ER to the
Golgi (Mellman, su ra). The COP coat consists of two major components, a GTP-
binding protein
(Arf or Sar) and coat protomer (coatomer). Coatomer is an equimolax complex of
seven proteins,
termed a-, ~i-, ~3'-, ~y-, ~-, E- and Z-COP. The coatomer complex binds to
dilysine motifs contained on
the cytoplasmic tails of integral membrane proteins. These include the
dilysine-containing retrieval
motif of membrane proteins of the ER and dibasic/diphenylamine motifs of
members of the p24 family.
The p24 family of type I membrane proteins represents the major membrane
proteins of COPI
vesicles. (Harter, C. and Wieland, F.T. (1998) Proc. Natl. Acad. Sci. USA
95:1.1649-11654.)
Vesicles can undergo homotypic,fusing with a same type vesicle, or
heterotypic, fusing with a
different type vesicle, fusion. Molecules required for appropriate targeting
and fusion of vesicles
include proteins in the vesicle membrane, the target membrane, and proteins
recruited from the
cytosol. During budding of the vesicle from the donor compartment, an integral
membrane protein,
VAMP (vesicle-associated membrane protein) is incorporated into the vesicle.
Soon after the vesicle
uncoats, a cytosolic prenylated GTP-binding protein, Rab, is inserted into the
vesicle membrane. The
amino acid sequence of Rab proteins reveals conserved GTP-binding domains
characteristic of Ras
superfamily members. In the vesicle membrane, GTP-bound Rab interacts with
VAMP. Once the
vesicle reaches the target membrane, a GTPase activating protein (GAP) in the
target membrane
converts the Rab protein to the GDP-bound form. A cytosolic protein, guanine-
nucleotide dissociation
inhibitor (GDI) then removes GDP-bound Rab from the vesicle membrane. Several
Rab isoforms
have been identified and appear to associate with specific compartments within
the cell. For example,
Rabs 4, 5, and 11 are associated with the early endosome, whereas Rabs 7 and 9
associate with the
late endosome. These differences may provide selectivity in the association
between vesicles and
their target membranes. (Novick, P., and Zerial, M. (1997) Cur. Opin. Cell
Biol. 9:496-504.)
i
Docking of the transport vesicle with the target membrane involves the
formation of a
complex between the vesicle SNAP receptor (v-SNARE), target membrane (t-)
SNARES, and
certain other membrane and cytosolic proteins. Many of these other proteins
have been identified
although their exact functions in the docking complex remain uncertain
(Tellam, J.T. et al. (1995) J.
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Biol. Chem. 270:5857-5863; Hata, Y. and Sudhof, T,C. (1995) J. Biol. Chem.
270:13022-13028). '
N-ethylmaleimide sensitive factor (NSF) and soluble NSF-attachment protein (a-
SNAP and (3-SNAP)
are two such proteins that are conserved from yeast to man and function in
most intracellular
membrane fusion reactions. Sec1 represents a family of yeast proteins that
function at many different
stages in the secretory pathway including membrane fusion. Recently, mammalian
homologs of Secl,
called Munc-18 proteins, have been identified (Katagiri, H. et al. (1995) J.
Biol. Chem. 270:4963-4966;
Hata et al. supra).
The SNARE complex involves three SNARE molecules, one in the vesicular
membrane and
two in the target membrane. Together they form a rod-shaped complex of four a-
helical coiled-coils.
l0 The membrane anchoring domains of all three SNAREs project from one end of
the rod. This
complex is similar to the rod-like structures formed by fusion proteins
characteristic of the enveloped
viruses, such as myxovirus, influenza, filovirus (Ebola), and the HIV and SIV
retroviruses (Skehel, J.J.,
and Wiley, D.C. (1998) Cell 95:871-874). It has been proposed that the SNARE
complex is sufficient
for membrane fusion, suggesting that the proteins which associate with the
complex provide regulation
over the fusion event (Weber, T. et al. (1998) Cell 92:759-772). For example,
in neurons, which
exhibit regulated exocytosis, docked vesicles do not fuse with the presynaptic
membrane until
depolarization, which leads to an influx of calcium (Bennett, M.K., and
Scheller, R.H. (1994) Annu.
Rev. Biochem. 63:63-100). Synaptotagmin, an integral membrane protein in the
synaptic vesicle,
associates with the t-SNARE syntaxin in the docking complex. Synaptotagmin
binds calcium in a
complex with negatively charged phospholipids, which allows the cytosolic SNAP
protein to displace
synaptotagmin from syntaxin and fusion to occur. Thus, synaptotagmin is a
negative regulator of
fusion in the neuron. (Littleton, J.T. et al. (1993) Cell 74:1125-1134.)
The most abundant membrane protein of synaptic vesicles appears to be the
glycoprotein
synaptophysin, a 38 kDa protein with four transmembrane domains and two
intravesicular loops.
Synaptophysin monomers associate into homopolymers which form channels in the
synaptic vesicle
membrane. Synaptophysin's calcium-binding ability, tyrosine phosphorylation,
and widespread
distribution in neural tissues suggest a potential role in neurosecretion
(Bennett, s_u~ra.).
The transport of proteins into and out of vesicles relies on interactions
between cell
membranes and a supporting membrane cytoskeleton consisting of spectrin and
other proteins. A
large family of related pxoteins called ankyrins participate in the transport
process by binding to the
membrane skeleton protein spectrin and to a protein in the cell membrane
called band 3, a component
of an anion channel in the cell membrane. Ankyrins therefore function as a
critical link between the
cytoskeleton and the cell membrane.
Originally found in association with erythroid cells, ankyrins are also found
in other tissues as
4
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well (Birkenmeier, C.S. et al. (1993) J. Biol. Chem. 268:9533-9540). Ankyrins
are large proteins
(--1800 amino acids) containing an N-terminal, 89 kDa domain that binds the
cell membrane proteins
band 3 and tubulin, a central 62 kDa domain that binds the cytoskeletal
proteins spectrin and vimentin,
and a C-terminal, 55 kDa regulatory domain that functions as a modifier of the
binding activities of the
other two domains. Individual genes for ankyrin are able to produce multiple
anky~in isoforms by
various insertions and deletions. These isoforms are of nearly identical size
but may have different
functions. In addition, smaller transcripts are produced which are missing
large regions of the coding
sequences from the N-terminal (band 3 binding), and central (spectrin binding)
domains. The
existence of such a large family of ankyrin proteins and the observation that
more than one type of
l0 ankyrin may be expressed in the same cell type suggests that ankyrins may
have more specialized
functions than simply binding the membrane skeleton to the plasma membrane
(Birkenmeier, supra).
In humans, two isoforms of ankyrin are expressed, alternatively, in developing
erythroids and
mature erythroids, respectively (Lambert, S. et. al. (1990) Proc. Natl. Acad.
Sci. USA 87:1730-1734).
A deficiency in erythroid spectrin and ankyrin has been associated with the
hemolytic anemia,
hereditary spherocytosis (Coetzer, T.L. et al. (1988) New Engl. J. Med.
318:230-234).
Correct trafficking of proteins is of particular importance for the proper
function of epithelial
cells, which are polarized into distinct apical and basolateral domains
containing different cell
membrane components such as lipids and membrane-associated proteins. Certain
proteins are flexible
and may be sorted to the basolateral or apical side depending upon cell type
or growth conditions. For
example, the kidney anion exchanger (kAE1) can be retargeted from the apical
to the basolateral
domain if cells are cultured at higher density. The protein kanadaptin was
isolated as a protein which
binds to the cytoplasmic domain of kAEl . It also colocalizes with kAE1 in
vesicles, but not in the
membrane, suggesting that kanadaptin's function is to guide kAE1-containing
vesicles to the
basolateral target membrane (Chen, J. et al. (1998) J. Biol. Chem. 273:1038-
1043).
Vesicle trafficking is crucial in the process of neurotransmission. Synaptic
vesicles carry
neurotransmitter molecules from the cytoplasm of .a neuron to the synapse.
Rab3's are a family of
GTP-binding proteins located on synaptic vesicles. The RIM family of proteins
are thought to be
effectors for Rab3's (Wang, Y. et al. (2000) J. Biol. Chem. 275:20033-20044).
Rabphilin-3 is a
synaptic vesicle protein. Granuphilins are proteins with homology to
rabphilins, and may have a unique
role in exocytosis (Wang, J. et al. (1999) J. Biol. Chem. 274:28542-28548).
The etiology of numerous human diseases and disorders can be attributed to
defects in the
trafficking of proteins to organelles or the cell surface. Defects in the
trafficking of membrane-bound
receptors and ion channels are associated with cystic fibrosis (cystic
fibrosis transmembrane
conductance regulator; CFTR), glucose-galactose malabsorption syndrome
(Na*/glucose
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cotransporter), hypercholesterolemia (low-density lipoprotein (LDL) receptor),
and forms of diabetes
mellitus (insulin receptor). Abnormal hormonal secretion is linked to
disorders including diabetes
insipidus (vasopressin), hyper- and hypoglycemia (insulin, glucagon), Grave's
disease and goiter
(thyroid hormone), and Cushing's and Addison's diseases (adrenocorticotropic
hormone; ACTH).
Cancer cells secrete excessive amounts of hormones or other biologically
active peptides.
Disorders related to excessive secretion of biologically active peptides by
tumor cells include: fasting
hypoglycemia due to increased insulin secretion from insulinoma-islet cell
tumors; hypertension due to
increased epinephrine and norepinephrine secreted from pheochromocytomas of
the adrenal medulla
and sympathetic paraganglia; and carcinoid syndrome, which includes abdominal
cramps, diarrhea, and
valvular heart disease, caused by excessive amounts of vasoactive substances
(serotonin, bradykinin,
histamine, prostaglandins, and polypeptide hormones) secreted from intestinal
tumors. Ectopic
synthesis and secretion of biologically active peptides (peptides not expected
from a tumor) includes
ACTH and vasopressin in~lung and pancreatic cancers; parathyroid hormone in
lung and bladder
cancers; calcitonin in lung and breast cancers; and thyroid-stimulating
hormone in medullary thyroid
carcinoma.
Various human pathogens alter host cell protein trafficking pathways to their
own advantage.
For example, the HIV protein Nef down-regulates cell surface expression of CD4
molecules by
accelerating their endocytosis through clathrin coated pits. This function of
Nef is important for the
spread of HIV from the infected cell (Harris, M. (1999) Curr. Biol. 9:8449-
8461). A recently
identified human protein, Nef associated factor 1 (Naf1), a protein with four
extended coiled-coil
domains, has been found to associate with Nef. Overexpression of Nafl
increased cell surface
expression of CD4, an effect which could be suppressed by Nef (Fukushi, M. et
al. (1999) FEBS Lett.
442:83-88).
The discovery of new secretion and trafficking molecules 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 vesicle trafficking, transport, neurological,
autoimmune/inflammatory, and
cell proliferative disorders, and in the assessment of the effects of
exogenous compounds on the
expression of nucleic acid and amino acid sequences of secretion and
trafficking molecules.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, secretion and trafficking
molecules, referred to
collectively as "SAT" and individually as "SAT-1," "SAT-2," "SAT-3," "SAT-4,"
"SAT-5," "SAT-6,"
"SAT-7," "SAT-8," and "SAT-9." In one aspect, the invention provides an
isolated polypeptide
selected from the group consisting of a) a polypeptide comprising an amino
acid sequence selected
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from the group consisting of SEQ ID NO;l-9, b) a polypeptide comprising a
naturally occurnng amino
acid sequence at least 90% identical to an amino acid sequence selected from
the group consisting of
SEQ ID N0:1-9, c) a biologically active fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ m NO:1-9, and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID N0:1-9. In
one alternative, the invention provides an isolated polypeptide comprising the
amino acid sequence of
SEQ m NO:l-9.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected from
the group consisting of a) a polypeptide comprising an amino acid sequence
selected from the group
l0 consisting of SEQ m NO:1-9, b) a polypeptide comprising a naturally
occurring amino acid sequence
at least 90% identical to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
9, c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-9, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID N0:1-9. In
one alternative, the
polynucleotide encodes a polypeptide selected from the group consisting of SEQ
m N0:1-9. In
another alternative, the polynucleotide is selected from the group consisting
of SEQ ID NO:10-18.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID N0:1-9, b) a polypeptide comprising a naturally occurring amino acid
sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO:1-9, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m N0:1-9, and d) an immunogenic fragment of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ ID N0:1-9. In one
alternative, the invention
provides a cell transformed with the recombinant polynucleotide. In another
alternative, the invention
provides a transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ m N0:1-9, b) a polypeptide comprising a naturally occurring amino acid
sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ m N0:1-9, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-9, and d) an immunogenic fragment of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-9. The method
comprises a) culturing
a cell under conditions suitable for expression of the polypeptide, wherein
said cell is transformed with
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a recombinant polynucleotide comprising a promoter sequence operably linked to
a polynucleotide
. encoding the polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid sequence
selected from the group consisting of SEQ m NO:1-9, b) a polypeptide
comprising a naturally
occurring amino acid sequence at least 90% identical to an amino acid sequence
selected from the
group consisting of SEQ m NO:l-9, c) a biologically active fragment of a
polypeptide having an amino
acid sequence selected from the group consisting of SEQ m NO:1-9, and d) an
immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
l0 m NO:1-9.
The invention fm.-ther provides an isolated polynucleotide selected from the
group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
m NO:10-18, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ~ N0:10-18,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
the polynucleotide of b), and e) an RNA equivalent of a)-d). In one
alternative, the polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a sample
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
m N0:10-18, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ m N0:10-18,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
the polynucleotide of b), and e) an RNA equivalent of a)-d). The method
comprises a) hybridizing the
sample with a probe comprising at least 20 contiguous nucleotides comprising a
sequence
complementary to said target polynucleotide in the sample, and which probe
specifically hybridizes to
said target polynucleotide, under conditions whereby a hybridization complex
is formed between said
probe and said target polynucleotide or fragments thereof, and b) detecting
the presence or absence of
said hybridization complex, and optionally, if present, the amount thereof. In
one alternative, the probe
comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a taxget polynucleotide
in a sample, said
target polynucleotide having a sequence of a polynucleotide selected from the
group consisting of a) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ m
N0:10-18, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
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identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:10-18, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) amplifying said
target polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b)
detecting the presence or absence of said amplified target polynucleotide or
fragment thereof, and,
optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of
a polypeptide
selected from the group consisting of a) a polypeptide comprising an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-9, b) a polypeptide comprising a
naturally occurring amino
l0 acid sequence at least 90% identical to an amino acid sequence selected
from the group consisting of
SEQ ID N0:1-9, c) a biologically active fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ m N0:1-9~ and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-9, and
a pharmaceutically acceptable excipient. In one embodiment, the composition
comprises an amino
acid sequence selected from the group consisting of SEQ m N0:1-9. The
invention additionally
provides a method of treating a disease or condition associated with decreased
expression of
functional SAT, comprising administering to a patient in need of such
treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino .
acid sequence selected from the group consisting of SEQ ID N0:1-9, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ m NO:1-9, c) a biologically active fragment
of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ m N0:1-9, and
d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-9. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional SAT, comprising
administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide composing an
amino acid sequence selected from the group consisting of SEQ m N0:1-9, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
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sequence selected from the group consisting of SEQ )D NO:l-9, c) a
biologically active fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ )D NO:1-9, and
d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-9. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a composition comprising an antagonist compound identified
by the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
SAT, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds to
a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID N0:1-9, b) a polypeptide
comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-9, c) a biologically active fragment
of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ ID NO:1-9,
and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID N0:1-9. The method comprises a) combining the polypeptide
with at least one
test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID N0:1-9, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ )D NO:1-9, c) a biologically active fragment
of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ ID NO:1-9,
and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ )D NO:l-9. 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
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selected from the group consisting of SEQ ID NO:10-18, the method comprising
a) exposing a sample
comprising the target polynucleotide to a compound, and b) detecting altered
expression of the target
polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound; b)
hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID NO:10-18, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18,
iii) a polynucleotide
having a sequence complementary to i), iv) a polynucleotide complementary to
the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions
whereby a specific
hybridization complex is formed between said probe and a target polynucleotide
in the biological
sample, said target polynucleotide selected from the group consisting of i) a
polynucleotide comprising
a polynucleotide sequence selected from the group consisting of SEQ ID N0:10-
18, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18,
iii) a polynucleotide
complementary to the polynucleotide of i), iv) a polynucleotide complementary
to the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Alternatively, the target
polynucleotide comprises a fragment
of a polynucleotide sequence selected from the group consisting of i)-v)
above; c) quantifying the
amount of hybridization complex; and d) comparing the amount of hybridization
complex in the treated
biological sample with the amount of hybridization complex in an untreated
biological sample, wherein
a difference in the amount of hybridization complex in the treated biological
sample is indicative of
toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability score for the match
between each
polypeptide and its GenBankhomolog is also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
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Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold parameters.
l0 DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to"the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing the
cell lines, protocols, reagents and vectors which are reported in the
publications and which might be
used in connection with the invention. Nothing herein is to be construed as an
admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"SAT" refers to the amino acid sequences of substantially purified SAT
obtained from any
species, particularly a mammalian species, including bovine, ovine, porcine,
marine, equine, and human,
and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
SAT. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
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compound or composition which modulates the activity of SAT either by directly
interacting with SAT
or by acting on components of the biological pathway in which S.AT
participates.
An "allelic variant" is an alternative form of the gene encoding SAT. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
S 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 SAT include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as SAT or a
polypeptide with at least one functional characteristic of SAT. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding SAT, and improper or unexpected hybridization to
allelic variants, with a
locus other than the normal chromosomal locus for the polynucleotide sequence
encoding SAT. 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 SAT. 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 SAT is retained. For example, negatively charged
amino acids may
include aspartic acid and glutamic acid, and positively charged amino acids
may include lysine and
arginine. Amino acids with uncharged polar side chains having similar
hydrophilicity values may
include: asparagine and glutamine; and serine and threonine. Amino acids with
uncharged side chains
having similar hydrophilicity values may include: leucine, isoleucine, and
valine; glycine and alanine;
and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid sequence
to the complete native amino acid sequence associated with the recited protein
molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well known
in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
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of SAT. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of SAT either by
directly interacting with SAT or by acting on components of the biological
pathway in which SAT
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind SAT 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 (KLI~. The coupled peptide is then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen used
to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic SAT, or of
any oligopeptide thereof, to
induce a specific immune response in appropriate animals or cells and to bind
with specific antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
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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 SAT or fragments of
SAT may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCl), detergents
(e.g., sodium dodecyl sulfate;
l0 SDS), and other components (e.g., Denhardt's solution, dry milk, salmon
sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to
repeated DNA sequence analysis to resolve uncalled bases, extended using the
XL-PCR kit (Applied
Biosystems, Foster City CA) in the 5' 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 axe
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 Be, Val
Lys Arg, Gin, GIu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
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Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
l0 absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
A "fragment" is a unique portion of SAT or the polynucleotide encoding SAT
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10, 15,
16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least S00 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
3o example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID NO:10-18 comprises a region of unique polynucleotide
sequence that
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specifically identifies SEQ ID N0:10-18, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID NO:10-18 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish SEQ
ID NO:10-18 from related polynucleotide sequences. The precise length of a
fragment of SEQ ID
N0:10-18 and the region of SEQ )D N0:10-18 to which the fragment coiresponds
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:1-9 is encoded by a fragment of SEQ ID N0:10-18. A
fragment
of SEQ >D NO:l-9 comprises a region of unique amino acid sequence that
specifically identifies SEQ
ID NO:1-9. For example, a fragment of SEQ ID N0:1-9 is useful as an
immunogenic peptide for the
development of antibodies that specifically recognize SEQ )D N0:1-9. The
precise length of a
fragment of SEQ ID N0:1-9 and the region of SEQ ID NO:l-9 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.
r
"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 Wl). 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
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Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available from
several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.govBLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
l0 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 fof° match: 1
Penalty for mismatch: -2
Opera Gap: 5 afad Extensior2 Gap: 2 penalties
Gap x drop-off.' SO
Expect: 10
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
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,
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explained in more detail above, generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=l, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
' example:
Matf-ix: BLOSUM62
Opera Gap: 11 af2d Extensiofa Gap: 1 penalties
Gap x drop-off 50
Expect: 10
Word Size: 3
Filtef°: 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
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hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill in
the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
l0 1 % (w/v) SDS, and about 100 ~g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (T~,~ for the
specific sequence at a defined ionic
strength and pH. The Tmis the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al. (1989)
Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor
Press, Plainview NY;
specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1 % SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used. SSC concentration may
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
sheared and denatured salmon sperm DNA at about 100-200 ~glml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
CA 02410679 2002-11-26
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to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of SAT
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
SAT 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 SAT. For example,
modulation may
cause an increase or a decrease in protein activity, binding characteristics,
or any other biological,
functional, or immunological properties of SAT,
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 antiserise 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 SAT may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in the
21
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art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary
by cell type depending on the enzymatic milieu of SAT.
"Probe" refers to nucleic acid sequences encoding SAT, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes. "Primers"
are short nucleic acids, usually DNA oligonucleotides, which may be annealed
to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
l0 identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas
South West Medical Center, Dallas TX) is capable of choosing specific primers
from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The
Primer3 primer
selection program (available to the public from the Whitehead Institute/MIT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
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avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for example,
as PCR or sequencing primers, microarray elements, or specific probes to
identify fully or partially
complementary polynucleotides in a sample of nucleic acids. Methods of
oligonucleotide selection are
not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for
example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
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
23
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instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing SAT,
nucleic acids encoding SAT, or fragments thereof may comprise a bodily fluid;
an extract from a cell,
chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA,
RNA, or cDNA, in
solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recognized by the
binding 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
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and plants, in Which one or more of the cells of the organism contains
heterologous nucleic acid
introduced by way of human intervention, such as by transgenic techniques well
known in the art. The
nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor of the cell,
by way of deliberate genetic manipulation, such as by microinjection or by
infection with a
recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in vitro
fertilization, but rather is directed to the introduction of a recombinant DNA
molecule. The transgenic
organisms contemplated in accordance with the present invention include
bacteria, cyanobacteria,
fungi, plants and animals. The isolated DNA of the present invention can be
introduced into the host
by methods known in the art, for example infection, transfection,
transformation or transconjugation.
Techniques for transferring the DNA of the present invention into such
organisms are widely known
and provided in references such as Sambrook et al. (1989), supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91
%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternative splicing of exons during mRNA processing.
The corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides will generally have significant amino acid
identity relative to each
other. A polymorphic variant is a variation in the polynucleotide sequence of
a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least
92%, at least 93%, at least
CA 02410679 2002-11-26
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94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human secretion and trafficking
molecules
(SAT), the polynucleotides encoding SAT, and the use of these compositions for
the diagnosis,
treatment, or prevention of vesicle trafficking, transport, neurological,
autoimmune/inflammatory, and
cell proliferative disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
l0 sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ ID NO:) and an
Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as
shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the
invention. Column 3
shows the GenBank identification number (Genbank ID NO:) of the nearest
GenBank homolog.
Column 4 shows the probability score for the match between each polypeptide
and its GenBank
homolog. Column 5 shows the annotation of the GenB ank homolog along with
relevant citations
where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and
2 show the polypeptide sequence identification number (SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention. Column
3 shows the number of amino acid residues in each polypeptide. Column 4 shows
potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the MOTIFS
program of the GCG sequence analysis software package (Genetics Computer
Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structure/function analysis and in some
cases, searchable
databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are secretion and
trafficking molecules. For
26
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example, SEQ ID N0:2 is 93% identical to mitsu8umin29 (GenBank ID 83077703), a
synaptophysin
family member, as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.)
The BLAST probability score is 2.9e-136, which indicates the probability of
obtainin8 the observed
polypeptide sequence ali8nment by chance. SEQ ID N0:2 also contains a
synaptophysin/synaptoporin
domain as determined by searching for statistically significant matches in the
hidden Markov model
(HMM)-based PFAM database of conserved protein family domains. (See Table 3.)
Data from
BLIMPS and PROFILESCAN analyses, and BLAST comparisons to protein signature
sequences in
the DOMO and PRODOM databases provide further corroborative evidence that SEQ
~ N0:2 is a
synaptophysin family member. SEQ ID N0:3 is 72% identical to rat apical
endosomal glycoprotein
(GenBank ID 8777776) with a BLAST probability score of 0Ø Data from BLAST
analyses against
the PRODOM database provide further corroborative evidence that SEQ ID N0:3 is
an apical
endosomal 8lycoprotein. SEQ ID N0:8 is 95% identical to Rattus norve~icus
synaptotagmin III
(GenBank ID 8484296) with a BLAST probability score of 0Ø SEQ ID N0:8 also
contains a C2
domain as determined by searching for statistically si8nificant matches in the
hidden Markov model
(HMM)-based PFAM database of conserved protein family domains. (See Table 3.)
Data from
BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative
evidence that SEQ
)D N0:8 is a C2 domain-containing protein, most likely a member of the
synaptota8min family. SEQ
ID N0:1, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID N0:7, and SEQ ID N0:9
were
analyzed and annotated in a similar manner. The algorithms and parameters for
the analysis of SEQ
ID N0:1-9 are described in Table 7.
As shown in Table 4, the full len8th polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from 8enomic
DNA, or any
combination of these two types of sequences. Columns 1 and 2 list the
polynucleotide sequence
identification number (Polynucleotide SEQ ID 1V0:) and the correspondin8
Incyte polynucleotide
consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide
of the invention.
Column 3 shows the length of each polynucleotide sequence in basepairs. Column
4 lists fragments of
the polynucleotide sequences which are useful, for example, in hybridization
or amplification
technolo8ies that identify SEQ ID N0:10-18 or that distinguish between SEQ ID
N0:10-18 and
related polynucleotide sequences. Column 5 shows identification numbers
corresponding to cDNA
sequences, codin8 sequences (exons) predicted from genomic DNA, and/or
sequence assemblages
comprised of both cDNA and 8enomic DNA. These sequences were used to assemble
the full length
polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the
nucleotide start (5')
and stop (3') positions of the cDNA and/or 8enomic sequences in column 5
relative to their respective
full len8th sequences.
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The identification numbers in Column Sof Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
1438701F1 is the
identification number of an Incyte cDNA sequence, and PANCNOT02 is the cDNA
library from
which it is derived. Incyte cDNAs for which cDNA libraries are not indicated
were derived from
pooled cDNA libraries (e.g., 70767606V1). Alternatively, the identification
numbers in column 5 may
refer to GenBank cDNAs or ESTs (e.g., 85810426) which contributed to the
assembly of the full
length polynucleotide sequences. In addition, the identification numbers in
column 5 may identify
sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UI~)
database (i.e., those
sequences including the designation "ENST"). Alternatively, the identification
numbers in column 5
may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.
e., those
sequences including the designation "NM" or "NT") or the NCBI RefSeq Protein
Sequence Records
(i. e., those sequences including the designation "NP"). Alternatively, the
identification numbers in
column 5 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together by
an "exon stitching" algorithm. For example, FL X~~~XXX N1 NZ_YYYYY N3 N4
represents a
"stitched" sequence in which X~XXX~ is the identification number of the
cluster of sequences to
which the algorithm was applied, and YYYYYis the number of the prediction
generated by the
algorithm, and N1,~,3..., if present, represent specific exons that may have
been manually edited during
analysis (See Example V). Alternatively, the identification numbers in column
5 may refer to
assemblages of exons brought together by an "exon-stretching" algorithm. For
example,
FLXXXXXX gAAAAA_gBBBBB_1 N is the identification number of a "stretched"
sequence, with
X~Ih:XXX being the Incyte project identification number, ~A_A_A_A_,4_ being
the GenBank identification
number of the human genomic sequence to which the "exon-stretching" algorithm
was applied,
gBBBBB being the GenBank identification number or NCBI RefSeq identification
number of the
nearest GenBank protein homolog, and N referring to specific exons (See
Example V). In instances
where a RefSeq sequence was used as a protein homolog for the "exon-
stretching" algorithm, a
RefSeq identifier (denoted by "NM," "NP," or "NT") may be used in place of the
GenBank identifier
(i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
Prefix ~ Type of analysis and/or examples of programs
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GNN, GFG,Exon prediction from genomic sequences using,
for example,
ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK)
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example ~.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in
column 5 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant Incyte
cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
l0 sequences which were assembled using Incyte cDNA sequences. The
representative cDNA library
is the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences
which were used to assemble and confirm the above polynucleotide sequences.
The tissues and
vectors which were used to construct the cDNA libraries shown in Table 5 are
described in Table 6.
The invention also encompasses SAT variants. A preferred SAT variant is one
which has at
15 least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid sequence
identity to the SAT amino acid sequence, and which contains at least one
functional or structural
characteristic of SAT.
The invention also encompasses polynucleotides which encode SAT. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
20 from the group consisting of SEQ ID N0:10-18, which encodes SAT. The
polynucleotide sequences
of SEQ ID NO:10-18, as presented in the Sequence Listing, embrace the
equivalent RNA sequences,
wherein occurrences of the nitrogenous base thymine are replaced with uracil,
and the sugar
backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
SAT. In
25 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 SAT. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:10-
18 which has at least about 70%, or alternatively at least about 85%, or even
at least about 95%
30 polynucleotide sequence identity to a nucleic acid sequence selected from
the group consisting of SEQ
ID NO:10-18. Any one of the polynucleotide variants described above can encode
an amino acid
sequence which contains at least one functional or structural characteristic
of SAT.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
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genetic code, a multitude of polynucleotide sequences encoding SAT, 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 SAT, and all such variations are to be considered as being
specifically disclosed.
Although nucleotide sequences which encode SAT and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring SAT under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding SAT or its
l0 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 SAT 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 SAT
and SAT
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 SAT or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
NO:10-18 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; I~immel, A.R. (1987) Methods
Enzymol. 152:507
511.) Hybridization conditions, including annealing and wash conditions, are
described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. 'The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or _
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
CA 02410679 2002-11-26
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(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley 8~ Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding SAT may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent
to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et
al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme digestions and
ligations may be used to insert an engineered double-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids
Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 primer analysis software
(National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in length,
to have a GC content of about 50% or more, and to anneal to the template at
temperatures of about
68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
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
31
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WO 02/02610 PCT/USO1/20704
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. Outputllight intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for 'sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode SAT may be cloned in recombinant DNA molecules that direct expression
of SAT, 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 SAT.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter SAT-encoding sequences for a variety of
purposes including, but not
limited to, modification of the cloning, processing, and/or expression of the
gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or improve
the biological properties of SAT, 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.
32
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WO 02/02610 PCT/USO1/20704
In another embodiment, sequences encoding SAT 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,
SAT 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 (Applied Biosystems). Additionally, the
amino acid sequence
of SAT, or any part thereof, may be altered during direct synthesis andlor
combined with sequences
from other proteins, or any part thereof, to produce a variant polypept'ide 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 SAT, the nucleotide sequences
encoding SAT or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which contains
the necessary elements for transcriptional and translational control of the
inserted coding sequence in
a suitable host. These elements include regulatory sequences, such as
enhancers, constitutive and
inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotide sequences
encoding SAT. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
SAT. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding SAT and its initiation codon and upstream regulatory
sequences axe 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. 20:225-162.)
Methods which axe well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding SAT and appropriate transcriptional and
translational control
elements. These methods include irl vitro recombinant DNA techniques,
synthetic techniques, and in
33
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. (1995)
C~.uTent Protocols in Molecular BioloQV, 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 SAT. 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, TM~ or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
i0 animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van
Heeke, G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw
Hill, New
York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and
Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di Nicola,
M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc.
Natl. Acad. Sci. USA
90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al. (1994)
Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-
242.) The
invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding SAT. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding SAT 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 SAT 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 SAT are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of SAT may be used. For
example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of SAT. A number of
vectors
34
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast SaccharomYces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et
al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of SAT. Transcription of
sequences encoding
SAT may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV
used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO
J.
l0 6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Brogue, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.) These
constl-ucts can be introduced into plant cells by direct DNA transformation or
pathogen-mediated
transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y
(1992) McGraw Hill.
New York NY, pp. 191-196.)
In mammalan cells, a number of viral-based expression systems may be utilized.
In cases
where an adenovirus is used as an expression vector, sequences encoding SAT
may be ugated into an
adenovirus transcription/translation complex consisting of the late promoter
and tripartite leader
sequence. Insertion in a non-essential El or E3 region of the viral genome may
be used to obtain
infective virus which expresses SAT in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalan 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
.25 DNA than can be contained in and expressed from a plasmid. HACs of about 6
kb to 10 Mb axe
constructed and delivered via conventional delivery methods (uposomes,
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 mammalan systems, stable
expression of
SAT in cell lines is preferred. For example, sequences encoding SAT can be
transformed into cell
ones 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 fox 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
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
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 vims thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tl~ 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,
dlafr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
l0 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),13 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest
is also present, the presence and expression of the gene may need to be
confirmed. For example, if
the sequence encoding SAT is inserted within a marker gene sequence,
transformed cells containing
sequences encoding SAT can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding SAT 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 SAT and
that express
SAT 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 SAT 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
36
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
monoclonal antibodies reactive to two non-interfering epitopes on SAT 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 hnmunolo~y, Crreene Pub.
Associates and Wiley-
Interscience, New York NY; and Pound, J.D. (1998) hnmunochemical 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 SAT
include oligolabeling,
nick translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the
sequences encoding SAT, 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 Wl), and
US Biochemical. Suitable reporter molecules or labels which may be used for
ease of detection
include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogeiuc
agents, as well as
substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding SAT 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 SAT may be designed to contain signal sequences
which direct
secretion of SAT 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
37
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
sequences encoding SAT 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 SAT
protein containing a
heterologous moiety that can be recognized by a commercially available
antibody may facilitate the
screening of peptide libraries for inhibitors of SAT 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,
l0 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 SAT encoding sequence and the heterologous protein
sequence, so that SAT 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 SAT may be
achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
SAT of the present invention or fragments thereof may be used to screen for
compounds that
specifically bind to SAT. At least one axed up to a plurality of test
compounds may be screened for
specific binding to SAT. 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
SAT, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunolo~y 1 (2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which SAT
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 SAT, either
as a secreted protein
or on the cell membrane. Preferred cells include cells from mammals, yeast,
Drosophila, or E. coli.
Cells expressing SAT or cell membrane fractions which contain SAT are then
contacted with a test
38
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
compound and binding, stimulation, or inhibition of activity of either SAT 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 SAT,
either in solution or
affixed to a solid support, and detecting the binding of SAT 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.
SAT of the present invention or fragments thereof may be used to screen for
compounds that
modulate the activity of SAT. Such compounds may include agonists,
antagonists, or partial or inverse
agonists. In one embodiment, an assay is performed under conditions permissive
for SAT activity,
wherein SAT is combined with at least one test compound, and the activity of
SAT in the presence of
a test compound is compared with the activity of SAT in the absence of the
test compound. A change
in the activity of SAT in the presence of the test compound is indicative of a
compound that modulates
the activity of SAT. Alternatively, a test compound is combined with an in
vitro or cell-free system
comprising SAT under conditions suitable for SAT activity, and the assay is
performed. In either of
these assays, a test compound which modulates the activity of SAT 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 SAT or their mammalian
homologs may be
"knocked out" in an animal model system using homologous recombination in
embryonic stem (ES)
cells. Such techniques are well known in the art and are useful for the
generation of animal models of
human disease. (See, e.g., U.S. Patent Number 5,175,383 and U.S. Patent Number
5,767,337.) For
example, mouse ES cells, such as the mouse 1291SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi,
M.R. (1989) Science 244:1288-1292). The vector integrates into the
corresponding region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using the
Cre-loxP system to knockout a gene of interest in a tissue- or developmental
stage-specific manner
(Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997)
Nucleic Acids Res.
25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell blastocysts such
as those from the C57BL16 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
39
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
therapeutic or toxic agents. .
Polynucleotides encoding SAT 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
fineages 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 SAT 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 SAT 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 SAT, e.g., by secreting SAT 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 SAT and secretion and trafficking molecules. In addition, the
expression of SAT is closely
associated with brain, spinal cord, lymphatic, and reproductive tissues.
Therefore, SAT appears to
play a role in vesicle trafficking, transport, neurological,
autoimmunelinflammatory, and cell
proliferative disorders. In the treatment of disorders associated with
increased SAT expression or
activity, it is desirable to decrease the expression or activity of SAT. In
the treatment of disorders
associated with decreased SAT expression or activity, it is desirable to
increase the expression or
activity of SAT.
Therefore, in one embodiment, SAT 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 SAT.
Examples of such disorders include, but are not limited to, a vesicle
trafficking disorder such as cystic
fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia,
diabetes mellitus, diabetes
insipidus, hyper- and hypoglycemia, Grave's disease, goiter, Cushing's
disease, and Addison's disease;
gastrointestinal disorders including ulcerative colitis, gastric and duodenal
ulcers; other conditions
associated with abnormal vesicle trafficking, including acquired
immunodeficiency syndrome (AIDS);
allergies including hay fever, asthma, and urticaria (hives); autoimmune
hemolytic anemia; proliferative
glomerulonephritis; inflammatory bowel disease; multiple sclerosis; myasthenia
gravis; rheumatoid and
osteoarthritis; scleroderma; Chediak-Higashi and Sjogren's syndromes; systemic
lupus erythematosus;
toxic shock syndrome; traumatic tissue damage; and viral, bacterial, fungal,
helminthic, and protozoal
CA 02410679 2002-11-26
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infections; a transport disorder such as akinesia, amyotrophic lateral
sclerosis, ataxia telangiectasia,
cystic fibrosis, Becker's muscular dystrophy, Bell's palsy, Charcot-Marie
Tooth disease, diabetes
mellitus, diabetes insipidus, diabetic neuropathy, Duchenne muscular
dystrophy, hyperkalemic periodic
paralysis, normokalemic periodic paralysis, Parkinson's disease, malignant
hyperthermia, multidrug
resistance, myasthenia gravis, myotonic dystrophy, catatonia, tardive
dyskinesia, dystonias, peripheral
neuropathy, cerebral neoplasms, prostate cancer; cardiac disorders associated
with transport, e.g.,
angina, bradyarrythrnia, tachyarrythmia, hypertension, Long QT syndrome,
myocarditis,
cardiomyopathy, nemaline myopathy, centronuclear myopathy, lipid myopathy,
mitochondrial
myopathy, thyrotoxic myopathy, ethanol myopathy, dermatomyositis, inclusion
body myositis, infectious
l0 myositis, polymyositis; neurological disorders associated with transport,
e.g., Alzheimer's disease,
amnesia, bipolar disorder, dementia, depression, epilepsy, Tourette's
disorder, paranoid psychoses, and
schizophrenia; and other disorders associated with transport, e.g.,
neurofibromatosis, postherpetic
neuralgia, trigeminal neuropathy, sarcoidosis, sickle cell anemia, Wilson's
disease, cataracts, infertility,
pulmonary artery stenosis, sensorineural autosomal deafness, hyperglycemia,
hypoglycemia, Grave's
disease, goiter, Cushing's disease, Addison's disease, glucose-galactose
malabsorption syndrome,
hypercholesterolemia, adrenoleukodystrophy, Zellweger syndrome, Menkes
disease, occipital horn
syndrome, von Gierke disease, cystinuria, iminoglycinuria, Hartup disease, and
Fanconi disease; 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
demyefinating 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
including Down syndrome, 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
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frontotemporal dementia; an autoimmune/inflammatory disorder such as such as
acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory
distress syndrome, allergies,
ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,
autoimmune hemolytic anemia,
autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy
(APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease,
atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel syndrome,
multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis,
osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and hehninthic
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. 1
In another embodiment, a vector capable of expressing SAT 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 SAT including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified SAT
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 SAT including, but not
limited to, those provided
above.
In still another embodiment, an agonist which modulates the activity of SAT
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of SAT including, but not limited to, those listed above.
In a further embodiment, an antagonist of SAT may be administered to a subject
to treat or
prevent a disorder associated with increased expression or activity of SAT.
Examples of such
disorders include, but are not limited to, those vesicle trafficking,
transport, neurological,
autoimmune/inflammatory, and cell proliferative disorders described above. In
one aspect, an antibody
42
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
which specifically binds SAT may be used directly as an antagonist or
indirectly as a targeting or
delivery mechanism for bringing a pharmaceutical agent to cells or tissues
which express SAT.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding SAT may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of SAT 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 SAT may be produced using methods which are generally known
in the art.
In particular, purified SAT may be used to produce antibodies or to screen
libraries of pharmaceutical
agents to identify those which specifically bind SAT. Antibodies to SAT 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 SAT 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 Calinette-Guerin) and Corynebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to SAT
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 SAT 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 SAT 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
43
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-
hybridoma
technique. (See, e.g., I~ohler, 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
SAT-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g., Burton,
D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter,
G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for SAT may also be
generated. For
example, such fragments include, but are not limited to, F(ab')2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281:)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
SAT and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two
non-interfering SAT 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 SAT. Affinity is
expressed as an association
constant, I~, which is defined as the molar concentration of SAT-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The I~
44
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
determined for a preparation of polyclonal antibodies, which are heterogeneous
in their affinities for
multiple SAT epitopes, represents the average affinity, or avidity, of the
antibodies for SAT. The Ka
determined for a preparation of monoclonal antibodies, which are monospecific
for a particular SAT
epitope, represents a true measure of affinity. High-affinity antibody
preparations with Ka ranging
from about 109 to 1012 L/mole are preferred for use in immunoassays in which
the SAT-antibody
complex must withstand rigorous manipulations. Low-affinity antibody
preparations with Ka ranging
from about 106 to 10' L/mole are preferred for use in immunopurification and
similar procedures
which ultimately require dissociation of SAT, preferably in active form, from
the antibody (Catty, D.
(1988) Antibodies, Volume I: A Practical Approach, IRI, Press, Washington DC;
Liddell, J.E. and A.
l0 Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
the quality and suitability of such preparations for certain downstream
applications. For example, a
polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of SAT-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, sera, and
Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding SAT, 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
SAT. 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 SAT.
(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
CA 02410679 2002-11-26
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al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding SAT 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)-Xl 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
l0 Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-
703), thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D. (1988)
Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA.
93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trypanosoma cruzi). In the
case where a genetic deficiency in SAT expression or regulation causes
disease, the expression of
SAT 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 SAT
are treated by constructing mammalian expression vectors encoding SAT and
introducing these
vectors by mechanical means into SAT-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; Ivies, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon (1998)
Curr. Opin. Biotechnol.
9:445-450).
Expression vectors that may be effective for the expression of SAT 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, PTI~-HYG (Clontech, Palo Alto CA). SAT may be
expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV),
Rous sarcoma virus
46
CA 02410679 2002-11-26
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(RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes), (ii) an
inducible promoter (e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998)
C~.wr. 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
FK506lrapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and Blau, H.M. su ra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding SAT from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845)'. The introduction of DNA to primary cells requires
modification of these
standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to SAT expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding SAT 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 fines producing high transducing efficiency
retroviral supernatant") discloses
a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirns 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)
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WO 02/02610 PCT/USO1/20704
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 SAT to cells which have one or more genetic
abnormalities with respect to
the expression of SAT. 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 Veima, 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 SAT to target cells which have one or more genetic
abnormalities with
respect to the expression of SAT. The use of herpes simplex virus (HSV)-based
vectors may be
i5 especially valuable for introducing SAT to cells of the central nervous
system, for which HSV has a
tropism. The construction and packaging of herpes-based vectors are well known
to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (
1999) Exp. Eye Res.
169:385-395). The construction of a HSV-7. 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 heipesvirus sequences, the generation of recombinant virus following
the transfection of
multiple plasmids containing different segments of the large herpesvirus
genomes, the growth and
propagation of herpesvirus, and the infection of cells with herpesvirus are
techniques well known to
those of ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding SAT 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) C~.uT. Opin. Biotechnol. 9:464-
469). During alphavirus
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CA 02410679 2002-11-26
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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 SAT into the
alphavirus genome in place
of the capsid-coding region results in the production of a large number of SAT-
coding RNAs and the
synthesis of high levels of SAT 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 (BHI~-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.
l0 (1997) Virology 228:74-83). The wide host range of alphaviruses will allow
the introduction of SAT
into a variety of cell types. The specific transduction of a subset of cells
in a population may require
the sorting of cells prior to transduction. The methods of manipulating
infectious cDNA clones of
alphaviruses, performing alphavirus cDNA and RNA transfections, and performing
alphavirus
infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions -10
and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can
be achieved using triple helix base-pairing methodology. Triple helix pairing
is useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in
Huber, B.E. and B.I. Carr,
Molecular and Immunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-
177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA
by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding SAT.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
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oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding SAT. 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.
l0 RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified
forms of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding SAT. 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 SAT
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding SAT may be therapeutically useful, and in the treatment of disorders
associated with
decreased SAT expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding SAT 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 taxget polynucleotide;
and selection from a
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library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding SAT 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
SAT 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 SAT. The amount of hybridization may be quantified,
thus forming the basis
for a comparison of the expression of the polynucleotide both with and without
exposure to one or
more test compounds. Detection of a change in the expression of a
polynucleotide exposed to a test
compound indicates that the test compound is effective in altering the
expression of the polynucleotide.
A screen for a compound effective in altering expression of a specific
polynucleotide can be earned
out, for example, using a 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).
5 A particular embodiment of the present invention involves screening a
combinatorial library of
oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide
nucleic acids, and modified
oligonucleotides) for antisense activity against a specific polynucleotide
sequence (Bruice, T.W. et al.
(1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No.
6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition which
generally comprises an active ingredient formulated with a pharmaceutically
acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and
proteins. Various
formulations are commonly known and are thoroughly discussed in the latest
edition of Remington's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist~of SAT,
antibodies to SAT, and mimetics, agonists, antagonists, or inhibitors of SAT.
The compositions utilized in this invention may be administered by any number
of routes
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including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-
acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and
proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the lung
have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton, J.S.
l0 et al., IT.S. Patent No. 5,997,848). Pulmonary delivery has the advantage
of administration without
needle injection, and obviates the need for potentially toxic penetration
enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising SAT or fragments thereof. For example, liposome
preparations.
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, SAT 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 SAT
or fragments thereof, antibodies of SAT, and agonists, antagonists or
inhibitors of SAT, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
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used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending ,upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity. of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and response
to therapy. Long-acting compositions may be administered every 3 to 4 days,
every week, or
biweekly depending on the half life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 ~cg, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind SAT may be used for
the diagnosis
of disorders characterized by expression of SAT, or in assays to monitor
patients being treated with
SAT or agonists, antagonists, or inhibitors of SAT. Antibodies useful for
diagnostic purposes may be
prepared in the same manner as descxibed above for therapeutics. Diagnostic
assays for SAT include
methods which utilize the antibody and a label to detect SAT 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 SAT, including ELISAs, RIAs, and FACS,
are known in
the art and provide a basis for diagnosing altered or abnormal levels of SAT
expression. Normal or
standard values for SAT expression are established by combining body fluids or
cell extracts taken
from normal mammalian subjects, for example, human subjects, with antibodies
to SAT under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of SAT
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.
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In another embodiment of the invention, the polynucleotides encoding SAT 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 SAT
may be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
SAT, and to monitor regulation of SAT levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding SAT or closely related
molecules may be used to
identify nucleic acid sequences which encode SAT. 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 SAT, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the SAT 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:10-18 or from
genomic sequences including promoters, enhancers, and introns of the SAT gene.
Means for producing specific hybridization probes for DNAs encoding SAT
include the
cloning of polynucleotide sequences encoding SAT or SAT derivatives into
vectors for the production
of mRNA probes. Such vectors are known in the art, are commercially available,
and may be used to
synthesize RNA probes in vitro by means of the addition of the appropriate RNA
polymerases and the
appropriate labeled nucleotides. Hybridization probes may be labeled by a
variety of reporter groups,
for example, by radionuclides such as 32P or 355, or by enzymatic labels, such
as alkaline phosphatase
coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotide sequences encoding SAT may be used for the diagnosis of
disorders
associated with expression of SAT. Examples of such disorders include, but are
not limited to, a
vesicle trafficking disorder such as cystic fibrosis, glucose-galactose
malabsorption syndrome,
hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper- and
hypoglycemia, Grave's disease,
goiter, Cushing's disease, and Addison's disease; gastrointestinal disorders
including ulcerative colitis,
gastric and duodenal ulcers; other conditions associated with abnormal vesicle
trafficking, including
acquired immunodeficiency syndrome (AIDS); allergies including hay fever,
asthma, and urticaria
(hives); autoimmune hemolytic anemia; proliferative glomerulonephritis;
inflammatory bowel disease;
multiple sclerosis; myasthenia gravis; rheumatoid and osteoarthritis;
scleroderma; Chediak-Higashi and
Sjogren's syndromes; systemic lupus erythematosus; toxic shock syndrome;
traumatic tissue damage;
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and viral, bacterial, fungal, hehninthic, and protozoal infections; a
transport disorder such as akinesia,
amyotrophic lateral sclerosis, ataxia telangiectasia, cystic fibrosis,
Becker's muscular dystrophy, Bell's
palsy, Charcot-Marie Tooth disease, diabetes mellitus, diabetes insipidus,
diabetic neuropathy,
Duchenne muscular dystrophy, hyperkalemic periodic paralysis, normokalemic
periodic paralysis,
Parkinson's disease, malignant hyperthermia, multidrug resistance, myasthenia
gravis, myotonic
dystrophy, catatonia, tardive dyskinesia, dystonias, peripheral neuropathy,
cerebral neoplasms, prostate
cancer; cardiac disorders associated with transport, e.g., angina,
bradyarrythmia, tachyarrythrnia,
hypertension, Long QT syndrome, myocarditis, cardiomyopathy, nemaline
myopathy, centronuclear
myopathy, lipid myopathy, mitochondrial myopathy, thyrotoxic myopathy, ethanol
myopathy,
dermatomyositis, inclusion body myositis, infectious myositis, polymyositis;
neurological disorders
associated with transport, e.g., Alzheimer's disease, amnesia, bipolar
disorder, dementia, depression,
'epilepsy, Tourette's disorder, paranoid psychoses, and schizophrenia; and
other disorders associated
with transport, e.g., neurofibromatosis, postherpetic neuralgia, trigeminal
neuropathy, sarcoidosis, sickle
cell anemia, Wilson's disease, cataracts, infertility, pulmonary artery
stenosis, sensorineural autosomal
deafness, hyperglycemia, hypoglycemia, Grave's disease, goiter, Cushing's
disease, Addison's disease,
glucose-galactose malabsorption syndrome, hypercholesterolemia,
adrenoleukodystrophy, Zellweger
syndrome, Menkes disease, occipital horn syndrome, von Gierke disease,
cystinuria, iminoglycinuria,
Hartup disease, and Fanconi disease; 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 including Down
syndrome, 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
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supranuclear palsy, corticobasal degeneration, and familial frontotemporal
dementia; an
autoimmune/inflammatory disorder such as such as acquired immunodeficiency
syndrome (AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis,
anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis, autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic
lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
l0 pericardial inflammation, osteoarrhritis, 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, primacy thrombocythemia, and cancers including adenocarcinoma,
leukemia, lymphoma,
melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of
the adrenal gland,
bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract; heart,
kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen,
testis, thymus, thyroid, and uterus. The polynucleotide sequences encoding SAT
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 SAT expression. Such qualitative or quantitative
methods are well known in
the art.
In a particular aspect, the nucleotide sequences encoding SAT may be useful in
assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding SAT 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
SAT 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
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the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of SAT,
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 SAT, under conditions suitable for hybridization or
amplification. Standard
hybridization may be quantified by comparing the values obtained from normal
subjects with values
from an experiment in which a known amount of a substantially purified
polynucleotide is used.
Standard values obtained in this manner may be compared with values obtained
from samples from
patients who are symptomatic for a disorder. Deviation from standard values is
used to establish the
l0 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 SAT
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 SAT, or a fragment of a polynucleotide complementary to the
polynucleotide encoding SAT,
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
3o encoding SAT may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic disease
in humans. Methods of SNP detection include, but are not limited to, single-
stranded conformation
polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP,
oligonucleotide primers
derived from the polynucleotide sequences encoding SAT are used to amplify DNA
using the
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polymerase chain reaction (PCR). The DNA may be dexived, for example, from
diseased or normal
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the
secondary and tertiary structures of PCR products in single-stranded form, and
these differences are
detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such as
DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico SNP
(isSNP), are capable of identifying polymorphisrris by comparing the sequence
of individual
overlapping DNA fragments which assemble into a common consensus sequence.
These computer-
based methods filter out sequence variations due to laboratory preparation of
DNA and sequencing
l0 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 SAT 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. Tmmunol. 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 below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic
profile.
In another embodiment, SAT, fragments of SAT, or antibodies specific for SAT
may be used
as elements on a microarray. The microarray may be used to monitor or measure
protein-protein
interactions, drug-target interactions, and gene expression profiles, as
described above.
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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
S a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a
signature similar to that of a compound with known toxicity, it is likely to
share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain
expression information
from a large number of genes and gene families. Ideally, a genome-wide
measurement of expression
provides the highest quality signature. Even genes whose expression is not
altered by any tested
compounds are important as well, as the levels of expression of these genes
are used to normalize the
rest of the expression data. The normalization procedure is useful for
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.
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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 axe
indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a proteome
can be subjected individually to further analysis. Proteome expression
patterns, or profiles, are
analyzed by quantifying the number of expressed proteins and their relative
abundance under given
conditions and at a given time. A profile of a cell's proteome may thus be
generated by separating
and analyzing the polypeptides of a particular tissue or cell type. In one
embodiment, the separation is
achieved using two-dimensional gel electrophoresis, in which proteins from a
sample are separated by
isoelectric focusing in the first dimension, and then according to molecular
weight by sodium dodecyl
sulfate slab gel electrophoresis in the second dimension (Steiner and
Anderson, supra). The proteins
are visualized in the gel as discrete and uniquely positioned spots, typically
by staining the gel with an
agent such as Coomassie Blue or silver or fluorescent stains. The optical
density of each protein spot
is generally proportional to the level of the protein in the sample. The
optical densities of equivalently
positioned protein spots from different samples, for example, from biological
samples either treated or
untreated with a test compound or therapeutic agent, are compared to identify
any changes in protein
spot density related to the treatment. The proteins in the spots are partially
sequenced using, for
example, standard methods employing chemical or enzymatic cleavage followed by
mass
spectrometry. The identity of the protein in a spot may be determined by
comparing its partial
sequence, preferably of at least 5 contiguous amino acid residues, to the
polypeptide sequences of the
present invention. In some cases, further sequence data may be obtained for
definitive protein
identification.
A proteomic profile may also be generated using antibodies specific for SAT to
quantify the
levels of SAT expression. In one embodiment, the antibodies are used as
elements on a microaxray,
and protein expression levels are quantified by exposing the microarray to the
sample and detecting
the levels of protein bound to each array element (Lueking, A. et al. (1999)
Anal. Biochem. 270:103-
111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a
variety of methods known in the art, for example, by reacting the proteins in
the sample with a thiol- or
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amino-reactive fluorescent compound and detecting the amount of fluorescence
bound at each array
element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
l0 In another embodiment, the toxicity of a test compound is assessed by
treating a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological sample.
A difference in the amount of protein between the two samples is indicative of
a toxic response to the
test compound in the treated sample. Individual proteins are identified by
sequencing the amino acid
residues of the individual proteins and comparing these partial sequences to
the polypeptides of the
present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared
with the amount in an untreated biological sample. A difference in the amount
of protein between the
two samples is indicative of a toxic response to the test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods.known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad.
Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalom D. et
al. (1995) PCT application W095135505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA
94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
Various types of
microarrays are well known and thoroughly described in DNA Microarrays: A
Practical Approach,
M. Schena, ed. (1999) Oxford University Press, London, hereby expressly
incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding SAT
may be used to
generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
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of a 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 P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
l0 (See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl.
Acad.~Sci. USA 83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding SAT on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to 11 q22-23, any
sequences mapping to that area may represent associated or regulatory genes
for further investigation.
(See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide
sequence of the instant
invention may also be used to detect differences in the chromosomal location
due to translocation,
inversion, etc., among normal, carrier, or affected individuals. ,
In another embodiment of the invention, SAT, 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 SAT 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
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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 SAT, or
fragments thereof,
and washed. Bound SAT is then detected by methods well known in the art.
Purified SAT 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 SAT specifically compete with a test compound
for binding SAT. In this
manner, antibodies can be used to detect the presence of any peptide which
shares one or more
antigenic determinants with SAT.
In additional embodiments, the nucleotide sequences which encode SAT may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are, therefore,
to be construed as merely illustrative, and not limitative of the remainder of
the disclosure in any way
whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below,
including U.S. Ser. No. 60/215,465, U.S. Ser. No. 60/239,384, and U.S. Ser.
No. 60/253,639, are
hereby expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some
tissues were
homogenized and lysed in guanidinium isothiocyanate, while others were
homogenized and lysed in
phenol or in a suitable mixture of denaturants, such as TRIZOL (Life
Technologies), a monophasic
solution of phenol and guanidine isothiocyanate. The resulting lysates were
centrifuged over CsCl
cushions or extracted with chloroform. RNA was precipitated from the lysates
with either isopropanol
or sodium acetate and ethanol, or by other routine methods,
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was
isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles
(QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively,
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RNA was isolated directly from tissue lysates using other RNA isolation kits,
e.g., the
POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the
UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life
Technologies), using
the recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra,
units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo Alto
CA), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli cells
including XL1-Blue, XLl-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 vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L,. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation such
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as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200
thermal cycler
(MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the .
techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public'
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein
family
databases such as PFAM. (HMM is a probabilistic approach which analyzes
consensus primary
structures of gene families. See, for example, Eddy, S.R. (1996) Curr. Opin.
Struct. Biol. 6:361-365.)
The queries were performed using programs based on BLAST, FASTA, BLIMPS, and
HMMER.
The Incyte cDNA sequences were assembled to produce full length polynucleotide
sequences.
Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched
sequences, or
Genscan-predicted coding sequences (see Examples IV and V) were used to extend
Incyte cDNA
assemblages to full length. Assembly was performed using programs based on
Phred, Phrap, and
Consed, and cDNA assemblages were screened for open reading frames using
programs based on
GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were
translated to derive
the corresponding full length polypeptide sequences. Alternatively, a
polypeptide of the invention may
. begin at any of the methionine residues of the full length translated
polypeptide. Full length polypeptide
sequences were subsequently analyzed by querying against databases such as the
GenBankprotein
databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and
hidden
Markov model (HMM)-based protein family databases such as PFAM. Full length
polynucleotide
sequences are also analyzed using MACDNASIS PRO software (Hitachi Software
Engineering,
South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and
polypeptide
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sequence alignments are generated using default parameters specified by the
CLUSTAL algorithm as
incorporated into the MEGALIGN multisequence alignment program (DNASTAR),
which also
calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and threshold
parameters. The first column of Table 7 shows the tools, programs, and
algorithms used, the second
column provides brief descriptions thereof, the third column presents
appropriate references, all of
which are incorporated by reference herein in their entirety, and the fourth
column presents, where
applicable, the scores, probability values, and other parameters used to
evaluate the strength of a
l0 match between two sequences (the higher the score or the lower the
probability value, the greater the
identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide and
polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ 1D
N0:10-18. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative secretion and trafficking molecules were initially identified by
running the Genscan
gene identification program against public genomic sequence databases (e.g.,
gbpri and gbhtg).
Genscan is a general-purpose gene identification program which analyzes
genomic DNA sequences
from a variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol.
268:78-94, and Burge,
C, and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program
concatenates predicted
exons to form an assembled cDNA sequence extending from a methionine to a stop
codon. The
output of Genscan is a FASTA database of polynucleotide and polypeptide
sequences. The maximum
range of sequence for Genscan to analyze at once was set to 30 kb. To
determine which of these
Genscan predicted cDNA sequences encode secretion and trafficking molecules,
the encoded
polypeptides were analyzed by querying against PFAM models for secretion and
trafficking
molecules. Potential secretion and trafficking molecules were also identified
by homology to Incyte
cDNA sequences that had been annotated as secretion and trafficking molecules.
These selected
Genscan-predicted sequences were then compared by BLAST analysis to the
genpept and gbpri
public databases. Where necessary, the Genscan-predicted sequences were then
edited by
comparison to the top BLAST hit from genpept to correct errors in the sequence
predicted by
Genscan, such as extra or omitted exons. BLAST analysis was also used to find
any Incyte cDNA or
public cDNA coverage of the Genscan-predicted sequences, thus providing
evidence for transcription.
When Incyte cDNA coverage was available, this information was used to correct
or confirm the
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Genscan predicted sequence. Full length polynucleotide sequences were obtained
by assembling
Genscan-predicted coding sequences with Incyte cDNA sequences and/or public
cDNA sequences
using the assembly process described in Example III. Alternatively, full
length polynucleotide
sequences were derived entirely from edited or unedited Genscan-predicted
coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Seguences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Seduences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBankprimate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenB ank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenB ank
protein homolog. The
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GenBank protein homolog, the chimeric protein, or both were used as probes to
search for homologous
genomic sequences from the public human genome databases. Partial DNA
sequences were
therefore "stretched" or extended by the addition of homologous genomic
sequences. The resultant
stretched sequences were examined to determine whether it contained a complete
gene.
VI. Chromosomal Mapping of SAT Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:10-18 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID NO:10-18 were assembled into clusters of contiguous and overlapping
sequences using
l0 assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances
are based on genetic markers mapped by Genethon which provide boundaries for
radiation hybrid
markers whose sequences were included in each of the clusters. Human genome
maps and other
resources available to the public, such as the NCBI "GeneMap' 99" World Wide
Web site
(http:/lwww.ncbi.nlm.nih.gov/genemapn, can be employed to determine if
previously identified disease
genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression .-
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
suura, ch. 7; Ausubel
(1995) supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. 'In addition, the
sensitivity of the computer
search can be modified to determine whether any particular match is
categorized as exact or similar.
The basis of the search is the product score, which is defined as:
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BLAST Score x Percent Identity
x minimum { length(Seq. 1), length(Seq. 2) }
5 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
l0 (HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity arid 100% overlap.
Alternatively, polynucleotide sequences encoding SAT are analyzed with respect
to the tissue
sources from which they were derived. For example, some full length sequences
are assembled, at
least in part, with overlapping Incyte cDNA sequences (see Example 111). Each
cDNA sequence is
derived from a cDNA library constructed from a human tissue. Each human tissue
is classified into
one of the following organ/tissue categories: cardiovascular system;
connective tissue; digestive
system; embryonic structures; endocrine system; exocrine glands; genitalia,
female; genitalia, male;
germ cells; heroic and immune system; liver; musculoskeletal system; nervous
system; pancreas;
respiratory system; sense organs; skin; stomatognathic system;
unclassified/mixed; or urinary tract.
The number of libraries in each category is counted and divided by the total
number of libraries across
all categories. Similarly, each human tissue is classified into one of the
following disease/condition
categories: cancer, cell line, developmental, inflammation, neurological,
trauma, cardiovascular, pooled,
and other, and the number of libraries in each category is counted and divided
by the total number of
libraries across all categories. The resulting percentages reflect the tissue-
arid disease-specific
expression of cDNA encoding SAT. cDNA sequences and cDNA library/tissue
information are
found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of SAT Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
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fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68 °C to about 72 °C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)2504,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE
enzyme (Life Technologies), and Pfu DNA polymerase (Stxatagene), with the
following parameters
for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 60°C, 1 min;
Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step
6: 68 °C, 5 min; Step 7: storage
at 4°C. In the alternative, the parameters for primer pair T7 and SK+
were as follows: Step 1: 94°C,
3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4:
68°C, 2 min; Step 5: Steps 2, 3, and 4
repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~cl to 10 ~l aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were religated using T4 lipase (New England Biolabs, Beverly MA) into
pUC 18 vector
(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to
fill-in restriction
CA 02410679 2002-11-26
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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
20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5'regulatory sequences using the above procedure along with
oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual hybridization Probes
Hybridization probes derived from SEQ ID NO:10-18 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~cCi of
~,~ 32p1 adenosine- triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 supe~ne size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases: Ase
I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40 °C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
X. Microarrays
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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, LTV, chemical, or mechanical bonding procedures.
A typical array may
be produced using available methods and machines well known to those of
ordinary skill in the art and
may contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science
270:467-470; Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and
J. Hodgson (1998)
Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, mieroarray 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/~1 oligo-(dT)
primer (2lmer), 1X ftrst
strand buffer, 0.03 units/~l RNase inhibitor, 500 ~M dATP, 500 ~M dGTP, 500 ~M
dTTP, 40 ~M
dCTP, 40 ~M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech).
The~reverse
transcription reaction is performed in a~ 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium
hydroxide and
incubated for 20 minutes at 85° C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
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CA 02410679 2002-11-26
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(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 ~15X 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 ~ g.
l0 Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia Biotech).
Pm-ified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a'110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522, incorporated herein by reference. 1 ~1 of the array
element DNA, at an average
concentration of 100 ng/~ 1, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINI~ER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubatiomof microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Tnc., 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 ~l of sample mixture consisting of 0.2 ~g
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide The chamber is kept at 100% humidity internally
by the addition of 140
~l of 5X SSC in a corner of the chamber. The chamber containing the arrays is
incubated for about
6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in
a first wash buffer (1X SSC, 0.1
SDS), three times for 10 minutes each at 45° C in a second wash buffer
(0.1X SSC), and dried.
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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.
l0 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
Biters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that location
to be correlated with a weight ratio of hybridizing species of 1:100,000. When
two samples from
different sources (e.g., representing test and control cells), each labeled
with a different fluorophore,
are hybridized to a single array for the purpose of identifying genes that are
differentially expressed,
the calibration is done by labeling samples of the calibrating cDNA with the
two fluorophores and
adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(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 axe excited and
' measured simultaneously, the data axe 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).
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XI. Complementary Polynucleotides
Sequences complementary to the SAT-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring SAT. 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 SAT. To
inhibit transcription, a
complementary oligonucleotide is designed from the most unique 5' sequence and
used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is
designed to prevent ribosomal binding to the SAT-encoding transcript.
XII. Expression of SAT
Expression and purification of SAT is achieved using bacterial or virus-based
expression
systems. For expression of SAT in bacteria, cDNA is subcloned into an
appropriate vector containing
an antibiotic resistance gene and an inducible promoter that directs high
levels of cDNA transcription.
Examples of such promoters include, but are not limited to, the trp-lac (tac)
hybrid promoter and the
TS or T7 bacteriophage promoter in conjunction with the lac operator
regulatory element.
Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21
(DE3). Antibiotic
resistant bacteria express SAT upon induction with isopropyl beta-D-
thiogalactopyranoside (IPTG).
Expression of SAT in eukaryotic cells is achieved by infecting insect or
mammalian cell lines with
recombinant Auto~raphica californica nuclear polyhedrosis virus (AcMNPV),
commonly known as
baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with
cDNA encoding SAT
by either homologous recombination or bacterial-mediated transposition
involving transfer plasmid
intermediates. Viral infectivity is maintained and the strong polyhedrin
promoter drives high levels of
cDNA transcription. Recombinant baculovirus is used to infect Spodoptera
fru~iperda (Sf9) insect
cells in most cases, or human hepatocytes, in some cases. Infection of the
latter requires additional
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, SAT is synthesized as a fusion protein with, e.g.,
glutathione S-
transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Schistosoma iaponicum, enables the purification of fusion proteins
on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Pharmacia
Biotech). Following purification, the GST moiety can be proteolytically
cleaved from SAT 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-
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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, sera,
ch. 10 and 16). Purified SAT obtained by these methods can be used directly in
the assays shown in
Examples XVI and XVII, where applicable.
XIII. Functional Assays
SAT function is assessed by expressing the sequences encoding SAT at
physiologically
elevated levels in mammalian cell culture systems, cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA),
both of which
contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations ox electroporation. 1-2 ,ug of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, Iaser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties, FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
The influence of SAT on gene expression can be assessed using highly purified
populations of
cells transfected with sequences encoding SAT 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 SAT and other genes of interest can be analyzed by
northern analysis
or microarray techniques.
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XIV. Production of SAT Specific Antibodies
SAT 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 SAT amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-KLH,complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide and anti-SAT activity by, for example, binding the peptide or SAT
to a substrate, blocking
with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-
iodinated goat anti-rabbit
IgG.
XV. Purification of Naturally Occurring SAT Using Specific Antibodies
Naturally occurring or recombinant SAT is substantially purified by
immunoaftinity
chromatography using antibodies specific for SAT. An immunoaffinity column is
constructed by
covalently coupling anti-SAT 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 SAT are passed over the immunoaffinity column, and the column
is washed
under conditions that allow the preferential absorbance of SAT (e.g., high
ionic strength buffers in the
presence of detergent). The column is eluted under conditions that disrupt
antibodylSAT 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
SAT is collected.
XVI. Identification of Molecules Which Interact with SAT
SAT, or biologically active fragments thereof, are labeled with 1~I Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a mufti-well plate are incubated with the
labeled SAT, washed, and
any wells with labeled SAT complex are assayed. Data obtained using different
concentrations of
SAT are used to calculate values for the number, affinity, and association of
SAT with the candidate
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CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
molecules. .
Alternatively, molecules interacting with SAT are analyzed using the yeast two-
hybrid system
as described in Fields, S. and 0. Song (1989) Nature 340:245-246, or using
commercially available kits
based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
SAT may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
XVII. Demonstration of SAT Activity
l0 SAT activity is measured by its inclusion in coated vesicles. SAT can be
expressed by
transforming a mammalian cell line such as COS7, HeLa, or CHO with an
eukaryotic expression
vector encoding SAT. Eukaryotic expression vectors are commercially available,
and the techniques
to introduce them into cells are well known to those skilled in the art. A
small amount of a second
plasmid, which expresses any one of a number of marker genes, such as (3-
galactosidase, is co-
transformed into the cells in order to allow rapid identification of those
cells which have taken up and
expressed the foreign DNA. The cells are incubated for 48-72 hours after
transformation under
conditions appropriate for the cell line to allow expression and accumulation
of SAT and ~3-
galactosidase.
Transformed cells are collected and cell lysates are assayed for vesicle
formation. A non-
hydrolyzable form of GTP, GTPYS, and an ATP regenerating system are added to
the lysate and the
mixture is incubated at 37 °C for 10 minutes. Under these conditions,
over 90% of the vesicles remain
coated (Orci, L. et al. (1989) Cell 56:357-368). Transport vesicles are salt-
released from the Golgi
membranes, loaded under a sucrose gradient, centrifuged, and fractions are
collected and analyzed by
SDS-PAGE. Co-localization of SAT with clathrin or COP coatamer is indicative
of SAT activity in
vesicle formation. The contribution of SAT in vesicle formation can be
confirmed by incubating
lysates with antibodies specific for SAT prior to GTPyS addition. The antibody
will bind to SAT and
interfere with its activity, thus preventing vesicle formation.
In the alternative, SAT activity is measured by its ability to alter vesicle
trafficking pathways.
Vesicle trafficking in cells transformed with SAT is examined using
fluorescence microscopy.
Antibodies specific for vesicle coat proteins or typical vesicle trafficking
substrates such as transferrin
or the mannose-6-phosphate receptor are commercially available. Various
cellular components such
as ER, Golgi bodies, peroxisomes, endosomes, lysosomes, and the plasmalemma
are examined.
Alterations in the numbers and locations of vesicles in cells transformed with
SAT as compared to
control cells are characteristic of SAT activity.
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CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
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.
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CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
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<110> INCYTE GENOMICS, INC.
LAL, Preeti
TANG, Y. Tom
YUE, Henry
WALIA, Narinder K.
BAUGHN, Mariah R.
DAS Debopriya
RAMKUMAR Jayala~ani
TRIBOULEY, Catherine M.
LU, Dyung Aina M.
HAFALIA, April
GANDHI, Ameena R.
LEE, Ernestine A.
XU, Yuming
BANDMAN, Olga
ELLIOT, Vlcki S.
NGUYEN, Danniel B.
BURRILL John D.
MARCUS, Gregory A.
ZINGLER, Kurt A.
LU, Yan
YAO Monique G.
GURURAJAN, Rajagopal
DING, Li
WARREN, Bridget A.
THANGAVELU, Kavitha
LEE, Sally
<120> SECRETION AND TRAFFICKING MOLECULES
<130> PF-0801 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/215,465; 60/239,384; 60/253,639
<151> 2000-06-29; 2000-10-10; 2000-11-28
<160> 18
<170> PERL Program
<210> 1
<211> 315
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 1577952CD1
<400> 1
Met Gln Arg Arg Ser Arg Gly Ile Asn Thr G1y Leu Ile Leu Leu
1 5 10 15
Leu Ser Gln Ile Phe His Val Gly Ile Asn Asn Ile Pro Pro Val
20 25 30
Thr Leu Ala Thr Leu Ala Leu Asn Ile Trp Phe Phe Leu Asn Pro
1/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
35 40 45
Gln Lys Pro Leu Tyr Ser Ser Cys Leu Ser Val Glu Lys Cys Tyr
50 55 60
Gln Gln Lys Asp Trp Gln Arg Leu Leu Leu Ser Pro Leu His His
65 70 75
Ala Asp Asp Trp His Leu Tyr Phe Asn Met Ala Ser Met Leu Trp
80 85 90
Lys Gly Ile Asn Leu Glu Arg Arg Leu Gly Ser Arg Trp Phe Ala
95 100 105
Tyr Val Ile Thr Ala Phe Ser Val Leu Thr Gly Val Val Tyr Leu
110 115 120
Leu Leu Gln Phe Ala Val Ala Glu Phe Met Asp Glu Pro Asp Phe
125 130 135
Lys Arg Ser Cys Ala Val Gly Phe Ser Gly Val Leu Phe Ala Leu
140 145 150
Lys Val Leu Asn Asn His Tyr Cys Pro Gly Gly Phe Val Asn Ile
155 160 165
Leu Gly Phe Pro Val Pro Asn Arg Phe Ala Cys Trp Val Glu Leu
170 175 180
Val Ala Ile His Leu Phe Ser Pro Gly Thr Ser Phe Ala Gly His
185 190 195
Leu Ala G1y Ile Leu Val Gly Leu Met Tyr Thr Gln Gly Pro Leu
200 205 210
Lys Lys Ile Met Glu Ala Cys A1a Gly Gly Phe Ser Ser Ser Val
215 220 225
Gly Tyr Pro Gly Arg Gln Tyr Tyr Phe Asn Ser Ser Gly Ser Ser
230 235 240
Gly Tyr Gln Asp Tyr Tyr Pro His Gly Arg Pro Asp His Tyr Glu
245 250 255
Glu Ala Pro Arg Asn Tyr Asp Thr Tyr Thr Ala Gly Leu Ser Glu
260 265 270
Glu Glu Gln Leu Glu Arg A1a Leu Gln Ala Ser Leu Trp Asp Arg
275 280 285
Gly Asn Thr Arg Asn Ser Pro Pro Pro Tyr Gly Phe His Leu Ser
290 295 300
Pro Glu G1u Met Arg Arg Gln Arg Leu His Arg,Phe Asp Ser Gln
305 310 315
<210> 2
<211> 272
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 4983705CD1
<400> 2
Met Ser Ser Thr Glu Ser A1a Gly Arg Thr Ala Asp Lys Ser Pro
1 5 10 15
Arg Gln Gln Val Asp Arg Leu Leu Val Gly Leu Arg Trp Arg Arg
20 25 30
Leu Glu Glu Pro Leu Gly Phe Ile Lys Val Leu Gln Trp Leu Phe
35 40 45
Ala Ile Phe Ala Phe Gly Ser Cys Gly Ser Tyr Ser Gly Glu Thr
2/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
50 55 ~ 60
Gly Ala Met Val Arg Cys Asn Asn Glu Ala Lys Asp Val Ser Ser
65 70 75
Ile Ile Val Ala Phe Gly Tyr Pro Phe Arg Leu His Arg Ile Gln
80 85 90
Tyr Glu Met Pro Leu Cys Asp Glu Glu Ser Ser Ser Lys Thr Met
95 100 105
His Leu Met Gly Asp Phe Ser Ala Pro Ala Glu Phe Phe Val Thr
110 l15 120
Leu Gly Ile Phe Ser Phe Phe Tyr Thr Met Ala Ala Leu Val Ile
125 130 135
Tyr Leu Arg Phe His Asn Leu Tyr Thr Glu Asn Lys Arg Phe Pro
140 145 150
Leu Val Asp Phe Cys Val Thr Val Ser Phe Thr Phe Phe Trp Leu
155 160 165
Val Ala Ala Ala Ala Trp Gly Lys Gly Leu Thr Asp Val Lys Gly
170 175 180
Ala Thr Arg Pro Ser Ser Leu Thr Ala Ala Met Ser Val Cys His
185 190 195
Gly Glu Glu Ala Val Cys Ser Ala Gly Ala Thr Pro Ser Met Gly
200 205 210
Leu Ala Asn Ile Ser Val Leu Phe Gly Phe Ile Asn Phe Phe Leu
215 220 225
Trp Ala Gly Asn Cys Trp Phe Val Phe Lys Glu Thr Pro Trp His
230 235 240
Gly Gln Gly Gln Gly Gln Asp Gln Asp Gln Asp Gln Asp Gln Gly
245 250 255.
Gln Gly Pro Ser Gln Glu Ser Ala Ala Glu Gln Gly Ala Val Glu
260 265 270
Lys Gln
<210> 3
<211> 1217
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1310465CD1
<400> 3
Met Pro Leu Ser Ser His Leu Leu Pro Ala Leu Val Leu Phe Leu
1 5 10 15
Ala Ala Gly Ser Ser Gly Trp Ala Trp Val Pro Asn His Cys Arg
20 25 30
Ser Pro Gly Gln Ala Val Cys Asn Phe Val Cys Asp Cys Arg Asp
35 40 45
Cys Ser Asp Glu Ala Gln Cys Gly Tyr His Gly Ala Ser Pro Thr
50 55 60
Leu Gly Ala Pro Phe Ala Cys Asp Phe Glu Gln Asp Pro Cys Gly
65 70 75
Trp Arg Asp Ile Ser Thr Ser Gly Tyr Ser Trp Leu Arg Asp Arg
80 85 90
Ala Gly Ala Ala Leu Glu Gly Pro Gly Pro His Ser Asp His Thr
95 100 105
3/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
Leu Gly Thr Asp Leu Gly Trp Tyr Met Ala Val Gly Thr His Arg
110 115 120
Gly Lys Glu Ala Ser Thr Ala Ala Leu Arg Ser Pro Thr Leu Arg
125 130 ~ 135
Glu Ala Ala Ser Ser Cys Lys Leu Arg Leu Trp Tyr His Ala Ala
140 145 150
Ser Gly Asp Val Ala Glu Leu Arg Val Glu Leu Thr His Gly Ala
155 160 165
Glu Thr Leu Thr Leu Trp Gln Ser Thr Gly Pro Trp Gly Pro Gly
170 175 180
Trp Gln Glu Leu Ala Val Thr Thr Gly Arg Ile Arg Gly Asp Phe
185 190 195
Arg Val Thr Phe Ser Ala Thr Arg Asn Ala Thr His Arg Gly Ala
200 205 210
Val A1a Leu Asp Asp Leu Glu Phe Trp Asp Cys Gly Leu Pro Thr
215 220 225
Pro Gln Ala Asn Cys Pro Pro Gly His His His Cys Gln Asn Lys
230 235 240
Val Cys Va1 Glu Pro Gln Gln Leu Cys Asp Gly Glu Asp Asn Cys
245 250 255
Gly Asp Leu Ser Asp G1u Asn Pro Leu Thr Cys Gly Arg His I1e
260 265 270
Ala Thr Asp Phe Glu Thr Gly Leu Gly Pro Trp Asn Arg Ser G1u
275 280 285
Gly Trp Ser Arg Asn His Arg Ala Gly Gly Pro Glu Arg Pro Ser
290 295 300
Trp Pro Arg Arg Asp His Ser Arg Asn Ser Ala Gln Gly Ser Phe
305 310 315
Leu Va1 Ser Val Ala Glu Pro Gly Thr Pro Ala Ile Leu Ser Ser
320 325 330
Pro Glu Phe Gln A1a Ser Gly Thr Ser Asn Cys Ser Leu Val Phe
335 340 345
Tyr Gln Tyr Leu Ser Gly Ser Glu Ala G1y Cys Leu Gln Leu Phe
350 355 360
Leu Gln Thr Leu Gly Pro Gly Ala Pro Arg Ala Pro Val Leu Leu
365 370 375
Arg Arg Arg Arg Gly Glu Leu Gly Thr Ala Trp Val Arg Asp Arg
380 385 390
Val Asp Ile Gln Ser Ala Tyr Pro Phe Gln Ile Leu Leu Ala Gly
395 400 405
Gln Thr Gly Pro Gly Gly Val Val Gly Leu Asp Asp Leu I1e Leu
410 415 420
Ser Asp His Cys Arg Pro Val Ser Glu Val Ser Thr Leu Gln Pro
425 430 435
Leu Pro Pro Gly Pro Arg Ala Pro Ala Pro Gln Pro Leu Pro Pro
440 445 450
Ser Ser Arg Leu Gln Asp Ser Cys Lys Gln Gly His Leu A1a Cys
455 460 465
Gly Asp Leu Cys Val Pro Pro Glu Gln Leu Cys Asp Phe Glu Glu
470 475 480
Gln Cys Ala Gly Gly Glu Asp Glu Gln Ala Cys Gly Thr Thr Asp
485 490 495
Phe Glu Ser Pro Glu Ala Gly Gly Trp Glu Asp Ala Ser Val Gly
500 505 510
Arg Leu Gln Trp Arg Arg Val Ser Ala Gln Glu Ser Gln Gly Ser
515 520 525
4/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
Ser Ala Ala Ala Ala Gly His Phe Leu Ser Leu Gln Arg Ala Trp
530 535 540
Gly Gln Leu Gly A1a Glu Ala Arg Va1 Leu Thr Pro Leu Leu Gly
545 550 555
Pro Ser Gly Pro Ser Cys Glu Leu His Leu Ala Tyr Tyr Leu Gln
560 565 570
Ser Gln Pro Arg Gly Phe Leu Ala Leu Val Val Val Asp Asn Gly
575 580 585
Ser Arg Glu Leu Ala Trp Gln Ala Leu Ser Ser Ser Ala Gly Ile
590 595 600
Trp Lys Val Asp Lys Val Leu Leu Gly Ala Arg Arg Arg Pro Phe
605 610 615
Arg Leu Glu Phe Val Gly Leu Val Asp Leu Asp Gly Pro Asp Gln
620 625 630
Gln Gly Ala Gly Val Asp Asn Val Thr Leu Arg Asp Cys Ser Pro
635 640 645
Thr Val Thr Thr Glu Arg Asp Arg Glu Val Ser Cys Asn Phe Glu
650 655 660
Arg Asp Thr Cys Ser Trp Tyr Pro Gly His Leu Ser Asp Thr His
665 670 675
Trp Arg Trp Val Glu Ser Arg Gly Pro Asp His Asp His Thr Thr
680 685 690
Gly Gln Gly His Phe Val Leu Leu Asp Pro Thr Asp Pro Leu Ala
695 700 705
Trp Gly His Ser Ala His Leu Leu Ser Arg Pro Gln Val Pro Ala
710 715 720
Ala Pro Thr Glu Cys Leu Ser Phe Trp Tyr His Leu His Gly Pro
725 730 735
Gln Ile Gly Thr Leu Arg Leu Ala Met Arg Arg Glu Gly Glu Glu
740 745 750
Thr His Leu Trp Ser Arg Ser G1y Thr Gln Gly Asn Arg Trp His
755 760 765
Glu Ala Trp Ala Thr Leu Ser His Gln Pro Gly Ser His Ala Gln
770 775 780
Tyr Gln Leu Leu Phe Glu Gly Leu Arg Asp Gly Tyr His Gly Thr
785 790 795
Met Ala Leu Asp Asp Val Ala Val Arg Pro Gly Pro Cys Trp Ala
800 805 810
Pro Asn Tyr Cys Ser Phe Glu Asp Ser Asp Cys Gly Phe Ser Pro
815 820 825
Gly Gly Gln Gly Leu Trp Arg Arg Gln Ala Asn Ala Ser Gly His
830 835 840
Ala Ala Trp Gly Pro Pro Thr Asp His Thr Thr Glu Thr Ala Gln
845 ' 850 855
Gly His Tyr Met Val Val Asp Thr Ser Pro Asp Ala Leu Pro Arg
860 865 870
Gly Gln Thr Ala Ser Leu Thr Ser Lys Glu His Arg Pro Leu Ala
875 880 885
Gln Pro Ala Cys Leu Thr Phe Trp Tyr His Gly Ser Leu Arg Ser
890 895 900
Pro Gly Thr Leu Arg Val Tyr Leu Glu Glu Arg Gly Arg His Gln
905 910 915
Val Leu Ser Leu Ser Ala His Gly Gly Leu Ala Trp Arg Leu Gly
920 925 930
Ser Met Asp Val Gln Ala Glu Arg Ala Trp Arg Val Val Phe Glu
935 940 945
5/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
Ala Val Ala Ala Gly Val Ala His Ser Tyr Val Ala Leu Asp Asp
950 955 960
Leu Leu Leu Gln Asp G1y Pro Cys Pro Gln Pro Gly Ser Cys Asp
965 970 975
Phe Glu Ser Gly Leu Cys Gly Trp Sex His Leu Ala Gly Pro Gly
980 985 990
Leu Gly Gly Tyr Ser Trp Asp Trp Gly Gly Gly Ala Thr Pro Ser
995 1000 1005
Arg Tyr Pro Gln Pro Pro Val Asp His Thr Leu Gly Thr Glu Ala
1010 1015 1020
Gly His Phe Ala Phe Phe Glu Thr Gly Val Leu Gly Pro Gly Gly
1025 1030 1035
Arg Ala Ala Trp Leu Arg Ser Glu Pro Leu Pro Ala Thr Pro Ala
1040 1045 1050
Ser Cys Leu Arg Phe Trp Tyr His Met Gly Phe Pro Glu His Phe
1055 1060 1065
Tyr Lys Gly Glu Leu Lys Val Leu Leu His Ser Ala Gln Gly Gln
1070 1075 1080
Leu Ala Val Trp Gly Ala Gly Gly His Arg Arg His Gln Trp Leu
1085 1090 1095
Glu Ala Gln Val Glu Val Ala Ser Ala Lys Glu Phe Gln Ile Val
1100 1105 1110
Phe Glu Ala Thr Leu Gly Gly Gln Pro Ala Leu Gly Pro Ile Ala
1115 - 1120 1125
Leu Asp Asp Val Glu Tyr Leu Ala Gly Gln His Cys Gln Gln Pro
1130 1135 1140
Ala Pro Ser Pro Gly Asn Thr Ala Ala Pro Gly Ser Va1 Pro Ala
1145 1150 1155
Val Val Gly Ser Ala Leu Leu Leu Leu Met Leu Leu Val Leu Leu
1160 1165 1170
Gly Leu Gly Gly Arg Arg Trp Leu Gln Lys Lys Gly Ser Cys Pro
1175 1180 1185
Phe Gln Ser Asn Thr Glu Ala Thr Ala Pro Gly Phe Asp Asn Ile
1190 1195 1200
Leu Phe Asn Ala Asp Gly Val Thr Leu Pro Ala Ser Val Thr Ser
1205 1210 1215
Asp Pro
<210> 4
<211> 589
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4291779CD1
<400> 4
Met Val Gly Gln Met Tyr Cys Tyr Pro Gly Ser His Leu Ala Arg
1 5 10 15
Ala Leu Thr Arg Ala Leu Ala Leu Ala Leu Va1 Leu Ala Leu Leu
20 25 30
Val Gly Pro Phe Leu Ser Gly Leu Ala Gly Ala Ile Pro Ala Pro
35 40 45
Gly Gly Arg Trp Ala Arg Asp Gly Pro Val Pro Pro Ala Ser Arg
6/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
50 55 60
Ser Arg Ser Val Leu Leu Asp Val Ser Ala G1y G1n Leu Leu Met
65 70 75
Val Asp Gly Arg His Pro Asp Ala Val Ala Trp Ala Asn Leu Thr
80 85 90
Asn Ala Ile Arg Glu Thr Gly Trp Ala Phe Leu Glu Leu Gly Thr
95 100 105
Ser Gly Gln Tyr Asn Asp Ser Leu Gln Ala Tyr Ala Ala Gly Val
110 115 120
Val Glu Ala Ala Val Ser Glu Glu Leu Ile Tyr Met His Trp Met
125 130 135
Asn Thr Val Val Asn Tyr Cys Gly Pro Phe Glu Tyr Glu Val Gly
140 145 150
Tyr Cys Glu Arg Leu Lys Ser Phe Leu Glu Ala Asn Leu Glu Trp
155 160 165
Met Gln Glu Glu Met Glu Ser Asn Pro Asp Ser Pro Tyr Trp His
170 175 180
Gln Val Arg Leu Thr Leu Leu Gln Leu Lys Gly~Leu Glu Asp Ser
185 190 195
Tyr Glu Gly Arg Val Ser Phe Pro Ala G1y Lys Phe Thr Tle Lys
200 205 210
Pro Leu Gly Phe Leu Leu Leu Gln Leu Ser Gly Asp Leu Glu Asp
215 220, 225
Leu Glu Leu Ala Leu Asn Lys Thr Lys Ile Lys Pro Ser Leu Gly
230 235 240
Ser Gly Ser Cys Ser Ala Leu Ile Lys Leu Leu Pro Gly Gln Ser
245 250 255
Asp Leu Leu Val Ala His Asn Thr Trp Asn Asn Tyr G1n His Met
260 265 270
Leu Arg Val Ile Lys Lys Tyr Trp Leu Gln Phe Arg Glu Gly Pro
275 280 285
Trp Gly Asp Tyr Pro Leu Val Pro Gly Asn Lys Leu Val Phe 'Ser
290 295 300
Ser Tyr Pro Gly Thr Ile Phe Ser Cys Asp Asp Phe Tyr Tle Leu
305 310 315
Gly Ser Gly Leu Val Thr Leu Glu Thr Thr Ile G1y Asn Lys Asn
320 325 330
Pro Ala Leu Trp Lys Tyr Val Arg Pro Arg Gly Cys Val Leu Glu
335 340 345
Trp Val Arg Asn Ile Val Ala Asn Arg Leu Ala Ser Asp Gly Ala
350 355 360
Thr Trp A1a Asp Ile Phe Lys Arg Phe Asn Ser Gly Thr Tyr Asn
365 370 375
Asn Gln Trp Met Ile Val Asp Tyr Lys Ala Phe Ile Pro Gly Gly
380 385 390
Pro Ser Pro G1y Ser Arg Val Leu Thr Ile Leu Glu Gln Ile Pro
395 400 405
Gly Met Val Val Va1 Ala Asp Lys Thr Ser G1u Leu Tyr Gln Lys
410 415 420
Thr Tyr Trp Ala Ser Tyr Asn Ile Pro Ser Phe Glu Thr Val Phe
425 430 435
Asn Ala Ser Gly Leu Gln Ala Leu Val Ala Gln Tyr Gly Asp Trp
440 445 450
Phe Ser Tyr Asp Gly Ser Pro Arg Ala Gln Ile Phe Arg Arg Asn
455 460 465
Gln Ser Leu Val Gln Asp Met Asp Ser Met Val Arg Leu Met Arg
7/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
470 475 480
Tyr Asn Asp Phe Leu His Asp Pro Leu Ser Leu Cys Lys Ala Cys
485 490 495
Asn Pro Gln Pro Asn G1y Glu Asn Ala Ile Ser Ala Arg Ser Asp
500 505 510
Leu Asn Pro Ala Asn Gly Ser Tyr Pro Phe Gln Ala Leu Arg Gln
515 520 525
Arg Ser His Gly Gly Ile Asp Val Lys Val Thr Ser Met Ser Leu
530 535 540
Ala Arg Ile Leu Ser Leu Leu Ala Ala Ser Gly Pro Thr Trp Asp
545 550 555
Gln Val Pro Pro Phe Gln Trp Ser Thr Ser Pro Phe Ser Gly Leu
560 565 570
Leu His Met Gly Gln Pro Asp Leu Trp Lys Phe Ala Pro Val Lys
575 580 585
Val Ser Trp Asp
<210> 5
<211> 671
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 4728247CD1
<400> 5
Met Ser Glu Leu Leu Asp Leu Ser Phe Leu Ser Glu Glu Glu Lys
1 5 10 15
Asp Leu Ile Leu Ser Val Leu Gln Arg Asp Glu G1u Val Arg Lys
20 25 . 30
Ala Asp Glu Lys Arg Ile Arg Arg Leu Lys Asn Glu Leu Leu Glu
35 40 45
Ile Lys Arg Lys Gly Ala Lys Arg Gly Ser Gln His Tyr Ser Asp
50 55 60
Arg Thr Cys Ala Arg Cys Gln Glu Ser Leu Gly Arg Leu Ser Pro
65 70 75
Lys Thr Asn Thr Cys Arg Gly Cys Asn His Leu Val Cys Arg Asp
80 85 90
Cys Arg Ile Gln Glu Ser Asn Gly Thr Trp Arg Cys Lys Val Cys
95 100 105
Ala Lys Glu Ile Glu Leu Lys Lys Ala Thr Gly Asp Trp Phe Tyr
110 115 120
Asp Gln Lys Val Asn Arg Phe Ala Tyr Arg Thr Gly Ser Glu Ile
125 130 135
Ile Arg Met Ser Leu Arg His Lys Pro Ala Val Ser Lys Arg Glu
140 145 150
Thr Val Gly Gln Ser Leu Leu His Gln Thr Gln Met G1y Asp Ile
155 160 165
Trp Pro Gly Arg Lys Ile Ile Gln Glu Arg Gln Lys Glu Pro Ser
170 175 180
Val Leu Phe Glu Val Pro Lys Leu Lys Ser Gly Lys Ser Ala Leu
185 190 195
Glu Ala Glu Ser Glu Ser Leu Asp Ser Phe Thr Ala Asp Ser Asp
200 205 210
8/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
Ser Thr Ser Arg Arg Asp Ser Leu Asp Lys Ser Gly Leu Phe Pro
215 220 225
Glu Trp Lys Lys Met Ser Ala Pro Lys Ser Gln Val Glu Lys Glu
230 235 240
Thr Gln Pro Gly Gly Gln Asn Val Val Phe Val Asp Glu Gly Glu
245 250 255
Met Ile Phe Lys Lys Asn Thr Arg Lys Ile Leu Arg Pro Ser Glu
260 265 270
Tyr Thr Lys Ser Val Ile Asp Leu Arg Pro Glu Asp Val Val His
275 280 285
Glu Ser Gly Ser Leu Gly Asp Arg Ser Lys Ser Val Pro Gly Leu
290 295 300
Asn Val Asp Met Glu Glu Glu Glu Glu Glu Glu Asp Ile Asp His
305 310 315
Leu Val Lys Leu His Arg Gln Lys Leu Ala Arg Ser Ser Met Gln
320 325 330
Ser Gly Ser Ser Met Ser Thr Ile Gly Ser Met Met Ser Ile Tyr
335 340 345
Ser G1u Ala Gly Asp Phe Gly Asn Ile Phe Val Thr Gly Arg Ile
350 355 360
Ala Phe Ser Leu Lys Tyr Glu Gln G1n Thr Gln Ser Leu Val Val
365 370 375
His Val Lys Glu Cys His Gln Leu Ala Tyr Ala Asp Glu Ala Lys
380 385 390
Lys Arg Ser Asn Pro Tyr Val Lys Thr Tyr Leu Leu Pro Asp Lys
395 400 405
Ser Arg Gln Gly Lys Arg Lys Thr Ser I1e Lys Arg Asp Thr Val
410 415 420
Asn Pro Leu Tyr Asp Glu Thr Leu Arg Tyr Glu Ile Pro Glu Ser
425 430 435
Leu Leu Ala Gln Arg Thr Leu Gln Phe Ser Val Trp His'His Gly
440 445 450
Arg Phe Gly Arg Asn Thr Phe Leu Gly Glu Ala Glu Ile Gln Met
455 460 465
Asp Ser Trp Lys Leu Asp Lys Lys Leu Asp His Cys Leu Pro Leu
470 475 480
His Gly Lys Ile Ser Ala G1u Ser Pro Thr G1y Leu Pro Ser His
485 490 495
Lys Gly Glu Leu Val Val Ser Leu Lys Tyr Ile Pro Ala Ser Lys
500 505 510
Thr Pro Val Gly Gly Asp Arg Lys Lys Ser Lys Gly Gly Glu Gly
515 520 525
Gly Glu Leu Gln Val Trp Ile Lys Glu Ala Lys Asn Leu Thr Ala
530 535 540
A1a Lys Ala Gly Gly Thr Ser Asp Ser Phe Val Lys Gly Tyr Leu
545 550 555
Leu Pro Met Arg Asn Lys Ala Ser Lys Arg Lys Thr Pro Val Met
560 565 570
Lys Lys Thr Leu Asn Pro His Tyr Asn His Thr Phe Val Tyr Asn
575 580 585
Gly Val Arg Leu Glu Asp Leu Gln His Met Cys Leu G1u Leu Thr
590 595 600
Val Trp Asp Arg Glu Pro Leu Ala Ser Asn Asp Phe Leu Gly Gly
605 610 615
Val Arg Leu Gly Val Gly Thr Gly Tle Ser Asn Gly Glu Val Val
620 ~ 625 630
9/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
Asp Trp Met Asp Ser Thr Gly Glu Glu Val Ser Leu Trp Gln Lys
635 640 645
Met Arg Gln Tyr Pro Gly Ser Trp Ala Glu Gly Thr Leu Gln Leu
650 655 660
Arg Ser Ser Met Ala Lys Gln Lys Leu Gly Leu
665 670
<210> 6
<211> 1519
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472259CD1
<400> 6
Met His Arg Glu Arg Asp Gly Val Val Arg Gln Ala Arg Glu Leu
1 5 10 15
Gln Arg Gln Leu Ala Glu Glu Leu Val Asn Arg Gly His Cys Ser
20 25 30
Arg Pro Gly Ala Ser Glu Val Ser Ala Ala Gln Cys Arg Cys Arg
35 40 ~ 45
Leu Gln Glu Val Leu Ala Gln Leu Arg Trp Gln Thr Asp Gly Glu
50 55 60
Gln Ala Ala Arg Ile Arg Tyr Leu Gln Ala Ala Leu Glu Val Glu
65 70 75
Arg Gln Leu Phe Leu Lys Tyr Ile Leu Ala His Phe Arg Gly His
80 85 90
Pro Ala Leu Ser Gly Ser Pro Asp Pro Gln Ala Val His Ser Leu
95 100 105
Glu Glu Pro Leu Pro Gln Thr Ser Ser Gly Ser Cys His Ala Pro
110 115 120
Lys Pro A1a Cys Gln Leu Gly Ser Leu Asp Ser Leu Ser Ala Glu
125 130 135
Val Gly Val Arg Ser Arg Ser Leu Gly Leu Val Ser Ser Ala Cys
140 145 150
Ser Ser Ser Pro Asp Gly Leu Leu Ser Thr His Ala Ser Ser Leu
155 160 165
Asp Cys Phe Ala Pro Ala Cys Ser Arg Ser Leu Asp Ser Thr Arg
170 175 180
Ser Leu Pro Lys Ala Ser Lys Ser Glu Glu Arg Pro Ser Ser Pro
185 190 195
Asp Thr Ser Thr Pro Gly'Ser Arg Arg Leu Ser Pro Pro Pro Ser
200 205 210
Pro Leu Pro Pro Pro Pro Pro Pro Ser Ala His Arg Lys Leu Ser
215 220 225
Asn Pro Arg Gly Gly Glu Gly Ser G1u Ser Gln Pro Cys Glu Val
230 235 240
Leu Thr Pro Ser Pro Pro Gly Leu Gly His His Glu Leu Ile Lys
245 250 255
Leu Asn Trp Leu Leu Ala Lys Ala Leu Trp Val Leu Ala Arg Arg
260 265 270
Cys Tyr Thr Leu Gln Glu Glu Asn Lys Gln Leu Arg Arg Ala Gly
275 280 285
Cys Pro Tyr Gln Ala Asp Glu Lys Val Lys Arg Leu Lys Val Lys
10/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
290 295 300
Arg Ala Glu Leu Thr Gly Leu Ala Arg Arg Leu Ala Asp Arg Ala
305 310 315
Arg Glu Leu Gln Glu Thr Asn Leu Arg Ala Val Ser Ala Pro Ile
320 325 330
Pro Gly Glu Ser Cys Ala Gly Leu Glu Leu Cys Gln Val Phe A1a
335 340 345
Arg Gln Arg Ala Arg Asp Leu Ser Glu Gln Ala Ser Ala Pro Leu
350 355 3&0
Ala Lys Asp Lys Gln Ile Glu Glu Leu Arg Gln Glu Cys His Leu
365 370 375
Leu Gln Ala Arg Val Ala Ser Gly Pro Cys Ser Asp Leu His Thr
380 385 390
Gly Arg Gly Gly Pro Cys Thr Gln Trp Leu Asn Val Arg Asp Leu
395 400 405
Asp Arg Leu Gln Arg Glu Ser Gln Arg Glu Val Leu Arg Leu Gln
410 415 420
Arg Gln Leu Met Leu Gln Gln G1y Asn Gly Gly Ala Trp Pro Glu
425 430 435
Ala Gly Gly Gln Ser Ala Thr Cys Glu Glu Val Arg Arg Gln Met
440 445 450
Leu Ala Leu Glu Arg Glu Leu Asp,Gln Arg Arg Arg Glu Cys Gln
455 460 465
~Glu Leu Gly Ala Gln Ala Ala Pro Ala Arg Arg Arg Gly Glu Glu
470 475 480
Ala Glu Thr Gln Leu Gln Ala Ala Leu Leu Lys Asn Ala Trp Leu
485 490 495
Ala Glu Glu Asn Gly Arg Leu Gln Ala Lys Thr Asp Trp Va1 Arg
500 505 510
Lys Val Glu A1a Glu Asn Ser Glu Val Arg Gly His Leu Gly Arg
515 520 525
Ala Cys Gln Glu Arg Asp Ala Ser Gly Leu Ile Ala Glu Gln Leu
530 535 540
Leu Gln Gln Ala Ala Arg Gly Gln Asp Arg Gln Gln Gln Leu Gln
545 550 555
Arg Asp Pro Gln Lys Ala Leu Cys Asp Leu His Pro Ser Trp Lys
560 565 570
Glu Ile Gln Ala Leu Gln Cys Arg Pro Gly His Pro Pro Glu Gln
575 580 585
Pro Trp Glu Thr Ser Gln Met Pro Glu Ser Gln Val Lys Gly Ser
590 595 600
Arg Arg Pro Lys Phe His Ala Arg Pro Glu Asp Tyr Ala Val Ser
605 610 615
Gln Pro Asn Arg Asp Ile Gln Glu Lys Arg Glu Ala Ser Leu Glu
&20 625 630
Glu Ser Pro Val Ala Leu Gly Glu Ser Ala Ser Val Pro Gln Val
635 640 645
Ser Glu Thr Val Pro A1a Ser Gln Pro Leu Ser Lys Lys Thr Ser
650 655 660
Ser Gln Ser Asn Ser Ser Ser Glu Gly Ser Met Trp A1a Thr Val
665 670 675
Pro Ser Ser Pro Thr Leu Asp Arg Asp Thr Ala Ser Glu Val Asp
680 685 690
Asp Leu Glu Pro Asp Ser Val Ser Leu Ala Leu Glu Met Gly Gly
695 700 705
Ser Ala Ala Pro Ala Ala Pro Lys Leu Lys Ile Phe Met Ala Gln
11/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
710 715 720
Tyr Asn Tyr Asn Pro Phe Glu Gly Pro Asn Asp His Pro Glu Gly
725 730 735
Glu Leu Pro Leu Thr Ala Gly Asp Tyr Ile Tyr Ile Phe Gly Asp
740 745 750
Met Asp Glu Asp Gly Phe Tyr Glu Gly Glu Leu Asp Asp Gly Arg
755 760 765
Arg Gly Leu Val Pro Ser Asn Phe Val Glu Gln Ile Pro Asp Ser
770 775 780
Tyr Ile Pro Gly Cys Leu Pro Ala Lys Ser Pro Asp Leu Gly Pro
785 790 795
Ser Gln Leu Pro Ala Gly GIn Asp Glu Ala Leu Glu Glu Asp Ser
800 805 810
Leu Leu Ser Gly Lys Ala Gln Gly Met Val Asp Arg Gly Leu Cys
815 820 825
Gln Met Val Arg Val Gly Ser Lys Thr G1u Val Ala Thr Glu Ile
830 835 840
Leu Asp Thr Lys Thr Glu Ala Cys Gln Leu Gly Leu Leu Gln Ser
845 850 855
Met G1y Lys Gln Gly Leu Ser Arg Pro Leu Leu Gly Thr Lys Gly
860 865 870
Val Leu Arg Met A1a Pro Met Gln Leu His Leu Gln Asn Val Thr
875 880 885
Ala Thr Ser Ala Asn Ile Thr Trp Val Tyr Ser Ser His Arg His
890 895 900
Pro His Val Val Tyr Leu Asp Asp Arg Glu His Ala Leu Thr Pro
905 910 915
Ala Gly Val Ser Cys Tyr Thr Phe Gln Gly Leu Cys Pro Gly Thr
920 925 930
His Tyr Arg Val Arg Val Glu Val Arg Leu Pro Trp Asp Leu Leu
935 940 945
Gln Val Tyr Trp Gly Thr Met Ser Ser Thr Val Thr Phe Asp Thr
950 955 960
Leu Leu Ala Gly Pro Pro Tyr Pro Pro Leu Asp Val Leu Val Glu
965 970 975
Arg His Ala Ser Pro Gly Val Leu Val Val Ser Trp Leu Pro Val
980 985 990
Thr Ile Asp Ser Ala Gly Ser Ser Asn Gly Val Gln Val Thr G1y
995 1000 1005
Tyr A1a Val Tyr Ala Asp Gly Leu Lys Val Cys Glu Val Ala Asp
1010 1015 1020
Ala Thr Ala Gly Ser Thr Val Leu Glu Phe Ser Gln Leu Gln Val
1025 1030 1035
Pro Leu Thr Trp Gln Lys Val Ser Val Arg Thr Met Ser Leu Cys
1040 1045 1050
Gly Glu Ser Leu Asp Ser Val Pro Ala Gln I1e Pro Glu Asp Phe
1055 1060 1065
Phe Met Cys His Arg Trp Pro Glu Thr Pro Pro Phe Ser Tyr Thr
1070 1075 1080
Cys Gly Asp Pro Ser Thr Tyr Arg Val Thr Phe Pro Val Cys Pro
1085 ' 1090 1095
Gln Lys Leu Ser Leu Ala Pro Pro Ser Ala Lys Ala Ser Pro His
1100 1105 1110
Asn Pro Gly Ser Cys Gly.Glu Pro Gln Ala Lys Phe Leu Glu Ala
1115 1120 1125
Phe Phe Glu Glu Pro Pro Arg Arg Gln Ser Pro Val Ser Asn Leu
12/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
1130 1135 1140
Gly Ser Glu Gly G1u Cys Pro Ser Ser Gly Ala Gly Ser Gln A1a
1145 1150 1155
Gln Glu Leu Ala Glu Ala Trp Glu Gly Cys Arg Lys Asp Leu Leu
1160 2165 2170
Phe Gln Lys Ser Pro Gln Asn His Arg Pro Pro Ser Va1 Ser Asp
1175 1180 1185
Gln Pro Gly Glu Lys Glu Asn Cys Tyr Gln His Met Gly Thr Ser
1190 1195 1200
Lys Ser Pro Ala Pro Gly Phe Ile His Leu Arg Thr Glu Cys Gly
1205 1210 1215
Pro Arg Lys Glu Pro Cys Gln Glu Lys Ala Ala Leu Glu Arg Val
1220 1225 1230
Leu Arg GIn Lys Gln Asp Ala Gln Gly Phe Thr Pro Pro Gln Leu
1235 1240 1245
Gly Ala Ser Gln Gln Tyr Ala Ser Asp Phe His Asn Val Leu Lys
1250 ~ 1255 1260
Glu Glu Gln Glu Ala Leu Cys Leu Asp Leu Trp Gly Thr Glu Arg
1265 1270 1275
Arg Glu Glu Arg Arg Glu Pro Glu Pro His Ser Arg Gln Gly Gln
1280 1285 1290
Ala Leu Gly Val Lys Arg Gly Cys Gln Leu His Glu Pro Ser Ser
1295 1300 1305
Ala Leu Cys Pro Ala Pro Ser Ala Lys Val Ile Lys Met Pro Arg
1310 1315 1320
Gly Gly Pro Gln Gln Leu Gly Thr Gly Ala Asn Thr Pro Ala Arg
1325 1330 1335
Val Phe Val Ala Leu Ser Asp Tyr Asn Pro Leu Val Met Ser Ala
1340 1345 1350
Asn Leu Lys Ala Ala Glu Glu Glu Leu Val Phe Gln Lys Arg Gln
2355 1360 1365
Leu Leu Arg Val Trp Gly Ser Gln Asp Thr His Asp Phe Tyr Leu
1370 1375 1380
Ser Glu Cys Asn Arg Gln Val Gly Asn Ile Pro Gly Arg Leu Val
1385 1390 1395
Ala Glu Met Glu Val Gly Thr Glu Gln Thr Asp Arg Arg Trp Arg
1400 1405 1410
Ser Pro Ala Gln Gly His Leu Pro Sex Val Ala His Leu Glu Asp
1415 1420 , 1425
Phe Gln Gly Leu Thr Ile Pro Gln Gly Ser Ser Leu Val Leu Gln
1430 1435 2440
Gly Asn Ser Lys Arg Leu Pro Leu Trp Thr Pro Lys Ile Met Ile
1445 1450 1455
Ala Ala Leu Asp Tyr Asp Pro G1y Asp Gly Gln Met Gly Gly Gln
1460 1465 1470
Gly Lys Gly Arg Leu Ala Leu Arg Ala Gly Asp Val Val Met Val
1475 2480 1485
Tyr Gly Pro Met Asp Asp Gln Gly Phe Tyr Tyr Gly Glu Leu Gly
1490 1495 1500
Gly His Arg Gly Leu Val Pro Ala His Leu Leu Asp His Met Ser
1505 1510 1515
Leu His Gly His
<210> 7
<212> 396
13/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476740CD1
<400> 7
Met Leu Ile Thr Val Tyr Cys Val Arg Arg Asp Leu Ser Glu Val
Z 5 10 15
Thr Phe Ser Leu Gln Val Ser Pro Asp Phe Glu Leu Arg Asn Phe
20 25 30
Lys Val Leu Cys Glu Ala Glu Ser Arg Val Pro Val Glu Glu Ile
35 ~ 40 45
Gln Ile Ile His Met Glu Arg Leu Leu Ile Glu Asp His Cys Ser
50 55 60
Leu Gly Ser Tyr Gly Leu Lys Asp Gly Asp Ile Val Val Leu Leu
65 70 75
Gln Lys Asp Asn Val Gly Pro Arg Ala Pro Gly Arg Ala Pro Asn
80 85 90
Gln Pro Arg Val Asp Phe Ser Gly IIe Ala Val Pro Gly Thr Ser
95 100 105
Ser Ser Arg Pro Gln His Pro Gly Gln Gln G1n Gln Arg Thr Pro
110 115 120
Ala Ala Gln Arg Ser Gln Gly Leu Ala Ser Gly Glu Lys Val Ala
125 130 135
Gly Leu Gln Gly Leu Gly Ser Pro Ala Leu Ile Arg Ser Met Leu
140 245 150
Leu Ser Asn Pro His Asp Leu Ser Leu Leu Lys Glu Arg Asn Pro
155 160 165
Pro Leu Ala Glu Ala Leu Leu Ser Gly Ser Leu Glu Thr Phe Ser
170 175 180
Gln Val Leu Met Glu G1n Gln Arg G1u Lys Ala Leu Arg Glu Gln
185 190 195
Glu Arg Leu Arg Leu Tyr Thr Ala Asp Pro Leu Asp Arg Glu Ala
200 205 210
Gln Ala Lys Ile Glu Glu Glu Ile Arg G1n Gln Asn Ile Glu Glu
215 220 225
Asn Met Asn Ile Ala Ile Glu Glu Ala Pro Glu Ser Phe Gly Gln
230 235 240
Val Thr Met Leu Tyr Ile Asn Cys Lys Val Asn Gly His Pro Leu
245 250 255
Lys Ala Phe Val Asp Ser Gly Ala Gln Met Thr Ile Met Ser Gln
260 265 270
Ala Cys Ala Glu Arg Cys Asn Ile Met Arg Leu Val Asp Arg Arg
275 280 285
Trp Ala Gly Val Ala Lys Gly Val Gly Thr Gln Arg Ile Ile Gly
290 295 300
Arg Val His Leu Ala Gln Ile Gln Ile G1u Gly Asp Phe Leu Gln
305 310 315
Cys Ser Phe Ser Ile Leu Glu Asp Gln Pro Met Asp Met Leu Leu
320 325 330
Gly Leu Asp Met Leu Arg Arg His Gln Cys Ser Ile Asp Leu Lys
335 340 345
Lys Asn Val Leu Val Ile Gly Thr Thr Gly Thr Gln Thr Tyr Phe
350 355 360
14/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
Leu Pro Glu Gly Glu Leu Pro Leu Cys Ser Arg Met Val Ser Gly
365 370 375
Gln Asp Glu Ser Ser Asp Lys Glu Ile Thr His Ser Val Met Asp
380 385 390
Ser Gly Arg Lys Glu His
395
<210> 8
<211> 590
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473774CD1
<400> 8
Met Ser Gly Asp Tyr Glu Asp Asp Leu Cys Arg Arg Ala Leu Ile
l 5 10 15
Leu Val Ser Asp Leu Cys Ala Arg Val Arg Asp Ala Asp Thr Asn
20 25 30
Asp Arg Cys Gln Glu Phe Asn Asp Arg Ile Arg Gly Tyr Pro Arg
35 40 45
Gly Pro Asp Ala Asp Ile Ser Val Ser Leu Leu Ser Val Ile Val
50 55 60
Thr Phe Cys Gly Ile Val Leu Leu Gly Val Ser Leu Phe Val Ser
65 70 75
Trp Lys Leu Cys Trp Val Pro Trp Arg Asp Lys Gly Gly Ser Ala
80 85 90
Val Gly Gly Gly Pro Leu Arg Lys Asp Leu Gly Pro Gly Val Gly
95 100 105
Leu Ala Gly Leu Val Gly Gly Gly Gly His His Leu A1a Ala Gly
110 115 120
Leu.Gly Gly His Pro Leu Leu Gly Gly Pro His His His Ala His
125 ~ 130 135
Ala Ala His His Pro Pro Phe Ala Glu Leu Leu Glu Pro GIy Ser
140 l45 150
Leu Gly Gly Ser Asp Thr Pro Glu Pro Ser Tyr Leu Asp Met Asp
155 160 165
Ser Tyr Pro Glu Ala Ala Ala Ala Ala Val Ala Ala Gly Val Lys
170 175 180
Pro Ser Gln Thr Ser Pro Glu Leu Pro Ser Glu Gly Gly Ala Gly
185 190 195
Ser Gly Leu Leu Leu Leu Pro Pro Ser Gly Gly Gly Leu Pro Ser
200 205 210
Ala Gln Ser His Gln Gln Val Thr Ser Leu Ala Pro Thr Thr Arg
215 220 225
Tyr Pro Ala Leu Pro Arg Pro Leu Thr Gln Gln Thr Leu Thr Ser
230 235 240
Gln Pro Asp Pro Ser Ser Glu Glu Arg Pro Pro Ala Leu Pro Leu
245 250 255
Pro Leu Pro Gly Gly Glu Glu Lys Ala Lys Leu Ile Gly Gln Ile
260 265 270
Lys Pro Glu Leu Tyr Gln Gly Thr Gly Pro Gly Gly Arg Arg Ser
275 280 285
Gly Gly Gly Pro Gly Ser Gly Glu Ala Gly Thr Gly Ala Pro Cys
15/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
290 295 300
G1y Arg Ile Ser Phe Ala Leu Arg Tyr Leu Tyr Gly Ser Asp Gln
305 310 315
Leu Val Val Arg Ile Leu Gln Ala Leu Asp Leu Pro Ala Lys Asp
320 325 330
Ser Asn Gly Phe Ser Asp Pro Tyr Val Lys Ile Tyr Leu Leu Pro
335 340 345
Asp Arg Lys Lys Lys Phe Gln Thr Lys Val His Arg Lys Thr Leu
350 355 3&0
Asn Pro Val Phe Asn Glu Thr Phe Gln Phe Ser Val Pro Leu Ala
365 370 375
Glu Leu Ala Gln Arg Lys Leu His Phe Ser Val Tyr Asp Phe Asp
380 385 390
Arg Phe Ser Arg His Asp Leu Ile Gly Gln Val Val Leu Asp Asn
395 400 405
Leu Leu Glu Leu Ala Glu Gln Pro Pro Asp Arg Pro Leu Trp Arg
41'0 415 420
Asp Ile Val Glu Gly Gly Ser Glu Lys Ala Asp Leu Gly Glu Leu
425 430 435
Asn Phe Ser Leu Cys Tyr Leu Pro Thr Ala Gly Arg Leu Thr Val
440 445 450
Thr Ile Ile Lys Ala Ser Asn Leu Lys Ala Met Asp Leu Thr Gly
455 460 465
Phe Ser Asp Pro Tyr Val Lys Ala Ser Leu Ile Ser Glu Gly Arg
470 475 480
Arg Leu Lys Lys Arg Lys Thr Ser Ile Lys Lys Asn Thr Leu Asn
485 490 495
Pro Thr Tyr Asn Glu Ala Leu Val Phe Asp Val Ala Pro Glu Ser
500 505 510
Val Glu Asn Va1 Gly Leu Sex Ile Ala Val Val Asp Tyr Asp Cys
515 520 525
Ile Gly His Asn Glu Val Ile Gly Val Cys Arg Val G1y Pro Asp
530 535 540
A1a Ala Asp Pro His G1y Arg Glu His Trp Ala Glu Met Leu Ala
545 550 555
Asn Pro Arg Lys Pro Val Glu His Trp His G1n Leu Val Glu Glu
560 565 570
Lys Thr Val Thr Ser Phe Thr Lys Gly Ser Lys Gly Leu Ser Glu
575 580 585
Lys Glu Asn Ser Glu
590
<210> 9
<211> 431
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7946329CD1
<400> 9
Met Ala Glu Ile Thr Asn Ile Arg Pro Ser Phe Asp Val Ser Pro
1 5 10 15
Val Val Ala Gly Leu Ile Gly Ala Ser Val Leu Val Val Cys Val
20 25 30
16/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
Ser Val Thr Val Phe Val Trp Ser Cys Cys His Gln Gln Ala Glu
35 40 45
Lys Lys His Lys Asn Pro Pro Tyr Lys Phe Ile His Met Leu Lys
50 55 60
Gly Ile Ser Ile Tyr Pro Glu Thr Leu Ser Asn Lys Lys Lys Ile
65 70 75
Ile Lys Val Arg Arg Asp Lys Asp Gly Pro Gly Arg Glu Gly Gly
80 85 90
Arg Arg Asn Leu Leu Val Asp Ala Ala Glu Ala Gly Leu Leu Ser
95 100 105
Arg Asp Lys Asp Pro Arg Gly Pro Ser Ser Gly Ser Cys Ile Asp
110 l15 120
Gln Leu Pro Ile Lys Met Asp Tyr Gly Glu Glu Leu Arg Ser Pro
125 130 135
Ile Thr Ser Leu Thr Pro Gly Glu Ser Lys Thr Thr Ser Pro Ser
140 145 150
Ser Pro Glu Glu Asp Val Met Leu G1y Ser Leu Thr Phe Ser Val
155 l60 . 165
Asp Tyr Asn Phe Pro Lys Lys Ala Leu Val Val Thr Ile Gln Glu
170 175 180
Ala His Gly Leu Pro Val Met Asp Asp Gln Thr Gln Gly Ser Asp
185 190 195.
Pro Tyr Ile Lys Met Thr Ile Leu Pro Asp Lys Arg His Arg Val
200 205 210
Lys Thr Arg Va1 Leu Arg Lys Thr Leu Asp Pro Val Phe Asp Glu
215 220 225
Thr Phe Thr Phe Tyr Gly Tle Pro Tyr Ser Gln Leu Gln Asp Leu
230 235 240
Val Leu His Phe Leu Val Leu Ser Phe Asp Arg Phe Ser Arg Asp
245 250 255
Asp Val Ile G1y Glu Val Met Val Pro Leu Ala Gly Val Asp Pro
260 265 270
Ser Thr Gly Lys Val Gln Leu Thr Arg Asp Ile Ile Lys Arg Asn
275 280 285
Ile Gln Lys Cys Ile Ser Arg Gly Glu Leu Gln Val Ser Leu Ser
290 295 300
Tyr Gln Pro Val Ala Gln Arg Met Thr Val Val Val Leu Lys Ala
305 310 315
Arg His Leu Pro Lys Met Asp Ile Thr Gly Leu Ser G1y Asn Pro
320 ' 325 330
Tyr Val Lys Val Asn Val Tyr Tyr Gly Arg Lys Arg Ile Ala Lys
335 340 345
Lys Lys Thr His Val Lys Lys Cys Thr Leu Asn Pro Ile Phe Asn
350 355 360
Glu Ser Phe Ile Tyr Asp Ile Pro Thr Asp Leu Leu Pro Asp Ile
365 370 375
Ser Ile Glu Phe Leu Val Ile Asp Phe Asp Arg Thr Thr Lys Asn
380 385 390
Glu Val Val Gly Arg Leu Ile Leu Gly Ala His Ser Va1 Thr Ala
395 400 405
Ser Gly Ala Glu His Trp Arg Glu Val Cys Glu Ser Pro Arg Lys
410 415 420
Pro Val Ala Lys Trp His Ser Leu Ser Glu Tyr
425 430
<210> 10
17/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
<211> 3424
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1577952CB1
<400> 10
cgcacgtgcg cgcgaagacg tggggacgca ggcgggtcgt agagagcgtt cagccgtctg 60
tatatctccc cagatacctg aaactgacca cctgagtacg ttttcccatt gctgagctgt 120
ttccctgata tctggccatg caacggagat caagagggat aaatactgga cttattctac 180
tcctttctca aatcttccat gttgggatca acaatattcc acctgtcacc ctagcaactt 240
tggccctcaa catctggttc ttcttgaacc ctcagaagcc actgtatagc tcctgc.ctta 300
gtgtggagaa gtgttaccag caaaaagact ggcagcgttt actgctctct ccccttcacc 360
atgctgatga ttggcatttg tatttcaata tggcatccat gctctggaaa ggaataaatc 420
tagaaagaag actgggaagt agatggtttg cctatgttat caccgcattt tctgtactta 480
ctggagtggt atacctgctc ttgcaatttg ctgttgccga atttatggat gaacctgact 540
tcaaaaggag ctgtgctgta ggtttctcag gagttttgtt tgctttgaaa gttcttaaca 600
accattattg ccctggaggc tttgtcaaca ttttgggctt tcctgtaccg aacagatttg 660
cttgttgggt cgaacttgtg gctattcatt tattctcacc agggacttcc ttcgctgggc 720
atctggctgg gattcttgtt ggactaatgt acactcaagg gcctctgaag aaaatcatgg 780
aagcatgtgc aggcggtttt tcctccagtg ttggttaccc aggacggcaa tactacttta 840
atagttcagg cagctctgga tatcaggatt attatccgca tggcaggcca gatcactatg 900
aagaagcacc caggaactat gacacgtaca cagcaggact gagtgaagaa gaacagctcg 960
agagagcatt acaagccagc ctctgggacc gaggaaatac cagaaatagc ccaccaccct 1020
acgggtttca tctctcacca gaagaaatga ggagacagcg gcttcacaga ttcgatagcc 1080
agtgaggtgg catcttggga agacatggcc tattcgtgta attattgccc atttggctca 1140
ttccccaagc ccctaattca ttttaattca ttttaaacaa aagcagagta caccggtatt 1200
gctccagatc gctcacatca cctgggacag tcccatggcc cctatgagtc aactcacagc 1260
ttgcggggag tgggccttct cctggccttg ttcttgctca taaacaggtc acttcctcca 1320
tgaagagacc agtttccacg ctcccatctc tcactgctga ctcagcgatg cetctgcctc 1380
ggtctgcttt tgaagactgt gaccttcacc aggaggtttt acttacacca gtcgggaaga 1440
ttagtccctc attctgcctg gagtgccccg tgtttgactt ggcagcgggt gtggagccat 1500
ccccgcgtcc tcctggcaca ttgccactgt ggctgtccag gaacaggatg tggctgcctt 1560
ggccgaatgt tgtcctactc tccccaaccc cggcgcctca gctcctcagc tcctcgggcc 1620
cctgcgtctg gctggtgttt gcagggcttt cgctctgctc tggtattgct ctgcctttat 1680
agaaagtctt attgaagaag tgtaagaaag acctaaggtg gggaagactc ctacacacac 1740
cattagtatc agtgacacca gcaatgtagg ttcccagccc cttcccagtg gcagcttgtg 1800
tgtccaggag ataggacatc atttaacgca tcagcaaagt agcagcagat gceacataca 1860
gagtagagcg aaggcatttg gtggatcggt cactagagat ctatcttgca gaaagtatgt 1920
ttttcctcat aaaagtgcct cttaattggc cattgtacca gccacttgtc ctagccaaat 1980
gtccaaaaca cgcccttggg ccccgccacg ttacaatcca cagattgtct gtctgagtcg 2040
tttaaggcat ttcctggtgc ttgtgttcca tgaataaaag gacaaagtca gaagatcact 2100
gatgtcttac tgtcaacaga gatattttaa aagagagaag caggaaaaga tcttcctttt 2160
ttgatctaca acttatatag ttttctgatt atgcacataa tagatatgcc ttccagatgc 2220
ataaggcaaa catctggaaa gaaatatacc caaatcttag caggggttat ctttgggagt 2280
ggagtacatg ggattttgct ttcttcattt ttataatttt atattactgt cttggaagat 2340
gtgtttatgt gtgtgtgtta cttttacaat caggaaaaca tatttaataa catatagtca 2400
agaaaacaga cttaaaaata aatactatgt gtccattgag aaaattcaca atataaacag 2460
aaatacaaat aaatacatac acaattttaa agtcacctgt agccctaccc ttagaggtac 2520
ccagggttaa cattttggtg gtattgtctt atcaattttt ccgttgatac attcagcaaa 2580
tttggagcac attgaccatg gagttttgtg tccaaatcca atctgaattt acctggaaga 2640
ggccttgaca cctgcatgga aatgagctaa gaaaaccact ggagccttgg gagctctttg 2700
gcctcctggc tggcccagta atatctgagc tcctttggtt aatttataac tgatataaaa 2760
ctacatcttc tttataatat aaattgtacc tgtgagtcta gaagctttaa atgtgtttaa 2820
18/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
attaaaatat tcaagctaaa tgttactgct ctctcccaaa ttctgtaagt ttgactcccg 2880
ttaccccaat tagaagtaac ttctttgttt catgccactt ttatagcatt tggtaattct 2940
gctataacac atcttgcccc tattattaac tgtgcacagc tacacaaagg tgtgccttct 3000
acgtgggaac atggattgtg aatgactctg taatgaggcc tgagtcttag ttatctttcc 3060
actcactccc cgtctcccct ttccaacccc aaaggctcac gataggggct cactaaatgt 3120
cagtgtttca ccaaagtatt ttttccattg tattaagagt ccagtcactg tatatggaag 3180
tattttattt tttatttttt tatatcactt gagtccacta gtagtacttc cttgctctgt 3240
ttgacttgtc agatacaaag acacgggatt agattttggg tggtaaaatt gtgatacgca 3300
tggctgttga tggagtggaa catcttagtg atgtgagaaa ggtcatttta gttataaatg 3360
taaaccaatt actttagcac aacaataaag atgttctgga aattaaaaaa aaaaaaaaaa 3420
3424
aagg
<210> 11
<211> 1033
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4983705CB1
<400> 11
agcggccgca gcctctgaga gcacgaacag cagcgccccc gcgtcccagc cagccagcca 60
gccagactgg actccggccc accgacggcc gctcgcgctc cggccccgct cgcctgctct 120
gccccggacc tgcagctccc cgctcccccg ccgtgtccgc cgcctcccgg ccagagagcc 180
aagcccccac gccgcgccca gcgctcgccg cgccagcatg tcctcgaccg agagcgccgg 240
ccgcacggcg gacaagtcgc cgcgccagca ggtggaccgc ctactcgtgg ggctgcgctg 300
gcggcggctg gaggagccgc tgggcttcat caaagttctc cagtggctct ttgctatttt 360
cgccttcggg tcctgtggct cctacagcgg ggagacagga gcaatggttc gctgcaacaa 420
cgaagccaag gacgtgagct ccatcatcgt tgcatttggc tatcccttca ggttgcaccg 480
gatccaatat gagatgcccc tctgcgatga agagtccagc tccaagacca tgcacctcat 540
gggggacttc tctgcacccg ccgagttctt cgtgaccctt ggcatctttt ccttcttcta 600
taccatggct gccctagtta tctacctgcg cttccacaac ctctacacag agaacaaacg 660
cttcccgctg gtggacttct gtgtgactgt ctccttcacc ttcttctggc tggtagctgc 720
agctgcctgg ggcaagggcc tgaccgatgt caagggggcc acacgaccat ccagcttgac 780
agcagccatg tcagtgtgcc atggagagga agcagtgtgc agtgccgggg ccacgccctc 840
tatgggcctg gccaacatct ccgtgctctt tggctttatc aacttcttcc tgtgggccgg 900
gaactgttgg tttgtgttca aggagacccc gtggcatgga cagggccagg gccaggacca 960
ggaccaggac caggaccagg gccagggtcc cagccaggag agtgcagctg agcagggagc 1020
agtggagaag cag 1033
<210> 12
<212> 3902
<212> DNA
<213> Homo Sapiens
<220>
<221> 'misc feature
<223> Incyte ID No: 1310465CB1
<400> 12
ctttccatca caggccgcac tgctccctct ggcccaacca tgcctctgtc cagccacctg 60
ctgcccgcct tggtcctgtt cctggcagca gggtcctcag gctgggcctg ggtccccaac 120
cactgcagga gccctggcca ggccgtgtgc aacttcgtgt gtgactgcag ggactgctca 180
gatgaggccc agtgtggtta ccacggggcc tcgcccaccc tgggcgcccc cttcgcctgt 240
gacttcgagc aggacccctg cggctggcgg gacattagta cctcaggcta cagctggctc 300
19/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
cgagacaggg caggggccgc actggagggt cctgggcctc actcagacca cacactgggc 360
accgacttgg gctggtacat ggccgttgga acccaccgag ggaaagaggc atccaccgca 420
gccctgcgct cgccaaccct gcgagaggca gcctcctctt gcaagctgag gctctggtac 480
cacgcggcct ctggagatgt ggctgaactg cgggtggagc tgacccatgg cgcagagacc 540
ctgaccctgt ggcagagcac agggccctgg ggccctggct ggcaggagtt ggcagtgacc 600
acaggccgca tccggggtga cttccgagtg accttctctg ccacccgaaa tgccacccac 660
aggggcgctg tggctctaga tgacctagag ttctgggact gtggtctgcc caccccccag 720
gccaactgtc ccccgggaca ccaccactgc cagaacaagg tctgcgtgga gccccagcag 780
ctgtgcgacg gggaagacaa ctgcggggac ctgtctgatg agaacccact cacctgtggc 840
cgccacatag ccaccgactt tgagacaggc ctgggcccat ggaaccgctc ggaaggctgg 900
tcccggaacc accgtgctgg tggtcctgag cgcccctcct ggccacgccg tgaccacagc 960
cggaacagtg cacagggctc cttcctggtc tccgtggccg agcctggcac ccctgctata 1020
ctctccagcc ccgaattcca agcctcaggc acctccaact gctcgctggt cttctatcag 1080
tacctgagtg ggtctgaggc tggctgcctc cagctgttcc tgcagactct ggggcccggc 1140
gccccccggg cccccgtcct gctgcggagg cgccgagggg agctggggac cgcctgggtc 1200
cgagaccgtg ttgacatcca gagcgcctac cccttccaga tcctcctggc cgggcagaca 1260
ggcccggggg gcgtcgtggg tctggacgac ctcatcctgt ctgaccactg cagaccagtc 1320
tcggaggtgt ccaccctgca gccgctgcct cctgggcccc gggccccagc cccccagccc 1380
ctgccgccca gctcgcggct ccaggattcc tgcaagcagg ggcatcttgc ctgcggggac 1440
ctgtgtgtgc ccccggaaca actgtgtgac ttcgaggagc agtgcgcagg gggcgaggac 1500
gagcaggcct gtggcaccac agactttgag tcccccgagg ctgggggctg ggaggacgcc 1560
agcgtggggc ggctgcagtg gcggcgtgtc tcagcccagg agagccaggg gtccagtgca 1620
gctgctgctg ggcacttcct gtctctgcag cgggcctggg ggcagctagg cgctgaggcc 1680
cgggtcctca cacccctcct tggcccttct ggccccagct gtgaactcca cctggcttat 1740
tatttacaga gccagccccg aggcttcctg gcactagttg tggtggacaa cggctcccgg 1800
gagctggcat ggcaggccct gagcagcagt gcaggcatct ggaaggtgga caaggtcctt 1860
ctaggggccc gccgccggcc cttccggctg gagtttgtcg gtttggtgga cttggatggc 1920
cctgaccagc agggagctgg ggtggacaac gtgaccctga gggactgtag ccccacagtg 1980
accaccgaga gagacagaga ggtctcctgt aactttgagc gggacacatg cagctggtac 2040
ccaggccacc tctcagacac acactggcgc tgggtggaga gccgcggccc tgaccacgac 2100
cacaccacag gccaaggcca ctttgtgctc ctggacccca cagaccccct ggcctggggc 2160
cacagtgccc acctgctctc caggccccag gtgccagcag cacccacgga gtgtctcagc 2220
ttctggtacc acctccatgg gccccagatt gggactctgc gcctagccat gagacgggaa 2280
ggggaggaga cacacctgtg gtcgcggtca ggcacccagg gcaaccgctg gcacgaggcc 2340
tgggccaccc tttcccacca gcctggctcc catgcccagt accagctgct gttcgagggc 2400
ctccgggacg gataccacgg caccatggcg ctggacgatg tggccgtgcg gccgggcccc 2460
tgctgggccc ctaattactg ctcctttgag gactcagact gcggcttctc ccctggaggc 2520
caaggtctct ggaggcggca ggccaatgcc tcgggccatg ctgcctgggg ccccccaaca 2580
gaccatacca ctgagacagc ccaagggcac tacatggtgg tggacacaag cccagacgca 2640
ctaccccggg gccagacggc ctccctgacc tccaaggagc acaggcccct ggcccagcct 2700
gcttgtctga ccttctggta ccacgggagc ctccgcagcc caggcaccct gcgggtctac 2760
ctggaggagc gcgggaggca ccaggtgctc agcctcagtg cccacggcgg gcttgcctgg 2820
cgcctgggca gcatggacgt gcaggccgag cgagcctgga gggtggtgtt tgaggcagtg 2880
gccgcaggcg tggcacactc ctacgtggct ctggatgatc tgctcctcca ggacgggccc 2940
tgccctcagc caggttcctg tgattttgag tctggcctgt gtggctggag ccacctggcc 3000
gggcccggcc tgggcggata cagctgggac tggggcgggg gagccacccc ctctcgttac 3060
ccccagcccc ctgtggacca caccctgggc acagaggcag gccactttgc cttctttgaa 3120
actggcgtgc tgggccccgg gggccgggcc gcctggctgc.gcagcgagcc tctgccggcc 3180
accccagcct cctgcctccg cttctggtac cacatgggtt ttcctgagca cttctacaag 3240
ggggagctga aggtactgct gcacagtgct cagggccagc tggctgtgtg gggcgcaggc 3300
gggcatcggc ggcaccagtg gctggaggcc caggtggagg tagccagtgc caaggagttc 3360
cagatcgtgt ttgaagccac tctgggcggc cagccagccc tggggcccat tgccctggat 3420
gacgtggagt atctggctgg gcagcattgc cagcagcctg cccccagccc ggggaacaca 3480
gccgcacccg ggtctgtgcc agctgtggtt ggcagtgccc tcctattgct catgctcctg 3540
gtgctgctgg gacttggggg acggcgctgg ctgcagaaga aggggagctg ccccttccag 3600
agcaacacag aggccacagc ccctggcttt gacaacatcc ttttcaatgc ggatggtgtc 3660
20/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
accctcccgg catctgtcac cagtgatccg tagaccaccc cagacaaggc cccgc~tcct 3720
cacgtgacat ccagcacttg gtcagaccct agccagggac cggacacctg ccccgcccag 3780
gctgggacag gctgcaggtc tcaggatatg ctgaggcctg ggcgttccct gccctgtgct 3840
gactctgttg ctctgtgaat aaacaccctg gcccatgagg gcagcccaaa aaaaaaaaaa 3900
as 3902
<210> 13
<211> 2574
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4291779CB1
<400> 13
cggctcgagg tgcggtcatg gtgggccaga tgtactgcta ccccggcagc cacctggccc 60
gggcgctgac gcgggcgctg gcgctggccc tggtgctggc cctgctggtc gggccgttcc 120
tgagcggcct ggcgggggcg atcccagcgc cggggggccg ctgggcgcgc gatgggccgg 180
tccctccagc ctcccgcagc cgctcggtgc tcctggacgt ctcggcgggc cagctgctta 240
tggtggacgg acgccaccct gacgccgtgg cctgggccaa cctcaccaac gccatccgcg 300
agactgggtg ggccttcctg gagctgggca caagtggcca atacaatgac agcttgcagg 360
cctatgcagc cggtgtggtg gaggctgctg tgtcggagga gctcatctac atgcactgga 420
tgaacacggt ggtgaattac tgcggcccct tcgagtatga agtcggctac tgcgagaggc 480
tgaagagctt cctggaggcc aacctagagt ggatgcagga agagatggag tcaaacccag 540
actcacctta ctggcaccag gtgcggctga ccctcctgca gctgaaaggc ctggaggaca 600
gctacgaagg ccgtgtgagc ttcccagctg ggaagttcac catcaaaccc ttggggttcc 660
tcctgctgca gctctctggg gacctggaag acctggagct ggccctgaac aagaccaaga 720
tcaaaccttc tctgggctct ggctcctgtt ctgccctcat caagctgctc cctggccaga 780
gtgacctcct ggttgcccac aacacctgga acaactacca gcacatgctg cgtgtcatca 840
agaagtactg gctccagttc cgggaaggcc cctgggggga ctacccgctg gttcccggca 900
acaagctggt cttctcctcc taccccggca ccatcttctc ctgcgacgac ttctacatcc 960
tgggcagtgg gctggtgaca ctggagacca ccattggcaa caagaaccca gccctgtgga 1020
agtatgtgcg gcccaggggc tgtgtgctgg agtgggtacg caacatcgtg gccaaccgcc 1080
tggcctcgga tggggccacc tgggcagaca tcttcaagag gttcaacagc ggcacgtata 1140
acaaccagtg gatgatcgtg gactacaagg cgttcatccc gggtgggccc agccccggga 1200
gccgggtgct taccatcctg gagcagatcc ccggcatggt ggtggtggct gacaagacct 1260
cggagctcta ccagaagacc tactgggcca gctacaacat accgtccttc gagactgtgt 1320
tcaatgccag tgggctgcag gccctagtgg cccagtatgg ggactggttt tcttatgacg 1380
ggagcccccg ggcccagatc ttccggcgga accagtcact ggtacaagac atggactcca 1440
tggtcaggct gatgaggtac aatgacttcc tccatgaccc tctgtcactg tgcaaagcct 1500
gcaaccccca gcccaatggg gagaatgcta tctccgcccg ctccgacctc aacccggcca 1560
atggctccta ccccttccag gccctgcgtc agcgctccca tgggggtatc gatgtgaagg 1620
tgaccagcat gtcactggcc aggatcctga gcctgctggc ggccagcggt cccacgtggg 1680
accaggtgcc cccgttccag tggagcacct cgcccttcag cggcctgctg cacatgggcc 1740
agccagacct ctggaagttc gcgcctgtca aggtttcatg ggactgaagt tctgtccctg 1800
ctctgctgct ttcgcccctg ctgaccctcg tcagggtcac ccccgtccca aggccaccgg 1860
acttctaact ccagcccctc ctgggggctt cgttctctga tctggggtct gagtcatctc 1920
ctcctagagt gggtcacgaa cctgatgggg ctcagaactg accccctctc tcccccgagg 1980
tgggtgggca ccgtggcgtc tcttctgccc tgccctaaat ctcccactct ctgtttctgt 2040
ctgtttccta ctgctgctct ctcaacctca ttcccacctc tggggcccct tcctcgtgct 2100
tctccttcct gagggtttgg gaaggtcctg gggcagactc tggggctccc atggggtgga 2160
aggagcctgt tccagcaccc ttctcccagc tgcattccca cgggtggccc tggagctggt 2220
gagctttgtc tgggcgttgt cttcggctgg cattgctcct cccagctctg gcccctctgc 2280
tccctcagga agcagtcccc tcgtctccct ttctgggcag cttccttgag gacagaaact 2340
tgaaaacaaa cacaaaccaa agtttctggc catctgtggc tggagggttc tgaatgtcct 2400
21/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
ctctccatgt caggcagagg gtcagccccc atgcttctgc ctcaggcccc accccacccc 2460
accccaggcc tgcccctcac ctcagggcca tacccacagc gccctgatgg aggaaccaga 2520
ccgcaggctg tgccaccatt aaacaagagc ggctgtgaaa aaaaaaaaaa aagg 2574
<210> 14
<211> 2878
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4728247CB1
<400> 14 ,
gtgaaagagg cgtgttgtct agtttcaaag gagaggagag aaggcaactc tggtagctct 60
ccttgtctgg ttgttttgaa gaaagaagag tagaagaaaa agttgagtaa atcatgtcgg 120
agttactgga cctttctttt ctgtctgagg aggaaaagga tttgattctc agtgttctac 180
agcgagatga agaggtccgg aaagcagatg agaaaaggat taggcgacta aagaatgagt 240
tactggagat aaaaaggaaa ggggccaaga ggggcagcca acactacagt gatcggacct 300
gtgcccggtg ccaggagagc ctgggccgtt tgagtcccaa aaccaatact tgtcggggtt 360
gtaatcacct ggtgtgtcgg gactgccgca tacaggaaag caatggtacc tggaggtgca 420
aggtgtgcgc caaggaaata gagttgaaga aagcaactgg ggactggttt tatgaccaga 480
aagtgaatcg ctttgcttac cgcacaggta gtgagataat caggatgtcc ctgcgccaca 540
aacctgcagt gagtaaaaga gagacagtgg gacagtccct ccttcatcag acacagatgg 600
gtgacatctg gccaggaaga aagatcattc aggagcggca gaaggagccc agtgtgctat 660
ttgaagtgcc aaagctgaaa agtggaaaga gtgcattgga agctgagagt gagagtctgg 720
atagcttcac agctgactcg gatagcacct ccaggagaga ctctctggat aaatctggcc 780
tctttccaga atggaagaag atgtctgctc ccaaatctca agtagaaaag gaaactcagc 840
ctggaggtca aaatgtggta tttgtggatg agggtgagat gatatttaag aagaacacca 900
gaaaaatcct caggccttca gagtacacta aatctgtgat agatcttcgc ccagaagatg 960
tggtacatga aagtggctcc ttgggagaca gaagcaaatc cgtcccaggc ctcaatgtgg 1020
atatggaaga ggaagaagaa gaagaagaca ttgaccacct agtgaagtta catcgccaga 1080
agctagccag aagcagcatg caaagtggct cctccatgag tacgatcggc agcatgatga 1140
gcatctacag tgaagctggt gatttcggga acatctttgt gactggcagg attgcctttt 1200
ccctgaagta tgagcagcaa acccagagtc tggttgtcca tgtgaaggag tgccatcagc 1260
tggcctatgc tgatgaagcc aagaagcgct ctaacccata tgtgaagact taccttctgc 1320
ctgacaagtc ccgccaagga aaaagaaaaa ccagcatcaa gcgggacact gttaatccac 1380
tatatgatga gacgctgagg tatgagatcc cagaatctct cctggcccag aggaccctgc 1440
agttct'cagt ttggcatcat ggtcgttttg gcagaaacac tttccttgga gaggcagaga 1500
tccagatgga ttcctggaag cttgataaga aactggatca ttgcctccct ttacatggaa 1560
agatcagtgc tgagtccccg actggcttgc catcacacaa aggcgagttg gtggtttcat 1620
tgaaatacat cccagcctcc aaaacccctg ttggaggtga ccggaaaaag agtaaaggtg 1680
gggaaggggg agagctccag gtgtggatca aagaagccaa gaacttgacg gctgccaaag 1740
caggagggac ttcagacagc tttgtcaagg gatacctcct tcccatgagg aacaaggcca 1800
gtaaacgtaa aactcctgtg atgaagaaga ccctgaatcc tcactacaac catacatttg 1860
tctacaatgg tgtgaggctg gaagatctac agcatatgtg cctggaactg actgtgtggg 1920
accgggagcc cctggccagc aatgacttcc tgggaggggt caggctgggt gttggcactg 1980
ggatcagtaa tggggaagtg gtggactgga tggactcgac tggggaagaa gtgagcctgt 2040
ggcagaagat gcgacagtac ccagggtctt gggcagaagg gactctgcag ctccgttcct 2100
caatggccaa gcagaagctg ggtttatgag tccctgtcct cttctgcagg tccagccctg 2160
gcgagggcag gtcagaggaa gtgaagaaat caagagcaaa gatttataat ttaatgtgta 2220
tgtgtgtatg tgtgtatgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtacaaaca 2280
tgtattttct gcaaatctca ttatgctggc tagagtgatg cagacttgtt cttcttttta 2340
aagcagtctc aagaataagc atttctttaa aatgtttctg tgtataatct agtttatttt 2400
cagagtccat tttttcttat gtctttataa ggttcactta acttaaaaac agcttttaaa 2460
acaacttttt atcttctgtc ttgctatcat tgttcctact tccctaggaa gccctggcta 2520
22/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
cctttcgcat taggaccagt ctgggtttta aggctctggg aagcagggtt ggttagtaaa 2580
gacaggaatg ttggggagag gtgagtagtt ccttcctctt tctcctctcc aatttatgct 2640
tttaacttat tttctacctg gataaacttc tggaacttgg cttttaaatt taacttttct 2700
agtttttaag cagtttccac cttgctttgg tctaatgctt ttctttgaaa tgctaacaga 2760
attcccaagc tttttccagt tctagatatc tttactagac ctttggggga ctcttataat 2820
ggagctgctt ttgaaaagca ctttaattag ataatgtatt ttgactaaat cacgagga 2878
<210> 15
<211> 5628
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472259CB1
<400> 15
atggccaagg actcgcccag ccccttgggc gcgtcgccca agaagccggg ctgctccagc 60
ccggcggcgg cagtgctgga gaaccagagg cgggagctgg agaagctacg ggcggagctg 120
gaggcggagc gggcaggctg gcgggcggaa cggcggcgct tcgctgcccg ggagcgccag 180
ctgcgtgagg aggccgagcg ggagcggcgg cagctggctg accatctgcg ctccaagtgg 240
gaggcacagc gcagccggga gttgcggcag ctgcaagagg agatgcagcg ggaacgcgag 300
gccgagatcc ggcagctgct gcgctggaac ggaggccgag cagcggcagc tgcagcagct 360
gcatgcaccg ggagcgcgat ggcgtggtgc gccaagcccg ggagctgcag cgccagctgg 420
ccgaggagct ggtgaaccgc ggccactgta gccgcccggg ggcgtccgag gtttccgcgg 480
cgcagtgccg ctgtcgcctg caggaagtgt tggcgcagct tcgctggcag actgacggcg 540
agcaggcggc gcgcatccgc tatctgcagg cggcgctgga ggtggagcgc cagctcttcc 600
tcaagtacat cctggcgcac ttccgcgggc acccggcttt gtcgggatca ccggaccccc 660
aagctgtgca ttccttggaa gaaccgctgc cccagacctc cagcggctct tgccacgccc 720
ccaaacccgc ctgccaactc ggatctctag acagcctgag tgctgaagtc ggtgtgcgct 780
cccgctcgct aggcctggtg tcctctgcgt gctccagctc cccagacggc ctgctctcca 840
cgcacgccag ctcccttgat tgcttcgcac ctgcgtgttc ccgctcgctt gacagcaccc 900
ggagcctccc caaggcctcc aaatccgagg agcggccctc ctcaccagac acctccaccc 960
ctggctcccg gaggctctcg ccgccaccat cgccactccc gccgccacca ccaccgtcag 1020
cccacaggaa actcagcaac ccgcggggag gagaaggctc tgagagccag ccctgcgaag 1080
tcctgactcc ctcacccccg ggcctgggcc accacgagct gataaagctg aactggctgc 1140
tggccaaggc gttgtgggtg ctggcgcgcc gctgttatac cctgcaagag gagaacaagc 1200
agctgcggcg tgcaggctgc ccctaccagg cagacgagaa ggtgaagcgg ctcaaggtaa 1260
agcgcgcgga gctgaccggg ctcgcgcggc gcctagctga ccgcgcccgc gagctgcagg 1320
agaccaacct ccgggccgtg agcgcgccta tacccggcga gagttgcgcc ggcctggagc 1380
tgtgccaagt ctttgcccgc cagcgcgctc gggacctgtc ggagcaggcg agcgcgccgc 1440
tggccaagga caagcagatc gaagagctgc ggcaggagtg ccacctcctg caggcgcgtg 1500
tcgcctcggg tccctgcagc gacctgcata ctggaagggg cggcccctgc acccagtggc 1560
tcaacgtcag agacttagac cgcctgcagc gcgagtccca gcgggaagtg ctgcgcctgc 1620
agaggcagtt gatgcttcag cagggcaacg gtggcgcttg gcccgaggcg ggcggccaga 1680
gcgcaacctg cgaggaggtg cgacggcaga tgctggcgct ggagcgcgag ctggaccagc 1740
ggcggcgcga gtgccaggag ctgggcgcgc aggcggcccc ggcgcggcga cgtggcgagg 1800
aggccgagac acagctgcag gcggcgctgc tcaaaaacgc ctggctggcg gaggagaatg 1860
ggcggctgca ggccaagacc gactgggtgc ggaaggtgga ggctgagaat agcgaagtgc 1920
gcggccacct gggccgcgcg tgtcaagagc gcgatgcctc cggcttgatc gccgaacagc 1980
tgctgcagca ggcggcgcgc gggcaggaca ggcagcagca gctgcaacgc gacccgcaga 2040
aggccctgtg tgacctccat ccttcctgga aggagataca ggcgctccag tgtcggcctg 2100
gtcaccctcc tgaacagccc tgggagacca gtcaaatgcc ggagtcccaa gttaaaggta 2160
gcagaaggcc caagttccac gcacggcctg aagactacgc agtgtcacag cccaacagag 2220
acatacagga gaaaagggaa gcctccctcg aggagagccc agttgccctt ggggagtcag 2280
ccagtgtccc ccaagtttca gagacagtcc ctgccagcca acctctgtcc aagaaaacca 2340
23/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
gctcccagtc aaactcctcc tctgaggggt cgatgtgggc caccgtgccg tcctccccta 2400
ctctggacag ggacacagcc agtgaggtgg atgacctgga gcctgacagc gtgtccctgg 2460
ccctggaaat ggggggctcg gcggctcctg ctgcccccaa gctcaagatc ttcatggctc 2520
agtataacta caacccattt gaggggccca atgatcaccc tgagggtgag ctgcccctca 2580
cagctgggga ctacatatat atcttcgggg acatggatga ggatggcttc tatgaggggg 2640
agcttgacga tggccggcgg gggctggtgc cctccaactt cgtggagcag attccggaca 2700
gctacatccc aggctgcctg cctgccaaat cccctgatct tggccccagt caactcccag 2760
cggggcagga tgaagctctg gaggaagaca gcttattatc tgggaaagcc cagggaatgg 2820
tggacagagg gctgtgccag atggtcaggg tgggctccaa gacagaagta gcaacagaga 2880
tcctggatac caagacggaa gcctgccagc tgggcttgct gcagagcatg gggaagcagg 2940
gcctctccag accccttctg gggaccaaag gggtgctccg tatggctccc atgcagctac 3000
acctgcagaa tgtcacagcc acatcagcca acatcacctg ggtctacagc agccaccgcc 3060
acccccatgt ggtatatctt gatgaccgag agcatgccct gaccccagcg ggcgtgagct 3120
gctacacctt ccagggcctg tgccccggca cgcactaccg ggtgcgggtg gaggtgcggc 3180
tgccatggga cttgctgcag gtgtattggg gaactatgtc ctccaccgtc accttcgaca 3240
cactcttggc aggacctccc tacccaccgc tggatgtgct ggtggagcgc catgcctcgc 3300
caggtgtcct'ggtggtcagc tggctccctg tgaccattga ctcagctggg tcctccaatg 3360
gagtccaggt caccggttat gctgtgtatg cagatgggct taaggtttgt gaggtcgccg 3420
atgccactgc tgggagcacc gtattggaat tctcccagct acaggtgccc ctcacgtggc 3480
agaaggtctc agtgagaacc atgtcactct gtggtgagtc cctggattca gtgcctgctc 3540
agatccccga ggacttcttc atgtgtcacc gatggccaga gactccaccc tttagctaca 3600
cttgtggcga cccatccacc tacagagtca ccttccccgt ctgcccccag aagctgtcac 3660
tggctcctcc gagtgccaag gccagccccc acaaccctgg aagctgcggg gagccccagg 3720
ccaagttcct agaagcattc tttgaagaac ccccaaggag gcaatcccca gtgtccaacc 3780
tgggctcaga aggagaatgt ccgagttcag gggctggcag ccaagcccag gagcttgcag 3840
aggcctggga gggctgtaga aaggacctgc tctttcagaa gagtccccag aaccacaggc 3900
caccttcagt cagtgaccag cctggggaga aggaaaattg ctaccagcac atgggcacca 3960
gcaaaagccc tgctccagga ttcatccatc tacgcaccga gtgtgggccc aggaaagaac 4020
cgtgtcagga aaaggctgcc cttgagaggg tacttcggca aaagcaagat gcccaagggt 4080
tcacacctcc ccagctgggc gccagccaac agtatgcatc tgacttccat aacgttttga 4140
aggaggagca ggaggcactg tgcttggatc tgtggggcac agagaggcga gaggagagga 4200
gggagcctga gccccacagc aggcaaggac aagctctggg ggtcaagaga gggtgccagc 4260
tccatgagcc cagctcggca ctgtgtccag ctccatccgc caaagtcatc aagatgccca 4320
ggggtggccc ccaacagctg gggacggggg ccaacactcc agccagggtc tttgtggccc 4380
tctctgatta caaccccctg gtgatgtctg ccaacctcaa ggctgcagag gaggagctgg 4440
tcttccagaa aaggcagttg ctaagagtgt ggggctctca ggacacccat gatttctacc 4500
tcagcgagtg caacaggcaa gtgggcaata tccccgggcg cctagtggct gagatggagg 4560
tggggacaga gcagactgat aggaggtggc gttctccggc ccaagggcac ctgccttctg 4620
tggcccacct cgaggacttt caggggctca ccatccccca gggttcctcc ctggtgctcc 4680
aggggaactc caagagactc ccactgtgga ctccaaagat catgatagca gctctggact 4740
atgatcctgg ggatgggcaa atggggggcc aggggaaggg caggctggcg ctgagggcag 4800
gagacgtggt catggtttac gggcccatgg atgaccaagg attctattat ggagagttgg 4860
gcggccacag gggcctggtt cctgcccacc tgctggatca catgtccctc catggacact 4920
gagcaagcat ccttgcccag gtagtggcct ctggctgctc acaccctgcc agaggagaag 4980
caagcgttca gaccctcaca ccagcacccc tcctcaccac cataagtagc atgtgctcca 5040
agtgccactg tgttaaactg atggtagtcc ttaagcgtcc cctaggctct gaaagtagca 5100
ggacttaagc ctgagttatt tgcaaaagca aacacaacaa gccaacccct gagagtctga 5160
gaagccattt caaagttgct gataactatg gcaggtatac ggagaagcgc ctttttctgt 5220
ggccaatgtg tgttttctct gggaggttaa ggttatctgt ccattgcctt gtacgaaagt 5280
ctcaagaaaa gtctacatct taaaaaagaa aaagcaatct gagtgttatt tttgggatgt 5340
gagggtgatc tggctgcgac atgtgtcacc ccattgatca tcagggttga ttcggctgat 5400
ctggctgact aggcgggtat ccccttcctc cctcaccact ccatgtgcgt ccctccagaa 5460
gctgtgtgct caatggaaga ggatgaccat ccccgataga ggacgatcgg tcttcagtca 5520
agagtataag agtagctgcg ctcccctgct agaacctcca aacgagctct cagaatgtta 5580
tttttctgtc ctatgtccaa cccctcatta aaatgttcat agaaaaaa 5628
24/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
<210> 16
<211> 1482
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476740CB1
<400> 16
tgggagacag cccccagaca gatgagtgtc gcgcctctct gagaggtgaa tgagcccgga 60
cggtccctac ctaccaagtc ctgaggagca gcggcaccaa cgacgcaggc ccgccccagc 120
ccgccagtga gccgcccatg ccctctgcta gcccggcccg cccgggcccc cgccatgctg 180
atcaccgtgt actgcgtgcg gagggacctc tccgaggtca ccttctctct ccaggtcagc 240
cccgactttg agctccgaaa cttcaaggtc ctctgcgaag cggagtccag agtccccgtc 300
gaagagatcc agatcatcca catggagcga ctcctcatcg aggaccactg ttccctgggc 360
tcctacggcc tcaaagatgg cgatatcgtg gttttactgc agaaggacaa tgtgggacct 420
cgggctccag ggcgtgcccc gaaccagcct cgtgtagact tcagtggcat tgcggtgcct 480
gggacgtcca gctcccgtcc acagcaccct ggacagcagc agcagcgcac acccgctgcc 540
cagcggtcac agggcttggc gtcaggagag aaggtggccg gcctgcaagg tctgggcagc 600
cccgccctga tccgcagcat gctgctctcc aacccccacg atctgtccct gctcaaggaa 660
cgcaaccctc ccttggcgga agccctgctc agcggaagcc ttgagacctt ttctcaggtg 720
ctgatggagc agcaaaggga aaaggccttg agagagcaag agaggcttcg tctctacaca 780
gccgacccac tggatcggga agctcaggcc aaaatagaag aggaaatccg gcagcaaaac 840
attgaagaaa acatgaatat agcgatagaa gaggcccccg agagttttgg acaagtgacg 900
atgctctaca ttaactgcaa agtgaatggg catcctttga aggcttttgt tgactcgggc 960
gcccagatga ccattatgag ccaggcttgt gccgagcgat gtaacatcat gaggctggtg 1020
gaccgacggt gggctggggt tgctaaagga gtgggcacac agagaattat tggccgtgtt 1080
catctagctc agattcaaat tgaaggtgat ttcttacagt gctctttctc catacttgag 1140
gatcaaccca tggatatgct tctaggccta gatatgctcc ggagacatca atgttccatc 1200
gatttgaaga aaaatgtgct ggtcatcggc accactggca cgcagactta ttttcttcct 1260
gagggagagt tgcccttatg ctctaggatg gtaagtgggc aagatgagtc ttcggacaag 1320
gaaattacac attcagtcat ggattcagga cgaaaagagc attaaagcac gttataaata 1380
tgttaccacc ttgagggagc ctcaggtccc cggcaattat aagttaagag cttactggca 1440
atgtaatcat taaaaaacat cagtaacaac taaaaaaaaa as 1482
<210> 17
<211> 2511
<212> DNA
<223> Homo sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 7473774CB1
<400> 17
gatgtgactg ttaagctgag ctttttctcc cggcctcagc ccctagatca gacattctct 60
ctcattaccc ggcgcgtggg aacgggtcca cagccccttg tccgccctag aacccccatg 120
ggctgccgcc cgccgccgcc tcggctgcca cccaggacac ggcagagata agcgcaggac 180
cagacggcca ccatgtcagg agactacgag gatgacctct gccggcgggc actcatcctg 240
gtctcggacc tctgtgcgcg ggtccgagat gctgacacca acgacaggtg ccaggagttc 300
aatgaccgaa tccgaggcta tccccggggt ccagatgcag acatctccgt gagcctgctg 360
tcggtcatcg tgacattctg tggcattgtc cttctgggtg tctctctctt cgtgtcctgg 420
aagttgtgct gggtgccctg gcgggacaag ggaggctcgg cagtgggcgg tggccccctg 480
cgcaaagacc taggccctgg tgtcgggctg gcaggcctgg taggcggagg cgggcaccac 540
ctggcggctg gcctgggtgg ccatcctctg ctgggcggcc cacaccacca tgcccatgcc 600
25/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
gcccaccatc caccctttgc tgagctgctg gagccaggca gcctgggggg ttctgacacc 660
cctgagccct cctacttgga catggactcg tatccagagg ctgcagcagc agcagtggcc 720
gctggggtca aaccgagcca aacatcccct gagctgccct ctgagggggg agcaggctct 780
gggttgctcc tgctgccccc cagtggtggg ggcttgccca gtgcccagtc acatcagcag 840
gtcacaagcc tggcacccac taccaggtac ccagccctgc cccgacccct cacccagcag 900
actctgacct cccagccgga ccccagcagt gaggagcgcc cacctgccct gcccttaccc 960
ctgcctggag gcgaggaaaa agccaaactc attgggcaga ttaagccaga gctgtaccag 1020
gggactggcc ctggtggccg gcggagcggt gggggcccag gctctggaga ggcaggcaca 1080
ggggcaccct gtggccgtat cagcttcgcc ctgcggtacc tctatggctc ggaccagctg 1140
gtggtgagga tcctgcaggc cctggacctc cctgccaagg actccaacgg cttctcagac 1200
ccctacgtca agatctacct gctgcctgac cgcaagaaaa agtttcagac caaggtgcac 1260
aggaagaccc tgaaccccgt cttcaatgag acgtttcaat tctcggtgcc cctggccgag 1320
ctggcccaac gcaaactgca cttcagcgtc tatgactttg accgcttctc gcggcacgac 1380
ctcatcggcc aggtggtgct ggacaacctc ctggagctgg ccgagcagcc ccctgaccgc 1440
ccgctctgga gggacatcgt ggagggcggc tcggaaaaag cagatcttgg ggagctcaac 1500
ttctcactct gctacctccc cacggccggg cgcctcaccg tgaccatcat caaagcctct 1560
aacctcaaag cgatggacct cactggcttc tcagacccct acgtgaaggc ctccctgatc 1620
agcgaggggc ggcgtctgaa gaagcggaaa acctccatca agaagaacac gctgaacccc 1680
acctataatg aggcgctggt gttcgacgtg gcccccgaga gcgtggagaa cgtggggctc 1740
agcatcgccg tggtggacta cgactgcatc gggcacaacg aggtgatcgg cgtgtgccgt 1800
gtgggccccg acgctgccga cccgcacggc cgcgagcact gggcagagat gctggccaat 1860
ccccgcaagc ccgtggagca ctggcatcag ctagtggagg aaaagactgt gaccagcttc 1920
acaaaaggca gcaaaggact atcagagaaa gagaactccg agtgaggggt ctggcctagg 1980
cccgggatcg gaccaggctc cctcaggacc ccatcctttc ctgcccggac cgtgaattca 2040
tctccttgaa gccataacgt ccgagctgot ggtgcggggc agccetggcc ctaggcttcc 2100
taaccctgga agcgagagga tgagaggagg ccggcccagc tccttctttc agggtggggg 2160
tcattcagcc tccactgtgt ctgtcttttc ttccctgggg ctccccctcg aggcgagggg 2220
ccatgcatgt ctgggggacc cctgcccccc aaaaccctct gtctgtctct gtctctttgc 2280
tgtttgtcca agactcagtg tcccgaccct tgttctcgcc gtgaatgtca atgggccaat 2340
cctctctgtc ctttcagaca cacacacacc tgtgtccacc ccttctgttc gccacaccct 2400
gcgtctggcc ggtcccccca ctgctgctgc tatcaacgcc agaataaaca cactctgtgg 2460
gtctcactcc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 2511
<210> 18
<211> 1680
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7946329CB1
<400> 18
cctacctctc atcaggacca gtctgactgc acctgcatcc ttagctcaga gcatccccgg 60
agcatcttaa gagctgagcg cagctgacaa ctaggggccg gaccgtcgca ggaggcgtcc 120
gctggatacc ttcccccttc cctgacctag agctctacag ctgctgcctc ggtactgacc 180
gagggttccc agagctgtct taccattgca aaaacgttat agcaacagcc tctgattacg 240
acatggctga gatcaccaat atccgaccta gctttgatgt gtcaccggtg gtggccggcc 300
tcatcggggc ctctgtgctg gtggtgtgtg tctcggtgac cgtctttgtc tggtcatgct 360
gccaccagca ggcagagaag aagcacaaga acccaccata caagtttatt cacatgctca 420
aaggcatcag catataccca gagaccctca gcaacaagaa gaaaatcatc aaagtgcgga 480
gagacaaaga tggtcctggg agggaaggtg gacgtaggaa cctgttggtg gacgcagcag 540
aggctggcct gctaagccga gacaaagatc ccagggggcc tagctctgga tcttgtatag 600
accaattacc catcaaaatg gactatgggg aagaactaag gagccctatt acaagcctga 660
cccctgggga gagcaaaacc acctctccat catctccaga ggaggatgtc atgctaggat 720
ccctcacctt ctcagtggac tataacttcc cgaaaaaagc cctggtggtg acaatccagg 780
26/27
CA 02410679 2002-11-26
WO 02/02610 PCT/USO1/20704
aggcccacgg gctgccagtg atggatgacc a~acccaggg atctgacccc tacatcaaaa 840
tgaccatcct tcctgacaaa cggcatcggg tgaagaccag agtgctgcgg aagaccctgg 900
accctgtgtt tgacgagacc ttcaccttct atggcatccc ctacagccag ctgcaggacc 960
tggtgctgca cttccttgtc ctcagctttg accgcttctc tcgggatgat gtcattggcg 1020
aggtcatggt gccactggca ggggtggacc ccagcacagg caaggtacaa ctgaccaggg 1080
acatcatcaa aaggaatatc cagaagtgca tcagcagagg ggagctccag gtgtctctgt 1140
catatcagcc tgtggcacag agaatgacag tggtggtcct caaagccaga cacttgccga 2200
agatggatat caccggtctc tcaggtaatc cttatgtcaa ggtgaacgtc tactacggca 1260
gaaagcgcat tgccaagaag aaaacccatg tgaagaagtg cactttgaac cccatcttca 1320
atgaatcttt catctacgac atccccactg acctcctgcc tgatatcagc atcgagttcc 1380
tcgttatcga cttcgatcgc accaccaaga atgaggtggt ggggaggctg atcctggggg 1440
cacacagtgt cacagccagt ggtgctgaac actggagaga ggtctgcgag agcccccgca 1500
agcctgtggc caagtggcac agtctgagcg agtactaatc ctgttcttct ctcctctaat 1560
ccccgggggc caagctgggg agggatgtgg aggggaaaaa gatgacagag aagtggactc 1620
caaacctcat tttagttgta gaagaaaatt tcttacaaaa caaattccac aaagaacacc 1680
27127
