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HUMAN TRANSPORT PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human
transport proteins
and to the use of these sequences in the diagnosis, treatment, and prevention
of transport, metabolic,
neurological, cardiovascular, reproductive, and immune disorders, and cell
proliferative disorders
including cancer.
BACKGROUND OF THE INVENTION
Eukaryotic cells are surrounded and subdivided into functionally distinct
organelles by
hydrophobic lipid bilayer membranes. These membranes act as a barrier to most
molecules, and
maintain the essential differences between the cytosol, the extracellular
environment, and the contents
of each intracellular organelle. Transport of essential nutrients, certain
metal ions, metabolic waste
products, cell signaling molecules, macromolecules, and proteins across lipid
membranes and
between organelles must be mediated by a variety of transport molecules.
Transport between the
cytoplasm and the extracellular environment, and between the cytoplasm and
lumenal spaces of
cellular organelles requires specific transport proteins. Each transport
protein carries a particular
class of molecule, such as ions, sugars, or amino acids, and often is specific
to a certain molecular
species of the class.
Cells and organelles require transport molecules to import and export
essential nutrients and
metal ions including K+, NH4+, P;, SO.~'--, sugars, and vitamins, as well as
various metabolic waste
products. Transport proteins also play roles in antibiotic resistance, toxin
secretion, ion balance,
synaptic neurotransmission, kidney function, intestinal absorption, tumor
growth, and other diverse
cell functions (Griffith, J. and C. Sansom (1998) The Transporter Facts Book,
Academic Press, San
Diego CA, pp. 3-29). Transport can occur by a passive concentration-dependent
mechanism, or can
be linked to an energy source such as ATP hydrolysis or an ion gradient.
Proteins that function in
transport include carrier proteins, which bind to a specific solute and
undergo a conformational
change that transfers the bound solute across the membrane, and channel
proteins, which form
hydrophilic pores that allow specific solutes to diffuse through the membrane
down an
electrochemical solute gradient.
Transport proteins are often mufti-pass transmembrane proteins, which either
actively
transport molecules across the membrane or passively allow them to cross.
Active transport involves
directional pumping of a solute across the membrane, usually against an
electrochemical gradient.
Active transport is tightly coupled to a source of metabolic energy, such as
ATP hydrolysis or an
electrochemically favorable ion gradient. Passive transport involves the
movement of a solute down
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its electrochemical gradient. Transport proteins can be further classified as
either Garner proteins or
channel proteins. Carrier proteins, which can function in active or passive
transport, bind to a
specific solute to be transported and undergo a conformational change which
transfers the bound
solute across the membrane. Channel proteins, which only function in passive
transport, form
hydrophilic pores across the membrane. When the pores open, specific solutes,
such as inorganic
ions, pass through the membrane and down the electrochemical gradient of the
solute. Examples
include facilitative transporters, the secondary active symporters and
antiporters driven by ion
gradients, and active ATP binding cassette transporters involved in multiple-
drug resistance and
targeting of antigenic peptides to MHC Class I molecules. Transported
substrates range from
nutrients and ions to a broad variety of drugs, peptides and proteins.
Information on the action of ARL-6 (ADP-ribosylation like factor), an
endoplasmic reticulum
transmembrane protein, can be found in Greenfield, J.J. and S. High ( 1999; J.
Cell Sci. 112:1477-
1486). Information on reduced folate carrier transporter proteins can be found
in Dixon, K.H. et al.
( 1994; J. Biol. Chem. 269:17-20) and Moscow, J.A. et al. ( 1995; Cancer Res.
55:5983-5987).
Carrier proteins which transport a single solute from one side of the membrane
to the other
are called uniporters. In contrast, coupled transporters link the transfer of
one solute with
simultaneous or sequential transfer of a second solute, either in the same
direction (symport) or in the
opposite direction (antiport). For example, intestinal and kidney epithelia
contain a variety of
symporter systems driven by the sodium gradient that exists across the plasma
membrane. Sodium
moves into the cell down its electrochemical gradient and brings the solute
into the cell with it. The
sodium gradient that provides the driving force for solute uptake is
maintained by the ubiquitous
Na+/K+ ATPase. Sodium-coupled transporters include the mammalian glucose
transporter (SGLTI ),
iodide transporter (NIS), and multivitamin transporter (SMVT). All three
transporters have twelve
putative transmembrane segments, extracellular glycosylation sites, and
cytoplasmically-oriented N-
and C-termini.
Mitochondrial carrier proteins are transmembrane-spanning proteins which
transport ions and
charged metabolites between the cytosol and the mitochondrial matrix. Examples
include the ADP,
ATP carrier protein; the 2-oxoglutarate/malate carrier; the phosphate carrier
protein; the brown fat
uncoupling protein which transports protons from the cytosol into the matrix;
the pyruvate carrier; the
dicarboxylate carrier which transports malate, succinate, fumarate, and
phosphate; the tricarboxylate
carrier which transports citrate and malate; and the Grave's disease carrier
protein, a protein
recognized by IgG in patients with active Grave's disease, an autoimmune
disorder resulting in
hyperthyroidism (Stryer, L. ( 1995) Biochemistry, W.H. Freeman and Company,
New York NY, p.
551; PROSITE PDOC00189 Mitochondrial energy transfer proteins signature;
Online Mendelian
Inheritance in Man (OMIM) *275000 Graves Disease).
This class of transporters also includes the mitochondrial uncoupling
proteins, which create
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proton leaks across the inner mitochondria) membrane, thus uncoupling
oxidative phosphorylation
from ATP synthesis. The result is energy dissipation in the form of heat.
Mitochondria) uncoupling
proteins have been implicated as modulators of thermoregulation and metabolic
rate, and have been
proposed as potential targets for drugs against metabolic diseases such as
obesity (Ricquier, D. et al.
( 1999) J. Int. Med. 245:637-642).
A number of metal ions such as iron, zinc, copper, cobalt, manganese,
molybdenum,
selenium, nickel, and chromium are important as cofactors for a number of
enzymes. For example,
zinc is required for the function of enzymes such as the extracellular matrix
metalloproteinases, and
zinc ions stabilize several motifs commonly found in transcription factors,
including zinc fingers, zinc
clusters, and LIM domains. Zinc and other metal ions must be provided in the
diet, and are absorbed
by transporters in the gastrointestinal tract. Plasma proteins transport the
metal ions to the liver and
other target organs, where specific transporters move the ions into cells and
cellular organelles as
needed. Imbalances in metal ion metabolism have been associated with a number
of disease states
(banks, D.M. (1986) J. Med. Genet. 23:99-106).
The largest and most diverse family of transport proteins known are the ATP-
binding cassette
(ABC) transporters. As a family, ABC transporters can transport substances
that differ markedly in
chemical structure and size, ranging from small molecules such as ions,
sugars, amino acids, peptides,
and phospholipids, to lipopeptides, large proteins, and complex hydrophobic
drugs. ABC proteins
consist of four modules: two nucleotide-binding domains (NBD), which hydrolyze
ATP to supply the
energy required for transport, and two membrane-spanning domains (MSD), each
containing six
putative transmembrane segments. These four modules may be encoded by a single
gene, as is the
case for the cystic fibrosis transmembrane regulator (CFTR), or by separate
genes. When encoded by
separate genes, each gene product contains a single NBD and MSD. These "half
molecules" form
homo- and heterodimers, such as Tapl and Tap2, the endoplasmic reticulum-based
major
histocompatibility (MHC) peptide transport system. Several genetic diseases
are attributed to defects
in ABC transporters, such as the following diseases and their corresponding
proteins: cystic fibrosis
(CFTR, an ion channel), adrenoleukodystrophy (adrenoleukodystrophy protein,
ALDP), Zellweger
syndrome (peroxisomal membrane protein-70, PMP70), and hyperinsulinemic
hypoglycemia
(sulfonylurea receptor, SUR). Overexpression of the multidrug resistance (MDR)
protein, another
ABC transporter, in human cancer cells makes the cells resistant to a variety
of cytotoxic drugs used
in chemotherapy (Taglicht, D. and S. Michaelis (1998) Methods Enzymol. 292:131-
163).
The nuclear pore complex (NPC) is a large multiprotein complex spanning the
nuclear
envelope which mediates the transport of proteins and RNA molecules between
the nucleus and the
cytoplasm, thus contributing to the regulation of gene expression. The NPC
allows passive diffusion
of ions, small molecules, and macromolecules under about 60kD, while larger
macromolecules are
transported by facilitated, energy-dependent pathways. Nuclear localization
signals (NLS), consisting
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of short stretches of amino acids enriched in basic residues, are found on
proteins that are targeted to
the nucleus, such as the glucocorticoid receptor. The NLS is recognized by the
NLS receptor,
importin, which then interacts with the monomeric GTP-binding protein Ran.
This NLS
protein/receptor/Ran complex navigates the nuclear pore with the help of the
homodimeric protein
nuclear transport factor 2 (NTF2) (Nakielny, S. and G. Dreyfuss ( 1997) Curr.
Opin. Cell Biol. 9:420-
429; Gorlich, D. ( 1997) Curr. Opin. Cell Biol. 9:412-419). Four O-linked
glycoproteins, p62, p58,
p54, and p45, exist as a stable "p62 complex" that forms a ring localized on
both nucleoplasmic and
cytoplasmic surfaces of the NPC. The p62, p58, and p54 proteins all interact
directly with the
cytosolic transport factors p97 and NTF2, suggesting that the p62 complex is
an important ligand
binding site near the central gated channel of the NPC (Hu, T. et al. (1996)
J. Cell Biol. 134:589-601).
Transport can also occur through intercellular bridges which connect the
cytoplasms of sister
cells, for example in the male and female germline of species ranging from
fruit flies to humans.
These bridges allow passage of cytoplasmic materials between cells during
development.
Intercellular bridges have also been found to connect somatic cells. The nurse
cells and oocyte of a
Drosophila egg chamber, which are derived from a single precursor cell through
four rounds of
mitosis, are connected to each other through intercellular bridges called ring
canals. The cells do not
completely separate after mitosis; the mitotic cleavage furrows are
transformed into ring canals by the
addition of an actin cytoskeleton lining the tunnels between the cells. The
Drosophila ketch protein
functions in organizing actin in the ring canal. Mutations in ketch cause
female sterility in
Dros~hila. Ketch contains four protein domains: the NTR domain at the N-
terminus, the BTB or
POZ domain, the IVR or intervening region; and the ketch repeat domain, which
contains six 50-
amino acid ketch repeats. The BTB or POZ domain, a 120-amino acid motif that
is also found in
several zinc-finger containing transcription factors, may be important in
dimerization of ketch. Ketch
repeats are found in other proteins as well and may be important for actin
binding (Robinson, D.N.
and L. Cooley (1997) J. Cell Biol. 138:799-810; Cooley, L. (1998) Cell 93:913-
915).
Ion Channels
The electrical potential of a cell is generated and maintained by controlling
the movement of
ions across the plasma membrane. The movement of ions requires ion channels,
which form an ion-
selective pore within the membrane. Ion channels share common structural and
mechanistic themes.
The channel consists of four or five subunits or protein monomers that are
arranged like a barrel in
the plasma membrane. Each subunit typically consists of six potential
transmembrane segments (S 1,
S2, S3, S4, S5, and S6). The center of the barrel forms a pore lined by a-
helices or ~3-strands. The
side chains of the amino acid residues comprising the a-helices or ~3-strands
establish the charge
(cation or anion) selectivity of the channel. The degree of selectivity, or
what specific ions are
allowed to pass through the channel, depends on the diameter of the narrowest
part of the pore. There
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are two basic types of ion channels, ion transporters and gated ion channels.
Ion transporters utilize
the energy obtained from ATP hydrolysis to actively transport an ion against
the ion's concentration
gradient. Gated ion channels allow passive flow of an ion down the ion's
electrochemical gradient
under restricted conditions. Together, these types of ion channels generate,
maintain, and utilize an
electrochemical gradient that is used in 1) electrical impulse conduction down
the axon of a nerve
cell, 2) transport of molecules into cells against concentration gradients, 3)
initiation of muscle
contraction, and 4) endocrine cell secretion.
Transmembrane ATPases are divided into three families. The phosphorylated (P)
class ion
transporters, including Na+-K+ ATPase, Caz+ ATPase, H'' ATPase, and Cu"'
ATPase, are activated by
a phosphorylation event. P-class ion transporters are responsible for
maintaining resting potential
distributions such that cytosolic concentrations of Na+ and Ca2+ are low and
cytosolic concentration
of K+ is high. The vacuolar (V) class of ion transporters include H+ pumps on
intracellular
organelles, such as lysosomes and Golgi. V-class ion transporters are
responsible for generating the
low pH within the lumen of these organelles that is required for function. The
coupling factor (F)
class consists of H+ pumps in the mitochondria. F-class ion transporters
utilize a proton gradient to
generate ATP from ADP and inorganic phosphate (P;).
Cu.~ ATPases export copper from cells (PROSITE PDOC00139 E1-E2 ATPases
phosphorylation site). Mutations in one Cu-'-' ATPase cause Wilson disease, in
which toxic amounts
of copper accumulate in a number of organs, particularly the liver and brain
(Tanzi, R.E. et al. ( 1993)
Nat. Genet. 5:344-350). Mutations in another Cu+' ATPase cause Menkes disease
and occipital horn
syndrome. Menkes disease mutations block export of copper from the
gastrointestinal tract, leading
to skeletal abnormalities, severe mental retardation, neurologic degeneration,
and mortality in early
childhood (Harrison, M.D. and C.T. Dameron (1999) J. Biochem. Mol. Toxicol.
13:93-106).
Occipital horn syndrome mutations cause connective tissue defects (Harrison,
suyra; Levinson, B. et
al. ( 1996) Hum. Mol. Genet. 5:1737-1742).
The coupling factor (F) class of ion transporters consists of H+ pumps in
mitochondria,
chloroplasts, and bacteria. For example, the FoF~ ATPase utilizes a proton
gradient across the inner
mitochondria) membrane to generate ATP from ADP and inorganic phosphate (P;).
The FoF, ATPase
is composed of the Fo complex, which is the transmembrane channel through
which protons flow, and
the F, complex, where ATP synthesis activity resides. Fn has three subunits, A
(also known as protein
6), B, and C (Lodish, H. et al. ( 1995) Molecular Cell BioloQV, Scientific
American Books, New York
NY, pp. 752-756; PROSITE PDOC00420 ATP synthase a subunit signature).
Voltage-gated Ca'-+ channels are involved in presynaptic neurotransmitter
release, and heart
and skeletal muscle contraction. The voltage-gated Ca'-+ channels from
skeletal muscle (L-type) and
brain (N-type) have been purified and, though their functions differ
dramatically, they have similar
subunit compositions. The channels are composed of three subunits. The a,
subunit forms the
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membrane pore and voltage sensor, while the a,=6 and (3 subunits modulate the
voltage-dependence,
gating properties, and the current amplitude of the channel. These subunits
are encoded by at least
six a,, one a"8, and four (3 genes. A fourth subunit, y, has been identified
in skeletal muscle (Walker,
D. et al. (1998) J. Biol. Chem. 273:2361-2367; and Jay, S.D. et al. (1990)
Science 248:490-492). The
human ~4 subunit is homologous to the mouse epilepsy gene lethargic, and is a
candidate for
involvement in neurological disorders including ataxia and absence epilepsy
(Escayg, A. et al. ( 1998)
Genomics 50:14-22).
Ligand-gated channels open their pores when an extracellular or intracellular
mediator binds
to the channel. Neurotransmitter-gated channels are channels that open when a
neurotransmitter
binds to their extracellular domain. These channels exist in the postsynaptic
membrane of nerve or
muscle cells. There are two types of neurotransmitter-gated channels. Sodium
channels open in
response to excitatory neurotransmitters, such as acetylcholine, glutamate,
and serotonin. This
opening causes an influx of Na+ and produces the initial localized
depolarization that activates the
voltage-gated channels and starts the action potential. Chloride channels open
in response to
inhibitory neurotransmitters, such as y-aminobutyric acid (GABA) and glycine,
leading to
hyperpolarization of the membrane and the subsequent generation of an action
potential.
Ion channels are expressed in a number of tissues where they are implicated in
a variety of
processes. CNG channels, while abundantly expressed in photoreceptor and
olfactory sensory cells,
are also found in kidney, lung, pineal, retinal ganglion cells, testis, aorta,
and brain. Calcium-activated
K+ channels may be responsible for the vasodilatory effects of bradykinin in
the kidney and for
shunting excess K+ from brain capillary endothelial cells into the blood. They
are also implicated in
repolarizing granulocytes after agonist-stimulated depolarization (Ishi, T.M.
et al. ( 1997) Proc. Natl.
Acad. Sci. USA 94:11651-11656). Another transmembrane protein, the leukotrine
B4 receptor (BLT)
appears to be involved in inflammation responses and host cell defense against
infection. BLT also
functions as an HIV coreceptor (Izumi, T. et al. ( 1997) Nature 387:620-624;
Martin, V. et al. ( 1999)
J. Biol. Chem. 274:8597-8603).
Ion channels have been the target for many drug therapies. Neurotransmitter-
gated channels
have been targeted in therapies for treatment of insomnia, anxiety,
depression, and schizophrenia.
Voltage-gated channels have been targeted in therapies for arrhythmia,
ischemic stroke, head trauma,
and neurodegenerative disease (Taylor, C.P. and L.S. Narasimhan ( 1997) Adv.
Pharmacol. 39:47-98).
K+ channels are located in all cell types, and may be regulated by voltage,
ATP
concentration, or second messengers such as Ca~ and cAMP. In non-excitable
tissue, K+ channels
are involved in protein synthesis, control of endocrine secretions, and the
maintenance of osmotic
equilibrium across membranes. In neurons and other excitable cells, in
addition to regulating action
potentials and repolarizing membranes, K+ channels are responsible for setting
resting membrane
potential. The cytosol contains non-diffusible anions and, to balance this net
negative charge, the cell
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contains a Na+-K+ pump and ion channels that provide the redistribution of
Na+, K+, and Cl-. The
pump actively transports Na+ out of the cell and K+ into the cell in a 3:2
ratio. Ion channels in the
plasma membrane allow K+ and CI ~ to flow by passive diffusion. Because of the
high negative charge
within the cytosol, CI - flows out of the cell. The flow of K' is balanced by
an electromotive force
pulling K+ into the cell, and a K+ concentration gradient pushing K+ out of
the cell. Thus, the resting
membrane potential is primarily regulated by K+flow (Salkoff, L. and T. Jegla
(1995) Neuron 15:489-
492). Information on NY-REN-45, a K+ channel integral membrane protein, can be
found in
Scanlan, M.J. et al. (1998; Int. J. Cancer 76:652-658). The emopamil-binding
protein (EBP) shares
structural features with both pro- and eukaryotic drug transport proteins
(Hanner, M. et al. ( 1995) J.
Biol. Chem. 270:7551-7557). The Na+ channel, transmembrane protein myelin
protein zero (MPZ)
may be responsible for some sporadic cases of Dejerine-Scottas disease
(hereditary motor and
sensory neuropathy type III) (Hayasaka, K. et al. ( 1993) Nat. Genet. 5:266-
268).
K+ pore-forming subunits generally have six transmembrane-spanning domains
with a short
region between the fifth and sixth transmembrane regions that senses membrane
potential; and the
amino and carboxy termini are located intracellularly. In mammalian heart, the
duration of
ventricular action potential is controlled by a K' current. Thus, the K+
channel is central to the
control of heart rate and rhythm. K+ channel dysfunctions are associated with
a number of renal
diseases including hypertension, hypokalemia, and the associated Banter's
syndrome and Getelman's
syndrome, as well as neurological disorders including epilepsy. K+ channels
have been implicated in
Alzheimer's disease by observations that a significant component of senile
plaques, beta amyloid or
A beta, also blocks voltage-gated potassium channels in hippocampal neurons
(Antes, L.M. et al.
(1998) Seminar Nephrol. 18:31-45; Stoffel, M. and L.Y. Jan (1998) Nat. Genet.
18:6-8; Madeja, M.
et al. (1997) Eur. J. Neurosci. 9:390-395; Good, T.A. et al. (1996) Biophys.
J. 70:296-304).
Gated ion channels control ion flow by regulating the opening and closing of
pores. These
channels are categorized according to the manner of regulating the gating
function. Mechanically-
gated channels open pores in response to mechanical stress, voltage-gated
channels open pores in
response to changes in membrane potential, and ligand-gated channels open
pores in the presence of a
specific ion, nucleotide, or neurotransmitter.
Voltage-gated Na+ channels are responsible for electrical excitability of
neurons, skeletal
muscle, heart, and neuroendocrine tissues. For example, the sequential opening
and closing of
voltage-gated Na+ channels results in the propagation of action potentials
down neuronal axons. Na+
channels isolated from rat brain tissue are heterotrimeric complexes composed
of a 260 kDa pore
forming a subunit that associates with two smaller auxiliary subunits, (31 and
(32. The ~i2 subunit is
an integral membrane glycoprotein that contains an extracellular Ig domain,
and its association with a
and (31 subunits correlates with increased function of the channel, a change
in the channel's gating
properties, as well as an increase in whole cell capacitance (Isom, L.L. et
al. ( 1995) Cell 83:433-442).
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Integral Membrane Proteins
The majority of known integral membrane proteins are transmembrane proteins
(TM) which
are characterized by an extracellular, a transmembrane, and an intracellular
domain. TM domains are
typically comprised of 15 to 25 hydrophobic amino acids which are predicted to
adopt an a-helical
conformation. TM proteins are classified as bitopic (Types I and II) and
polytopic (Types III and IV)
(Singer, S.J. (1990) Annu. Rev. Cell Biol. 6:247-96). Bitopic proteins span
the membrane once while
polytopic proteins contain multiple membrane-spanning segments. TM proteins
that act as cell-
surface receptor proteins involved in signal transduction include growth and
differentiation factor
receptors, and receptor-interacting proteins such as Drosophila pecanex and
frizzled proteins, LIV-1
protein, NF2 protein, and GNS1/SUR4 eukaryotic integral membrane proteins. TM
proteins also act
as transporters of ions or metabolites, such as gap junction channels
(connexins) and ion channels,
and as cell anchoring proteins, such as lectins, integrins, and fibronectins.
TM proteins act as vesicle
organelle-forming molecules, such as calveolins, or as cell recognition
molecules, such as cluster of
differentiation (CD) antigens, glycoproteins, and mucins. Information on
connexin can be found in
Kanter, H.L. et al. (1994; J. Mol. Cell. Cardiol. 26:861-868).
Many membrane proteins (MPs) contain amino acid sequence motifs that target
these proteins
to specific subcellular sites. Examples of these motifs include PDZ domains,
KDEL, RGD, NGR, and
GSL sequence motifs, von Willebrand factor A (vWFA) domains, and EGF-like
domains. RGD,
NGR, and GSL motif-containing peptides have been used as drug delivery agents
in cancer treatments
which target tumor vasculature (Arap, W. et al. ( 1998) Science, 279:377-380.)
Furthermore, MPs
may also contain amino acid sequence motifs, such as the carbohydrate
recognition domain (CRD),
also known as the C-type lectin domain, that mediate interactions with
extracellular or intracellular
molecules.
G-protein coupled receptors (GPCR) comprise a superfamily of integral membrane
proteins
which transduce extracellular signals. GPCRs include receptors for biogenic
amines, lipid mediators
of inflammation, peptide hormones, and sensory signal mediators. The structure
of these
highly-conserved receptors consists of seven hydrophobic transmembrane
regions, an extracellular
N-terminus, and a cytoplasmie C-terminus. Three extracellular loops alternate
with three intracellular
loops to link the seven transmembrane regions. The most conserved parts of
these proteins are the
transmembrane regions and the first two cytoplasmic loops. Cysteine disulfide
bridges connect the
second and third extracellular loops. A conserved, acidic-Arg-aromatic residue
triplet present in the
second cytoplasmic loop may interact with G proteins. A GPCR consensus pattern
is characteristic of
most proteins belonging to this superfamily (ExPASy PROSITE document PS00237;
and Watson,
S. and S. Arkinstall (1994) The G-protein Linked Receytor Facts Book, Academic
Press, San Diego
CA, pp 2-6). Mutations and changes in transcriptional activation of GPCR-
encoding genes have been
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associated with neurological disorders such as schizophrenia, Parkinson's
disease, Alzheimer's
disease, drug addiction, and feeding disorders.
Cytochromes are electron-transferring proteins that contain a heme prosthetic
group, a
porphyrin ring containing a tightly bound iron atom. Cytochromes act as
oxidoreductases in such
diverse cellular processes as respiration, photosynthesis, fatty acid
metabolism, and neurotransmitter
biosynthesis. The heme iron atom serves as the actual electron carrier by
changing from the ferric to
the ferrous oxidation state when accepting an electron. Cytochromes accept
electrons from one
substrate such as NADH or ascorbate and donate them to other electron carriers
such as other
cytochromes, ubiquinone, or semidehydroascorbic acid (Lodish, H. et al. (
1995) Molecular Cell
BioloQV, Scientific American Books, New York NY, pp. 759-770, 786-797;
Sperling, P. et al. (1995)
Eur. J. Biochem. 232:798-805; and Online Mendelian Inheritance in Man (OMIM)
*600019
Cytochrome b561, CYB561).
Cytochrome b5 is an electron donor in membrane-linked redox enzyme systems
involved in
lipid and drug metabolism. Cytochrome b5 has been found in Golgi, plasma,
outer mitochondrial,
endoplasmic reticulum (ER), and microbody membranes. Conserved amino acids in
cytochrome b5
include eight invariant amino acids at W34, H51, P52, G53, G54, G63, F70, and
H74, and fifteen
conserved amino acids at L24, I35, S36, V41, Y42, N43, T45, W47, A48, L58,
D65, T67, L85, T87,
and G88 (numbering based on the sunflower cytochrome b5/delta-6 desaturase
fusion protein; GI
1040729, Sperling, supra). The invariant residues HS 1PGG are involved in heme-
binding.
Cytochrome b5-like domains have also been found linked to other enzymes. For
example,
cytochrome b5-like domains are part of delta-9 fatty acid desaturases in yeast
and Histoplasma
capsulatum, nitrate reductase, sulfite reductase, flavocytochrome b2,
Arabido~sis thaliana acyl lipid
desaturase, and Borago officinalis (borage) and Helianthus annuus (sunflower)
delta-6 desaturases
(Sperling, supra; Sayanova, O. et al (1997) Proc. Natl. Acad. Sci. USA 94:4211-
4216; and Mitchell,
A.G. and C.E. Martin (1997) J. Biol. Chem. 272:28281-28288).
Signal peptides are found on proteins that are targeted to the endoplasmic
reticulum (ER).
Signal peptides consist of stretches of amino acids enriched in hydrophobic
residues. Signal peptides
are usually found at the extreme N-terminus of the protein and are recognized
by a cytosolic signal-
recognition peptide (SRP). The SRP binds to the signal peptide and to an SRP
receptor, an integral
membrane protein in the ER. Once bound to the SRP receptor, the newly formed
protein containing
the signal peptide is translocated across the ER membrane. Proteins containing
signal peptides may
end up inserted into the lipid bilayer, or they may end up in the lumen of an
organelle or secreted from
the cell.
Disease Correlation
The etiology of numerous human diseases and disorders can be attributed to
defects in the
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transport of molecules across membranes. Defects in the trafficking of
membrane-bound transporters
and ion channels are associated with several disorders, e.g. cystic fibrosis,
glucose-galactose
malabsorption syndrome, hypercholesterolemia, von Gierke disease, and certain
forms of diabetes
mellitus. Single-gene defect diseases resulting in an inability to transport
small molecules across
membranes include, e.g., cystinuria, iminoglycinuria, Hartup disease, and
Fanconi disease (van't Hoff,
W.G. ( 1996) Exp. Nephrol. 4:253-262; Talente, G.M. et al. ( 1994) Ann.
Intern. Med. 120:218-226;
and Chillon, M. et al. (1995) New Engl. J. Med. 332:1475-1480).
Cystinuria is an inherited disease that results from the inability to
transport cystine, the
disulfide-linked dimer of cysteine, from the urine into the blood.
Accumulation of cystine in the
urine leads to the formation of cystine stones in the kidneys.
Transthyretin (TTR), present in human plasma, binds to and transports the
thyroid hormone
thyroxine. Mutations in TTR result in the conversion of TTR to amyloid, an
insoluble fibrillar
structure. The resulting amyloid plaques have been shown to be the causative
agent in the
development of familial amyloid polyneuropathy and senile systemic amyloidosis
(Miroy, G.J. et al.
IS (1996) Proc. Natl. Acad. Sci. USA 93:15051-15056).
Stomatin, a 31-kDa erythrocyte integral membrane protein has been linked to
the hereditary
anemia stomatocytosis. This anemia is characterized by red blood cells that
lack stomatin and leak
Na+ and K+. Thus, stomatin is presumed to play a role in the regulation of ion
transport. Red blood
cell ion transport defects are also linked to other disorders such as
hypertension (Stewart, G.W.
(1997) Int. J. Biochem. Cell Biol. 29:271-274).
The discovery of new human transport proteins and the polynucleotides encoding
them
satisfies a need in the art by providing new compositions which are useful in
the diagnosis,
prevention, and treatment of transport, metabolic, neurological,
cardiovascular, reproductive, and
immune disorders, and cell proliferative disorders including cancer.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, human transport proteins,
referred to
collectively as "TPPT" and individually as "TPPT-1," "TPPT-2," "TPPT-3," "TPPT-
4.," "TPPT-5,"
"TPPT-6," ..~P.h-7," ".LPph-8 " ".LpPT-9,,~ ".hpPT-lp,,> "TPPT-11,~, ".hPPT-12
" "Z.pp.L-13 ,>
"TPPT-14," "TPPT-15," "TPPT-16," "TPPT-17," "TPPT-18," "TPPT-19," "TPPT-20,"
"TPPT-21,"
"TPPT-22," "TPPT-23," "TPPT-24," "TPPT-25," "TPPT-26," "TPPT-27," "TPPT-28,"
"TPPT-29,"
"TPPT-30," "TPPT-31," "TPPT-32," "TPPT-33," "TPPT-34," "TPPT-35," "TPPT-36,"
"TPPT-37,"
"TPPT-38," "TPPT-39," "TPPT-40," "TPPT-41," "TPPT-42," and "TPPT-43." In one
aspect, the
invention provides an isolated polypeptide comprising an amino acid sequence
selected from the
group consisting of a) an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
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43, b) a naturally occurring amino acid sequence having at least 90% sequence
identity to an amino
acid sequence selected from the group consisting of SEQ ID NO:I-43, c) a
biologically active
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-43, and d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-43. In one alternative, the invention provides an isolated polypeptide
comprising the amino
acid sequence of SEQ ID NO:1-43.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising
an amino acid sequence selected from the group consisting of a) an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-43, b) a naturally occurring amino
acid sequence having at
least 90% sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-43, c) a biologically active fragment of an amino acid sequence selected
from the group
consisting of SEQ >D NO:1-43, and d) an immunogenic fragment of an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-43. In one alternative, the
polynucleotide encodes a
polypeptide selected from the group consisting of SEQ ID NO:1-43. In another
alternative, the
polynucleotide is selected from the group consisting of SEQ ID N0:44-86.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising
an amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ B7 NO:1-43, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-43,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ )I7 NO:1-43, and d) an immunogenic fragment of an amino acid sequence
selected from the
group consisting of SEQ )D NO:1-43. In one alternative, the invention provides
a cell transformed
with the recombinant polynucleotide. In another alternative, the invention
provides a transgenic
organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising an
amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-43, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-43,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ ID NO:1-43, and d) an immunogenic fragment of an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-43. The method comprises a) culturing a cell
under conditions
suitable for expression of the polypeptide, wherein said cell is transformed
with a recombinant
polynucleotide comprising a promoter sequence operably linked to a
polynucleotide encoding the
polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
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polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO' 1-43, b) a
naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-43, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-43, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-43.
The invention further provides an isolated polynucleotide comprising a
polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:44-86, b) a naturally occurnng polynucleotide sequence
having at least 70%
sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID N0:44-
86, c) a polynucleotide sequence complementary to a), d) a polynucleotide
sequence complementary
to b), and e) an RNA equivalent of a)-d). In one alternative, the
polynucleotide comprises at least 60
contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
comprising a polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:44-86, b) a naturally occurring polynucleotide
sequence having at least 70%
sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ m N0:44-
86, c) a polynucleotide sequence complementary to a), d) a polynucleotide
sequence complementary
to b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing
the sample with a
probe comprising at least 20 contiguous nucleotides comprising a sequence
complementary to said
target polynucleotide in the sample, and which probe specifically hybridizes
to said target
polynucleotide, under conditions whereby a hybridization complex is formed
between said probe and
said target polynucleotide or fragments thereof, and b) detecting the presence
or absence of said
hybridization complex, and optionally, if present, the amount thereof. In one
alternative, the probe
comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide comprising a
polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:44-86, b) a naturally occurring polynucleotide
sequence having at least 70%
sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID N0:44-
86, c) a polynucleotide sequence complementary to a), d) a polynucleotide
sequence complementary
to b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying
said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting
the presence or absence of said amplified target polynucleotide or fragment
thereof, and, optionally, if
present, the amount thereof.
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The invention further provides a pharmaceutical composition comprising an
effective amount
of a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO:I-43, b) a
naturally occurring
amino acid sequence having at least 90% sequence identity to an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-43, c) a biologically active fragment of
an amino acid sequence
selected from the group consisting of SEQ )T7 NO:1-43, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-43, and a
pharmaceutically
acceptable excipient. In one embodiment, the pharmaceutical composition
comprises an amino acid
sequence selected from the group consisting of SEQ ID NO:1-43. The invention
additionally provides
a method of treating a disease or condition associated with decreased
expression of functional TPPT,
comprising administering to a patient in need of such treatment the
pharmaceutical composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-43,
b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-43, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ m NO:1-43, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-43. The
method comprises a) exposing a sample comprising the polypeptide to a
compound, and b) detecting
agonist activity in the sample. In one alternative, the invention provides a
pharmaceutical
composition comprising an agonist compound identified by the method and a
pharmaceutically
acceptable excipient. In another alternative, the invention provides a method
of treating a disease or
condition associated with decreased expression of functional TPPT, comprising
administering to a
patient in need of such treatment the pharmaceutical composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide comprising an amino acid sequence selected from
the group consisting
of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-
43, b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ )D NO:1-43, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-43, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-43. The
method comprises a) exposing a sample comprising the polypeptide to a
compound, and b) detecting
antagonist activity in the sample. In one alternative, the invention provides
a pharmaceutical
composition comprising an antagonist compound identified by the method and a
pharmaceutically
acceptable excipient. In another alternative, the invention provides a method
of treating a disease or
condition associated with overexpression of functional TPPT, comprising
administering to a patient in
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need of such treatment the pharmaceutical composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-43, b) a
naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-43, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-43, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-43. The method
comprises a)
combining the polypeptide with at least one test compound under suitable
conditions, and b)
detecting binding of the polypeptide to the test compound, thereby identifying
a compound that
specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-43,
b) a naturally
occurnng amino acid sequence having at least 90% sequence identity to an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-43, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-43, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-43. The
method comprises a) combining the polypeptide with at least one test compound
under conditions
permissive for the activity of the polypeptide, b) assessing the activity of
the polypeptide in the
presence of the test compound, and c) comparing the activity of the
polypeptide in the presence of the
test compound with the activity of the polypeptide in the absence of the test
compound, wherein a
change in the activity of the polypeptide in the presence of the test compound
is indicative of a
compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected from the group consisting of SEQ ID N0:44-86, the method
comprising a)
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs),
clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments
used to assemble full-
length sequences encoding TPPT.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of TPPT.
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Table 3 shows selected fragments of each nucleic acid sequence; the tissue-
specific
expression patterns of each nucleic acid sequence as determined by northern
analysis; diseases,
disorders, or conditions associated with these tissues; and the vector into
which each cDNA was
cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA clones
encoding TPPT were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection with the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"TPPT" refers to the amino acid sequences of substantially purified TPPT
obtained from any
species, particularly a mammalian species, including bovine, ovine, porcine,
murine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
TPPT. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of TPPT either by
directly interacting with
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TPPT or by acting on components of the biological pathway in which TPPT
participates.
An "allelic variant" is an alternative form of the gene encoding TPPT. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurnng 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
m a given sequence.
"Altered" nucleic acid sequences encoding TPPT include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as TPPT or a
polypeptide with at least one functional characteristic of TPPT. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding TPPT, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
TPPT. 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 TPPT. 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 TPPT is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid
sequence to the complete native amino acid sequence associated with the
recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerise chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of TPPT. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of TPPT either by
directly interacting with TPPT or by acting on components of the biological
pathway in which TPPT
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participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, Flab' ),, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind TPPT polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used
to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from
the translation of RNA,
or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly used
carriers that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and
keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
IS 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 TPPT, or of
any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
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amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding TPPT or fragments of
TPPT may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(PE Biosystems,
Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which
has been assembled from
one or more overlapping cDNA, EST, or genomic DNA fragments using a computer
program for
fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison
WI) or Phrap
(University of Washington, Seattle WA). Some sequences have been both extended
and assembled to
produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile Leu Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
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A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide sequence can include, for example,
replacement of
hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative
polynucleotide encodes a
polypeptide which retains at least one biological or immunological function of
the natural molecule.
A derivative polypeptide is one modified by glycosylation, pegylation, or any
similar process that
retains at least one biological or immunological function of the polypeptide
from which it was
derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
A "fragment" is a unique portion of TPPT or the polynucleotide encoding TPPT
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10, 15,
16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide)
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:44-86 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:44-86, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:44-86 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:44-86 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:44-86 and the region of SEQ ID N0:44-86 to which the fragment
corresponds are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID NO:I-43 is encoded by a fragment of SEQ ID N0:44-86. A
fragment
of SEQ ID NO: I-43 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO: I-43. For example, a fragment of SEQ ID NO:1-43 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:I-43.
The precise length of
a fragment of SEQ ID NO:I-43 and the region of SEQ ID NO:I-43 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
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purpose for the fragment.
A "full-length" polynucleotide sequence is one containing at least a
translation initiation
codon (e.g., methionine) followed by an open reading frame and a translation
termination codon. A
"full-length" polynucleotide sequence encodes a "full-length" polypeptide
sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available from
several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
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Open Gap: S and Extension Gap: 2 penalties
Gap x drop-off: SO
Expect: 70
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
IS in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the charge
and hydrophobicity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (Apr-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off. SO
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Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1 % (w/v) SDS, and about 100 pg/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.,
1989, Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring
Harbor Press,
Plainview NY; specifically see volume 2, chapter 9.
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High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1 % SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used. SSC concentration may
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
sheared and denatured salmon sperm DNA at about 100-200 pg/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of TPPT
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 TPPT 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 TPPT. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of TPPT.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
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polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation,
and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an TPPT may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in
the art. These processes may occur synthetically or biochemically. Biochemical
modifications will
vary by cell type depending on the enzymatic milieu of TPPT.
"Probe" refers to nucleic acid sequences encoding TPPT, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerise enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerise chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al., 1989, Molecular Cloning: A Laborator~Manual, 2"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et a1.,1987, Current
Protocols in Molecular
Bioloey, 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
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can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The
Primer3 primer
selection program (available to the public from the Whitehead Institute/MIT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
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
CA 02375493 2001-12-11
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expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding TPPT, or fragments thereof, or TPPT itself, may comprise a
bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a cell; a cell;
genomic DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" refers to the collective pattern of gene expression by a
particular cell
type or tissue under given conditions at a given time.
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"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or
viral infection, electroporation, heat shock, lipofection, and particle
bombardment. The term
"transformed" cells includes stably transformed cells in which the inserted
DNA is capable of
replication either as an autonomously replicating plasmid or as part of the
host chromosome, as well
as transiently transformed cells which express the inserted DNA or RNA for
limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to animals
and plants, in which one or more of the cells of the organism contains
heterologous nucleic acid
introduced by way of human intervention, such as by transgenic techniques well
known in the art.
The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor of
the cell, by way of deliberate genetic manipulation, such as by microinjection
or by infection with a
recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants, and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook et al. (1989),
supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
95% or at least 98% or
greater sequence identity over a certain defined length. A variant may be
described as, for example,
an "allelic" (as defined above), "splice," "species," or "polymorphic"
variant. A splice variant may
have significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternative splicing of exons during mRNA processing.
The corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides generally will have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
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polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least
98% or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human transport proteins
(TPPT), the
polynucleotides encoding TPPT, and the use of these compositions for the
diagnosis, treatment, or
prevention of transport, metabolic, neurological, cardiovascular,
reproductive, and immune disorders,
and cell proliferative disorders including cancer.
IS Table I lists the Incyte clones used to assemble full length nucleotide
sequences encoding
TPPT. Columns I and 2 show the sequence identification numbers (SEQ ID NOs) of
the polypeptide
and nucleotide sequences, respectively. Column 3 shows the clone IDs of the
Incyte clones in which
nucleic acids encoding each TPPT were identified, and column 4 shows the cDNA
libraries from
which these clones were isolated. Column 5 shows Incyte clones and their
corresponding cDNA
libraries. Clones for which cDNA libraries are not indicated were derived from
pooled cDNA
libraries. In some cases, GenBank sequence identifiers are also shown in
column 5. The Incyte
clones and GenBank cDNA sequences, where indicated, in column 5 were used to
assemble the
consensus nucleotide sequence of each TPPT and are useful as fragments in
hybridization
technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; column 6
shows homologous sequences as identified by BLAST analysis; and column 7 shows
analytical
methods and in some cases, searchable databases to which the analytical
methods were applied. The
methods of column 7 were used to characterize each polypeptide through
sequence homology and
protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding TPPT. The first column of Table
3 lists the nucleotide
SEQ )D NOs. Column 2 lists fragments of the nucleotide sequences of column 1.
These fragments
are useful, for example, in hybridization or amplification technologies to
identify SEQ ID N0:44-86
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and to distinguish between SEQ ID N0:44-86 and related polynucleotide
sequences. The
polypeptides encoded by these fragments are useful, for example, as
immunogenic peptides. Column
3 lists tissue categories which express TPPT as a fraction of total tissues
expressing TPPT. Column 4
lists diseases, disorders, or conditions associated with those tissues
expressing TPPT as a fraction of
total tissues expressing TPPT. Column 5 lists the vectors used to subclone
each cDNA library.
Of particular interest is the expression of SEQ )D NO:50 exclusively in
cardiovascular tissue,
the expression of SEQ ID N0:56 in nervous and gastrointestinal tissues, the
expression of SEQ ID
N0:57 in gastrointestinal tissues, and the expression of SEQ ID N0:66 in
nervous system tissues. Of
particular note is the tissue-specific expression of SEQ ID N0:75. Over 71 %
of the cDNA libraries
expressing SEQ )D N0:75 are derived from lung tissue.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
from which cDNA clones encoding TPPT were isolated. Column 1 references the
nucleotide SEQ ID
NOs, column 2 shows the cDNA libraries from which these clones were isolated,
and column 3 shows
the tissue origins and other descriptive information relevant to the cDNA
libraries in column 2.
SEQ ID N0:44 maps to chromosome 7 within the interval from 38.80 to 42.10
centiMorgans.
SEQ ID N0:48 maps to chromosome X within the interval from 107.90 to 122.80
centiMorgans.
SEQ ID N0:60 maps to chromosome 2 within the interval from 157.0 to 167.0
centiMorgans. SEQ
ID N0:65 maps to chromosome 2 within the interval from 17.4 to 40.7
centiMorgans and to
chromosome 5 within the interval from 61.1 to 69.6 centiMorgans. The interval
on chromosome 5
from 61.1 to 69.6 centiMorgans also contains genes associated with Cockayne
syndrome. SEQ ID
N0:69 maps to chromosome 3 within the interval from 157.40 to 162.00
centiMorgans. SEQ ID
N0:70 maps to chromosome 3 within the interval from 176.40 to 179.80
centiMorgans. SEQ ID
N0:71 maps to chromosome 18 within the interval from the p-terminus to 52.30
centiMorgans. SEQ
ID N0:73 maps to chromosome 17 within the interval from 75.70 to 84.20
centiMorgans, and to
chromosome 2 within the interval from 204.70 to 209.30 centiMorgans. SEQ ID
N0:76 maps to
chromosome 20 within the interval from 79.00 to 94.40 centiMorgans. SEQ ID
N0:80 maps to
chromosome 18 within the interval from 1.60 to 6.20 centiMorgans, and to
chromosome 11 within the
interval from 117.90 to 126.00 centiMorgans. SEQ ID N0:83 maps to chromosome
17 within the
interval from 67.60 to 69.30 centiMorgans, and from 83.8 centiMorgans to the q-
terminus, and to
chromosome 7 within the interval from 105.20 to 114.50 centiMorgans.
The invention also encompasses TPPT variants. A preferred TPPT variant is one
which has
at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the TPPT amino acid sequence, and which contains at least
one functional or
structural characteristic of TPPT.
The invention also encompasses polynucleotides which encode TPPT. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
29
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
from the group consisting of SEQ ID N0:44-86, which encodes TPPT. The
polynucleotide sequences
of SEQ ID N0:44-86, 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
TPPT. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at
least about 85%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding TPPT. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:44-
86 which has at least about 70%, or alternatively at least about 85%, or even
at least about 95%
polynucleotide sequence identity to a nucleic acid sequence selected from the
group consisting of
SEQ )D N0:44-86. Any one of the polynucleotide variants described above can
encode an amino acid
sequence which contains at least one functional or structural characteristic
of TPPT.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynucleotide sequences encoding TPPT, 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 TPPT, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode T'PPT and its variants are
generally capable of
hybridizing to the nucleotide sequence of the naturally occurring TPPT under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding TPPT or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding TPPT 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 TPPT
and
TPPT 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 TPPT or any fragment thereof.
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:44-86 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger ( 1987) Methods Enzymol. 152:399-407; Kimmel, A.R. ( 1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(PE
Biosystems, Foster City CA), thermostable T7 polymerase (Amersham Pharmacia
Biotech,
Piscataway NJ), or combinations of polymerases and proofreading exonucleases
such as those found
in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
Preferably,
sequence preparation is automated with machines such as the MICROLAB 2200
liquid transfer
system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA)
and ABI
CATALYST 800 thermal cycler (PE Biosystems). Sequencing is then carried out
using either the
ABI 373 or 377 DNA sequencing system (PE Biosystems), the MEGABACE 1000 DNA
sequencing
system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art.
The resulting
sequences are analyzed using a variety of algorithms which are well known in
the art. (See, e.g.,
Ausubel, F.M. ( 1997) Short Protocols in Molecular Biology, John Wiley & Sons,
New York NY, unit
7.7; Meyers, R.A. (1995) Molecular Biolow and Biotechnolo~y, Wiley VCH, New
York NY, pp.
856-853.)
The nucleic acid sequences encoding TPPT 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
31
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 Primer Analysis software
(National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the
template at temperatures of
about 68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode TPPT may be cloned in recombinant DNA molecules that direct expression
of TPPT, 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 TPPT.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter TPPT-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
32
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WO 00/78953 PCT/US00/16668
improve the biological properties of TPPT, such as its biological or enzymatic
activity or its ability to
bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner
In another embodiment, sequences encoding TPPT 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,
TPPT itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide
synthesis can be performed using various solution-phase or solid-phase
techniques. (See, e.g.,
Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. ( 1995) Science 269:202-204.) Automated
synthesis may be achieved
using the ABI 431A peptide synthesizer (PE Biosystems). Additionally, the
amino acid sequence of
TPPT, or any part thereof, may be altered during direct synthesis and/or
combined with sequences
from other proteins, or any part thereof, to produce a variant polypeptide or
a polypeptide having a
sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier ( 1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active TPPT, the nucleotide sequences
encoding TPPT 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 TPPT. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
TPPT. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding TPPT and its initiation codon and upstream regulatory
sequences are inserted into
33
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
the appropriate expression vector, no additional transcriptional or
translational control signals may be
needed. However, in cases where only coding sequence, or a fragment thereof,
is inserted, exogenous
translational control signals including an in-frame ATG initiation codon
should be provided by the
vector. Exogenous translational elements and initiation codons may be of
various origins, both
natural and synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers
appropriate for the particular host cell system used. (See, e.g., Scharf, D.
et al. ( 1994) Results Probl.
Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding TPPT and appropriate transcriptional and
translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. ( 1995)
Current Protocols in Molecular 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 TPPT. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra; Ausubel, sera; Van Heeke, G.
and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Bitter, G.A. et al. (1987) Methods
Enzymol. 153:516-544;
Scorer, C.A. et al. (1994) Bio/Technology 12:181-184; Engelhard, E.K. et al.
(1994) Proc. Natl. Acad.
Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945;
Takamatsu, N.
(1987) EMBO J. 6:307-311; Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;
Brogue, R. et al. (1984)
Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ.
17:85-105; The McGraw Hill
Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-
196; Logan, J.
and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659; and Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355.) Expression vectors derived from retroviruses,
adenoviruses, or herpes or
vaccinia viruses, or from various bacterial plasmids, may be used for delivery
of nucleotide sequences
to the targeted organ, tissue, or cell population. (See, e.g., Di Nicola, M.
et al. ( 1998) Cancer Gen.
Ther. 5(6):350-356; Yu, M. et al., ( 1993) Proc. Natl. Acad. Sci. USA 90(
13):6340-6344; Buller, R.M.
et al. (1985) Nature 317(6040):813-815; McGregor, D.P. et al. (1994) Mol.
Immunol. 31(3):219-226;
and Verma, LM. and N. Somia ( 1997) Nature 389:239-242.) The invention is not
limited by the host
cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding TPPT. For example,
routine cloning,
34
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
subcloning, and propagation of polynucleotide sequences encoding TPPT 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 TPPT 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 TPPT are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of TPPT may be used.
For example, vectors
containing the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of TPPT. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomvces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, supra; and Scorer, supra.)
Plant systems may also be used for expression of TPPT. Transcription of
sequences encoding
TPPT may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV
used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO
J. 6:307-311).
Alternatively, plant promoters such as the small subunit of RUBISCO or heat
shock promoters may be
used. (See, e.g., Coruzzi, supra; Broglie, supra; and Winter, supra.) These
constructs can be
introduced into plant cells by direct DNA transformation or pathogen-mediated
transfection. (See,
e.g., The McGraw Hill Yearbook of Science and Technolo~y ( 1992) McGraw Hill,
New York NY,
pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding TPPT
may be ligated into an
adenovirus transcription/translation complex consisting of the late promoter
and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses TPPT in host cells. (See, e.g., Logan, J. and
T. Shenk ( 1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat. Genet.
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
TPPT in cell lines is preferred. For example, sequences encoding TPPT can be
transformed into cell
lines using expression vectors which may contain viral origins of replication
and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media
before being switched to selective media. The purpose of the selectable marker
is to confer resistance
to a selective agent, and its presence allows growth and recovery of cells
which successfully express
the introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and apr cells, respectively.
(See, e.g., Wigler, M. et al.
( 1977) Cell 11:223-232; Lowy, I. et al. ( 1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic, or
herbicide resistance can be used as the basis for selection. For example, dhfr
confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. ( 1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. ( 1981 )
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan ( 1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech), B glucuronidase and its substrate B-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding TPPT is inserted within a marker gene sequence, transformed
cells containing
sequences encoding TPPT can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding TPPT 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 TPPT
and that express
TPPT 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
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CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
Immunological methods for detecting and measuring the expression of TPPT using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on TPPT 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) Seroloeical Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in ImmunoloQV, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. ( 1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding TPPT
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding TPPT, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding TPPT 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 TPPT may be designed to contain signal
sequences which
direct secretion of TPPT 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
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CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural. modified, or recombinant
nucleic acid
sequences encoding TPPT 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 TPPT protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of TPPT activity.
Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available
affinity matrices. Such moieties include, but are not limited to, glutathione
S-transferase (GST),
maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their cognate
fusion proteins on immobilized glutathione, maltose, phenylarsine oxide,
calmodulin, and metal-
chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffinity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies
that specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the TPPT encoding sequence and the
heterologous protein
sequence, so that TPPT 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 TPPT 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.
TPPT of the present invention or fragments thereof may be used to screen for
compounds that
specifically bind to TPPT. At least one and up to a plurality of test
compounds may be screened for
specific binding to TPPT. 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
TPPT, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, Coligan, J.E. et al. ( 1991 ) Current Protocols
in Immunoloay 1 (2):
Chapter S.) Similarly, the compound can be closely related to the natural
receptor to which TPPT
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 TPPT,
either as a secreted
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protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing TPPT or cell membrane fractions which contain TPPT are
then contacted with
a test compound and binding, stimulation, or inhibition of activity of either
TPPT 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
TPPT, either in
solution or affixed to a solid support, and detecting the binding of TPPT 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.
TPPT of the present invention or fragments thereof may be used to screen for
compounds that
modulate the activity of TPPT. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for TPPT
activity, wherein TPPT is combined with at least one test compound, and the
activity of TPPT in the
presence of a test compound is compared with the activity of TPPT in the
absence of the test
compound. A change in the activity of TPPT in the presence of the test
compound is indicative of a
compound that modulates the activity of TPPT. Alternatively, a test compound
is combined with an
in vitro or cell-free system comprising TPPT under conditions suitable for
TPPT activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of TPPT
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 TPPT or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem
(ES) cells. Such techniques are well known in the art and are useful for the
generation of animal
models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent
No. 5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding
region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. ( 1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (
1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
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to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding TPPT may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding TPPT 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 TPPT 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 TPPT, e.g., by secreting TPPT 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 TPPT and human transport proteins. In addition, the
expression of TPPT is
closely associated with neurological, cardiovascular, reproductive,
gastrointestinal, and
hematopoietic/immune tissues, and inflammation, cell proliferation, and
cancer. Therefore, TPPT
appears to play a role in transport, metabolic, neurological, cardiovascular,
reproductive, and immune
disorders, and cell proliferative disorders including cancer. In the treatment
of disorders associated
with increased TPPT expression or activity, it is desirable to decrease the
expression or activity of
TPPT. In the treatment of disorders associated with decreased TPPT expression
or activity, it is
desirable to increase the expression or activity of TPPT.
Therefore, in one embodiment, TPPT 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 TPPT.
Examples of such disorders include, but are not limited to, 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,
tachyarrythmia, hypertension, Long QT syndrome, myocarditis, cardiomyopathy,
nemaline
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myopathy, centronuclear myopathy, lipid myopathy, mitochondria) 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
metabolic disorder such as Addison's disease, cerebrotendinous xanthomatosis,
congenital adrenal
hyperplasia, coumarin resistance, cystic fibrosis, diabetes, fatty
hepatocirrhosis,
fructose-1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma,
glycogen storage diseases,
hereditary fructose intolerance, hyperadrenalism, hypoadrenalism,
hyperparathyroidism,
hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia,
hypothyroidism,
hyperlipidemia, hyperlipemia, lipid myopathies, lipodystrophies, lysosomal
storage diseases,
mannosidosis, neuraminidase deficiency, obesity, pentosuria phenylketonuria,
and pseudovitamin D-
deficiency rickets; a neurological disorder such as epilepsy, ischemic
cerebrovascular disease, stroke,
cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease,
dementia, Parkinson's
disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and
other motor neuron
disorders, progressive neural muscular atrophy, retinitis pigmentosa,
hereditary ataxias, multiple
sclerosis and other demyelinating diseases, bacterial and viral meningitis,
brain abscess, subdural
empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis
and radiculitis, viral
central nervous system disease, prion diseases including kuru, Creutzfeldt-
Jakob disease, and
Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional
and metabolic diseases
of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous
system disorders, cranial
nerve disorders, spinal cord diseases, muscular dystrophy and other
neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and polymyositis,
inherited, metabolic,
endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental
disorders including
mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD),
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial
frontotemporal dementia; a cardiovascular disorder such as arteriovenous
fistula, atherosclerosis,
hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections,
varicose veins,
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thrombophlebitis and phlebothrombosis, vascular tumors, and complications of
thrombolysis, balloon
angioplasty, vascular replacement, and coronary artery bypass graft surgery,
congestive heart failure,
ischemic heart disease, angina pectoris, myocardial infarction, hypertensive
heart disease,
degenerative valvular heart disease, calcific aortic valve stenosis,
congenitally bicuspid aortic valve,
mitral annular calcification, mitral valve prolapse, rheumatic fever and
rheumatic heart disease,
infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of
systemic lupus
erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis,
pericarditis, neoplastic heart
disease, congenital heart disease, and complications of cardiac
transplantation, congenital lung
anomalies, atelectasis, pulmonary congestion and edema, pulmonary embolism,
pulmonary
hemorrhage, pulmonary infarction, pulmonary hypertension, vascular sclerosis,
obstructive pulmonary
disease, restrictive pulmonary disease, chronic obstructive pulmonary disease,
emphysema, chronic
bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viral and
mycoplasmal pneumonia,
lung abscess, pulmonary tuberculosis, diffuse interstitial diseases,
pneumoconioses, sarcoidosis,
idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis,
hypersensitivity pneumonitis,
pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse
pulmonary
hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary
hemosiderosis, pulmonary
involvement in collagen-vascular disorders, pulmonary alveolar proteinosis,
lung tumors,
inflammatory and noninflammatory pleural effusions, pneumothorax, pleural
tumors, drug-induced
lung disease, radiation-induced lung disease, and complications of lung
transplantation; a
reproductive disorder such as a disorder of prolactin production, infertility,
including tubal disease,
ovulatory defects, and endometriosis, a disruption of the estrous cycle, a
disruption of the menstrual
cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an
endometrial or ovarian
tumor, a uterine fibroid, autoimmune disorders, an ectopic pregnancy, and
teratogenesis; cancer of the
breast, fibrocystic breast disease, and galactorrhea; a disruption of
spermatogenesis, abnormal sperm
physiology, cancer of the testis, cancer of the prostate, benign prostatic
hyperplasia, prostatitis,
Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia;
an immune disorder
such as inflammation, actinic keratosis, acquired immunodeficiency syndrome
(AIDS), Addison's
disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia,
arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune thyroiditis,
autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),
bronchitis, bursitis,
cholecystitis, cirrhosis, 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, paroxysmal nocturnal hemoglobinuria,
hepatitis, hypereosinophilia,
irritable bowel syndrome, mixed connective tissue disease (MCTD), multiple
sclerosis, myasthenia
gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis,
osteoporosis,
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pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis,
scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus
erythematosus, systemic
sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections, trauma, and
hematopoietic cancer including
lymphoma, leukemia, and myeloma; and a cell proliferative disorder such as
actinic keratosis,
arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed
connective tissue disease
(MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,
psoriasis, primary
thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma,
melanoma,
myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal
gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal
tract, heart, kidney, liver,
lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,
skin, spleen, testis,
thymus, thyroid, and uterus.
In another embodiment, a vector capable of expressing TPPT 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 TPPT including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
TPPT 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 TPPT
including, but not
limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of TPPT
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of TPPT including, but not limited to, those listed above.
In a further embodiment, an antagonist of TPPT may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of TPPT.
Examples of such
disorders include, but are not limited to, those transport, metabolic,
neurological, cardiovascular,
reproductive, and immune disorders, and cell proliferative disorders including
cancer described
above. In one aspect, an antibody which specifically binds TPPT 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 TPPT.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding TPPT may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of TPPT 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
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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 TPPT may be produced using methods which are generally known
in the art.
In particular, purified TPPT may be used to produce antibodies or to screen
libraries of
pharmaceutical agents to identify those which specifically bind TPPT.
Antibodies to TPPT 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 TPPT or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants
used in humans, BCG
(bacilli Calmette-Guerin) and Corvnebacterium~arvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to TPPT
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 TPPT
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 TPPT may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
technique. (See, e.g., Kohler, G. et al. ( 1975) Nature 256:495-497; Kozbor,
D. et al. ( 1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. ( 1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
TPPT-specific single
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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 TPPT 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
TPPT and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering TPPT epitopes is generally used, but a
competitive binding assay may
also be employed (Pound, su ra .
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for TPPT. Affinity
is expressed as an
association constant, Ka, which is defined as the molar concentration of TPPT-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium conditions.
The Ka determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple TPPT epitopes, represents the average affinity, or
avidity, of the antibodies for
TPPT. The Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular TPPT epitope, represents a true measure of affinity. High-affinity
antibody preparations
with Ka ranging from about 109 to 10'z L/mole are preferred for use in
immunoassays in which the
TPPT-antibody complex must withstand rigorous manipulations. Low-affinity
antibody preparations
with K~ ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of TPPT, preferably in active
form, from the
antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer ( 1991 ) A Practical Guide to Monoclonal
Antibodies, John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
CA 02375493 2001-12-11
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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 TPPT-
antibody complexes. Procedures for evaluating antibody specificity, titer, and
avidity, and guidelines
for antibody quality and usage in various applications, are generally
available. (See, e.g., Catty,
supra, and Coligan et al., supra.)
In another embodiment of the invention, the polynucleotides encoding TPPT, 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
TPPT. 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
TPPT. (See, e.g., Agrawal, S., ed. ( 1996) Antisense Therapeutics, Humana
Press Inc., Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. ( 1995) Br. Med. Bull. 51 (
1 ):217-225; Boado, R.J. et
al. ( 1998) J. Pharm. Sci. 87( 11 ):1308-1315; and Moms, M.C. et al. ( 1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding TPPT 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)-X 1 disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. ( 1993) Cell 75:207-216; Crystal, R.G. et
al. ( 1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and Somia, N. (1997) Nature
389:239-242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
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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-I 1399),
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 TPPT expression or regulation causes
disease, the expression of
TPPT 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
TPPT are treated by constructing mammalian expression vectors encoding TPPT
and introducing
these vectors by mechanical means into TPPT-deficient cells. Mechanical
transfer technologies for
use with cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii)
ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv)
receptor-mediated gene
transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson
(1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of TPPT include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen,
Carlsbad CA),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON,
PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). TPPT may be expressed
using (i) a
constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40
virus, thymidine kinase (TK), or (3-actin genes), (ii) an inducible promoter
(e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA
89:5547-5551; Gossen, M. et al. ( 1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau ( 1998)
Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the
ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and H.M. Blau, su ra ), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding TPPT from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb ( 1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of
these standardized mammalian transfection protocols.
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In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to TPPT expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding TPPT under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. ( 1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
IS discloses a method for obtaining retrovirus packaging cell lines and is
hereby incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-cells),
and the return of transduced cells to a patient are procedures well known to
persons skilled in the art
of gene therapy and have been well documented (Ranga, U. et al. (1997) J.
Virol. 71:7020-7029;
Bauer, G. et al. ( 1997) Blood 89:2259-2267; Bonyhadi, M.L. ( 1997) J. Virol.
71:4707-4716; Ranga,
U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood
89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding TPPT to cells which have one or more genetic
abnormalities with respect to
the expression of TPPT. The construction and packaging of adenovirus-based
vectors are well known
to those with ordinary skill in the art. Replication defective adenovirus
vectors have proven to be
versatile for importing genes encoding immunoregulatory proteins into intact
islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. ( 1999) Annu.
Rev. Nutr. 19:511-544; and Verma, LM. and N. Somia ( 1997) Nature 18:389:239-
242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding TPPT to target cells which have one or more genetic
abnormalities with
respect to the expression of TPPT. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing TPPT 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
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WO 00/78953 PCT/US00/16668
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (
1999) Exp. Eye
Res.169:385-395). The construction of a HSV-1 virus vector has also been
disclosed in detail in U.S.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is
hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use
of recombinant
HSV d92 which consists of a genome containing at least one exogenous gene to
be transferred to a
cell under the control of the appropriate promoter for purposes including
human gene therapy. Also
taught by this patent are the construction and use of recombinant HSV strains
deleted for ICP4, ICP27
and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol.
73:519-532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The
manipulation of cloned
herpesvirus sequences, the generation of recombinant virus following the
transfection of multiple
plasmids containing different segments of the large herpesvirus genomes, the
growth and propagation
of herpesvirus, and the infection of cells with herpesvirus are techniques
well known to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding TPPT to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li ( 1998) Curr. Opin. Biotech. 9:464-
469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full-length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
TPPT into the alphavirus
genome in place of the capsid-coding region results in the production of a
large number of TPPT-
coding RNAs and the synthesis of high levels of TPPT in vector transduced
cells. While alphavirus
infection is typically associated with cell lysis within a few days, the
ability to establish a persistent
infection in hamster normal kidney cells (BHK-21 ) with a variant of Sindbis
virus (SIN) indicates that
the lytic replication of alphaviruses can be altered to suit the needs of the
gene therapy application
(Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of
alphaviruses will allow the
introduction of TPPT 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
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been described in the literature. (See, e.g., Gee, J.E. et al. ( 1994) in
Huber, B.E. and B.I. Carr,
Molecular and Immunolo i~ c Approaches, Futura Publishing, Mt. Kisco NY, pp.
163-177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA
by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding TPPT.
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
IS candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding TPPT. Such DNA sequences may be incorporated into a wide
variety of vectors
with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these
cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines,
cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half-
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified
forms of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding TPPT. 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
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
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 TPPT
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding TPPT may be therapeutically useful, and in the treament of disorders
associated with
decreased TPPT expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding TPPT may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding TPPT 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
TPPT 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 TPPT. The amount of hybridization may be quantified,
thus forming the
basis for a comparison of the expression of the polynucleotide both with and
without exposure to one
or more test compounds. Detection of a change in the expression of a
polynucleotide exposed to a
test compound indicates that the test compound is effective in altering the
expression of the
polynucleotide. A screen for a compound effective in altering expression of a
specific polynucleotide
can be carried out, for example, using a Schizosaccharomyces pombe gene
expression system
(Atkins, D. et al. (1999) U.S. Patent No. 5.932,435; Arndt, G.M. et al. (2000)
Nucleic Acids Res.
28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000)
Biochem. Biophys. Res.
Commun. 268:8-13). A particular embodiment of the present invention involves
screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
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
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using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. ( 1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
pharmaceutical
composition which generally comprises an active ingredient formulated with a
pharmaceutically
acceptable excipient. Excipients may include, for example, sugars, starches,
celluloses, gums, and
proteins. Various formulations are commonly known and are thoroughly discussed
in the latest
edition of Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA).
Such
pharmaceutical compositions may consist of TPPT, antibodies to TPPT, and
mimetics, agonists,
antagonists, or inhibitors of TPPT.
The pharmaceutical compositions utilized in this invention may be administered
by any
number of routes including, but not limited to, oral, intravenous,
intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, pulmonary, transdermal,
subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, or rectal means.
Pharmaceutical compositions for pulmonary administration may be prepared in
liquid or dry
powder form. These compositions are generally aerosolized immediately prior to
inhalation by the
patient. In the case of small molecules (e.g. traditional low molecular weight
organic drugs), aerosol
delivery of fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g.
larger peptides and proteins), recent developments in the field of pulmonary
delivery via the alveolar
region of the lung have enabled the practical delivery of drugs such as
insulin to blood circulation
(see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary
delivery has the advantage of
administration without needle injection, and obviates the need for potentially
toxic penetration
enhancers.
Pharmaceutical compositions suitable for use in the invention include
compositions wherein
the active ingredients are contained in an effective amount to achieve the
intended purpose. The
determination of an effective dose is well within the capability of those
skilled in the art.
Specialized forms of pharmaceutical compositions may be prepared for direct
intracellular
delivery of macromolecules comprising TPPT or fragments thereof. For example,
liposome
preparations containing a cell-impermeable macromolecule may promote cell
fusion and intracellular
delivery of the macromolecule. Alternatively, TPPT 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
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WO 00/78953 PCT/US00/16668
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 TPPT
or fragments thereof, antibodies of TPPT, and agonists, antagonists or
inhibitors of TPPT, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDso (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio.
Pharmaceutical compositions
which exhibit large therapeutic indices are preferred. The data obtained from
cell culture assays and
animal studies are used to formulate a range of dosage for human use. The
dosage contained in such
compositions is preferably within a range of circulating concentrations that
includes the EDSO with
little or no toxicity. The dosage varies within this range depending upon the
dosage form employed,
the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4
days, every week, or biweekly depending on the half-life and clearance rate of
the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 fig, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
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 TPPT may be used for
the
diagnosis of disorders characterized by expression of TPPT, or in assays to
monitor patients being
treated with TPPT or agonists, antagonists, or inhibitors of TPPT. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for TPPT include methods which utilize the antibody and a label to detect TPPT
in human body fluids
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WO 00/78953 PCT/US00/16668
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 TPPT, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
TPPT expression. Normal
or standard values for TPPT expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibody to TPPT under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of TPPT
expressed in subject,
control, and disease samples from biopsied tissues are compared with the
standard values. Deviation
between standard and subject values establishes the parameters for diagnosing
disease.
In another embodiment of the invention, the polynucleotides encoding TPPT 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 TPPT may
be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
TPPT, and to monitor regulation of TPPT levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding TPPT or closely related
molecules may be used to
identify nucleic acid sequences which encode TPPT. 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 TPPT, 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 TPPT encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:44-86 or from
genomic sequences including promoters, enhaneers, and introns of the TPPT
gene.
Means for producing specific hybridization probes for DNAs encoding TPPT
include the
cloning of polynucleotide sequences encoding TPPT or TPPT 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 3ZP or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding TPPT may be used for the diagnosis of
disorders
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associated with expression of TPPT. Examples of such disorders include, but
are not limited to, 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,
S 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, tachyarrythmia, 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, Addispn'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 metabolic disorder such as Addison's disease,
cerebrotendinous xanthomatosis,
congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis,
diabetes, fatty hepatocirrhosis,
fructose-1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma,
glycogen storage diseases,
hereditary fructose intolerance, hyperadrenalism, hypoadrenalism,
hyperparathyroidism,
hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia,
hypothyroidism,
hyperlipidemia, hyperlipemia, lipid myopathies, lipodystrophies, lysosomal
storage diseases,
mannosidosis, neuraminidase deficiency, obesity, pentosuria phenylketonuria,
and pseudovitamin D-
deficiency rickets; a neurological disorder such as epilepsy, ischemic
cerebrovascular disease, stroke,
cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease,
dementia, Parkinson's
disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and
other motor neuron
disorders, progressive neural muscular atrophy, retinitis pigmentosa,
hereditary ataxias, multiple
sclerosis and other demyelinating diseases, bacterial and viral meningitis,
brain abscess, subdural
empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis
and radiculitis, viral
central nervous system disease, prion diseases including kuru, Creutzfeldt-
Jakob disease, and
Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional
and metabolic diseases
of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous
system disorders, cranial
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nerve disorders, spinal cord diseases, muscular dystrophy and other
neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and polymyositis,
inherited, metabolic,
endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental
disorders including
mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD),
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial
frontotemporal dementia; a cardiovascular disorder such as arteriovenous
fistula, atherosclerosis,
hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections,
varicose veins,
thrombophlebitis and phlebothrombosis, vascular tumors, and complications of
thrombolysis, balloon
angioplasty, vascular replacement, and coronary artery bypass graft surgery,
congestive heart failure,
ischemic heart disease, angina pectoris, myocardial infarction, hypertensive
heart disease,
degenerative valvular heart disease, calcific aortic valve stenosis,
congenitally bicuspid aortic valve,
mural annular calcification, mitral valve prolapse, rheumatic fever and
rheumatic heart disease,
infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of
systemic lupus
erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis,
pericarditis, neoplastic heart
disease, congenital heart disease, and complications of cardiac
transplantation, congenital lung
anomalies, atelectasis, pulmonary congestion and edema, pulmonary embolism,
pulmonary
hemorrhage, pulmonary infarction, pulmonary hypertension, vascular sclerosis,
obstructive
pulmonary disease, restrictive pulmonary disease, chronic obstructive
pulmonary disease,
emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, bacterial
pneumonia, viral and
mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuse
interstitial diseases,
pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative
interstitial pneumonitis,
hypersensitivity pneumonitis, pulmonary eosinophilia bronchiolitis obliterans-
organizing pneumonia,
diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic
pulmonary
hemosiderosis, pulmonary involvement in collagen-vascular disorders, pulmonary
alveolar
proteinosis, lung tumors, inflammatory and noninflammatory pleural effusions,
pneumothorax,
pleural tumors, drug-induced lung disease, radiation-induced lung disease, and
complications of lung
transplantation; a reproductive disorder such as a disorder of prolactin
production, infertility,
including tubal disease, ovulatory defects, and endometriosis, a disruption of
the estrous cycle, a
disruption of the menstrual cycle, polycystic ovary syndrome, ovarian
hyperstimulation syndrome, an
endometrial or ovarian tumor, a uterine fibroid, autoimmune disorders, an
ectopic pregnancy, and
teratogenesis; cancer of the breast, fibrocystic breast disease, and
galactorrhea; a disruption of
spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of
the prostate, benign
prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma
of the male breast, and
gynecomastia; an immune disorder such as inflammation, actinic keratosis,
acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory
distress syndrome,
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allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis,
asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
polyendocrinopathy-
candidiasis-ectodermal dystrophy (APECED), bronchitis, bursitis,
cholecystitis, cirrhosis, 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, paroxysmal nocturnal hemoglobinuria, hepatitis,
hypereosinophilia, irritable bowel
syndrome, mixed connective tissue disease (MCTD), multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, myelofibrosis, osteoarthritis,
osteoporosis, pancreatitis,
polycythemia vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid
arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis, primary
thrombocythemia, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner
syndrome,
complications of cancer, hemodialysis, and extracorporeal circulation, viral,
bacterial, fungal,
parasitic, protozoal, and helminthic infections, trauma, and hematopoietic
cancer including
lymphoma, leukemia, and myeloma; and a cell proliferative disorder such as
actinic keratosis,
arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed
connective tissue disease
(MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,
psoriasis, primary
thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma,
melanoma,
myeloma, sarcoma, teratocarcinoma, and, in particular, 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 TPPT 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 TPPT expression. Such qualitative or quantitative
methods are well known
in the art.
In a particular aspect, the nucleotide sequences encoding TPPT may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding TPPT 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 TPPT in the
sample indicates the presence of the associated disorder. Such assays may also
be used to evaluate
the efficacy of a particular therapeutic treatment regimen in animal studies,
in clinical trials, or to
monitor the treatment of an individual patient.
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In order to provide a basis for the diagnosis of a disorder associated with
expression of TPPT,
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 TPPT, under conditions suitable for hybridization
or amplification.
Standard hybridization may be quantified by comparing the values obtained from
normal subjects
with values from an experiment in which a known amount of a substantially
purified polynucleotide is
used. Standard values obtained in this manner may be compared with values
obtained from samples
from patients who are symptomatic for a disorder. Deviation from standard
values is used to establish
the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding TPPT
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 TPPT, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
TPPT, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding TPPT 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 TPPT are used to amplify
DNA using the
polymerase chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the
secondary and tertiary structures of PCR products in single-stranded form, and
these differences are
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detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers
are fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such
as DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico
SNP (isSNP), are capable of identifying polymorphisms by comparing the
sequence of individual
overlapping DNA fragments which assemble into a common consensus sequence.
These computer-
based methods filter out sequence variations due to laboratory preparation of
DNA and sequencing
errors using statistical models and automated analyses of DNA sequence
chromatograms. In the
alternative, SNPs may be detected and characterized by mass spectrometry
using, for example, the
high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
Methods which may also be used to quantify the expression of TPPT include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid
quanritation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described in Seilhamer, J.J. et al.,
"Comparative Gene Transcript
Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference. The
microarray may also be
used to identify genetic variants, mutations, and polymorphisms. This
information may be used to
determine gene function, to understand the genetic basis of a disorder, to
diagnose a disorder, to
monitor progression/regression of disease as a function of gene expression,
and to develop and
monitor the activities of therapeutic agents in the treatment of disease. In
particular, this information
may be used to develop a pharmacogenomic profile of a patient in order to
select the most appropriate
and effective treatment regimen for that patient. For example, therapeutic
agents which are highly
effective and display the fewest side effects may be selected for a patient
based on his/her
pharmacogenomic profile.
In another embodiment, antibodies specific for TPPT, or TPPT or fragments
thereof may be
used as elements on a microarray. The microarray may be used to monitor or
measure protein-protein
interactions, drug-target interactions, and gene expression profiles, as
described above.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. ( 1995) U.S. Patent No. 5,474,796; Schena, M. et al. (
1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. ( 1995) PCT application W095/251116;
Shalom D. et al.
( 1995) PCT application W095/35505; Heller, R.A. et al. ( 1997) Proc. Natl.
Acad. Sci. USA 94:2150-
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2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are well
known and thoroughly described in DNA Microarra~ys: 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 TPPT
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a mufti-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial Pl
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
(See, e.g., Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA
83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding TPPT on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse, may
reveal associated markers even if the exact chromosomal locus is not known.
This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to l 1q22-23,
any sequences mapping to that area may represent associated or regulatory
genes for further
investigation. (See, e.g., Gatti, R.A. et al. ( 1988) Nature 336:577-580.) The
nucleotide sequence of
the instant invention may also be used to detect differences in the
chromosomal location due to
translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, TPPT, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
CA 02375493 2001-12-11
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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 TPPT and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. ( 1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with TPPT, or
fragments thereof,
and washed. Bound TPPT is then detected by methods well known in the art.
Purified TPPT 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 TPPT specifically compete with a test compound
for binding TPPT. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with TPPT.
In additional embodiments, the nucleotide sequences which encode TPPT may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below, in
particular U.S. Ser. No. 60/139,923, U.S. Ser. No. 60/148,177, U.S. Ser. No.
60/149,357, and U.S.
Ser. No. 60/162,287, are hereby expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some
tissues were homogenized and lysed in guanidinium isothiocyanate, while others
were homogenized
and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL
(Life Technologies), a
monophasic solution of phenol and guanidine isothiocyanate. The resulting
lysates were centrifuged
over CsCI cushions or extracted with chloroform. RNA was precipitated from the
lysates with either
isopropanol or sodium acetate and ethanol, or by other routine methods.
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
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using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-1000
bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORTI plasmid (Life Technologies),
pcDNA2.1 plasmid
(Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA).
Recombinant
plasmids were transformed into competent E. coli cells including XLI-Blue, XL1-
BIueMRF, or
SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DHIOB 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 as the ABI CATALYST 800 (PE Biosystems) thermal cycler or the PTC-200
thermal cycler (MJ
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Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or
the MICROLAB
2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were
prepared using reagents
provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such
as the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
Electrophoretic
separation of cDNA sequencing reactions and detection of labeled
polynucleotides were carried out
using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI
PRISM 373 or
377 sequencing system (PE Biosystems) in conjunction with standard ABI
protocols and base calling
software; or other sequence analysis systems known in the art. Reading frames
within the cDNA
sequences were identified using standard methods (reviewed in Ausubel, 1997,
supra, unit 7.7). Some
of the cDNA sequences were selected for extension using the techniques
disclosed in Example VI.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilled in the
art. Table 5 summarizes the tools, programs, and algorithms used and provides
applicable
descriptions, references, and threshold parameters. The first column of Table
5 shows the tools,
IS programs, and algorithms used, the second column provides brief
descriptions thereof, the third
column presents appropriate references, all of which are incorporated by
reference herein in their
entirety, and the fourth column presents, where applicable, the scores,
probability values, and other
parameters used to evaluate the strength of a match between two sequences (the
higher the score, the
greater the homology between two sequences). Sequences were analyzed using
MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software
(DNASTAR). Polynucleotide and polypeptide sequence alignments were generated
using the default
parameters specified by the clustal algorithm as incorporated into the
MEGALIGN multisequence
alignment program (DNASTAR), which also calculates the percent identity
between aligned
sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
were then queried
against a selection of public databases such as the GenBank primate, rodent,
mammalian, vertebrate,
and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences
were assembled
into full length polynucleotide sequences using programs based on Phred,
Phrap, and Consed, and
were screened for open reading frames using programs based on GeneMark, BLAST,
and FASTA.
The full length polynucleotide sequences were translated to derive the
corresponding full length
amino acid sequences, and these full length sequences were subsequently
analyzed by querying
against databases such as the GenBank databases (described above), SwissProt,
BLOCKS, PRINTS,
DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family
databases such
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as PFAM. HMM is a probabilistic approach which analyzes consensus primary
structures of gene
families. (See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide and
amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:44-86. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies were described in The Invention section above.
IV. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel,
1995, supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
search can be modified to determine whether any particular match is
categorized as exact or similar.
The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum { length(Seq. 1 ), length(Seq. 2) {
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by gaps).
If there is more than one HSP, then the pair with the highest BLAST score is
used to calculate the
product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding TPPT occurred. Analysis involved the categorization of
cDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
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reproductive, and urologic. The disease/condition categories included cancer,
inflammation, trauma,
cell proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
sequence of interest was counted and divided by the total number of libraries
across all categories.
Percentage values of tissue-specific and disease- or condition-specific
expression are reported in
Table 3.
V. Chromosomal Mapping of TPPT Encoding Polynucleotides
The cDNA sequences which were used to assemble SEQ ID N0:44-49 and SEQ ID
N0:54-86
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 )D N0:44-49 and SEQ ID N0:54-86 were assembled into
clusters of
contiguous and overlapping sequences using assembly algorithms such as Phrap
(Table 5). Radiation
hybrid and genetic mapping data available from public resources such as the
Stanford Human
Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and
Genethon were
used to determine if any of the clustered sequences had been previously
mapped. Inclusion of a
mapped sequence in a cluster resulted in the assignment of all sequences of
that cluster, including its
particular SEQ ID NO:, to that map location.
The genetic map locations of SEQ ID N0:44, SEQ ID N0:48, SEQ ID N0:60, SEQ ID
N0:65, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:73, SEQ ID N0:76,
SEQ ID
N0:80,and SEQ ID N0:83 are described in The Invention as ranges, or intervals,
of human
chromosomes. More than one map location is reported for SEQ ID N0:65, SEQ ID
N0:73, SEQ ID
N0:80, and SEQ ID N0:83, indicating that previously mapped sequences having
similarity, but not
complete identity, to SEQ ID N0:65, SEQ ID N0:73, SEQ ID N0:80, and SEQ ID
N0:83 were
assembled into their respective clusters. The map position of an interval, in
centiMorgans, is
measured relative to the terminus of the chromosome's p-arm. (The centiMorgan
(cM) is a unit of
measurement based on recombination frequencies between chromosomal markers. On
average, 1 cM
is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can
vary widely due to
hot and cold spots of recombination.) The cM distances are based on genetic
markers mapped by
Genethon which provide boundaries for radiation hybrid markers whose sequences
were included in
each of the clusters. Diseases associated with the public and Incyte sequences
located within the
indicated intervals are also reported in the Invention section where
applicable. Human genome maps
and other resources available to the public, such as the NCBI "GeneMap'99"
World Wide Web site
(http://www.ncbi.nlm.nih.govlgenemap/), can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
VI. Extension of TPPT Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:44-86 were produced by
extension of
an appropriate fragment of the full length molecule using oligonucleotide
primers designed from this
CA 02375493 2001-12-11
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fragment. One primer was synthesized to initiate 5' extension of the known
fragment, and the other
primer, to initiate 3' extension of the known fragment. The initial primers
were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg'-+, (NH4)ZS04,
and (3-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 p1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~1 to 10 /c1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose mini-gel to determine which reactions were
successful in extending
the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37°C in
384-well plates in LB/2x Garb liquid media.
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The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
S quantified by PICOGREEN reagent (Molecular Probes) as described above.
Samples with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with
20% dimethysulfoxide ( 1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
In like manner, the polynucleotide sequences of SEQ )D N0:44-86 are used to
obtain 5'
regulatory sequences using the procedure above, along with oligonucleotides
designed for such
extension, and an appropriate genomic library.
VII. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:44-86 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of-the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ,uCi of
[y-3zP] adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech). An
aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases: Ase
I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VIII. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena ( 1999),
supra). Suggested substrates include silicon, silica, glass slides, glass
chips, and silicon wafers.
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Alternatively, a procedure analogous to a dot or slot blot may also be used to
arrange and link
elements to the surface of a substrate using thermal, UV, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinary skill in the art and may contain any appropriate number of
elements. (See, e.g.,
Schena, M. et al. ( 1995) Science 270:467-470; Shalon, D. et al. ( 1996)
Genome Res. 6:639-645;
Marshall, A. and J. Hodgson ( 1998) Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof
may comprise the elements of the microarray. Fragments or oligomers suitable
for hybridization can
be selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described
in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/pl oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/pl RNase inhibitor, 500 ~M dATP, 500 LiM dGTP, 500
L~M dTTP, 40 E.iM
dCTP, 40 E.~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 O.SM sodium
hydroxide and
incubated for 20 minutes at 85 °C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen ( 1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 ~1 SX SSC/0.2% SDS.
Microarrav 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
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uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
pg. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522, incorporated herein by reference. 1 p1 of the array
element DNA, at an average
concentration of 100 ng/pl, is loaded into the open capillary printing element
by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60
°C followed by washes in 0.2%
SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 p1 of sample mixture consisting of 0.2 pg
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX 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 cm' coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of
140 p1 of SX SSC in a corner of the chamber. The chamber containing the arrays
is incubated for
about 6.5 hours at 60 °C. The arrays are washed for 10 min at 45
°C in a first wash buffer ( 1X SSC,
0.1 % SDS), three times for 10 minutes each at 45 °C in a second wash
buffer (0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
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.
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In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are differentially
expressed, the calibration is done by labeling samples of the calibrating cDNA
with the two
fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
IX. Complementary Polynucleotides
Sequences complementary to the TPPT-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring TPPT. 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 TPPT. 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 TPPT-encoding transcript.
X. Expression of TPPT
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Expression and purification of TPPT is achieved using bacterial or virus-based
expression
systems. For expression of TPPT 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 TPPT upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of TPPT in eukaryotic cells is
achieved by infecting insect
or mammalian cell lines with recombinant Auto~aphica californica nuclear
polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding TPPT 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 in~erda (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, TPPT 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
TPPT at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity
purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues, enables
purification on metal-chelate
resins (QIAGEN). Methods for protein expression and purification are discussed
in Ausubel ( 1995,
supra, ch. 10 and 16). Purified TPPT obtained by these methods can be used
directly in the assays
shown in Examples XI and XV.
XI. Demonstration of TPPT Activity
TPPT transport activity is assayed by measuring uptake of labeled substrates
into Xenopus
laevis oocytes. Oocytes at stages V and VI are injected with TPPT mRNA (10 ng
per oocyte) and
incubated for 3 days at 18°C in OR2 medium (82.SmM NaCI, 2.5 mM KCI,
1mM CaCI,, 1mM MgCI,,
1mM Na=HP04, 5 mM Hepes, 3.8 mM NaOH , SOpg/ml gentamycin, pH 7.8) to allow
expression of
TPPT. Oocytes are then transferred to standard uptake medium ( 100mM NaCI, 2
mM KCI, 1 mM
CaCI,, 1mM MgCI=, 10 mM Hepes/T'ris pH 7.5). Uptake of various substrates
(e.g., amino acids,
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sugars, drugs, ions, and neurotransmitters) is initiated by adding labeled
substrate (e.g. radiolabeled
with jH, fluorescently labeled with rhodamine, etc.) to the oocytes. After
incubating for 30 minutes,
uptake is terminated by washing the oocytes three times in Na+-free medium,
measuring the
incorporated label, and comparing with controls. TPPT activity is proportional
to the level of
internalized labeled substrate.
XII. Functional Assays
TPPT function is assessed by expressing the sequences encoding TPPT at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid
(Invitrogen), both of which
contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ~g of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. ( 1994) Flow Cvtometr_y, Oxford, New York NY.
The influence of TPPT on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding TPPT 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 TPPT and other genes of interest can be analyzed
by northern analysis
or microarray techniques.
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XIII. Production of TPPT Specific Antibodies
TPPT 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 TPPT amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with
the oligopeptide-
KL.H complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-
TPPT activity by, for example, binding the peptide or TPPT to a substrate,
blocking with 1 % BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
XIV. Purification of Naturally Occurring TPPT Using Specific Antibodies
Naturally occurring or recombinant TPPT is substantially purified by
immunoaffmity
chromatography using antibodies specific for TPPT. An immunoaffinity column is
constructed by
covalently coupling anti-TPPT 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 TPPT are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of TPPT (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/TPPT 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 TPPT is collected.
XV. Identification of Molecules Which Interact with TPPT
TPPT, or biologically active fragments thereof, are labeled with '25I 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 TPPT, washed, and
any wells with labeled TPPT complex are assayed. Data obtained using different
concentrations of
TPPT are used to calculate values for the number, affinity, and association of
TPPT with the
candidate molecules.
Alternatively, molecules interacting with TPPT are analyzed using the yeast
two-hybrid
system as described in Fields, S. and O. Song ( 1989, Nature 340:245-246), or
using commercially
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CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
TPPT may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S. Patent
No. 6,057,101 ).
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the invention.
Although the invention has been described in connection with certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which are obvious
to those skilled in molecular biology or related fields are intended to be
within the scope of the
following claims.
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CA 02375493 2001-12-11
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CA 02375493 2001-12-11
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SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
LAL, Preeti
YANG, Junming
YUE, Henry
HILLMAN, Jennifer L.
TANG, Y. Tom
BANDMAN, Olga
BURFORD, Neil
BAUGHN, Mariah R.
AZIMZAI, Yalda
LU, Dyung Aina M.
AU-YOUNG, Janice
PATTERSON, Chandra
<120> HUMAN TRANSPORT PROTEINS
<130> PF-0709 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/139,923; 60/148,177; 60/149,357; 60/162,287
<151> 1999-06-17; 1999-08-10; 1999-08-18; 1999-10-28
<160> 86
<170> PERL Program
<210> 1
<211> 623
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 264114CD1
<400> 1
Met Ser Thr Gln Asp Glu Arg Gln Ile Asn Thr Glu Tyr Ala Val
1 5 10 15
Ser Leu Leu Glu Gln Leu Lys Leu Phe Tyr Glu Gln Gln Leu Phe
20 25 30
Thr Asp Ile Val Leu Ile Val Glu Gly Thr Glu Phe Pro Cys His
35 40 45
Lys Met Val Leu Ala Thr Cys Ser Ser Tyr Phe Arg Ala Met Phe
50 55 60
Met Ser Gly Leu Ser Glu Ser Lys Gln Thr His Val His Leu Arg
65 70 75
Asn Val Asp Ala Ala Thr Leu Gln Ile Ile Ile Thr Tyr Ala Tyr
80 85 90
Thr Gly Asn Leu Ala Met Asn Asp Ser Thr Val Glu Gln Leu Tyr
95 100 105
Glu Thr Ala Cys Phe Leu Gln Val Glu Asp Val Leu Gln Arg Cys
110 115 120
Arg Glu Tyr Leu Ile Lys Lys Ile Asn Ala Glu Asn Cys Val Arg
125 130 135
Leu Leu Ser Phe Ala Asp Leu Phe Ser Cys Glu Glu Leu Lys Gln
140 145 150
Ser Ala Lys Arg Met Val Glu His Lys Phe Thr Ala Val Tyr His
155 160 165
Gln Asp Ala Phe Met Gln Leu Ser His Asp Leu Leu Ile Asp Ile
170 175 180
Leu Ser Ser Asp Asn Leu Asn Val Glu Lys Glu Glu Thr Val Arg
185 190 195
Glu Ala Ala Met Leu Trp Leu Glu Tyr Asn Thr Glu Ser Arg Ser
1 /60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
200 205 210
Gln Tyr Leu Ser Ser Val Leu Ser Gln Ile Arg Ile Asp Ala Leu
215 220 225
Ser Glu Val Thr Gln Arg Ala Trp Phe Gln Gly Leu Pro Pro Asn
230 235 240
Asp Lys Ser Val Val Val Gln Gly Leu Tyr Lys Ser Met Pro Lys
245 250 255
Phe Phe Lys Pro Arg Leu_Gly Met Thr Lys Glu Glu Met Met Ile
260 265 270
Phe Ile Glu Ala Ser Ser Glu Asn Pro Cys Ser Leu Tyr Ser Ser
275 280 285
Val Cys Tyr Ser Pro Gln Ala Glu Lys Val Tyr Lys Leu Cys Ser
290 295 300
Pro Pro Ala Asp Leu His Lys Val Gly Thr Val Val Thr Pro Asp
305 310 315
Asn Asp Ile Tyr Ile Ala Gly Gly Gln Val Pro Leu Lys Asn Thr
320 325 330
Lys Thr Asn His Ser Lys Thr Ser Lys Leu Gln Thr Ala Phe Arg
335 340 345
Thr Val Asn Cys Phe Tyr Trp Phe Asp Ala Gln Gln Asn Thr Trp
350 355 360
Phe Pro Lys Thr Pro Met Leu Phe Val Arg Ile Lys Pro Ser Leu
365 370 375
Val Cys Cys Glu Gly Tyr Ile Tyr Ala Ile Gly Gly Asp Ser Val
380 385 390
Gly Gly Glu Leu Asn Arg Arg Thr Val Glu Arg Tyr Asp Thr Glu
395 400 405
Lys Asp Glu Trp Thr Met Val Ser Pro Leu Pro Cys Ala Trp Gln
410 415 420
Trp Ser Ala Ala Val Val Val His Asp Cys Ile Tyr Val Met Thr
425 430 435
Leu Asn Leu Met Tyr Cys Tyr Phe Pro Arg Ser Asp Ser Trp Val
440 445 450
Glu Met Ala Met Arg Gln Thr Ser Arg Ser Phe Ala Ser Ala Ala
455 460 465
Ala Phe Gly Asp Lys Ile Phe Tyr Ile Gly Gly Leu His Ile Ala
470 475 480
Thr Asn Ser Gly Ile Arg Leu Pro Ser Gly Thr Val Asp Gly Ser
485 490 495
Ser Val Thr Val Glu Ile Tyr Asp Val Asn Lys Asn Glu Trp Lys
500 505 510
Met Ala Ala Asn Ile Pro Ala Lys Arg Tyr Ser Asp Pro Cys Val
515 520 525
Arg Ala Val Val Ile Ser Asn Ser Leu Cys Val Phe Met Arg Glu
530 535 540
Thr His Leu Asn Glu Arg Ala Lys Tyr Val Thr Tyr Gln Tyr Asp
545 550 555
Leu Glu Leu Asp Arg Trp Ser Leu Arg Gln His Ile Ser Glu Arg
560 565 570
Val Leu Trp Asp Leu Gly Arg Asp Phe Arg Cys Thr Val Gly Lys
575 580 585
Leu Tyr Pro Ser Cys Leu Glu Glu Ser Pro Trp Lys Pro Pro Thr
590 595 600
Tyr Leu Phe Ser Thr Asp Gly Thr Glu Glu Phe Glu Leu Asp Gly
605 610 615
Glu Met Val Ala Leu Pro Pro Val
620
<210> 2
<211> 99
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1455669CD1
<400> 2
Met Ala Ala Pro Ala Glu Pro Cys Ala Gly Gln Gly Val Trp Asn
2/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
1 5 10 15
Gln Thr Glu Pro Glu Pro Ala Ala Thr Ser Leu Leu Ser Leu Cys
20 25 30
Phe Leu Arg Thr Ala Gly Val Trp Val Pro Pro Met Tyr Leu Trp
35 40 45
Val Leu Gly Pro Ile Tyr Leu Leu Phe Ile His His His Gly Arg
50 55 60
Gly Tyr Leu Arg Met Ser Pro Leu Phe Lys Ala Lys Met Val Ala
65 70 75
Ala Ile Pro Gly Ser Leu Glu Pro Gly Asn Val Arg Gly Arg Gln
80 85 90
Gly Thr Gly Trp Asn Leu Val Lys Ser
<210> 3
<211> 374
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2084989CD1
<400> 3
Met Glu Ser Lys Met Gly Glu Leu Pro Leu Asp Ile Asn Ile Gln
1 5 10 15
Glu Pro Arg Trp Asp Gln Ser Thr Phe Leu Gly Arg Ala Arg His
20 25 30
Phe Phe Thr Val Thr Asp Pro Arg Asn Leu Leu Leu Ser Gly Ala
35 40 45
Gln Leu Glu Ala Ser Arg Asn Ile Val Gln Asn Tyr Arg Ala Gly
50 55 60
Val Val Thr Pro Gly Ile Thr Glu Asp Gln Leu Trp Arg Ala Lys
65 70 75
Tyr Val Tyr Asp Ser Ala Phe His Pro Asp Thr Gly Glu Lys Val
so a5 90
Val Leu Ile Gly Arg Met Ser Ala Gln Val Pro Met Asn Met Thr
95 100 105
Ile Thr Gly Cys Met Leu Thr Phe Tyr Arg Lys Thr Pro Thr Val
110 115 120
Val Phe Trp Gln Trp Val Asn Gln Ser Phe Asn Ala Ile Val Asn
125 130 135
Tyr Ser Asn Arg Ser Gly Asp Thr Pro Ile Thr Val Arg Gln Leu
140 145 150
Gly Thr Ala Tyr Val Ser Ala Thr Thr Gly Ala Val Ala Thr Ala
155 160 165
Leu Gly Leu Lys Ser Leu Thr Lys His Leu Pro Pro Leu Val Gly
170 175 180
Arg Phe Val Pro Phe Ala Ala Val Ala Ala Ala Asn Cys Ile Asn
185 190 195
Ile Pro Leu Met Arg Gln Arg Glu Leu Gln Val Gly Ile Pro Val
200 205 210
Ala Asp Glu Ala Gly Gln Arg Leu Gly Tyr Ser Val Thr Ala Ala
215 220 225
Lys Gln Gly Ile Phe Gln Val Val Ile Ser Arg Ile Cys Met Ala
230 235 240
Ile Pro Ala Met Ala Ile Pro Pro Leu Ile Met Asp Thr Leu Glu
245 250 255
Lys Lys Asp Phe Leu Lys Val Gly Asp Cys Thr Ser Leu Val Leu
260 265 270
Glu Trp Ala Met Ala Gly Arg Ser Asp Gln Ala Pro Thr Leu Ser
275 280 285
Pro Ala Ser Pro Asp Ser Leu Arg Leu Ala Ser Pro Ser Pro Asp
290 295 300
Pro Cys Thr Ala Ser Ser Thr Phe Val His Ser Ala Arg Met Asn
305 310 315
Trp Ala Gly Val Lys Glu Leu Cys Arg Gly Arg Arg Arg Gly Gln
320 325 330
Arg Lys Glu Thr Asn Phe Ile Ser Val Thr Pro Val Ala Ser Asp
3/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
335 340 345
Thr Gln Lys Gly Thr Val Ile Val Met Leu Asp Leu Met Leu Ile
350 355 360
Leu Leu Pro Pro Ser Ala Ser Ile Leu Arg Gly Thr His Gly
365 370
<210> 4
<211> 271
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2501034CD1
<400> 4
Met Gly Asn Gly Gly Arg Ser Gly Leu Gln Gln Gly Lys Gly Asn
1 5 10 15
Val Asp Gly Val Ala Ala Thr Pro Thr Ala Ala Ser Ala Ser Cys
20 25 30
Gln Tyr Arg Cys Ile Glu Cys Asn Gln Glu Ala Lys Glu Leu Tyr
35 40 45
Arg Asp Tyr Asn His Gly Val Leu Lys Ile Thr Ile Cys Lys Ser
50 55 60
Cys Gln Lys Pro Val Asp Lys Tyr Ile Glu Tyr Asp Pro Val Ile
65 70 75
Ile Leu Ile Asn Ala Ile Leu Cys Lys Ala Gln Ala Tyr Arg His
80 85 90
Ile Leu Phe Asn Thr Gln Ile Asn Ile His Gly Lys Leu Cys Ile
95 100 105
Phe Cys Leu Leu Cys Glu Ala Tyr Leu Arg Trp Trp Gln Leu Gln
110 115 120
Asp Ser Asn Gln Asn Thr Ala Pro Asp Asp Leu Ile Arg Tyr Ala
125 130 135
Lys Glu Trp Asp Phe Tyr Arg Met Phe Ala Ile Ala Ala Leu Glu
140 145 150
Gln Thr Ala Tyr Phe Ile Gly Ile Phe Thr Phe Leu Trp Val Glu
155 160 165
Arg Pro Met Thr Ala Lys Lys Lys Pro Asn Phe Ile Leu Leu Leu
170 175 180
Lys Ala Leu Leu Leu Ser Ser Tyr Gly Lys Leu Leu Leu Ile Pro
185 190 195
Ala Val Ile Trp Glu His Asp Tyr Thr Ser Val Cys Leu Lys Leu
200 205 210
Ile Lys Val Phe Val Leu Thr Ser Asn Phe Gln Ala Ile Arg Val
215 220 225
Thr Leu Asn Ile Asn Arg Lys Leu Ser Phe Leu Ala Val Leu Ser
230 235 240
Gly Leu Leu Leu Glu Ser Ile Met Val Tyr Phe Phe Gln Ser Met
245 250 255
Glu Trp Asp Val Gly Ser Asp Tyr Ala Ile Phe Lys Ser Gln Asp
260 265 270
Phe
<210> 5
<211> 323
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2745212CD1
<400> 5
Met Ala Pro Lys Gln Asp Pro Lys Pro Lys Phe Gln Glu Gly Glu
1 5 10 15
Arg Val Leu Cys Phe His Gly Pro Leu Leu Tyr Glu Ala Lys Cys
20 25 30
Val Lys Val Ala Ile Lys Asp Lys Gln Val Lys Tyr Phe Ile His
4/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
35 40 45
Tyr Ser Gly Trp Asn Lys Asn Trp Asp Glu Trp Val Pro Glu Ser
50 55 60
Arg Val Leu Lys Tyr Val Asp Thr Asn Leu Gln Lys Gln Arg Glu
65 70 75
Leu Gln Lys Ala Asn Gln Glu Gln Tyr Ala Glu Gly Lys Met Arg
80 85 90
Gly Ala Ala Pro Gly Lys Lys Thr Ser Gly Leu Gln Gln Lys Asn
95 100 105
Val Glu Val Lys Thr Lys Lys Asn Lys Gln Lys Thr Pro Gly Asn
110 115 120
Gly Asp Gly Gly Ser Thr Ser Glu Thr Pro Gln Pro Pro Arg Lys
125 130 135
Lys Arg Ala Arg Val Asp Pro Thr Val Glu Asn Glu Glu Thr Phe
140 145 150
Met Asn Arg Val Glu Val Lys Val Lys Ile Pro Glu Glu Leu Lys
155 160 165
Pro Trp Leu Val Asp Asp Trp Asp Leu Ile Thr Arg Gln Lys Gln
170 175 180
Leu Phe Tyr Leu Pro Ala Lys Lys Asn Val Asp Ser Ile Leu Glu
185 190 195
Asp Tyr Ala Asn Tyr Lys Lys Ser Arg Gly Asn Thr Asp Asn Lys
200 205 210
Glu Tyr Ala Val Asn Glu Val Val Ala Gly Ile Lys Glu Tyr Phe
215 220 225
Asn Val Met Leu Gly Thr Gln Leu Leu Tyr Lys Phe Glu Arg Pro
230 235 240
Gln Tyr Ala Glu Ile Leu Ala Asp His Pro Asp Ala Pro Met Ser
245 250 255
Gln Val Tyr Gly Ala Pro His Leu Leu Arg Leu Phe Val Arg Ile
260 265 270
Gly Ala Met Leu Ala Tyr Thr Pro Leu Asp Glu Lys Ser Leu Ala
275 280 285
Leu Leu Leu Asn Tyr Leu His Asp Phe Leu Lys Tyr Leu Ala Lys
290 295 300
Asn Ser Ala Thr Leu Phe Ser Ala Ser Asp Tyr Glu Val Ala Pro
305 310 315
Pro Glu Tyr His Arg Lys Ala Val
320
<210> 6
<211> 274
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4833111CD1
<400> 6
Met Asp Arg Pro Gly Phe Val Ala Ala Leu Val Ala Gly Gly Val
1 5 10 15
Ala Gly Val Ser Val Asp Leu Ile Leu Phe Pro Leu Asp Thr Ile
20 25 30
Lys Thr Arg Leu Gln Ser Pro Gln Gly Phe Ser Lys Ala Gly Gly
35 40 45
Phe His Gly Ile Tyr Ala Gly Val Pro Ser Ala Ala Ile Gly Ser
50 55 60
Phe Pro Asn Ala Ala Ala Phe Phe Ile Thr Tyr Glu Tyr Val Lys
65 70 75
Trp Phe Leu His Ala Asp Ser Ser Ser Tyr Leu Thr Pro Met Lys
80 85 90
His Met Leu Ala Ala Ser Ala Gly Glu Val Val Ala Cys Leu Ile
95 100 105
Arg Val Pro Ser Glu Val Val Lys Gln Arg Ala Gln Val Ser Ala
110 115 120
Ser Thr Arg Thr Phe Gln Ile Phe Ser Asn Ile Leu Tyr Glu Glu
125 130 135
Gly Ile Gln Gly Leu Tyr Arg Gly Tyr Lys Ser Thr Val Leu Arg
5/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
140 145 150
Glu Ile Pro Phe Ser Leu Val Gln Phe Pro Leu Trp Glu Ser Leu
155 160 165
Lys Ala Leu Trp Ser Trp Arg Gln Asp His Val Val Asp Ser Trp
170 175 180
Gln Ser Ala Val Cys Gly Ala Phe Ala Gly Gly Phe Ala Ala Ala
185 190 195
Val Thr Thr Pro Leu Asp Val Ala Lys Thr Arg Ile Thr Leu Ala
200 205 210
Lys Ala Gly Ser Ser Thr Ala Asp Gly Asn Val Leu Ser Val Leu
215 220 225
His Gly Val Trp Arg Ser Gln Gly Leu Ala Gly Leu Phe Ala Gly
230 235 240
Val Phe Pro Arg Met Ala Ala Ile Ser Leu Gly Gly Phe Ile Phe
245 250 255
Leu Gly Ala Tyr Asp Arg Thr His Ser Leu Leu Leu Glu Val Gly
260 265 270
Arg Lys Ser Pro
<210> 7
<211> 291
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 876677CD1
<400> 7
Met Asp Ser Arg Val Ser Ser Pro Glu Lys Gln Asp Lys Glu Asn
1 5 10 15
Phe Val Gly Val Asn Asn Lys Arg Leu Gly Val Cys Gly Trp Ile
20 25 30
Leu Phe Ser Leu Ser Phe Leu Leu Val Ile Ile Thr Phe Pro Ile
35 40 45
Ser Ile Trp Met Cys Leu Lys Ile Ile Lys Glu Tyr Glu Arg Ala
50 55 60
Val Val Phe Arg Leu Gly Arg Ile Gln Ala Asp Lys Ala Lys Gly
65 70 75
Pro Gly Leu Ile Leu Val Leu Pro Cys Ile Asp Val Phe Val Lys
80 85 90
Val Asp Leu Arg Thr Val Thr Cys Asn Ile Pro Pro Gln Glu Ile
95 100 105
Leu Thr Arg Asp Ser Val Thr Thr Gln Val Asp Gly Val Val Tyr
110 115 120
Tyr Arg Ile Tyr Ser Ala Val Ser Ala Val Ala Asn Val Asn Asp
125 130 135
Val His Gln Ala Thr Phe Leu Leu Ala Gln Thr Thr Leu Arg Asn
140 145 150
Val Leu Gly Thr Gln Thr Leu Ser Gln Ile Leu Ala Gly Arg Glu
155 160 165
Glu Ile Ala His Ser Ile Gln Thr Leu Leu Asp Asp Ala Thr Glu
170 175 180
Leu Trp Gly Ile Arg Val Ala Arg Val Glu Ile Lys Asp Val Arg
185 190 195
Ile Pro Val Gln Leu Gln Arg Ser Met Ala Ala Glu Ala Glu Ala
200 205 210
Thr Arg Glu Ala Arg Ala Lys Val Leu Ala Ala Glu Gly Glu Met
215 220 225
Asn Ala Ser Lys Ser Leu Lys Ser Ala Ser Met Val Leu Ala Glu
230 235 240
Ser Pro Ile Ala Leu Gln Leu Arg Tyr Leu Gln Thr Leu Ser Thr
245 250 255
Val Ala Thr Glu Lys Asn Ser Thr Ile Val Phe Pro Leu Pro Met
260 265 270
Asn Ile Leu Glu Gly Ile Gly Gly Val Ser Tyr Asp Asn His Lys
275 280 285
Lys Leu Pro Asn Lys Ala
6/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
290
<210> 8
<211> 381
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2326143CD1
<400> 8
Met Ser Arg His Glu Gly Val Ser Cys Asp Ala Cys Leu Lys Gly
1 5 10 15
Asn Phe Arg Gly Arg Arg Tyr Lys Cys Leu Ile Cys Tyr Asp Tyr
20 25 30
Asp Leu Cys Ala Ser Cys Tyr Glu Ser Gly Ala Thr Thr Thr Arg
35 40 45
His Thr Thr Asp His Pro Met Gln Cys Ile Leu Thr Arg Val Asp
50 55 60
Phe Asp Leu Tyr Tyr Gly Gly Glu Ala Phe Ser Val Glu Gln Pro
65 70 75
Gln Ser Phe Thr Cys Pro Tyr Cys Gly Lys Met Gly Tyr Thr Glu
80 85 90
Thr Ser Leu Gln Glu His Val Thr Ser Glu His Ala Glu Thr Ser
95 100 105
Thr Glu Val Ile Cys Pro Ile Cys Ala Ala Leu Pro Gly Gly Asp
110 115 120
Pro Asn His Val Thr Asp Asp Phe Ala Ala His Leu Thr Leu Glu
125 130 135
His Arg Ala Pro Arg Asp Leu Asp Glu Ser Ser Gly Val Arg His
140 145 150
Val Arg Arg Met Phe His Pro Gly Arg Gly Leu Gly Gly Pro Arg
155 160 165
Ala Arg Arg Ser Asn Met His Phe Thr Ser Ser Ser Thr Gly Gly
170 175 180
Leu Ser Ser Ser Gln Ser Ser Tyr Ser Pro Ser Asn Arg Glu Ala
185 190 195
Met Asp Pro Ile Ala Glu Leu Leu Ser Gln Leu Ser Gly Val Arg
200 205 210
Arg Ser Ala Gly Gly Gln Leu Asn Ser Ser Gly Pro Ser Ala Ser
215 220 225
Gln Leu Gln Gln Leu Gln Met Gln Leu Gln Leu Glu Arg Gln His
230 235 240
Ala Gln Ala Ala Arg Gln Gln Leu Glu Thr Ala Arg Asn Ala Thr
245 250 255
Arg Arg Thr Asn Thr Ser Ser Val Thr Thr Thr Ile Thr Gln Ser
260 265 270
Thr Ala Thr Thr Asn Ile Ala Asn Thr Glu Ser Ser Gln Gln Thr
275 280 285
Leu Gln Asn Ser Gln Phe Leu Leu Thr Arg Leu Asn Asp Pro Lys
290 295 300
Met Ser Glu Thr Glu Arg Gln Ser Met Glu Ser Glu Arg Ala Asp
305 310 315
Arg Ser Leu Phe Val Gln Glu Leu Leu Leu Ser Thr Leu Val Arg
320 325 330
Glu Glu Ser Ser Ser Ser Asp Glu Asp Asp Arg Gly Glu Met Ala
335 340 345
Asp Phe Gly Ala Met Gly Cys Val Asp Ile Met Pro Leu Asp Val
350 355 360
Ala Leu Glu Asn Leu Asn Leu Lys Glu Ser Asn Lys Gly Asn Glu
365 370 375
Pro Pro Pro Pro Pro Leu
380
<210> 9
<211> 190
<212> PRT
<213> Homo sapiens
7/60
CA 02375493 2001-12-11
WO 00178953 PCT/US00/16668
<220>
<221> misc_feature
<223> Incyte ID No: 2786302CD1
<400> 9
Met Lys Tyr Gly Asn Glu Ile Met Asn Lys Asp Pro Val Phe Arg
1 5 10 15
Ile Ser Pro Arg Ser Arg Glu Thr His Pro Asn Pro Glu Glu Pro
20 25 30
Glu Glu Glu Asp Glu Asp Val Gln Ala Glu Arg Val Gln Ala Ala
35 40 45
Asn Ala Leu Thr Ala Pro Asn Leu Glu Glu Glu Pro Val Ile Thr
50 55 60
Ala Ser Cys Leu His Lys Glu Tyr Tyr Glu Thr Lys Lys Ser Cys
65 70 75
Phe Ser Thr Arg Lys Lys Lys Ile Ala Ile Arg Asn Val Ser Phe
80 85 90
Cys Val Lys Lys Gly Glu Val Leu Gly Leu Leu Gly His Asn Gly
95 100 105
Ala Gly Lys Ser Thr Ser Ile Lys Met Ile Thr Gly Cys Thr Lys
110 115 120
Pro Thr Ala Gly Val Val Val Leu Gln Gly Ser Arg Ala Ser Val
125 130 135
Arg Gln Gln His Asp Asn Ser Leu Lys Phe Leu Gly Tyr Cys Pro
140 145 150
Gln Glu Asn Ser Leu Trp Pro Lys Leu Thr Met Lys Glu His Leu
155 160 165
Glu Leu Tyr Ala Ala Val Glu Arg Leu Gly Gln Lys Arg Cys Cys
170 175 180
Ser Gln Tyr Phe Thr Ile Gly Gly Arg Ser
185 190
<210> 10
<211> 297
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3735780CD1
<400> 10
Met Met Asp Ser Glu Ala His Glu Lys Arg Pro Pro Ile Leu Thr
1 5 10 15
Ser Ser Lys Gln Asp Ile Ser Pro His Ile Thr Asn Val Gly Glu
20 25 30
Met Lys His Tyr Leu Cys Gly Cys Cys Ala Ala Phe Asn Asn Val
35 40 45
Ala Ile Thr Phe Pro Ile Gln Lys Val Leu Phe Arg Gln Gln Leu
50 55 60
Tyr Gly Ile Lys Thr Arg Asp Ala Ile Leu Gln Leu Arg Arg Asp
65 70 75
Gly Phe Arg Asn Leu Tyr Arg Gly Ile Leu Pro Pro Leu Met Gln
80 85 90
Lys Thr Thr Thr Leu Ala Leu Met Phe Gly Leu Tyr Glu Asp Leu
95 100 105
Ser Cys Leu Leu His Lys His Val Ser Ala Pro Glu Phe Ala Thr
110 115 120
Ser Gly Val Ala Ala Val Leu Ala Gly Thr Thr Glu Ala Ile Phe
125 130 135
Thr Pro Leu Glu Arg Val Gln Thr Leu Leu Gln Asp His Lys His
140 145 150
His Asp Lys Phe Thr Asn Thr Tyr Gln Ala Phe Lys Ala Leu Lys
155 160 165
Cys His Gly Ile Gly Glu Tyr Tyr Arg Gly Leu Val Pro Ile Leu
170 175 180
Phe Arg Asn Gly Leu Ser Asn Val Leu Phe Phe Gly Leu Arg Gly
185 190 195
Pro Ile Lys Glu His Leu Pro Thr Ala Thr Thr His Ser Ala His
8/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
200 205 210
Leu Val Asn Asp Phe Ile Cys Gly Gly Leu Leu Gly Ala Met Leu
215 220 225
Gly Phe Leu Phe Phe Pro Ile Asn Val Val Lys Thr Arg Ile Gln
230 235 240
Ser Gln Ile Gly Gly Glu Phe Gln Ser Phe Pro Lys Val Phe Gln
245 250 255
Lys Ile Trp Leu Glu Arg Asp Arg Lys Leu Ile Asn Leu Phe Arg
260 265 270
Gly Ala His Leu Asn Tyr His Arg Ser Leu Ile Ser Trp Gly Ile
275 280 285
Ile Asn Ala Thr Tyr Glu Phe Leu Leu Lys Val Ile
290 295
<210> 11
<211> 89
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 039026CD1
<400> 11
Met Ala Ala Gln Ile Pro Glu Ser Asp Gln Ile Lys Gln Phe Lys
1 5 10 15
Glu Phe Leu Gly Thr Tyr Asn Lys Leu Thr Glu Thr Cys Phe Leu
20 25 30
Asp Cys Val Lys Asp Phe Thr Thr Arg Glu Val Lys Pro Glu Glu
35 40 45
Thr Thr Cys Ser Glu His Cys Leu Gln Lys Tyr Leu Lys Met Thr
50 55 60
Gln Arg Ile Ser Met Arg Phe Gln Glu Tyr His Ile Gln Gln Asn
65 70 75
Glu Ala Leu Ala Ala Lys Ala Gly Leu Leu Gly Gln Pro Arg
80 85
<210> 12
<211> 115
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 260607CD1
<400> 12
Met Ala Leu Ile Pro Ser Arg Val Trp Leu Pro Phe Ala Val Trp
1 5 10 15
Val Val Asp Ser Ala Pro Val Arg Gly Leu Val Arg Arg Glu Pro
20 25 30
Phe Leu Arg Thr Gly Ser Phe Ile Ala Leu Phe Tyr Phe Pro Pro
35 40 45
Leu Leu Pro Val Leu Ile Asn Leu Phe Ser Phe Phe Leu Thr Pro
50 55 60
Ser Phe Trp Arg Gln Leu Gly Ala Ile Leu Val Tyr Ala Ser Leu
65 70 75
Leu Ala Glu Lys Thr Pro Phe Lys Thr Gln Arg Thr Leu Glu Gly
80 85 90
Asp Ala Leu Val Gly Ser Val Ser Ile Phe Leu Cys Ala Lys Asp
95 100 105
Arg Gln Thr Glu Ala Glu Arg Gly Cys Ser
110 115
<210> 13
<211> 675
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
9/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
<223> Incyte ID No: 1429651CD1
<400> 13
Met Glu Ser Gly Thr Ser Ser Pro Gln Pro Pro Gln Leu Asp Pro
1 5 10 15
Leu Asp Ala Phe Pro Gln Lys Gly Leu Glu Pro Gly Asp Ile Ala
20 25 30
Val Leu Val Leu Tyr Phe Leu Phe Val Leu Ala Val Gly Leu Trp
35 40 45
Ser Thr Val Lys Thr Lys Arg Asp Thr Val Lys Gly Tyr Phe Leu
50 55 60
Ala Gly Gly Asp Met Val Trp Trp Pro Val Gly Ala Ser Leu Phe
65 70 75
Ala Ser Asn Val Gly Ser Gly His Phe Ile Gly Leu Ala Gly Ser
80 85 90
Gly Ala Ala Thr Gly Ile Ser Val Ser Ala Tyr Glu Leu Asn Gly
95 100 105
Leu Phe Ser Val Leu Met Leu Ala Trp Ile Phe Leu Pro Ile Tyr
110 115 120
Ile Ala Gly Gln Val Thr Thr Met Pro Glu Tyr Leu Arg Lys Arg
125 130 135
Phe Gly Gly Ile Arg Ile Pro Ile Ile Leu Ala Val Leu Tyr Leu
140 145 150
Phe Ile Tyr Ile Phe Thr Lys Ile Ser Val Asp Met Tyr Ala Gly
155 160 165
Ala Ile Phe Ile Gln Gln Ser Leu His Leu Asp Leu Tyr Leu Ala
170 175 180
Ile Val Gly Leu Leu Ala Ile Thr Ala Val Tyr Thr Val Ala Gly
185 190 195
Gly Leu Ala Ala Val Ile Tyr Thr Asp Ala Leu Gln Thr Leu Ile
200 205 210
Met Leu Ile Gly Ala Leu Thr Leu Met Gly Tyr Ser Phe Ala Ala
215 220 225
Val Gly Gly Met Glu Gly Leu Lys Glu Lys Tyr Phe Leu Ala Leu
230 235 240
Ala Ser Asn Arg Ser Glu Asn Ser Ser Cys Gly Leu Pro Arg Glu
245 250 255
Asp Ala Phe His Ile Phe Arg Asp Pro Leu Thr Ser Asp Leu Pro
260 265 270
Trp Pro Gly Val Leu Phe Gly Met Ser Ile Pro Ser Leu Trp Tyr
275 280 285
Trp Cys Thr Asp Gln Val Ile Val Gln Arg Thr Leu Ala Ala Lys
290 295 300
Asn Leu Ser His A1a Lys Gly Gly Ala Leu Met Ala Ala Tyr Leu
305 310 315
Lys Val Leu Pro Leu Phe Ile Met Val Phe Pro Gly Met Val Ser
320 325 330
Arg Ile Leu Phe Pro Asp Gln Val Ala Cys Ala Asp Pro Glu Ile
335 340 345
Cys Gln Lys Ile Cys Ser Asn Pro Ser Gly Cys Ser Asp Ile Ala
350 355 360
Tyr Pro Lys Leu Val Leu Glu Leu Leu Pro Thr Gly Leu Arg Gly
365 370 375
Leu Met Met Ala Val Met Val Ala Ala Leu Met Ser Ser Leu Thr
380 385 390
Ser Ile Phe Asn Ser Ala Ser Thr Ile Phe Thr Met Asp Leu Trp
395 400 405
Asn His Leu Arg Pro Arg Ala Ser Glu Lys Glu Leu Met Ile Val
410 415 420
Gly Arg Val Phe Val Leu Leu Leu Val Leu Val Ser Ile Leu Trp
425 430 435
Ile Pro Val Val Gln Ala Ser Gln Gly Gly Gln Leu Phe Ile Tyr
440 445 450
Ile Gln Ser Ile Ser Ser Tyr Leu Gln Pro Pro Val Ala Val Val
455 460 465
Phe Ile Met Gly Cys Phe Trp Lys Arg Thr Asn Glu Lys Gly Ala
470 475 480
Phe Trp Gly Leu Ile Ser Gly Leu Leu Leu Gly Leu Val Arg Leu
10/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
485 490 495
Val Leu Asp Phe Ile Tyr Val Gln Pro Arg Cys Asp Gln Pro Asp
500 505 510
Glu Arg Pro Val Leu Val Lys Ser Ile His Tyr Leu Tyr Phe Ser
515 520 525
Met Ile Leu Ser Thr Val Thr Leu Ile Thr Val Ser Thr Val Ser
530 535 540
Trp Phe Thr Glu Pro Pro Ser Lys Glu Met Val Ser His Leu Thr
545 550 555
Trp Phe Thr Arg His Asp Pro Val Val Gln Lys Glu Gln Ala Pro
560 565 570
Pro Ala Ala Pro Leu Ser Leu Thr Leu Ser Gln Asn Gly Met Pro
575 580 585
Glu Ala Ser Ser Ser Ser Ser Val Gln Phe Glu Met Val Gln Glu
590 595 600
Asn Thr Ser Lys Thr His Ser Cys Asp Met Thr Pro Lys Gln Ser
605 610 615
Lys Val Val Lys Ala Ile Leu Trp Leu Cys Gly Ile Gln Glu Lys
620 625 630
Gly Lys Glu Glu Leu Pro Ala Arg Ala Glu Ala Ile Ile Val Ser
635 640 645
Leu Glu Glu Asn Pro Leu Val Lys Thr Leu Leu Asp Val Asn Leu
650 655 660
Ile Phe Cys Val Ser Cys Ala Ile Phe Ile Trp Gly Tyr Phe Ala
665 670 675
<210> 14
<211> 320
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2069971CD1
<400> 14
Met Tyr His Cys His Ser Gly Ser Lys Pro Thr Glu Lys Gly Ala
1 5 10 15
Asn Glu Tyr Ala Tyr Ala Lys Trp Lys Leu Cys Ser Ala Ser Ala
20 25 30
Ile Cys Phe Ile Phe Met Ile Ala Glu Val Val Gly Gly His Ile
35 40 45
Ala Gly Ser Leu Ala Val Val Thr Asp Ala Ala His Leu Leu Ile
50 55 60
Asp Leu Thr Ser Phe Leu Leu Ser Leu Phe Ser Leu Trp Leu Ser
65 70 75
Ser Lys Pro Pro Ser Lys Arg Leu Thr Phe Gly Trp His Arg Ala
80 85 90
Glu Ile Leu Gly Ala Leu Leu Ser Ile Leu Cys Ile Trp Val Val
95 100 105
Thr Gly Val Leu Val Tyr Leu Ala Cys Glu Arg Leu Leu Tyr Pro
110 115 120
Asp Tyr Gln Ile Gln Ala Thr Val Met Ile Ile Val Ser Ser Cys
125 130 135
Ala Val Ala Ala Asn Ile Val Leu Thr Val Val Leu His Gln Arg
140 145 150
Cys Leu Gly His Asn His Lys Glu Val Gln Ala Asn Ala Ser Val
155 160 165
Arg Ala Ala Phe Val His Ala Leu Gly Asp Leu Phe Gln Ser Ile
170 175 180
Ser Val Leu Ile Ser Ala Leu Ile Ile Tyr Phe Lys Pro Glu Tyr
185 190 195
Lys Ile Ala Asp Pro Ile Cys Thr Phe Ile Phe Ser Ile Leu Val
200 205 210
Leu Ala Ser Thr Ile Thr Ile Leu Lys Asp Phe Ser Ile Leu Leu
215 220 225
Met Glu Gly Val Pro Lys Ser Leu Asn Tyr Ser Gly Val Lys Glu
230 235 240
11 /60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Leu Ile Leu Ala Val Asp Gly Val Leu Ser Val His Ser Leu His
245 250 255
Ile Trp Ser Leu Thr Met Asn Gln Val Ile Leu Ser Ala His Val
260 265 270
Ala Thr Ala Ala Ser Arg Asp Ser Gln Val Val Arg Arg Glu Ile
275 280 285
Ala Lys Ala Leu Ser Lys Ser Phe Thr Met His Ser Leu Thr Ile
290 295 300
Gln Met Glu Ser Pro Val Asp Gln Asp Pro Asp Cys Leu Phe Cys
305 310 315
Glu Asp Pro Cys Asp
320
<210> 15
<211> 462
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2329339CD1
<400> 15
Met Ala Glu Glu Gln Glu Phe Thr Gln Leu Cys Lys Leu Pro Ala
1 5 10 15
Gln Pro Ser His Pro His Cys Val Asn Asn Thr Tyr Arg Ser Ala
20 25 30
Gln His Ser Gln Ala Leu Leu Arg Gly Leu Leu Ala Leu Arg Asp
35 40 45
Ser Gly Ile Leu Phe Asp Val Val Leu Val Val Glu Gly Arg His
50 55 60
Ile Glu Ala His Arg Ile Leu Leu Ala Ala Ser Cys Asp Tyr Phe
65 70 75
Arg Gly Met Phe Ala Gly Gly Leu Lys Glu Met Glu Gln Glu Glu
80 85 90
Val Leu Ile His Gly Val Ser Tyr Asn Ala Met Cys Gln Ile Leu
95 100 105
His Phe Ile Tyr Thr Ser Glu Leu Glu Leu Ser Leu Ser Asn Val
110 115 120
Gln Glu Thr Leu Val Ala Ala Cys Gln Leu Gln Ile Pro Glu Ile
125 130 135
Ile His Phe Cys Cys Asp Phe Leu Met Ser Trp Val Asp Glu Glu
140 145 150
Asn Ile Leu Asp Val Tyr Arg Leu Ala Glu Leu Phe Asp Leu Ser
155 160 165
Arg Leu Thr Glu Gln Leu Asp Thr Tyr Ile Leu Lys Asn Phe Val
170 175 180
Ala Phe Ser Arg Thr Asp Lys Tyr Arg Gln Leu Pro Leu Glu Lys
185 190 195
Val Tyr Ser Leu Leu Ser Ser Asn Arg Leu Glu Val Ser Cys Glu
200 205 210
Thr Glu Val Tyr Glu Gly Ala Leu Leu Tyr His Tyr Ser Leu Glu
215 220 225
Gln Val Gln Ala Asp Gln Ile Ser Leu His Glu Pro Pro Lys Leu
230 235 240
Leu Glu Thr Val Arg Phe Pro Leu Met Glu Ala Glu Val Leu Gln
245 250 255
Arg Leu His Asp Lys Leu Asp Pro Ser Pro Leu Arg Asp Thr Val
260 265 270
Ala Ser Gly Leu Met Tyr His Arg Asn Glu Ser Leu Gln Pro Ser
275 280 285
Leu Gln Ser Pro Gln Thr Glu Leu Arg Ser Asp Phe Gln Cys Val
290 295 300
Val Gly Phe Gly Gly Ile His Ser Thr Pro Ser Thr Val Leu Ser
305 310 315
Asp Gln Ala Lys Tyr Leu Asn Pro Leu Leu Gly Glu Trp Lys His
320 325 330
Phe Thr Ala Ser Leu Ala Pro Arg Met Ser Asn Gln Gly Ile Ala
335 340 345
12/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Val Leu Asn Asn Phe Val Tyr Leu Ile Gly Gly Asp Asn Asn Val
350 355 360
Gln Gly Phe Arg Ala Glu Ser Arg Cys Trp Arg Tyr Asp Pro Arg
365 370 375
His Asn Arg Trp Phe Gln Ile Gln Ser Leu Gln Gln Glu His Ala
380 385 390
Asp Leu Ser Val Cys Val Val Gly Arg Tyr Ile Tyr Ala Val Ala
395 400 405
Gly Arg Asp Tyr His Asn Asp Leu Asn Ala Val Glu Arg Tyr Asp
410 415 420
Pro Ala Thr Asn Ser Trp Ala Tyr Val Ala Pro Leu Lys Arg Glu
425 430 435
Val Tyr Ala His Ala Gly Ala Thr Leu Glu Gly Lys Met Tyr Ile
440 445 450
Thr Cys Gly Arg Lys Leu Ile Pro Phe Ser Glu Gly
455 460
<210> 16
<211> 98
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2540219CD1
<400> 16
Met Arg Ala Cys Ala Val Trp Leu Ala Gly Gly Met Ala Gly Ala
1 5 10 15
Ile Ser Trp Gly Thr Ala Thr Pro Met Asp Val Val Lys Ser Arg
20 25 30
Leu Gln Ala Asp Gly Val Tyr Leu Asn Lys Tyr Lys Gly Val Leu
35 40 45
Asp Cys Ile Ser Gln Ser Tyr Gln Lys Glu Gly Leu Lys Val Phe
50 55 60
Phe Arg Gly Ile Thr Val Asn Ala Val Arg Gly Phe Pro Met Ser
65 70 75
Ala Ala Met Phe Leu Gly Tyr Glu Leu Ser Leu Gln Ala Ile Arg
80 85 90
Gly Asp His Ala Val Thr Ser Pro
<210> 17
<211> 748
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2722462CD1
<400> 17
Met Asn Tyr Gln Glu Ala Ala Ile Tyr Leu Gln Glu Gly Glu Asn
1 5 10 15
Asn Asp Lys Phe Phe Thr His Pro Lys Asp Ala Lys Ala Leu Ala
20 25 30
Ala Tyr Leu Phe Ala His Asn His Leu Phe Tyr Leu Met Glu Leu
35 40 45
Ala Thr Ala Leu Leu Leu Leu Leu Leu Ser Leu Cys Glu Ala Pro
50 55 60
Ala Val Pro Ala Leu Arg Leu Gly Ile Tyr Val His Ala Thr Leu
65 70 75
Glu Leu Phe Ala Leu Met Val Val Val Phe Glu Leu Cys Met Lys
80 85 90
Leu Arg Trp Leu Gly Leu His Thr Phe Ile Arg His Lys Arg Thr
95 100 105
Met Val Lys Thr Ser Val Leu Val Val Gln Phe Val Glu Ala Ile
110 115 120
Val Val Leu Val Arg Gln Met Ser His Val Arg Val Thr Arg Ala
125 130 135
13/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Leu Arg Cys Ile Phe Leu Val Asp Cys Arg Tyr Cys Gly Gly Val
140 145 150
Arg Arg Asn Leu Arg Gln Ile Phe Gln Ser Leu Pro Pro Phe Met
155 160 165
Asp Ile Leu Leu Leu Leu Leu Phe Phe Met Ile Ile Phe Ala Ile
170 175 180
Leu Gly Phe Tyr Leu Phe Ser Pro Asn Pro Ser Asp Pro Tyr Phe
185 190 195
Ser Thr Leu Glu Asn Ser Ile Val Ser Leu Phe Val Leu Leu Thr
200 205 210
Thr Ala Asn Phe Pro Asp Val Met Met Pro Ser Tyr Ser Arg Asn
215 220 225
Pro Trp Ser Cys Val Phe Phe Ile Val Tyr Leu Ser Ile Glu Leu
230 235 240
Tyr Phe Ile Met Asn Leu Leu Leu Ala Val Val Phe Asp Thr Phe
245 250 255
Asn Asp Ile Glu Lys Arg Lys Phe Lys Ser Leu Leu Leu His Lys
260 265 270
Arg Thr Ala Ile Gln His Ala Tyr Arg Leu Leu Ile Ser Gln Arg
275 280 285
Arg Pro Ala Gly Ile Ser Tyr Arg Gln Phe Glu Gly Leu Met Arg
290 295 300
Phe Tyr Lys Pro Arg Met Ser Ala Arg Glu Arg Tyr Leu Thr Phe
305 310 315
Lys Ala Leu Asn Gln Asn Asn Thr Pro Leu Leu Ser Leu Lys Asp
320 325 330
Phe Tyr Asp Ile Tyr Glu Val Ala Ala Leu Lys Trp Lys Ala Lys
335 340 345
Lys Asn Arg Glu His Trp Phe Asp Glu Leu Pro Arg Thr Ala Leu
350 355 360
Leu Ile Phe Lys Gly Ile Asn Ile Leu Val Lys Ser Lys Ala Phe
365 370 375
Gln Tyr Phe Met Tyr Leu Val Val Ala Val Asn Gly Val Trp Ile
380 385 390
Leu Val Glu Thr Phe Met Leu Lys Gly Gly Asn Phe Phe Ser Lys
395 400 405
His Val Pro Trp Ser Tyr Leu Val Phe Leu Thr Ile Tyr Gly Val
410 415 420
Glu Leu Phe Leu Lys Val Ala Gly Leu Gly Pro Val Glu Tyr Leu
425 430 435
Ser Ser Gly Trp Asn Leu Phe Asp Phe Ser Val Thr Val Phe Ala
440 445 450
Phe Leu Gly Leu Leu Ala Leu Ala Leu Asn Met Glu Pro Phe Tyr
455 460 465
Phe Ile Val Val Leu Arg Pro Leu Gln Leu Leu Arg Leu Phe Lys
470 475 480
Leu Lys Glu Arg Tyr Arg Asn Val Leu Asp Thr Met Phe Glu Leu
485 490 495
Leu Pro Arg Met Ala Ser Leu Gly Leu Thr Leu Leu Ile Phe Tyr
500 505 510
Tyr Ser Phe Ala Ile Val Gly Met Glu Phe Phe Cys Gly Ile Val
515 520 525
Phe Pro Asn Cys Cys Asn Thr Ser Thr Val Ala Asp Ala Tyr Arg
530 535 540
Trp Arg Asn His Thr Val Gly Asn Arg Thr Val Val Glu Glu Gly
545 550 555
Tyr Tyr Tyr Leu Asn Asn Phe Asp Asn Ile Leu Asn Ser Phe Val
560 565 570
Thr Leu Phe Glu Leu Thr Val Val Asn Asn Trp Tyr Ile Ile Met
575 580 585
Glu Gly Val Thr Ser Gln Thr Ser His Trp Ser Arg Leu Tyr Phe
590 595 600
Met Thr Phe Tyr Ile Val Thr Met Val Val Met Thr Ile Ile Val
605 610 615
Ala Phe Ile Leu Glu Ala Phe Val Phe Arg Met Asn Tyr Ser Arg
620 625 630
Lys Asn Gln Asp Ser Glu Val Asp Gly Gly Ile Thr Leu Glu Lys
635 640 645
14/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Glu Ile Ser Lys Glu Glu Leu Val Ala Val Leu Glu Leu Tyr Arg
650 655 660
Glu Ala Arg Gly Ala Ser Ser Asp Val Thr Arg Leu Leu Glu Thr
665 670 675
Leu Ser Gln Met Glu Arg Tyr Gln Gln His Ser Met Val Phe Leu
680 685 690
Gly Arg Arg Ser Arg Thr Lys Ser Asp Leu Ser Leu Lys Met Tyr
695 700 705
Gln Glu Glu Ile Gln Glu Trp Tyr Glu Glu His Ala Arg Glu Gln
710 715 720
Glu Gln Gln Arg Gln Leu Ser Ser Ser Ala Ala Pro Ala Ala Gln
725 730 735
Gln Pro Pro Gly Ser Arg Gln Arg Ser Gln Thr Val Thr
740 745
<210> 18
<211> 507
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2739264CD1
<400> 18
Met Ala Phe Asn Phe Gly Ala Pro Ser Gly Thr Ser Gly Thr Ala
1 5 10 15
Ala Ala Thr Ala Ala Pro Ala Gly Gly Phe Gly Gly Phe Gly Thr
20 25 30
Thr Ser Thr Thr Ala Gly Ser Ala Phe Ser Phe Ser Ala Pro Thr
35 40 45
Asn Thr Gly Thr Thr Gly Leu Phe Gly Gly Thr Gln Asn Lys Gly
50 55 60
Phe Gly Phe Gly Thr Gly Phe Gly Thr Thr Thr Gly Thr Ser Thr
65 70 75
Gly Leu Gly Thr Gly Leu Gly Thr Gly Leu Gly Phe Gly Gly Phe
80 85 90
Asn Thr Gln Gln Gln Gln Gln Thr Thr Leu Gly Gly Leu Phe Ser
95 100 105
Gln Pro Thr Gln Ala Pro Thr Gln Ser Asn Gln Leu Ile Asn Thr
110 115 120
Ala Ser Ala Leu Ser Ala Pro Thr Leu Leu Gly Asp Glu Arg Asp
125 130 135
Ala Ile Leu Ala Lys Trp Asn Gln Leu Gln Ala Phe Trp Gly Thr
140 145 150
Gly Lys Gly Tyr Phe Asn Asn Asn Ile Pro Pro Val Glu Phe Thr
155 160 165
Gln Glu Asn Pro Phe Cys Arg Phe Lys Ala Val Gly Tyr Ser Cys
170 175 180
Met Pro Ser Asn Lys Asp Glu Asp Gly Leu Val Val Leu Val Phe
185 190 195
Asn Lys Lys Glu Thr Glu Ile Arg Ser Gln Gln Gln Gln Leu Val
200 205 210
Glu Ser Leu His Lys Val Leu Gly Gly Asn Gln Thr Leu Thr Val
215 220 225
Asn Val Glu Gly Thr Lys Thr Leu Pro Asp Asp Gln Thr Glu Val
230 235 240
Val Ile Tyr Val Val Glu Arg Ser Pro Asn Gly Thr Ser Arg Arg
245 250 255
Val Pro Ala Thr Thr Leu Tyr Ala His Phe Glu Gln Ala Asn Ile
260 265 270
Lys Thr Gln Leu Gln Gln Leu Gly Val Thr Leu Ser Met Thr Arg
275 280 285
Thr Glu Leu Ser Pro Ala Gln Ile Lys G1n Leu Leu Gln Asn Pro
290 295 300
Pro Ala Gly Val Asp Pro Ile Ile Trp Glu Gln Ala Lys Val Asp
305 310 315
Asn Pro Asp Ser Glu Lys Leu Ile Pro Val Pro Met Val Gly Phe
320 325 330
15/60
Met Thr Phe Tyr Ile Val Thr Met Val Val Met Thr Ile Ile V
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Lys Glu Leu Leu Arg Arg Leu Lys Val Gln Asp Gln Met Thr Lys
335 340 345
Gln His Gln Thr Arg Leu Asp Ile Ile Ser Glu Asp Ile Ser Glu
350 355 360
Leu Gln Lys Asn Gln Thr Thr Ser Val Ala Lys Ile Ala Gln Tyr
365 370 375
Lys Arg Lys Leu Met Asp Leu Ser His Arg Thr Leu Gln Val Leu
380 385 390
Ile Lys Gln Glu Ile Gln Arg Lys Ser Gly Tyr Ala Ile Gln Ala
395 400 405
Asp Glu Glu Gln Leu Arg Val Gln Leu Asp Thr Ile Gln Gly Glu
410 415 420
Leu Asn Ala Pro Thr Gln Phe Lys Gly Arg Leu Asn Glu Leu Met
425 430 435
Ser Gln Ile Arg Met Gln Asn His Phe Gly Ala Val Arg Ser Glu
440 445 450
Glu Arg Tyr Tyr Ile Asp Ala Asp Leu Leu Arg Glu Ile Lys Gln
455 460 465
His Leu Lys Gln Gln Gln Glu Gly Leu Ser His Leu Ile Ser Ile
470 475 480
Ile Lys Asp Asp Leu Glu Asp Ile Lys Leu Val Glu His Gly Leu
485 490 495
Asn Glu Thr Ile His Ile Arg Gly Gly Val Phe Ser
500 505
<210> 19
<211> 592
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2758310CD1
<400> 19
Met Trp Phe Cys Gly Gln Ser Thr Pro Phe Gly Cys Glu Leu His
1 5 10 15
Asp Thr Cys Val Gln Leu Cys His Phe His Ser Ala Leu Leu His
20 25 30
Arg Arg Gln Lys Pro Trp Pro Ser Pro Ala Val Phe Phe Arg Arg
35 40 45
Asn Val Arg Gly Leu Pro Pro Arg Phe Ser Ser Pro Thr Pro Leu
50 55 60
Trp Arg Lys Val Leu Ser Thr Ala Val Val Gly Ala Pro Leu Leu
65 70 75
Leu Gly Ala Arg Tyr Val Met Ala Glu Ala Arg Glu Lys Arg Arg
80 85 90
Met Arg Leu Val Val Asp Gly Met Gly Arg Phe Gly Arg Ser Leu
95 100 105
Lys Val Gly Leu Gln Ile Ser Leu Asp Tyr Trp Trp Cys Thr Asn
110 115 120
Val Val Leu Arg Gly Trp Lys Ser Pro Gly Tyr Leu Glu Val Met
125 130 135
Ser Ala Cys His Gln Arg Ala Ala Asp Ala Leu Val Ala Gly Ala
140 145 150
Ile Ser Asn Gly Gly Leu Tyr Val Lys Leu Gly Gln Gly Leu Cys
155 160 165
Ser Phe Asn His Leu Leu Pro Pro Glu Tyr Thr Arg Thr Leu Arg
170 175 180
Val Leu Glu Asp Arg Ala Leu Lys Arg Gly Phe Gln Glu Val Asp
185 190 195
Glu Leu Phe Leu Glu Asp Phe Gln Ala Leu Pro His Glu Leu Phe
200 205 210
Gln Glu Phe Asp Tyr Gln Pro Ile Ala Ala Ala Ser Leu Ala Gln
215 220 225
Val His Arg Ala Lys Leu His Asp Gly Thr Ser Val Ala Val Lys
230 235 240
Val Gln Tyr Ile Asp Leu Arg Asp Arg Phe Asp Gly Asp Ile His
245 250 255
16/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Thr Leu Glu Leu Leu Leu Arg Leu Val Glu Val Met His Pro Ser
260 265 270
Phe Gly Phe Ser Trp Val Leu Gln Asp Leu Lys Gly Thr Leu Ala
275 280 285
Gln Glu Leu Asp Phe Glu Asn Glu Gly Arg Asn Ala Glu Arg Cys
290 295 300
Ala Arg Glu Leu Ala His Phe Pro Tyr Val Val Val Pro Arg Val
305 310 315
His Trp Asp Lys Ser Ser Lys Arg Val Leu Thr Ala Asp Phe Cys
320 325 330
Ala Gly Cys Lys Val Asn Asp Val Glu Ala Ile Arg Ser Gln Gly
335 340 345
Leu Ala Val His Asp Ile Ala Glu Lys Leu Ile Lys Ala Phe Ala
350 355 360
Glu Gln Ile Phe Tyr Thr Gly Phe Ile His Ser Asp Pro His Pro
365 370 375
Gly Asn Val Leu Val Arg Lys Gly Pro Asp Gly Lys Ala Glu Leu
380 385 390
Val Leu Leu Asp His Gly Leu Tyr Gln Phe Leu Glu Glu Lys Asp
395 400 405
Arg Ala Ala Leu Cys Gln Leu Trp Arg Ala Ile Ile Leu Arg Asp
410 415 420
Asp Ala Ala Met Arg Ala His Ala Ala Ala Leu Gly Val Gln Asp
425 430 435
Tyr Leu Leu Phe Ala Glu Met Leu Met Gln Arg Pro Val Arg Leu
440 445 450
Gly Gln Leu Trp Gly Ser His Leu Leu Ser Arg Glu Glu Ala Ala
455 460 465
Tyr Met Val Asp Met Ala Arg Glu Arg Phe Glu Ala Val Met Ala
470 475 480
Val Leu Arg Glu Leu Pro Arg Pro Met Leu Leu Val Leu Arg Asn
485 490 495
Ile Asn Thr Val Arg Ala Ile Asn Val Ala Leu Gly Ala Pro Val
500 505 510
Asp Arg Tyr Phe Leu Met Ala Lys Arg Ala Val Arg Gly Trp Ser
515 520 525
Arg Leu Ala Gly Ala Thr Tyr Arg Gly Val Tyr Gly Thr Ser Leu
530 535 540
Leu Arg His Ala Lys Val Val Trp Glu Met Leu Lys Phe Glu Val
545 550 555
Ala Leu Arg Leu Glu Thr Leu Ala Met Arg Leu Thr Ala Leu Leu
560 565 570
Ala Arg Ala Leu Val His Leu Ser Leu Val Pro Pro Ala Glu Glu
575 580 585
Leu Tyr Gln Tyr Leu Glu Thr
590
<210> 20
<211> 841
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2762348CD1
<400> 20
Met Ala Ser Val Phe Arg Ser Glu Glu Met Cys Leu Ser Gln Leu
1 5 10 15
Phe Leu Gln Val Glu Ala Ala Tyr Cys Cys Val Ala Glu Leu Gly
20 25 30
Glu Leu Gly Leu Val Gln Phe Lys Asp Leu Asn Met Asn Val Asn
35 40 45
Ser Phe Gln Arg Lys Phe Val Asn Glu Val Arg Arg Cys Glu Ser
50 55 60
Leu Glu Arg Ile Leu Arg Phe Leu Glu Asp Glu Met Gln Asn Glu
65 70 75
Ile Val Val Gln Leu Leu Glu Lys Ser Pro Leu Thr Pro Leu Pro
80 85 90
17/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Arg Glu Met Ile Thr Leu Glu Thr Val Leu Glu Lys Leu Glu Gly
95 100 105
Glu Leu Gln Glu Ala Asn Gln Asn Gln Gln Ala Leu Lys Gln Ser
110 115 120
Phe Leu Glu Leu Thr Glu Leu Lys Tyr Leu Leu Lys Lys Thr Gln
125 130 135
Asp Phe Phe Glu Thr Glu Thr Asn Leu Ala Asp Asp Phe Phe Thr
140 145 150
Glu Asp Thr Ser Gly Leu Leu Glu Leu Lys Ala Val Pro Ala Tyr
155 160 165
Met Thr Gly Lys Leu Gly Phe Ile Ala Gly Cys Asp Pro Thr Gly
170 175 180
Lys Arg Met Ala Ser Phe Glu Arg Leu Leu Trp Arg Val Cys Arg
185 190 195
Gly Asn Val Tyr Leu Lys Phe Ser Glu Met Asp Ala Pro Leu Glu
200 205 210
Asp Pro Val Thr Lys Glu Glu Ile Gln Lys His Ile Phe Ile Ile
215 220 225
Phe Tyr Gln Gly Glu Gln Leu Arg Gln Lys Ile Lys Lys Ile Cys
230 235 240
Asp Gly Phe Arg Ala Thr Val Tyr Pro Cys Pro Glu Pro Ala Val
245 250 255
Glu Arg Arg Glu Met Leu Glu Ser Val Asn Val Arg Leu Glu Asp
260 265 270
Leu Ile Thr Val Ile Thr Gln Thr Glu Ser His Arg Gln Arg Leu
275 280 285
Leu Gln Glu Ala Ala Ala Asn Trp His Ser Trp Leu Ile Lys Val
290 295 300
Gln Lys Met Lys Ala Val Tyr His Ile Leu Asn Met Cys Asn Ile
305 310 315
Asp Val Thr Gln Gln Cys Val Ile Ala Glu Ile Trp Phe Pro Val
320 325 330
Ala Asp Ala Thr Arg Ile Lys Arg Ala Leu Glu Gln Gly Met Glu
335 340 345
Leu Ser Gly Ser Ser Met Ala Pro Ile Met Thr Thr Val Gln Ser
350 355 360
Lys Thr Ala Pro Pro Thr Phe Asn Arg Thr Asn Lys Phe Thr Ala
365 370 375
Gly Phe Gln Asn Ile Val Asp Ala Tyr Gly Val Gly Ser Tyr Arg
380 385 390
Glu Ile Asn Pro Ala Pro Tyr Thr Ile Ile Thr Phe Pro Phe Leu
395 400 405
Phe Ala Val Met Phe Gly Asp Cys Gly His Gly Thr Val Met Leu
410 415 420
Leu Ala Ala Leu Trp Met Ile Leu Asn Glu Arg Arg Leu Leu Ser
425 430 435
Gln Lys Thr Asp Asn Glu Ile Trp Asn Thr Phe Phe His Gly Arg
440 445 450
Tyr Leu Ile Leu Leu Met Gly Ile Phe Ser Ile Tyr Thr Gly Leu
455 460 465
Ile Tyr Asn Asp Cys Phe Ser Lys Ser Leu Asn Ile Phe Gly Ser
470 475 480
Ser Trp Ser Val Gln Pro Met Phe Arg Asn Gly Thr Trp Asn Thr
485 490 495
His Val Met Glu Glu Ser Leu Tyr Leu Gln Leu Asp Pro Ala Ile
500 505 510
Pro Gly Val Tyr Phe Gly Asn Pro Tyr Pro Phe Gly Ile Asp Pro
515 520 525
Ile Trp Asn Leu Ala Ser Asn Lys Leu Thr Phe Leu Asn Ser Tyr
530 535 540
Lys Met Lys Met Ser Val Ile Leu Gly Ile Val Gln Met Val Phe
545 550 555
Gly Val Ile Leu Ser Leu Phe Asn His Ile Tyr Phe Arg Arg Thr
560 565 570
Leu Asn Ile Ile Leu Gln Phe Ile Pro Glu Met Ile Phe Ile Leu
575 580 585
Cys Leu Phe Gly Tyr Leu Val Phe Met Ile Ile Phe Lys Trp Cys
590 595 600
18/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Cys Phe Asp Val His Val Ser Gln His Ala Pro Ser Ile Leu Ile
605 610 615
His Phe Ile Asn Met Phe Leu Phe Asn Tyr Ser Asp Ser Ser Asn
620 625 630
Ala Pro Leu Tyr Lys His Gln Gln Glu Val Gln Ser Phe Phe Val
635 640 645
Val Met Ala Leu Ile Ser Val Pro Trp Met Leu Leu Ile Lys Pro
650 655 660
Phe Ile Leu Arg Ala Ser His Arg Lys Ser Gln Leu Gln Ala Ser
665 670 675
Arg Ile Gln Glu Asp Ala Thr Glu Asn Ile Glu Gly Asp Ser Ser
680 685 690
Ser Pro Ser Ser Arg Ser Gly Gln Arg Thr Ser Ala Asp Thr His
695 700 705
Gly Ala Leu Asp Asp His Gly Glu Glu Phe Asn Phe Gly Asp Val
710 715 720
Phe Val His Gln Ala Ile His Thr Ile Glu Tyr Cys Leu Gly Cys
725 730 735
Ile Ser Asn Thr Ala Ser Tyr Leu Arg Leu Trp Ala Leu Ser Leu
740 745 750
Ala His Ala Gln Leu Ser Glu Val Leu Trp Thr Met Val Met Asn
755 760 765
Ser Gly Leu Gln Thr Arg Gly Trp Gly Gly Ile Val Gly Val Phe
770 775 780
Ile Ile Phe Ala Val Phe Ala Val Leu Thr Val Ala Ile Leu Leu
785 790 795
Ile Met Glu Gly Leu Ser Ala Phe Leu His Ala Leu Arg Leu His
800 805 810
Trp Val Glu Phe Gln Asn Lys Phe Tyr Val Gly Asp Gly Tyr Lys
815 820 825
Phe Ser Pro Phe Ser Phe Lys His Ile Leu Asp Gly Thr Ala Glu
830 835 840
Glu
<210> 21
<211> 253
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3715961CD1
<400> 21
Met Ser Glu Cys Pro Leu Ile Leu Tyr Ile His Lys His Ile Asp
1 5 10 15
Thr Tyr Ser Gln Ser Tyr Leu Phe Asn Asp Leu Phe Tyr Pro Val
20 25 30
Tyr Ser Gly Gly Arg Met Val Thr Tyr Glu His Leu Arg Glu Val
35 40 45
Val Phe Gly Lys Ser Glu Asp Glu His Tyr Pro Leu Trp Lys Ser
50 55 60
Val Ile Gly Gly Met Met Ala Gly Val Ile Gly Gln Phe Leu Ala
65 70 75
Asn Pro Thr Asp Leu Val Lys Val Gln Met Gln Met Glu Gly Lys
80 85 90
Arg Lys Leu Glu Gly Lys Pro Leu Arg Phe Arg Gly Val His His
95 100 105
Ala Phe Ala Lys Ile Leu Ala Glu Gly Gly Ile Arg Gly Leu Trp
110 115 120
Ala Gly Trp Val Pro Asn Ile Gln Arg Ala Ala Leu Val Asn Met
125 130 135
Gly Asp Leu Thr Thr Tyr Asp Thr Val Lys His Tyr Leu Val Leu
140 145 150
Asn Thr Pro Leu Glu Asp Asn Ile Met Thr His Gly Leu Ser Ser
155 160 165
Leu Cys Ser Gly Leu Val Ala Ser Ile Leu Gly Thr Pro Ala Asp
170 175 180
19/60
CA 02375493 2001-12-11
WO 00/78953 PCT/~JS00/16668
Val Ile Lys Ser Arg Ile Met Asn Gln Pro Arg Asp Lys Gln Gly
185 190 195
Arg Gly Leu Leu Tyr Lys Ser Ser Thr Asp Cys Leu Ile Gln Ala
200 205 210
Val Gln Gly Glu Gly Phe Met Ser Leu Tyr Lys Gly Phe Leu Pro
215 220 225
Ser Trp Leu Arg Met Thr Pro Trp Ser Met Val Phe Trp Leu Thr
230 235 240
Tyr Glu Lys Ile Arg Glu Met Ser Gly Val Ser Pro Phe
245 250
<210> 22
<211> 229
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5108194CD1
<400> 22
Met Gly Asn Gly Val Lys Glu Gly Pro Val Arg Leu His Glu Asp
1 5 10 15
Ala Glu Ala Val Leu Ser Ser Ser Val Ser Ser Lys Arg Asp His
20 25 30
Arg Gln Val Leu Ser Ser Leu Leu Ser Gly Ala Leu Ala Gly Ala
35 40 45
Leu Ala Lys Thr Ala Val Ala Pro Leu Asp Arg Thr Lys Ile Ile
50 55 60
Phe Gln Val Ser Ser Lys Arg Phe Ser Ala Lys Glu Ala Phe Arg
65 70 75
Val Leu Tyr Tyr Thr Tyr Leu Asn Glu Gly Phe Leu Ser Leu Trp
80 85 90
Arg Gly Asn Ser Ala Thr Met Val Arg Val Val Pro Tyr Ala Ala
95 100 105
Ile Gln Phe Ser Ala His Glu Glu Tyr Lys Arg Ile Leu Gly Ser
110 115 120
Tyr Tyr Gly Phe Arg Gly Glu Ala Leu Pro Pro Trp Pro Arg Leu
125 130 135
Phe Ala Gly Ala Leu Ala Gly Thr Thr Ala Ala Ser Leu Thr Tyr
140 145 150
Pro Leu Asp Leu Val Arg Ala Arg Met Ala Val Thr Pro Lys Glu
155 160 165
Met Tyr Ser Asn Ile Phe His Val Phe Ile Arg Ile Ser Arg Glu
170 175 180
Glu Gly Leu Lys Thr Leu Tyr His Gly Phe Met Pro Thr Val Leu
185 190 195
Gly Val Ile Pro Tyr Ala Gly Leu Ser Phe Phe Thr Tyr Glu Thr
200 205 210
Leu Lys Ser Leu His Arg Glu Tyr Ser Gly Arg Lys Leu Ile Pro
215 220 225
Phe Ser Glu Gly
<210> 23
<211> 170
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5503122CD1
<400> 23
Met Tyr Asp Asn Leu Tyr Leu His Gly Ile Glu Asp Ser Glu Ala
1 5 10 15
Gly Ser Ala Asp Ser Tyr Thr Ser Arg Pro Ser Asp Ser Asp Val
20 25 30
Ser Leu Glu Glu Asp Arg G1u Ala Ile Arg Gln Glu Arg Glu Gln
35 40 45
20/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Gln Ala Ala Ile Gln Leu Glu Arg Ala Lys Ser Lys Pro Val Ala
50 55 60
Phe Ala Val Lys Thr Asn Val Ser Tyr Cys Gly Ala Leu Asp Glu
65 70 75
Asp Val Pro Val Pro Ser Thr Ala Ile Ser Phe Asp Ala Lys Asp
80 85 90
Phe Leu His Ile Lys Glu Lys Tyr Asn Asn Asp Trp Trp Ile Gly
95 100 105
Arg Leu Val Lys Glu Gly Cys Glu Ile Gly Phe Ile Pro Ser Pro
110 115 120
Leu Arg Leu Glu Asn Ile Arg Ile Gln Gln Glu Gln Lys Arg Gly
125 130 135
Arg Phe His Gly Gly Lys Ser Ser Gly Asn Ser Ser Ser Ser Leu
140 145 150
Gly Glu Met Val Ser Gly Thr Phe Arg Ala Thr Pro Thr Ser Thr
155 160 165
Gly Glu Gly Cys Ser
170
<210> 24
<211> 655
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5517972CD1
<400> 24
Met Ser Ser Ser Asn Val Glu Val Phe Ile Pro Val Ser Gln Gly
1 5 10 15
Asn Thr Asn Gly Phe Pro Ala Thr Ala Ser Asn Asp Leu Lys Ala
20 25 30
Phe Thr Glu Gly Ala Val Leu Ser Phe His Asn Ile Cys Tyr Arg
35 40 45
Val Lys Leu Lys Ser Gly Phe Leu Pro Cys Arg Lys Pro Val Glu
50 55 60
Lys Glu Ile Leu Ser Asn Ile Asn Gly Ile Met Lys Pro Gly Leu
65 70 75
Asn Ala Ile Leu Gly Pro Thr Gly Gly Gly Lys Ser Ser Leu Leu
80 85 90
Asp Val Leu Ala Ala Arg Lys Asp Pro Ser Gly Leu Ser Gly Asp
95 100 105
Val Leu Ile Asn Gly Ala Pro Arg Pro Ala Asn Phe Lys Cys Asn
110 115 120
Ser Gly Tyr Val Val Gln Asp Asp Val Val Met Gly Thr Leu Thr
125 i30 135
Val Arg Glu Asn Leu Gln Phe Ser Ala Ala Leu Arg Leu Ala Thr
140 145 150
Thr Met Thr Asn His Glu Lys Asn Glu Arg Ile Asn Arg Val Ile
155 160 165
Gln Glu Leu Gly Leu Asp Lys Val Ala Asp Ser Lys Val Gly Thr
170 175 180
Gln Phe Ile Arg Gly Val Ser Gly Gly Glu Arg Lys Arg Thr Ser
185 190 195
Ile Gly Met Glu Leu Ile Thr Asp Pro Ser Ile Leu Phe Leu Asp
200 205 210
Glu Pro Thr Thr Gly Leu Asp Ser Ser Thr Ala Asn Ala Val Leu
215 220 225
Leu Leu Leu Lys Arg Met Ser Lys Gln Gly Arg Thr Ile Ile Phe
230 235 240
Ser Ile His Gln Pro Arg Tyr Ser Ile Phe Lys Leu Phe Asp Ser
245 250 255
Leu Thr Leu Leu Ala Ser Gly Arg Leu Met Phe His Gly Pro Ala
260 265 270
Gln Glu Ala Leu Gly Tyr Phe Glu Ser Ala Gly Tyr His Cys Glu
275 280 285
Ala Tyr Asn Asn Pro Ala Asp Phe Phe Leu Asp Ile Ile Asn Gly
290 295 300
21 /60
Val Gln Gly Glu Gly Phe Met Ser Leu Tyr Lys Gly Phe Leu Pro
215
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Asp Ser Thr Ala Val Ala Leu Asn Arg Glu Glu Asp Phe Lys Ala
305 310 315
Thr Glu Ile Ile Glu Pro Ser Lys Gln Asp Lys Pro Leu Ile Glu
320 325 330
Lys Leu Ala Glu Ile Tyr Val Asn Ser Ser Phe Tyr Lys Glu Thr
335 340 345
Lys Ala Glu Leu His Gln Leu Ser Gly Gly Glu Lys Lys Lys Lys
350 355 360
Ile Thr Val Phe Lys Glu Ile Ser Tyr Thr Thr Ser Phe Cys His
365 370 375
Gln Leu Arg Trp Val Ser Lys Arg Ser Phe Lys Asn Leu Leu Gly
380 385 390
Asn Pro Gln Ala Ser Ile Ala Gln Ile Ile Val Thr Val Val Leu
395 400 405
Gly Leu Val Ile Gly Ala Ile Tyr Phe Gly Leu Lys Asn Asp Ser
410 415 420
Thr Gly Ile Gln Asn Arg Ala Gly Val Leu Phe Phe Leu Thr Thr
425 430 435
Asn Gln Cys Phe Ser Ser Val Ser Ala Val Glu Leu Phe Val Val
440 445 450
Glu Lys Lys Leu Phe Ile His Glu Tyr Ile Ser Gly Tyr Tyr Arg
455 460 465
Val Ser Ser Tyr Phe Leu Gly Lys Leu Leu Ser Asp Leu Leu Pro
470 475 480
Met Arg Met Leu Pro Ser Ile Ile Phe Thr Cys Ile Val Tyr Phe
485 490 495
Met Leu Gly Leu Lys Pro Lys Ala Asp Ala Phe Phe Val Met Met
500 505 510
Phe Thr Leu Met Met Val Ala Tyr Ser Ala Ser Ser Met Ala Leu
515 520 525
Ala Ile Ala Ala Gly Gln Ser Val Val Ser Val Ala Thr Leu Leu
530 535 540
Met Thr Ile Cys Phe Val Phe Met Met Ile Phe Ser Gly Leu Leu
545 550 555
Val Asn Leu Thr Thr Ile Ala Ser Trp Leu Ser Trp Leu Gln Tyr
560 565 570
Phe Ser Ile Pro Arg Tyr Gly Phe Thr Ala Leu Gln His Asn Glu
575 580 585
Phe Leu Gly Gln Asn Phe Cys Pro Gly Leu Asn Ala Thr Gly Asn
590 595 600
Asn Pro Cys Asn Tyr Ala Thr Cys Thr Gly Glu Glu Tyr Leu Val
605 610 615
Lys Gln Gly Ile Asp Leu Ser Pro Trp Gly Leu Trp Lys Asn His
620 625 630
Val Ala Leu Ala Cys Met Ile Val Ile Phe Leu Thr Ile Ala Tyr
635 640 645
Leu Lys Leu Leu Phe Leu Lys Lys Tyr Ser
650 655
<210> 25
<211> 184
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5593114CD1
<400> 25
Met Trp Val Phe Gly Tyr Gly Ser Leu Ile Trp Lys Val Asp Phe
1 5 10 15
Pro Tyr Gln Asp Lys Leu Val Gly Tyr Ile Thr Asn Tyr Ser Arg
20 25 30
Arg Phe Trp Gln Gly Ser Thr Asp His Arg Gly Val Pro Gly Lys
35 40 45
Pro Gly Arg Val Val Thr Leu Val Glu Asp Pro Ala Gly Cys Val
50 55 60
Trp Gly Val Ala Tyr Arg Leu Pro Val Gly Lys Glu Glu Glu Val
65 70 75
22/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Lys Ala Tyr Leu Asp Phe Arg Glu Lys Gly Gly Tyr Arg Thr Thr
80 85 90
Thr Val Ile Phe Tyr Pro Lys Asp Pro Thr Thr Lys Pro Phe Ser
95 100 105
Val Leu Leu Tyr Ile Gly Thr Cys Asp Asn Pro Asp Tyr Leu Gly
110 115 120
Pro Ala Pro Leu Glu Asp Ile Ala Glu Gln Ile Phe Asn Ala Ala
125 130 135
Gly Pro Ser Gly Arg Asn Thr Glu Tyr Leu Phe Glu Leu Ala Asn
140 145 150
Ser Ile Arg Asn Leu Val Pro Glu Glu Ala Asp Glu His Leu Phe
155 160 165
Ala Leu Glu Lys Leu Val Lys Glu Arg Leu Glu Gly Lys Gln Asn
170 175 180
Leu Asn Cys Ile
<210> 26
<211> 154
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 044775CD1
<400> 26
Met Gly Ala Phe Glu Cys Val Arg Lys Val Tyr Gln Thr Asp Gly
1 5 10 15
Leu Lys Gly Phe Tyr Arg Gly Met Ser Ala Ser Tyr Ala Gly Ile
20 25 30
Ser Glu Thr Val Ile His Phe Val Ile Tyr Glu Ser Ile Lys Gln
35 40 45
Lys Leu Leu Glu Tyr Lys Thr Ala Ser Thr Met Glu Asn Asp Glu
50 55 60
Glu Ser Val Lys Glu Ala Ser Asp Phe Val Gly Met Met Leu Ala
65 70 75
Ala Ala Thr Ser Lys Thr Cys Ala Thr Thr Ile Ala Tyr Pro His
80 85 90
Glu Val Val Arg Thr Arg Leu Arg Glu Glu Gly Thr Lys Tyr Arg
95 100 105
Ser Phe Phe Gln Thr Leu Ser Leu Leu Val Gln Glu Glu Gly Tyr
110 115 120
Gly Ser Leu Tyr Arg Gly Leu Thr Thr His Leu Val Arg Gln Ile
125 130 135
Pro Asn Thr Ala Ile Met Met Ala Thr Tyr Glu Leu Val Val Tyr
140 145 150
Leu Leu Asn Gly
<210> 27
<211> 438
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 116588CD1
<400> 27
Met Leu Leu Val Thr Pro Arg Pro Glu Arg Gly Gly Arg Gly Thr
1 5 10 15
Glu Leu Gly Glu Phe Cys Gly Thr Pro Leu Leu Phe Ser Ser Tyr
20 25 30
Phe Cys Tyr Asp Asn Pro Ala Ala Leu Gln Thr Gln Val Lys Arg
35 40 45
Asp Met Gln Val Asn Thr Thr Lys Phe Met Leu Leu Tyr Ala Trp
50 55 60
Tyr Ser Trp Pro Asn Val Val Leu Cys Phe Phe Gly Gly Phe Leu
65 70 75
23/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Ile Asp Arg Val Phe Gly Ile Arg Trp Gly Thr Ile Ile Phe Ser
80 85 90
Cys Phe Val Cys Ile Gly Gln Val Val Phe Ala Leu Gly Gly Ile
95 100 105
Phe Asn Ala Phe Trp Leu Met Glu Phe Gly Arg Phe Val Phe Gly
110 115 120
Ile Gly Gly Glu Ser Leu Ala Val Ala Gln Asn Thr Tyr Ala Val
125 130 135
Ser Trp Phe Lys Gly Lys Glu Leu Asn Leu Val Phe Gly Leu Gln
140 145 150
Leu Ser Met Ala Arg Ile Gly Ser Thr Val Asn Met Asn Leu Met
155 160 165
Gly Trp Leu Tyr Ser Lys Ile Glu Ala Leu Leu Gly Ser Ala Gly
170 175 180
His Thr Thr Leu Gly Ile Thr Leu Met Ile Gly Gly Val Thr Cys
185 190 195
Ile Leu Ser Leu Ile Cys Ala Leu Ala Leu Ala Tyr Leu Asp Gln
200 205 210
Arg Ala Glu Arg Ile Leu His Lys Glu Gln Gly Lys Thr Gly Glu
215 220 225
Val Ile Lys Leu Thr Asp Val Lys Asp Phe Ser Leu Pro Leu Trp
230 235 240
Leu Ile Phe Ile Ile Cys Val Cys Tyr Tyr Val Ala Val Phe Pro
245 250 255
Phe Ile Gly Leu Gly Lys Val Phe Phe Thr Glu Lys Phe Gly Phe
260 265 270
Ser Ser Gln Ala Ala Ser Ala Ile Asn Ser Val Val Tyr Val Ile
275 280 285
Ser Ala Pro Met Ser Pro Val Phe Gly Leu Leu Val Asp Lys Thr
290 295 300
Gly Lys Asn Ile Ile Trp Val Leu Cys Ala Val Ala Ala Thr Leu
305 310 315
Val Ser His Met Met Leu Ala Phe Thr Met Trp Asn Pro Trp Ile
320 325 330
Ala Met Cys Leu Leu Gly Leu Ser Tyr Ser Leu Leu Ala Cys Ala
335 340 345
Leu Trp Pro Met Val Ala Phe Val Val Pro Glu His Gln Leu Gly
350 355 360
Thr Ala Tyr Gly Phe Met Gln Ser Ile Gln Asn Leu Gly Leu Ala
365 370 375
Ile Ile Ser Ile Ile Ala Gly Met Ile Leu Asp Ser Arg Gly Tyr
380 385 390
Leu Phe Leu Glu Val Phe Phe Ile Ala Cys Val Ser Leu Ser Leu
395 400 405
Leu Ser Val Val Leu Leu Tyr Leu Val Asn Arg Ala Gln Gly Gly
410 415 420
Asn Leu Asn Tyr Ser Ala Arg Gln Arg Glu Glu Ile Lys Phe Ser
425 430 435
His Thr Glu
<210> 28
<211> 237
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 875369CD1
<400> 28
Met Ala His Val Gly Ser Arg Lys Arg Ser Arg Ser Arg Ser Arg
1 5 10 15
Ser Arg Gly Arg Gly Ser Glu Lys Arg Lys Lys Lys Ser Arg Lys
20 25 30
Asp Thr Ser Arg Asn Cys Ser Ala Ser Thr Ser Gln Gly Arg Lys
35 40 45
Ala Ser Thr Ala Pro Gly Ala Glu Ala Ser Pro Ser Pro Cys Ile
50 55 60
24/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Thr Glu Arg Ser Lys Gln Lys Ala Arg Arg Arg Thr Arg Ser Ser
65 70 75
Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser
80 85 90
Ser Ser Ser Ser Ser Ser Ser Ser Asp Gly Arg Lys Lys Arg Gly
95 100 105
Lys Tyr Lys Asp Lys Arg Arg Lys Lys Lys Lys Lys Arg Lys Lys
110 115 120
Leu Lys Lys Lys Gly Lys Glu Lys Ala Glu Ala Gln Gln Val Glu
125 130 135
Ala Leu Pro Gly Pro Ser Leu Asp Gln Trp His Arg Ser Ala Gly
140 145 150
Glu Glu Glu Asp Gly Pro Val Leu Thr Asp Glu Gln Lys Ser Arg
155 160 165
Ile Gln Ala Met Lys Pro Met Thr Lys Glu Glu Trp Asp Ala Arg
170 175 180
Gln Ser Ile Ile Arg Lys Val Val Asp Pro Glu Thr Gly Arg Thr
185 190 195
Arg Leu Ile Lys Gly Asp Gly Glu Val Leu Glu Glu Ile Val Thr
200 205 210
Lys Glu Arg His Arg Glu Ile Asn Lys Gln Ala Thr Arg Gly Asp
215 220 225
Cys Leu Ala Phe Gln Met Arg Ala Gly Leu Leu Pro
230 235
<210> 29
<211> 219
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1325518CD1
<400> 29
Met Lys Leu Leu Leu Trp Ala Cys Ile Val Cys Val Ala Phe Ala
1 5 10 15
Arg Lys Arg Arg Phe Pro Phe Ile Gly Glu Asp Asp Asn Asp Asp
20 25 30
Gly His Pro Leu His Pro Ser Leu Asn Ile Pro Tyr Gly Ile Arg
35 40 45
Asn Leu Pro Pro Pro Leu Tyr Tyr Arg Pro Val Asn Thr Val Pro
50 55 60
Ser Tyr Pro Gly Asn Thr Tyr Thr Asp Thr Gly Leu Pro Ser Tyr
65 70 75
Pro Trp Ile Leu Thr Ser Pro Gly Phe Pro Tyr Val Tyr His Ile
80 85 90
Arg Gly Phe Pro Leu Ala Thr Gln Leu Asn Val Pro Pro Leu Pro
95 100 105
Pro Arg Gly Phe Pro Phe Val Pro Pro Ser Arg Phe Phe Ser Ala
110 115 120
Ala Ala Ala Pro Ala Ala Pro Pro Ile Ala Ala Glu Pro Ala Ala
125 130 135
Ala Ala Pro Leu Thr Ala Thr Pro Val Ala Ala Glu Pro Ala Ala
140 145 150
Gly Ala Pro Val Ala Ala Glu Pro Ala Ala Glu Ala Pro Val Gly
155 160 165
Ala Glu Pro Ala Ala Glu Ala Pro Val Ala Ala Glu Pro Ala Ala
170 175 180
Glu Ala Pro Val Gly Val Glu Pro Ala Ala Glu Glu Pro Ser Pro
185 190 195
Ala Glu Pro Ala Thr Ala Lys Pro Ala Ala Pro Glu Pro His Pro
200 205 210
Ser Pro Ser Leu Glu Gln Ala Asn Gln
215
<210> 30
<211> 707
<212> PRT
<213> Homo Sapiens
25/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
<220>
<221> misc_feature
<223> Incyte ID No: 2060987CD1
<400> 30
Met Ala Ala Ala Ala Thr Ala Ala Glu Gly Val Pro Ser Arg Gly
1 5 10 15
Pro Pro Gly Glu Val Ile His Leu Asn Val Gly Gly Lys Arg Phe
20 25 30
Ser Thr Ser Arg Gln Thr Leu Thr Trp Ile Pro Asp Ser Phe Phe
35 40 45
Ser Ser Leu Leu Ser Gly Arg Ile Ser Thr Leu Lys Asp Glu Thr
50 55 60
Gly Ala Ile Phe Ile Asp Arg Asp Pro Thr Val Phe Ala Pro Ile
65 70 75
Leu Asn Phe Leu Arg Thr Lys Glu Leu Asp Pro Arg Gly Val His
80 85 90
Gly Ser Ser Leu Leu His Glu Ala Gln Phe Tyr Gly Leu Thr Pro
95 100 105
Leu Val Arg Arg Leu Gln Leu Arg Glu Glu Leu Asp Arg Ser Ser
110 115 120
Cys Gly Asn Val Leu Phe Asn Gly Tyr Leu Pro Pro Pro Val Phe
125 130 135
Pro Val Lys Arg Arg Asn Arg His Ser Leu Val Gly Pro Gln Gln
140 145 150
Leu Gly Gly Arg Pro Ala Pro Val Arg Arg Ser Asn Thr Met Pro
155 160 165
Pro Asn Leu Gly Asn Ala Gly Leu Leu Gly Arg Met Leu Asp Glu
170 175 180
Lys Thr Pro Pro Ser Pro Ser Gly Gln Pro Glu Glu Pro Gly Met
185 190 195
Val Arg Leu Val Cys Gly His His Asn Trp Ile Ala Val Ala Tyr
200 205 210
Thr Gln Phe Leu Val Cys Tyr Arg Leu Lys Glu Ala Ser Gly Trp
215 220 225
Gln Leu Val Phe Ser Ser Pro Arg Leu Asp Trp Pro Ile Glu Arg
230 235 240
Leu Ala Leu Thr Ala Arg Val His Gly Gly Ala Leu Gly Glu His
245 250 255
Asp Lys Met Val Ala Ala Ala Thr Gly Ser Glu Ile Leu Leu Trp
260 265 270
Ala Leu Gln Ala Glu Gly Gly Gly Ser Glu Ile Gly Val Phe His
275 280 285
Leu Gly Val Pro Val Glu Ala Leu Phe Phe Val Gly Asn Gln Leu
290 295 300
Ile Ala Thr Ser His Thr Gly Arg Ile Gly Val Trp Asn Ala Val
305 310 315
Thr Lys His Trp Gln Val Gln Glu Val Gln Pro Ile Thr Ser Tyr
320 325 330
Asp Ala Ala Gly Ser Phe Leu Leu Leu Gly Cys Asn Asn Gly Ser
335 340 345
Ile Tyr Tyr Val Asp Val Gln Lys Phe Pro Leu Arg Met Lys Asp
350 355 360
Asn Asp Leu Leu Val Ser Glu Leu Tyr Arg Asp Pro Ala Glu Asp
365 370 375
Gly Val Thr Ala Leu Ser Val Tyr Leu Thr Pro Lys Thr Ser Asp
380 385 390
Ser Gly Asn Trp Ile Glu Ile Ala Tyr Gly Thr Ser Ser Gly Gly
395 400 405
Val Arg Val Ile Val Gln His Pro Glu Thr Val Gly Ser Gly Pro
410 415 420
Gln Leu Phe Gln Thr Phe Thr Val His Arg Ser Pro Val Thr Lys
425 430 435
Ile Met Leu Ser Glu Lys His Leu Ile Ser Val Cys Ala Asp Asn
440 445 450
Asn His Val Arg Thr Trp Ser Val Thr Arg Phe Arg Gly Met Ile
455 460 465
Ser Thr Gln Pro Gly Ser Thr Pro Leu Ala Ser Phe Lys Ile Leu
26/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
470 475 480
Ala Leu Glu Ser Ala Asp Gly His Gly Gly Cys Ser Ala Gly Asn
485 490 495
Asp Ile Gly Pro Tyr Gly Glu Arg Asp Asp Gln Gln Val Phe Ile
500 505 510
Gln Lys Val Val Pro Ser Ala Ser Gln Leu Phe Val Arg Leu Ser
515 520 525
Ser Thr Gly Gln Arg Val Cys Ser Val Arg Ser Val Asp Gly Ser
530 535 540
Pro Thr Thr Ala Phe Thr Val Leu Glu Cys Glu Gly Ser Arg Arg
545 550 555
Leu Gly Ser Arg Pro Arg Arg Tyr Leu Leu Thr Gly Gln Ala Asn
560 565 570
Gly Ser Leu Ala Met Trp Asp Leu Thr Thr Ala Met Asp Gly Leu
575 580 585
Gly Gln Ala Pro Ala Gly Gly Leu Thr Glu Gln Glu Leu Met Glu
590 595 600
Gln Leu Glu His Cys Glu Leu Ala Pro Pro Ala Pro Ser Ala Pro
605 610 615
Ser Trp Gly Cys Leu Pro Ser Pro Ser Pro Arg Ile Ser Leu Thr
620 625 630
Ser Leu His Ser Ala Ser Ser Asn Thr Ser Leu Ser Gly His Arg
635 640 645
Gly Ser Pro Ser Pro Pro Gln Ala Glu Ala Arg Arg Arg Gly Gly
650 655 660
Gly Ser Phe Val Glu Arg Cys Gln Glu Leu Val Arg Ser Gly Pro
665 670 675
Asp Leu Arg Arg Pro Pro Thr Pro Ala Pro Trp Pro Ser Ser Gly
680 685 690
Leu Gly Thr Pro Leu Thr Pro Pro Lys Met Lys Leu Asn Glu Thr
695 700 705
Ser Phe
<210> 31
<211> 279
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2172064CD1
<400> 31
Met Cys Gly Arg Phe Leu Arg Arg Leu Leu Ala Glu Glu Ser Arg
1 5 10 15
Arg Ser Thr Pro Val Gly Arg Leu Leu Leu Pro Val Leu Leu Gly
20 25 30
Phe Arg Leu Val Leu Leu Ala Ala Ser Gly Pro Gly Val Tyr Gly
35 40 45
Asp Glu Gln Ser Glu Phe Val Cys His Thr Gln Gln Pro Gly Cys
50 55 60
Lys Ala Ala Cys Phe Asp Ala Phe His Pro Leu Ser Pro Leu Arg
65 70 75
Ser Trp Val Phe Gln Val Ile Leu Val Ala Val Pro Ser Ala Leu
80 85 90
Tyr Met Gly Phe Thr Leu Tyr His Val Ile Trp His Trp Glu Leu
95 100 105
Ser Gly Lys Gly Lys Glu Glu Glu Thr Leu Ile Gln Gly Arg Glu
110 115 120
Gly Asn Thr Asp Val Pro Gly Ala Gly Ser Leu Arg Leu Leu Trp
125 130 135
Ala Tyr Val Ala Gln Leu Gly Ala Arg Leu Val Leu Glu Gly Ala
140 145 150
Ala Leu Gly Leu Gln Tyr His Leu Tyr Gly Phe Gln Met Pro Ser
155 160 165
Ser Phe Ala Cys Arg Arg Glu Pro Cys Leu Gly Ser Ile Thr Cys
170 175 180
Asn Leu Ser Arg Pro Ser Glu Lys Thr Ile Phe Leu Lys Thr Met
27/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
185 190 195
Phe Gly Val Ser Gly Phe Cys Leu Leu Phe Thr Phe Leu Glu Leu
200 205 210
Val Leu Leu Gly Leu Gly Arg Trp Trp Arg Thr Trp Lys His Lys
215 220 225
Ser Ser Ser Ser Lys Tyr Phe Leu Thr Ser Glu Ser Thr Arg Arg
230 235 240
His Lys Lys Ala Thr Asp Ser Leu Pro Val Val Glu Thr Lys Glu
245 250 255
Gln Phe Gln Glu Ala Val Pro Gly Arg Ser Leu Ala Gln Glu Lys
260 265 270
Gln Arg Pro Val Gly Pro Arg Asp Ala
275
<210> 32
<211> 154
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2219267CD1
<400> 32
Met Val Thr Gly Leu Ala Ser Leu Leu Leu Leu Ala Gly Ala Gln
1 5 10 15
Tyr Leu Pro Gly Trp Thr Val Leu Phe Leu Ser Val Leu Gly Leu
20 25 30
Leu Ala Ser Arg Ala Val Ser Ala Leu Ser Ser Leu Phe Ala Ala
35 40 45
Glu Val Phe Pro Thr Val Ile Arg Gly Ala Gly Leu Gly Leu Val
50 55 60
Leu Gly Ala Gly Phe Leu Gly Gln Ala Ala Gly Pro Leu Asp Thr
65 70 75
Leu His Gly Arg Gln Gly Phe Phe Leu Gln Gln Val Val Phe Ala
80 85 90
Ser Leu Ala Val Leu Ala Leu Leu Cys Val Leu Leu Leu Pro Glu
95 100 105
Ser Arg Ser Arg Gly Leu Pro Gln Ser Leu Gln Asp Ala Asp Arg
110 115 120
Leu Arg Arg Ser Pro Leu Leu Arg Gly Arg Pro Arg Gln Asp His
125 130 135
Leu Pro Leu Leu Pro Pro Ser Asn Ser Tyr Trp Ala Gly His Thr
140 145 150
Pro Glu Gln His
<210> 33
<211> 289
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2308629CD1
<400> 33
Met Val Ala Gly Ala Val Ala Gly Ile Leu Glu His Cys Val Met
1 5 10 15
Tyr Pro Ile Asp Cys Val Lys Thr Arg Met Gln Ser Leu Gln Pro
20 25 30
Asp Pro Ala Ala Arg Tyr Arg Asn Val Leu Glu Ala Leu Trp Arg
35 40 45
Ile Ile Arg Thr Glu Gly Leu Trp Arg Pro Met Arg Gly Leu Asn
50 55 60
Val Thr Ala Thr Gly Ala Gly Pro Ala His Ala Leu Tyr Phe Ala
65 70 75
Cys Tyr Glu Lys Leu Lys Lys Thr Leu Ser Asp Val Ile His Pro
80 85 90
Gly Gly Asn Ser His Ile Ala Asn Gly Ala Ala Gly Cys Val Ala
28/60
CA 02375493 2001-12-11
WO 00/78953 PCT/LTS00/16668
95 100 105
Thr Leu Leu His Asp Ala Ala Met Asn Pro Ala Glu Val Val Lys
110 115 120
Gln Arg Met Gln Met Tyr Asn Ser Pro Tyr His Arg Val Thr Asp
125 130 135
Cys Val Arg Ala Val Trp Gln Asn Glu Gly Ala Gly Ala Phe Tyr
140 145 150
Arg Ser Tyr Thr Thr Gln Leu Thr Met Asn Val Pro Phe Gln Ala
155 160 165
Ile His Phe Met Thr Tyr Glu Phe Leu Gln Glu His Phe Asn Pro
170 175 180
Gln Arg Arg Tyr Asn Pro Ser Ser His Val Leu Ser Gly Ala Cys
185 190 195
Ala Gly Ala Val Ala Ala Ala Ala Thr Thr Pro Leu Asp Val Cys
200 205 210
Lys Thr Leu Leu Asn Thr Gln Glu Ser Leu Ala Leu Asn Ser His
215 220 225
Ile Thr Gly His Ile Thr Gly Met Ala Ser Ala Phe Arg Thr Val
230 235 240
Tyr Gln Val Gly Gly Val Thr Ala Tyr Phe Arg Gly Val Gln Ala
245 250 255
Arg Val Ile Tyr Gln Ile Pro Ser Thr Ala Ile Ala Trp Ser Val
260 265 270
Tyr Glu Phe Phe Lys Tyr Leu Ile Thr Lys Arg Gln Glu Glu Trp
275 280 285
Arg Ala Gly Lys
<210> 34
<211> 300
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2660038CD1
<400> 34
Met Asp Phe Leu Met Ser Gly Leu Ala Ala Cys Gly Ala Cys Val
1 5 10 15
Phe Thr Asn Pro Leu Glu Val Val Lys Thr Arg Met Gln Leu Gln
20 25 30
Gly Glu Leu Gln Ala Pro Gly Thr Tyr Gln Arg His Tyr Arg Asn
35 40 45
Val Phe His Ala Phe Ile Thr Ile Gly Lys Val Asp Gly Leu Ala
50 55 60
Ala Leu Gln Lys Gly Leu Ala Pro Ala Leu Leu Tyr Gln Phe Leu
65 70 75
Met Asn Gly Ile Arg Leu Gly Thr Tyr Gly Leu Ala Glu Ala Gly
80 85 90
Gly Tyr Leu His Thr Ala Glu Ala Thr His Ser Pro Ala Arg Ser
95 100 105
Ala Ala Ala Gly Ala Met Ala Gly Val Met Gly Ala Tyr Leu Gly
110 115 120
Ser Pro Ile Tyr Met Val Lys Thr His Leu Gln Ala Gln Ala Ala
125 130 135
Ser Glu Ile Ala Val Gly His Gln Tyr Lys His Gln Gly Met Phe
140 145 150
Gln Ala Leu Thr Glu Ile Gly Gln Lys His Gly Leu Val Gly Leu
155 160 165
Trp Arg Gly Ala Leu Gly Gly Leu Pro Arg Val Ile Val Gly Ser
170 175 180
Ser Thr Gln Leu Cys Thr Phe Ser Ser Thr Lys Asp Leu Leu Ser
185 190 195
Gln Trp Glu Ile Phe Pro Pro Gln Ser Trp Lys Leu Ala Leu Val
200 205 210
Ala Ala Met Met Ser Gly Ile Ala Val Val Leu Ala Met Ala Pro
215 220 225
Phe Asp Val Ala Cys Thr Arg Leu Tyr Asn Gln Pro Thr Asp Ala
29/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
230 235 240
Gln Gly Lys Gly Leu Met Tyr Arg Gly Ile Leu Asp Ala Leu Leu
245 250 255
Gln Thr Ala Arg Thr Glu Gly Ile Phe Gly Met Tyr Lys Gly Ile
260 265 270
Gly Ala Ser Tyr Phe Arg Leu Gly Pro His Thr Ile Leu Ser Leu
275 280 285
Phe Phe Trp Asp Gln Leu Arg Ser Leu Tyr Tyr Thr Asp Thr Lys
290 295 300
<210> 35
<211> 382
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2670745CD1
<400> 35
Met Leu Arg Trp Thr Val His Leu Glu Gly Gly Pro Arg Arg Val
1 5 10 15
Asn His Ala Ala Val Ala Val Gly His Arg Val Tyr Ser Phe Gly
20 25 30
Gly Tyr Cys Ser Gly Glu Asp Tyr Glu Thr Leu Arg Gln Ile Asp
35 40 45
Val His Ile Phe Asn Ala Val Ser Leu Arg Trp Thr Lys Leu Pro
50 55 60
Pro Val Lys Ser Ala Ile Arg Gly Gln Ala Pro Val Val Pro Tyr
65 70 75
Met Arg Tyr Gly His Ser Thr Val Leu Ile Asp Asp Thr Val Leu
80 85 90
Leu Trp Gly Gly Arg Asn Asp Thr Glu Gly Ala Cys Asn Val Leu
95 100 105
Tyr Ala Phe Asp Val Asn Thr His Lys Trp Phe Thr Pro Arg Val
110 115 120
Ser Gly Thr Val Pro Gly Ala Arg Asp Gly His Ser Ala Cys Val
125 130 135
Leu Gly Lys Ile Met Tyr Ile Phe Gly Gly Tyr Glu Gln Gln Ala
140 145 150
Asp Cys Phe Ser Asn Asp Ile His Lys Leu Asp Thr Ser Thr Met
155 160 165
Thr Trp Thr Leu Ile Cys Thr Lys Gly Ser Pro Ala Arg Trp Arg
170 175 180
Asp Phe His Ser Ala Thr Met Leu Gly Ser His Met Tyr Val Phe
185 190 195
Gly Gly Arg Ala Asp Arg Phe Gly Pro Phe His Ser Asn Asn Glu
200 205 210
Ile Tyr Cys Asn Arg Ile Arg Val Phe Asp Thr Arg Thr Glu Ala
215 220 225
Trp Leu Asp Cys Pro Pro Thr Pro Val Leu Pro Glu Gly Arg Arg
230 235 240
Ser His Ser Ala Phe Gly Tyr Asn Gly Glu Leu Tyr Ile Phe Gly
245 250 255
Gly Tyr Asn Ala Arg Leu Asn Arg His Phe His Asp Leu Trp Lys
260 265 270
Phe Asn Pro Val Ser Phe Thr Trp Lys Lys Ile Glu Pro Lys Gly
275 280 285
Lys Gly Pro Cys Pro Arg Arg Arg Gln Cys Cys Cys Ile Val Gly
290 295 300
Asp Lys Ile Val Leu Phe Gly Gly Thr Ser Pro Ser Pro Glu Glu
305 310 315
Gly Leu Gly Asp Glu Phe Asp Leu Ile Asp His Ser Asp Leu His
320 325 330
Ile Leu Asp Phe Ser Pro Ser Leu Lys Thr Leu Cys Lys Leu Ala
335 340 345
Val Ile Gln Tyr Asn Leu Asp Gln Ser Cys Leu Pro His Asp Ile
350 355 360
30/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Arg Trp Glu Leu Asn Ala Met Thr Thr Asn Ser Asn Ile Ser Arg
365 370 375
Pro Ile Val Ser Ser His Gly
380
<210> 36
<211> 287
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2676443CD1
<400> 36
Met Ala Ala Glu Ala Arg Val Ser Arg Trp Tyr Phe Gly Gly Leu
1 5 10 15
Ala Ser Cys Gly Ala Ala Cys Cys Thr His Pro Leu Asp Leu Leu
20 25 30
Lys Val His Leu Gln Thr Gln Gln Glu Val Lys Leu Arg Met Thr
35 40 45
Gly Met Ala Leu Arg Val Val Arg Thr Asp Gly Ile Leu Ala Leu
50 55 60
Tyr Ser Gly Leu Ser Ala Ser Leu Cys Arg Gln Met Thr Tyr Ser
65 70 75
Leu Thr Arg Phe Ala Ile Tyr Glu Thr Val Arg Asp Arg Val Ala
80 85 90
Lys Gly Ser Gln Gly Pro Leu Pro Phe His Glu Lys Val Leu Leu
95 100 105
Gly Ser Val Ser Gly Leu Ala Gly Gly Phe Val Gly Thr Pro Ala
110 115 120
Asp Leu Val Asn Val Arg Met Gln Asn Asp Val Lys Leu Pro Gln
125 130 135
Gly Gln Arg Arg Asn Tyr Ala His Ala Leu Asp Gly Leu Tyr Arg
140 145 150
Val Ala Arg Glu Glu Gly Leu Arg Arg Leu Phe Ser Gly Ala Thr
155 160 165
Met Ala Ser Ser Arg Gly Ala Leu Val Thr Val Gly Gln Leu Ser
170 175 180
Cys Tyr Asp Gln Ala Lys Gln Leu Val Leu Ser Thr Gly Tyr Leu
185 190 195
Ser Asp Asn Ile Phe Thr His Phe Val Ala Ser Phe Ile Ala Gly
200 205 210
Gly Cys Ala Thr Phe Leu Cys Gln Pro Leu Asp Val Leu Lys Thr
215 220 225
Arg Leu Met Asn Ser Lys Gly Glu Tyr Gln Gly Val Phe His Cys
230 235 240
Ala Val Glu Thr Ala Lys Leu Gly Pro Leu Ala Phe Tyr Lys Gly
245 250 255
Leu Val Pro Ala Gly Ile Arg Leu Ile Pro His Thr Val Leu Thr
260 265 270
Phe Val Phe Leu Glu Gln Leu Arg Lys Asn Phe Gly Ile Lys Val
275 280 285
Pro Ser
<210> 37
<211> 497
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3295764CD1
<400> 37
Met Asp Val Pro Gly Pro Val Ser Arg Arg Ala Ala Ala Ala Ala
1 5 10 15
Ala Thr Val Leu Leu Arg Thr Ala Arg Val Arg Arg Glu Cys Trp
20 25 30
31/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
Phe Leu Pro Thr Ala Leu Leu Cys Ala Tyr Gly Phe Phe Ala Ser
35 40 45
Leu Arg Pro Ser Glu Pro Phe Leu Thr Pro Tyr Leu Leu Gly Pro
50 55 60
Asp Lys Asn Leu Thr Glu Arg Glu Val Phe Asn Glu Ile Tyr Pro
65 70 75
Val Trp Thr Tyr Ser Tyr Leu Val Leu Leu Phe Pro Val Phe Leu
80 85 90
Ala Thr Asp Tyr Leu Arg Tyr Lys Pro Val Val Leu Leu Gln Gly
95 100 105
Leu Ser Leu Ile Val Thr Trp Phe Met Leu Leu Tyr Ala Gln Gly
110 115 120
Leu Leu Ala Ile Gln Phe Leu Glu Phe Phe Tyr Gly Ile Ala Thr
125 130 135
Ala Thr Glu Ile Ala Tyr Tyr Ser Tyr Ile Tyr Ser Val Val Asp
140 145 150
Leu Gly Met Tyr Gln Lys Val Thr Ser Tyr Cys Arg Ser Ala Thr
155 160 165
Leu Val Gly Phe Thr Val Gly Ser Val Leu Gly Gln Ile Leu Val
170 175 180
Ser Val Ala Gly Trp Ser Leu Phe Ser Leu Asn Val Ile Ser Leu
185 190 195
Thr Cys Val Ser Val Ala Phe Ala Val Ala Trp Phe Leu Pro Met
200 205 210
Pro Gln Lys Ser Leu Phe Phe His His Ile Pro Ser Thr Cys Gln
215 220 225
Arg Val Asn Gly Ile Lys Val Gln Asn Gly Gly Ile Val Thr Asp
230 235 240
Thr Pro Ala Ser Asn His Leu Pro Gly Trp Glu Asp Ile Glu Ser
245 250 255
Lys Ile Pro Leu Asn Met Glu Glu Pro Pro Val Glu Glu Pro Glu
260 265 270
Pro Lys Pro Asp Arg Leu Leu Val Leu Lys Val Leu Trp Asn Asp
275 280 285
Phe Leu Met Cys Tyr Ser Ser Arg Pro Leu Leu Cys Trp Ser Val
290 295 300
Trp Trp Ala Leu Ser Thr Cys Gly Tyr Phe Gln Val Val Asn Tyr
305 310 315
Thr Gln Gly Leu Trp Glu Lys Val Met Pro Ser Arg Tyr Ala Ala
320 325 330
Ile Tyr Asn Gly Gly Val Glu Ala Val Ser Thr Leu Leu Gly Ala
335 340 345
Val Ala Val Phe Ala Val Gly Tyr Ile Lys Ile Ser Trp Ser Thr
350 355 360
Trp Gly Glu Met Thr Leu Ser Leu Phe Ser Leu Leu Ile Ala Ala
365 370 375
Ala Val Tyr Ile Met Asp Thr Val Gly Asn Ile Trp Val Cys Tyr
380 385 390
Ala Ser Tyr Val Val Phe Arg Ile Ile Tyr Met Leu Leu Ile Thr
395 400 405
Ile Ala Thr Phe Gln Ile Ala Ala Asn Leu Ser Met Glu Arg Tyr
410 415 420
Ala Leu Val Phe Gly Val Asn Thr Phe Ile Ala Leu Ala Leu Gln
425 430 435
Thr Leu Leu Thr Leu Ile Val Val Asp Ala Ser Gly Leu Gly Leu
440 445 450
Glu Ile Thr Thr Gln Phe Leu Ile Tyr Ala Ser Tyr Phe Ala Leu
455 460 465
Ile Ala Val Val Phe Leu Ala Ser Gly Ala Val Ser Val Met Lys
470 475 480
Lys Cys Arg Lys Leu Glu Asp Pro Gln Ser Ser Ser Gln Val Thr
485 490 495
Thr Ser
<210> 38
<211> 228
<212> PRT
<213> Homo sapiens
32/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
<220>
<221> misc_feature
<223> Incyte ID No: 3438320CD1
<400> 38
Met Pro Arg Arg Gly Leu Val Ala Gly Pro Asp Leu Glu Tyr Phe
1 5 10 15
Gln Arg Arg Tyr Phe Thr Pro Ala Glu Val Ala Gln His Asn Arg
20 25 30
Pro Glu Asp Leu Trp Val Ser Tyr Leu Gly Arg Val Tyr Asp Leu
35 40 45
Thr Ser Leu Ala Gln Glu Tyr Lys Gly Asn Leu Leu Leu Lys Pro
50 55 60
Ile Val Glu Val Ala Gly Gln Asp Ile Ser His Trp Phe Asp Pro
65 70 75
Lys Thr Arg Asp Ile Arg Lys His Ile Asp Pro Leu Thr Gly Cys
80 85 90
Leu Arg Tyr Cys Thr Pro Arg Gly Arg Phe Val His Val Pro Pro
95 100 105
Gln Leu Pro Cys Ser Asp Trp Ala Asn Asp Phe Gly Lys Pro Trp
110 115 120
Trp Gln Gly Ser Tyr Tyr Glu Val Gly Arg Leu Ser Ala Lys Thr
125 130 135
Arg Ser Ile Arg Ile Ile Asn Thr Leu Thr Ser Gln Glu His Thr
140 145 150
Leu Glu Val Gly Val Leu Glu Ser Ile Trp Glu Ile Leu His Arg
155 160 165
Tyr Leu Pro Tyr Asn Ser His Ala Ala Ser Tyr Thr Trp Lys Tyr
170 175 180
Glu Gly Lys Asn Leu Asn Met Asp Phe Thr Leu Glu Glu Asn Gly
185 190 195
Ile Arg Asp Glu Glu Glu Glu Phe Asp Tyr Leu Ser Met Asp Gly
200 205 210
Thr Leu His Thr Pro Ala Ile Leu Leu Tyr Phe Asn Asp Asp Leu
215 220 225
Thr Glu Leu
<210> 39
<211> 273
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3986488CD1
<400> 39
Met Ala Ala Thr Ile Met Ile Leu Tyr Val Ser Lys Leu Asn Lys
1 5 10 15
Ile Ile His Phe Pro Asp Phe Asp Lys Lys Ile Pro Val Lys Leu
20 25 30
Phe Pro Leu Pro Leu Leu Tyr Val Gly Asn His Ile Ser Gly Leu
35 40 45
Ser Ser Thr Ser Lys Leu Ser Leu Pro Met Phe Thr Val Leu Arg
50 55 60
Lys Phe Thr Ile Pro Leu Thr Leu Leu Leu Glu Thr Ile Ile Leu
65 70 75
Gly Lys Gln Tyr Ser Leu Asn Ile Ile Leu Ser Val Phe Ala Ile
80 85 90
Ile Leu Gly Ala Phe Ile Ala Ala Gly Ser Asp Leu Ala Phe Asn
95 100 105
Leu Glu Gly Tyr Ile Phe Val Phe Leu Asn Asp Ile Phe Thr Ala
110 115 120
Ala Asn Gly Val Tyr Thr Lys Gln Lys Met Asp Pro Lys Glu Leu
125 130 135
Gly Lys Tyr Gly Val Leu Phe Tyr Asn Ala Cys Phe Met Ile Ile
140 145 150
Pro Thr Leu Ile Ile Ser Val Ser Thr Gly Asp Leu Gln Gln Ala
33/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
155 160 165
Thr Glu Phe Asn Gln Trp Lys Asn Val Val Phe Ile Leu Gln Phe
170 175 180
Leu Leu Ser Cys Phe Leu Gly Phe Leu Leu Met Tyr Ser Thr Val
185 190 195
Leu Cys Ser Tyr Tyr Asn Ser Ala Leu Thr Thr Ala Val Val Gly
200 205 210
Ala Ile Lys Asn Val Ser Val Ala Tyr Ile Gly Ile Leu Ile Gly
215 220 225
Gly Asp Tyr Ile Phe Ser Leu Leu Asn Phe Val Gly Leu Asn Ile
230 235 240
Cys Met Ala Gly Gly Leu Arg Tyr Ser Phe Leu Thr Leu Ser Ser
245 250 255
Gln Leu Lys Pro Lys Pro Val Gly Glu Glu Asn Ile Cys Leu Asp
260 265 270
Leu Lys Ser
<210> 40
<211> 206
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4378816CD1
<400> 40
Met Gly Ala Glu Trp Glu Leu Gly Ala Glu Ala Gly Gly Ser Leu
1 5 10 15
Leu Leu Cys Ala Ala Leu Leu Ala Ala Gly Cys Ala Leu Gly Leu
20 25 30
Arg Leu Gly Arg Gly Gln Gly Ala Ala Asp Arg Gly Ala Leu Ile
35 40 45
Trp Leu Cys Tyr Asp Ala Leu Val His Phe Ala Leu Glu Gly Pro
50 55 60
Phe Val Tyr Leu Ser Leu Val Gly Asn Val Ala Asn Ser Asp Gly
65 70 75
Leu Ile Ala Ser Leu Trp Lys Glu Tyr Gly Lys Ala Asp Ala Arg
80 85 90
Trp Val Tyr Phe Asp Pro Thr Ile Val Ser Val Glu Ile Leu Thr
95 100 105
Val Ala Leu Asp Gly Ser Leu Ala Leu Phe Leu Ile Tyr Ala Ile
110 115 120
Val Lys Glu Lys Tyr Tyr Arg His Phe Leu Gln Ile Thr Leu Cys
125 130 135
Val Cys Glu Leu Tyr Gly Cys Trp Met Thr Phe Leu Pro Glu Trp
140 145 150
Leu Thr Arg Ser Pro Asn Leu Asn Thr Ser Asn Trp Leu Tyr Cys
155 160 165
Trp Leu Tyr Leu Phe Phe Phe Asn Gly Val Trp Val Leu Ile Pro
170 175 180
Gly Leu Leu Leu Trp Gln Ser Trp Leu Glu Leu Lys Lys Met His
185 190 195
Gln Lys Glu Thr Ser Ser Val Lys Lys Phe Gln
200 205
<210> 41
<211> 235
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4797137CD1
<400> 41
Met Gln Gln Arg Gly Ala Ala Gly Ser Arg Gly Cys Ala Leu Phe
1 5 10 15
Pro Leu Leu Gly Val Leu Phe Phe Gln Gly Val Tyr Ile Val Phe
34/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
20 25 30
Ser Leu Glu Ile Arg Ala Asp Ala His Val Arg Gly Tyr Val Gly
35 40 45
Glu Lys Ile Lys Leu Lys Cys Thr Phe Lys Ser Thr Ser Asp Val
50 55 60
Thr Asp Lys Leu Thr Ile Asp Trp Thr Tyr Arg Pro Pro Ser Ser
65 70 75
Ser His Thr Val Ser Ile Phe His Tyr Gln Ser Phe Gln Tyr Pro
80 85 90
Thr Thr Ala Gly Thr Phe Arg Asp Arg Ile Ser Trp Val Gly Asn
95 100 105
Val Tyr Lys Gly Asp Ala Ser Ile Ser Ile Ser Asn Pro Thr Ile
110 115 120
Lys Asp Asn Gly Thr Phe Ser Cys Ala Val Lys Asn Pro Pro Asp
125 130 135
Val His His Asn Ile Pro Met Thr Glu Leu Thr Val Thr Glu Arg
140 145 150
Gly Phe Gly Thr Met Leu Ser Ser Val Ala Leu Leu Ser Ile Leu
155 160 165
Val Phe Val Pro Ser Ala Val Val Val Ala Leu Leu Leu Val Arg
170 175 180
Met Gly Arg Lys Ala Ala Gly Leu Lys Lys Arg Ser Arg Ser Gly
185 190 195
Tyr Lys Lys Ser Ser Ile Glu Val Ser Asp Asp Thr Asp Gln Glu
200 205 210
Glu Glu Glu Ala Cys Met Ala Arg Leu Cys Val Arg Cys Ala Glu
215 220 225
Cys Leu Asp Ser Asp Tyr Glu Glu Thr Tyr
230 235
<210> 42
<211> 147
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5470806CD1
<400> 42
Met Ala Ser Leu Arg Leu Phe Leu Leu Cys Leu Ala Val Leu Ile
1 5 10 15
Phe Ala Ser Glu Ala Gly Pro Gly Gly Ala Gly Glu Ser Lys Cys
20 25 30
Pro Leu Met Val Lys Val Leu Asp Ala Val Arg Gly Ser Pro Ala
35 40 45
Val Asp Val Ala Val Lys Val Phe Lys Lys Thr Ala Asp Gly Ser
50 55 60
Trp Glu Pro Phe Ala Ser Gly Lys Thr Ala Glu Ser Gly Glu Leu
65 70 75
His Gly Leu Thr Thr Asp Glu Lys Phe Thr Glu Gly Val Tyr Arg
80 85 90
Val Glu Leu Asp Thr Lys Ser Tyr Trp Lys Ala Leu Gly Ile Ser
95 100 105
Pro Phe His Glu Tyr Ala Glu Val Val Phe Thr Ala Asn Asp Ser
110 115 120
Gly His Arg His Tyr Thr Ile Ala Ala Leu Leu Ser Pro Tyr Ser
125 130 135
Tyr Ser Thr Thr Ala Val Val Ser Asn Pro Gln Asn
140 145
<210> 43
<211> 147
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5473242CD1
35/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
<400> 43
Met Val His Leu Thr Asp Ala Glu Lys Ala Thr Val Asn Gly Leu
1 5 10 15
Trp Gly Lys Val Asn Pro Val Glu Ile Gly Ala Glu Ser Leu Ala
20 25 30
Ser Leu Leu Ile Val Tyr Pro Trp Thr Gln Arg Tyr Phe Ser Lys
35 40 45
Phe Gly Asp Leu Ser Ser Val Ser Ala Ile Met Gly Asn Pro Gln
50 55 60
Val Lys Ala His Gly Glu Lys Val Ile Asn Ala Phe Asp Asp Gly
65 70 75
Leu Lys His Leu Asp Asn Leu Lys Gly Thr Phe Ala Ser Leu Ser
80 85 90
Glu Leu His Cys Asp Lys Leu His Val Asp Pro Glu Asn Phe Arg
95 100 105
Leu Leu Gly Asn Met Ile Val Ile Met Met Gly His His Leu Gly
110 115 120
Lys Glu Phe Thr Pro Ser Ala Gln Ala Ala Phe Gln Lys Val Val
125 130 135
Ala Gly Val Ala Ser Ala Leu Ala His Lys Tyr His
140 145
<210> 44
<211> 2701
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 264114CB1
<400> 44
gcggcggcgc cagcttcctc ggccggaggg gaggcgagac cccaggcgag gccgcggcgg 60
gagggccacg cccccgacgc cgcgccggag gggcccagtg tggacggggc caccggctgg 120
agcggatccc acacctccgg accgagggac gcggttactc cacaggatcc gctgaacata 180
ggatgttgcc acaaaatcta cctcgtgtat ttttctcttt cactcatgag ctgcacaatt 240
gcagatttga gcacaatgtc tgcagactgt gttgaaaaac tctgaagaac ctaattaaca 300
caggatgacc taggagtgat tctaagtctg tgtaacaaga tattactcat tagtgaatgt 360
gtcagtcttg gtactgaatg ctgcagataa cagcaagtag gttctccttt atttctgaag 420
tattcacttg accttccatc agtaagacgg acttttctaa tctgttcctg gagatattaa 480
tggaatacag tcatgtccac tcaagacgag aggcagatca atactgaata tgctgtgtca 540
ttgttggaac agttgaaact gttttatgaa cagcagttgt ttactgacat agtgttaatt 600
gttgagggca ctgaattccc ttgtcataag atggttcttg caacatgtag ctcttatttc 660
agggccatgt ttatgagtgg actaagtgaa agcaaacaaa cccatgtaca cctgaggaat 720
gtcgatgctg ccaccttaca gataataata acttatgcat acacgggtaa cttggcaatg 780
aatgacagca ctgtagaaca gctttatgaa acagcttgct tcctacaggt agaagatgtg 840
ttacaacgtt gtcgagaata tttaattaaa aaaataaatg cagagaattg tgtacgattg 900
ttgagttttg ctgatctctt cagttgtgag gaattaaaac agagtgctaa aagaatggtg 960
gagcacaagt tcactgctgt gtatcatcag gacgcgttca tgcagctgtc acatgaccta 1020
ctgatagata ttctcagtag tgacaattta aatgtagaaa aggaagaaac cgttcgagaa 1080
gctgctatgc tgtggctaga gtataacaca gaatcacgat cccagtattt gtcttctgtt 1140
cttagccaaa tcagaattga tgcactttca gaagtaacac agagagcttg gtttcaaggt 1200
ctgccaccca atgataagtc agtggtggtt caaggtctgt ataagtccat gcccaagttt 1260
ttcaaaccaa gacttgggat gactaaagag gaaatgatga ttttcattga agcatcttca 1320
gaaaatcctt gtagtcttta ctcttctgtc tgttacagcc cccaagcaga aaaagtttac 1380
aagttatgta gcccaccagc tgatttgcat aaggttggga ccgttgtaac tcctgataat 1440
gatatctaca tagcaggggg tcaagttcct ctgaaaaaca caaaaacaaa tcacagtaaa 1500
acaagcaaac ttcagactgc cttcagaact gtgaattgct tttattggtt tgatgcacag 1560
caaaatacct ggtttccaaa gaccccaatg ctttttgtcc gcataaagcc atctttggtt 1620
tgctgtgaag gctatatcta tgcaattgga ggagatagcg taggtggaga acttaatcgg 1680
aggaccgtag aaagatacga cactgagaaa gatgagtgga cgatggtaag ccctttacct 1740
tgtgcttggc aatggagtgc agcagttgtg gttcatgact gcatttatgt gatgacactg 1800
aacctcatgt actgttattt tccaaggtct gactcatggg tagaaatggc catgagacag 1860
actagtaggt cctttgcttc agctgcagct tttggtgata aaattttcta tattggaggg 1920
ttgcatattg ctaccaattc cggcataaga ctcccctctg gcactgtaga tgggtcttca 1980
gtaactgtgg aaatttatga tgtgaataaa aatgagtgga aaatggcagc caacatccct 2040
gctaagaggt actctgaccc ctgtgttaga gctgttgtga tctcaaattc tctatgtgtg 2100
tttatgcgag aaacccactt aaatgagcga gctaaatacg tcacctacca atatgacctg 2160
gaacttgacc ggtggtctct gcggcagcat atatctgaac gtgtactgtg ggacttgggg 2220
36/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
agagattttc gatgcactgt ggggaaactc tatccatcct gccttgaaga gtctccatgg 2280
aaaccaccaa cttatctttt ttcaacggat gggacagaag agtttgaact ggatggagaa 2340
atggttgcac taccacctgt atagtgggga agttcaggga gtgcacgcct gagttatgtg 2400
ctttgtcatt ttctttgcta aacaaaagag gctatgaaag aactaaatat gagtacataa 2460
aattctatct ttgataaatt ttatttttat gccctactta atatttgcat cagtataata 2520
tatatcagtg agtcttacag aaagatatgc ttccataata tgaaatagat tattcaataa 2580
ttgagaaact ttatgtgtaa tcatgagagt ataagaatct ggattatcta acattgttag 2640
ccctgtgtat gtacagttca aaaagttcat ttataaaagt agtttcctgt tcctagttga 2700
t 2701
<210> 45
<211> 736
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1455669CB1
<400> 45
gagacttagc gacagacaga cgctgggacc cacgacgaca gaaggcgccg atggccgcgc 60
ctgctgagcc ctgcgcgggg cagggggtct ggaaccagac agagcctgaa cctgccgcca 120
ccagcctgct gagcctgtgc ttcctgagaa cagcaggggt ctgggtaccc cccatgtacc 180
tctgggtcct tggtcccatc tacctcctct tcatccacca ccatggccgg ggctacctcc 240
ggatgtcccc actcttcaaa gccaagatgg tagctgccat ccctgggagc ctggaaccag 300
gcaatgttcg ggggaggcag gggacaggct ggaacctggt gaagtcttaa agtagactcc 360
tcctatcggg gtgtagaagg gaatctgtta atcaaacaga gcaatattag aaaggctaca 420
gaggtcaact cagtggaaca cggttctccc aaacagattt tgtaattccg aaaatccacg 480
catgcgcaaa catacgcata cactcccatg ttcctggaca gtttatagct accataacct 540
ggcattttcc aaaacatacc atgtagactc ttggatacac aaggtaattt tagagccaca 600
ttaggatgaa ccttttaaaa agttatgcat ttatttttat gttcccccac tggctgtatt 660
ataggacaat ttttatatgt gatatgtatt taccttagtg tgttaaataa acactggcat 720
tccaagtgtg aaaaaa 736
<210> 46
<211> 1826
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2084989CB1
<400> 46
ccttaggcgc cagggacagc cgagcgttac ctggtcccgg gcagcggagt tctttaccca 60
ccccagttct ggttctgacg ccctagctca ttccgcaaat ttagggcttg ggtctggctt 120
gttcccctcc ggctcgaacc acctcttctc tgagccgagc cagctaccgg ggctcctgga 180
attgccaccc ctccctgggc acccttgagg cctccgtgga gggacgtcac ggggcagagc 240
gggacgtgag cctgagtttg ctgcaggcgt gctctgtgtg gtggctgggt tctgccaatc 300
cccgtgccca ccgggtgggc gcggccggga agctcctgcc cctccctgct ggtcggcgtc 360
acgcgtgacg tcccgcgtga tggctgggag ggcccggcgg cgacagcgga ggcagagagg 420
aaggcggttc tgagagcttc agagagcgat ggaaagcaaa atgggtgaat tgcctttaga 480
catcaacatc caggaacctc gctgggacca aagtactttc ctgggcagag cccggcactt 540
tttcactgtt actgatcctc gaaatctgct gctgtccggg gcacagctgg aagcttctcg 600
gaacatcgtg cagaactaca gggccggcgt ggtgacccca gggatcaccg aggaccagct 660
gtggagggcc aagtatgtgt atgactccgc cttccatccg gacacagggg agaaggtggt 720
cctgattggc cgcatgtcag cccaggtgcc catgaacatg accatcactg gctgcatgct 780
cacattctac aggaagaccc caaccgtggt gttctggcag tgggtgaatc agtccttcaa 840
tgccattgtt aactactcca accgcagtgg tgacactccc atcactgtga ggcagctggg 900
gacagcctat gtgagtgcca ccactggagc tgtggccacg gccctgggac tcaaatccct 960
caccaagcac ctgcccccct tggtcggcag atttgtgccc tttgcagcag tggcagctgc 1020
caactgcatc aacatccccc tgatgaggca gagagagctg caggtgggca tcccggtggc 1080
tgatgaggca ggtcagaggc ttggctactc ggtgactgca gccaagcagg gaatcttcca 1140
ggtggtgatt tcaagaatct gcatggcgat tcctgccatg gccatcccac cactgatcat 1200
ggacactctg gagaagaaag acttcctgaa ggtaggcgac tgtacctctc ttgtcctgga 1260
atgggcgatg gctgggagaa gtgaccaggc cccaactctc tctccagcct cgcctgattc 1320
tctaagactt gccagccctt ctcctgaccc ctgcaccgcc tcctccacct tcgttcattc 1380
agcaagaatg aactgggctg gggtgaagga actctgcagg ggcaggagga gaggacaaag 1440
37/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
gaaggaaacc aacttcatca gtgttactcc agtggcttct gacacacaga aggggactgt 1500
catagtcatg cttgatctca tgctcattct tttaccccct agtgcctcca tactgagagg 1560
tacacacggg tgaacacgca cacacagaca tgaacaggac acgaaagcaa agcacaggaa 1620
caagctctgg ctcattcaca gaatcattta ttcacaaatg tattgagtgc catgcaccag 1680
gcatgtttta gggctgagga gatggcactg aacacaatgg ttatggcccc tgtcctcatg 1740
aagtttatag tctgatgcag. aaaccaataa acaaggaggc acccacataa atacattctt 1800
agaaagtgta aaaataaaaa aaaaaa 1826
<210> 47
<211> 1325
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2501034CB1
<400> 47
ccacgggtcc ggttctggac tgcagttgag tggaaatggg caacggcggg cggagcggcc 60
tgcagcaggg gaaggggaac gtggatgggg tggcagcgac tcctactgct gcctcggcct 120
cctgccagta caggtgcatc gaatgcaacc aggaggccaa agagttgtac cgagactata 180
accacggtgt gctgaagata accatctgta aatcctgcca gaaacctgta gacaaatata 240
tcgagtatga tcctgttatc atcttgatta atgctatatt gtgcaaagct caggcctaca 300
gacatattct tttcaatact caaataaata tccatggaaa actctgcata ttttgtttgc 360
tttgtgaagc atacctgagg tggtggcagc ttcaagattc caaccagaat actgcccctg 420
atgacttgat cagatatgct aaggaatggg atttctatag aatgtttgcg attgctgctt 480
tagaacaaac tgcctatttt attggcattt ttaccttcct gtgggtagaa cggcccatga 540
cggcaaaaaa aaagcccaac ttcattttgc tgctgaaagc attattatta tctagctacg 600
gaaaactctt gctgattcca gctgtcattt gggaacatga ctacacatct gtgtgcctca 660
aactcattaa agtatttgtt cttacatcaa attttcaggc aattagagtg accctaaaca 720
tcaaccgtaa gctctccttc ttggccgtgt tgagtggctt actgctggaa agcatcatgg 780
tctacttctt ccagagtatg gaatgggatg ttggaagtga ttatgccatc tttaaatctc 840
aggacttctg aagagtttta ttcttcttca ctatctgtgg catgaccagc tgtatctgaa 900
agagaaaaga catgaaatat aaaccaacct cctcatttct gttgagtaaa atgaagcaaa 960
gattggaaac actttctgaa aaagaaagca atgataatag cggtggatac ccacccccac 1020
aaatgcaccc aagagacaag ccatttacat acagatattc acagtcacac atagaaacac 1080
ccacatggac acaaggaatg ttgctgcaga gactgaatga catgcaacag gtgaaggttt 1140
atacgttata cacaaggcca ggtaagcgct cataattcac acataataaa acatctaggt 1200
ttcattcctt tgacatgttt atatcttttt aatttaaatg ttgttactgg cttaaaatat 1260
tttgtgttct tacaatagaa acgcttttaa taaagtcttt cagaataaac caaaaaaaaa 1320
aaaaa 1325
<210> 48
<211> 1832
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2745212CB1
<400> 48
tgggctgtcg ttggctggag cagcggctgc gcgggtcgcg gtgctgtgag gtctgcgggc 60
gctggcaaat ccggcccagg atgtagagct ggcagtgcct gacggcgcgt ctgacgcgga 120
gttgggtggg gtagagagta gggggcggta gtcgggggtg gtgggagaag gaggaggcgg 180
cgaatcactt ataaatggcg ccgaagcagg acccgaagcc taaattccag gagggtgagc 240
gagtgctgtg ctttcatggg cctcttcttt atgaagcaaa gtgtgtaaag gttgccataa 300
aggacaaaca agtgaaatac ttcatacatt acagtggttg gaataaaaat tgggatgaat 360
gggttccgga gagcagagta ctcaaatacg tggacaccaa tttgcagaaa cagcgagaac 420
ttcaaaaagc caatcaggag cagtatgcag aggggaagat gagaggggct gccccaggaa 480
agaagacatc tggtctgcaa cagaaaaatg ttgaagtgaa aacgaaaaag aacaaacaga 540
aaacacctgg aaatggagat ggtggcagta ccagtgagac ccctcagcct cctcggaaga 600
aaagggcccg ggtagatcct actgttgaaa atgaggaaac attcatgaac agagttgaag 660
ttaaagtaaa gattcctgaa gagctaaaac cgtggcttgt tgatgactgg gacttaatta 720
ccaggcaaaa acagctcttt tatcttcctg ccaagaagaa tgtggattcc attcttgagg 780
attatgcaaa ttacaagaaa tctcgtggaa acacagataa taaggagtat gcggttaatg 840
aagttgtggc agggataaaa gaatacttca acgtaatgtt gggtacccag ctactctata 900
aatttgagag accacagtat gctgaaattc ttgcagatca tcccgatgca cccatgtccc 960
3 8/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
aggtgtatgg agcgccacat ctcctgagat tatttgtacg aattggagca atgttggctt 1020
atacacctct ggatgagaag agccttgctt tattactcaa ttatcttcac gatttcctaa 1080
agtacctggc aaagaattct gcaactttgt tcagtgccag cgattatgaa gtggctcctc 1140
ctgagtacca tcggaaagct gtgtgagagg cactctcact cacttatgtt tggatctccg 1200
taaacacatt tttgttctta gtctatctct tgtacaaacg atgtgctttg aagatgttag 1260
tgtataacaa ttgatgtttg ttttctgttt gattttaaac agagaaaaaa taaaaggggg 1320
taatagctcc ttttttcttc tttctttttt tttttcattt caaaattgct gccagtgttt 1380
tcaatgatgg acaacagagg gatatgctgt agagtgtttt attgcctagt tgacaaagct 1440
gcttttgaat gctggtggtt ctattccttt gacactacgc acttttataa tacatgttaa 1500
tgctatatga caaaatgctc tgattcctag tgccaaaggt tcaattcagt gtatataact 1560
gaacacactc atccatttgt gcttttgttt ttttttatgg tgcttaaagt aaagagccca 1620
tcctttgcaa gtcatccatg ttgttactta ggcattttat cttggctcaa attgttgaag 1680
aatggtggct tgtttcatgg tttttgtatt tgtgtctaat gcacgtttta acatgataga 1740
cgcaatgcat tgtgtagcta gttttctgga aaagtcaatc ttttaggaat tgtttttcag 1800
atcttcaata aattttttct ttaaatttca as 1832
<210> 49
<211> 1211
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4833111CB1
<400> 49
gacagacccg cctcaaacat ggcggcgccc agcgcgcgag gacgtgatcc gcttctgctc 60
cggcttggat tgtagccttg acgaggtctg agcgaccatg gaccggccgg ggttcgtggc 120
agcgctggtg gctggtgggg tagcaggtgt ttctgttgac ttgatattat ttcctctgga 180
taccattaaa accaggctgc agagtcccca aggatttagt aaggctggtg gttttcatgg 240
aatatatgct ggcgttcctt ctgctgctat tggatccttt cctaatgctg ctgcattttt 300
tatcacctat gaatatgtga agtggttttt gcatgctgat tcatcttcgt atttgacacc 360
tatgaaacat atgttggctg cctctgctgg agaagtggtt gcctgcctga ttcgagttcc 420
atctgaagtg gttaagcaga gggcacaggt atctgcttct acaagaacat ttcagatttt 480
ctctaacatc tr.atatgaag agggtatcca agggttgtat cgaggctata aaagcacagt 540
tttaagagag attccttttt ctttggtcca gtttccctta tgggagtcct taaaagccct 600
ctggtcctgg aggcaggatc atgtggtgga ttcttggcag tcagcagtct gtggagcttt 660
tgcaggtgga tttgccgctg cagtcaccac ccctctagac gtggcaaaga caagaattac 720
gctggcaaag gctggctcca gcactgctga tgggaatgtg ctctctgtcc tgcatggggt 780
ctggcggtca caggggctgg caggattatt tgcaggtgtc ttccctcgaa tggcagccat 840
cagtctggga ggtttcatct ttctgggggc ttatgaccga acgcacagct tgctgttgga 900
agttggcaga aagagtcctt gaagcagaga caagcctcac ctccacttct gtcaagagag 960
gggcctgcag tgcaaaccct cttccgctga gcagctgtct gaactatagg ccccagtgct 1020
gaagaccagt tgtgctaaga taccggcatg gagattgtgc catccgtggt ataggctggc 1080
tggtatgaag tcattggcct gtatgccaga gagctaagag aagaaaacgg ggtctgtggc 1140
ggtactctga acaatttcct cagaacctct taataaataa gtttggtaat gctgagaaaa 1200
aaaaaaaaaa a 1211
<210> 50
<211> 1046
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 876677CB1
<400> 50
cccacgcgtc cgggaatgtc tttcactctc tcatactttc tcctctcccc tctcccaagc 60
acatctgagt tgctgcctgt tcttcacact tagctccaaa cccatgaaaa attgccaagt 120
ataaaagctt ctcaagaatg agatggattc tagggtgtct tcacctgaga agcaagataa 180
agagaatttc gtgggtgtca acaataaacg gcttggtgta tgtggctgga tcctgttttc 240
cctctctttc ctgttggtga tcattacctt ccccatctcc atatggatgt gcttgaagat 300
cattaaggag tatgaacgtg ctgttgtatt ccgtctggga cgcatccaag ctgacaaagc 360
caaggggcca ggtttgatcc tggtcctgcc atgcatagat gtgtttgtca aagttgacct 420
ccgaacagtt acttgcaaca ttcctccaca agagatcctc accagagact ccgtaactac 480
tcaggtagat ggagttgtct attacagaat ctatagtgct gtctcagcag tggctaatgt 540
caacgatgtc catcaagcaa catttctgct ggctcaaacc actctgagaa atgtcttagg 600
39/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
gacacagacc ttgtcccaga tcttagctgg acgagaagag atcgcccata gcatccagac 660
tttacttgat gatgccaccg aactgtgggg gatccgggtg gcccgagtgg aaatcaaaga 720
tgttcggatt cccgtgcagt tgcagagatc catggcagcc gaggctgagg ccacccggga 780
agcgagagcc aaggtccttg cagctgaagg agaaatgaat gcttccaaat ccctgaagtc 840
agcctccatg gtgctggctg agtctcccat agctctccag ctgcgctacc tgcagacctt 900
gagcacggta gccaccgaga agaattctac gattgtgttt cctctgccca tgaatatact 960
agagggcatt ggtggcgtca gctatgataa ccacaagaag cttccaaata aagcctgagg 1020
tcctcttgcg gtagtcagct attgca 1046
<210> 51
<211> 1660
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2326143C81
<400> 51
gctcccctgc ccaccccgcc cccgtggccg agcccgggag tcgagtggga gtcggccggc 60
cggcgcgggc agccgtgacc cccgcggggg acactgcagc cggagcccgg gaggggccgc 120
gccgccaccg tctgaactag gatgtcccga catgaaggtg tcagctgtga tgcatgttta 180
aaaggaaatt ttcgaggtcg cagatataag tgtttaattt gctacgatta cgatctttgt 240
gcatcttgtt atgaaagtgg tgcaacaaca acaaggcata caactgacca cccaatgcag 300
tgcatattaa caagggtaga ttttgattta tactatggtg gggaagcttt ctctgtagag 360
cagccacagt cttttacttg tccctattgt ggaaaaatgg gctatacgga gacatctctt 420
caagaacatg ttacttctga acatgcagaa acatcaacag aagtgatttg tccaatatgt 480
gcagcgttac ctggaggcga tcctaatcat gtcacggatg actttgcagc tcatcttaca 540
cttgaacaca gagcccctag agatttagat gaatcgagtg gtgttcgaca tgtacgtaga 600
atgtttcacc ctggccgggg attaggaggt cctcgtgctc gtagatcaaa catgcacttt 660
actagcagtt ctactggtgg actttcttct tctcagagtt catattctcc aagcaatagg 720
gaagccatgg atcctatagc tgagctttta tctcagttat caggagtgag acgttctgca 780
ggaggacagc ttaattcctc tggcccttcc gcttctcagt tacaacaact gcagatgcag 840
ctgcagctag aacggcagca tgcccaggca gcacggcaac aactggagac cgcacgcaac 900
gcaacccggc gtactaacac aagcagtgtc accactacaa tcacacaatc cacagcaaca 960
accaacatag ctaatacaga aagcagtcag cagactctac agaattccca gtttctttta 1020
acaaggttga atgatcctaa aatgtctgaa acggagcgcc agtccatgga aagcgagcgt 1080
gcagaccgca gcctgtttgt ccaagagctc cttctgtcca ctttagtgcg tgaagagagc 1140
tcatcctcag atgaggatga tcggggggag atggcagatt ttggtgctat gggctgtgta 1200
gatattatgc ctttagatgt tgctttagaa aacctaaatt taaaagagag taataaagga 1260
aatgagcctc caccacctcc tctttgatga catcccaatt cgcagacaat gtcctctgtg 1320
ctgtatttgc caatgaaagt ggacaacaac tatcttgggt ttgtttggtg attgtaattt 1380
caggtctgtc actcttgtta cattgtgtac attcaaaagg aagagagaaa atatatatga 1440
taatcatttc cacttaacta atttttactt ctagcaggta aatgtaggta gcagtgcagg 1500
ggtgatctct gcttcctgta ccttgacatg caaaaggctc tcctaatact ccacattcaa 1560
actgaagagg aaaattgaaa tctctaatga agctgctgtg tgtatttatg aatattaatg 1620
aataaaaact gcttggatgg tttaccttaa aaaaaaaaaa 1660
<210> 52
<211> 1110
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2786302CB1
<400> 52
cctttattag agaaagagtg acctatctac cccttacttg gggaagactc tctgcttcaa 60
cagtttcttc tgtcttctgc tcctggacat ggcatgaaaa cggaagtttg agtatttcaa 120
ggataaagaa taactctagg acaagagttg ctttggggct aaacctatct cctgtattca 180
tttagatttg gctttaaaag cccatactat tttattagct cttctatgct ttttaaaagt 240
ttttaaaaaa attaatgtgg gctttttaat tctttctcag tgggagggtg agtttgaata 300
aacctttctt ccacatgaga agtattttac aagttgcttg tcaaatttaa aagaaaatga 360
tcaaatcttc aagaaaatga tcaaatcttc tgtgacaaaa aaatggacaa atattcacca 420
ttgagtgtga atgccatggt gatgctaatt ctgattattt ttcttttctt ttagccatac 480
cttcagagtg ttattttcct ttttgtcata aggtgtctgg aaatgaagta tggaaatgaa 540
ataatgaata aagacccagt tttcagaatc tctccacgga gtagagaaac tcatcccaat 600
40/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
ccggaagagc ccgaagaaga agatgaagat gttcaagctg aaagagtcca agcagcaaat 660
gcactcactg ctccaaactt ggaggaggaa ccagtcataa ctgcaagctg tttacacaag 720
gaatattatg agacaaagaa aagttgcttt tcaacaagaa agaagaaaat agccatcaga 780
aatgtttcct tttgtgttaa aaaaggtgaa gttttgggat tactaggaca caatggagct 840
ggtaaaagta cttccattaa aatgataact gggtgcacaa agccaactgc aggagtggtg 900
gtgttacaag gcagcagagc atcagtaagg caacagcatg acaacagcct caagttcttg 960
gggtactgcc ctcaggagaa ctcactgtgg cccaagctta ccatgaaaga gcacttggag 1020
ttgtatgcag ctgtggaaag actggggcaa aaaagatgct gctctcagta tttcacgatt 1080
ggtgggaggt cttaagctcc aggaacaact 1110
<210> 53
<211> 1120
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3735780CB1
<400> 53
gaaatccagt tatcaaaatt gactcaagaa gagagaacct aacagaacaa taacaatgga 60
agaaattggg aacattatca caaagctatc atcctgccaa actccaggct cagatgtcac 120
aggttaaaaa aagtccttca tgaaaaagaa agatcttaag cagcatgatg gattcagaag 180
ctcatgaaaa gaggccacca atactaacat cttcaaaaca agatatatca cctcatatta 240
caaatgttgg tgagatgaag cattacttgt gtggctgctg tgcagccttc aacaatgtcg 300
caatcacatt tcccattcag aaggtcctct ttcgacaaca gctgtatggc atcaaaaccc 360
gggatgcaat acttcagttg agaagggatg gatttcgaaa tttgtatcgt ggaatccttc 420
ccccattgat gcagaagaca actacgcttg cacttatgtt tggtctgtat gaggatttat 480
cctgccttct ccacaagcat gtcagtgctc cagagtttgc aaccagtggc gtggcggcag 540
tgcttgcagg gacaacagaa gcaattttca ctccactgga aagagttcag acattgcttc 600
aagaccacaa acatcatgac aaatttacca acacttacca ggctttcaag gcactgaaat 660
gtcatggaat tggagagtat tatcgaggct tggtgcccat tcttttccgg aatggactca 720
gcaatgtctt gtttttcggc cttcgaggtc ccattaagga gcatctgcct accgcaacga 780
ctcacagtgc tcatctggtc aatgatttta tctgtggagg tctattgggt gccatgttgg 840
gattcttgtt ttttccaatt aatgttgtaa aaactcgcat acagtctcag attggtgggg 900
aatttcagtc tttccccaag gttttccaaa aaatctggct ggaacgggac agaaaactga 960
taaatctttt cagaggtgcc catctgaatt accatcggtc cctcatctct tggggcataa 1020
tcaatgcaac ttatgagttc ttgttaaagg ttatatgaaa aaaccatcag ttaagtgcca 1080
tttatcaact gaatagacct tctaagaaga aaaaaaaaaa 1120
<210> 54
<211> 886
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 039026CB1
<400> 54
ggccgcggct cctgtccaga ccctgaccct ccctcccaag gctcaaccgt cccccaacaa 60
ccgccagcct tgtactgatg tcggatgcga gagcctgtgc ttaagtaaga atcaggcctt 120
attggagaca ttcaagcaaa ggttggacaa ctacttttcc agaacagaaa ggaaactcat 180
gcatcagaaa aggtgactaa taaaggtacc agaagaatat ggctgcacaa ataccagaat 240
ctgatcagat aaaacagttt aaggaatttc tggggaccta caataaactt acagagacct 300
gctttttgga ctgtgttaaa gacttcacaa caagagaagt aaaacctgaa gagaccacct 360
gttcagaaca ttgcttacag aaatatttaa aaatgacaca aagaatatcc atgagatttc 420
aggaatatca tattcagcag aatgaagccc tggcagccaa agcaggactc cttggccaac 480
cacgatagag aagtcctgat ggatgaactt ttgatgaaag attgccaaca gctgctttat 540
tggaaatgag gactcatctg atagaatccc ctgaaagcag tagccaccat gttcaaccat 600
ctgtcatgac tgtttggcaa atggaaaccg ctggagaaac aaaattgcta tttaccagga 660
ataatcacaa tagaaggtct tattgttcag tgaaataata agatgcaaca tttgttgagg 720
ccttatgatt cagcagcttg gtcacttgat tagaaaaata aaccattgtt tcttcaattg 780
tgactgttaa ttttaaagca acttatgtgt tcgatcatgt atgagataga aaaattttta 840
ttactcaaag taaaataaat ggaaatatca ctgaaaaaaa aaaaaa 886
<210> 55
<211> 2336
41/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 260607CB1
<400> 55
taatacgctc actataggga atttggccct cgagcagtaa ttcggcacga ggaccatctc 60
tttaggatat atttttaaat tctttgaaac acataaccaa aatggtttga ttcactgact 120
gactttgaag ctgcatctgc cagttacacc ccaaatggct ttaatcccct ctcgggtctg 180
gttgcctttt gcagtttggg ttgtggactc agctcctgtg aggggtctgg ttaggagaga 240
gccattttta aggacaggga gttttatagc ccttttctac tttcctcccc tcctcccagt 300
ccttatcaat cttttttcct ttttcctgac cccctccttc tggaggcagt tgggagctat 360
ccttgtttat gcctcactat tggcagaaaa gaccccattt aaaacccaga gaacactgga 420
gggggatgct ctagttggtt ctgtgtccat tttcctctgt gccaaagaca gacagacaga 480
ggctgagaga ggctgttcct gaatcaaagc aatagccagc tttcgacaca tacctggctg 540
tctgaggagg aaggcctcct ggaaactggg agctaagggc gaggcccttc ccttcagagg 600
ctcctggggg attagggtgt ggtgtttgcc aagccaaggg gtagggagcc gagaaattgg 660
tctgtcggct cctggttgca ctttggggaa ggagaggaag tttggggctc caggtagctc 720
cctgttgtgg gactgctctg tcccctgccc ctactgcaga gatagcactg ccgagttccc 780
ttcaggcctg gcagacgggc agtgaggagg ggcctcagtt agctctcaag ggtgccttcc 840
cctcctccca acccagacat accctctgcc aaactgggaa ccagcagtgc tagtaactac 900
ctcacagagc cccagagggc ctgcttgagc cttcttgctc cacaggagaa gctggtgcct 960
ctaggcaacc ccttcctccc acctctcatc aggggtgggg gttctccttt ctttcccctg 1020
aagtgtttat ggggagatcc tagtggcttt gccattcaaa ccactcgact gtttgcctgt 1080
ttcttgaaaa ccagtagaag ggaaacagca cagcctgtca cagtaattgc aggaagattg 1140
aagaaaaatc ctcatcaatg ccaggggaca taaaagccat ttcccttcca aatactcgac 1200
aatttagatg cagaacattt ctctgtattc agacttagag taacaccagc tgaaaactgc 1260
agtttctttc ctttggatac ataaggcttc tctatcgggg tacgggacag ggaggaggcc 1320
tcatgtctga agggggattt aggggcgaga gccgcagccc tgaccctcgg tcctgtgcac 1380
cgctttgggg cacagtctga tggcgccttt gctggcgcct tagtatggtt gactccggat 1440
ggacaaaaga aaaaaaattt tttttcttga atgaaatagc aggaagctcc tcgggagcat 1500
gtgttttgat taaccgtagt gatggatgct acgagtataa atggattaac tacctcaatc 1560
cttacagtaa gattggaact aagggcaggg actcatgcat aagggtatga atcccagcca 1620
ggacaagtga gttgaggctt gtgccacaaa aggtttgtcc ttggggaaca ggcaggcctg 1680
ccaggatccc ccccatatcg attgggctgg gagggctggc cgtgaggtcc ccactttctg 1740
ctttccttgc ccatgtgtca cccctttggc ctccagcttg tccctctctc actttctata 1800
gctttgttgg accagatggt gaggaaagga atggcctctt cccttctaga gggggctggc 1860
tggagtgaga cctggggctt ggcctggaac ccaccacaca gccccaaagt caggaagcct 1920
ggggaaacca gagctgagac ctcttcaaca gggtttcttt gagatcctac acctccattg 1980
ggcccttttt cagtcttcaa tgggggccca gttggctcta gaaggagaag aggtgaagca 2040
ggatcctttg ccctggggga gtctgagggc gcggtccttg gactcattca ggccgtcttt 2100
gtagttgggg gagttccact gggcgatccc agcccctccc cacccaccct ctaatggacc 2160
tcctcataga agccccattt cacttttgtt ttatctacct cttagcaaaa caatagataa 2220
attaggtagt ggcagctcca cttgcttagg ttaggggggg aaaaagattt ctttttccaa 2280
aggaaaaaaa tattaccttg agaatacttt ccaaaaaata aaatttaaaa aaaaaa 2336
<210> 56
<211> 2200
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1429651CB1
<400> 56
gagaacttta cgcctggatc tcatctaact gacacagaaa ccctgtaagg atccagaggt 60
ctcgttcagg accatggaga gcggcaccag cagccctcag cctccacagt tagatcccct 120
ggatgcgttt ccccagaagg gcttggagcc tggggacatc gcggtgctag ttctgtactt 180
cctctttgtc ctggctgttg gactatggtc cacagtgaag accaaaagag acacagtgaa 240
aggctacttc ctggctggag gggacatggt gtggtggcca gtgggtgcat ccttgtttgc 300
cagcaatgtt ggaagtggac atttcattgg cctggcaggg tcaggtgctg ctacgggcat 360
ttctgtatca gcttatgaac ttaatggctt gttttctgtg ctgatgttgg cctggatctt 420
cctacccatc tacattgctg gtcaggtcac cacgatgcca gaatacctac ggaagcgctt 480
cggtggcatc agaatcccca tcatcctggc tgtactctac ctatttatct acatcttcac 540
caagatctcg gtagacatgt atgcaggtgc catcttcatc cagcagtctt tgcacctgga 600
42/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
tctgtacctg gccatagttg ggctactggc catcactgct gtatacacgg ttgctggtgg 660
cctggctgct gtgatctaca cggatgccct gcagacgctg atcatgctta taggagcgct 720
caccttgatg ggctacagtt tcgccgcggt tggtgggatg gaaggactga aggagaagta 780
cttcttggcc ctggctagca accggagtga gaacagcagc tgcgggctgc cccgggaaga 840
tgccttccat attttccgag atccgctgac atctgatctc ccgtggccgg gggtcctatt 900
tggaatgtcc atcccatccc tctggtactg gtgcacggat caggtgattg tccagcggac 960
tctggctgcc aagaacctgt cccatgccaa aggaggtgct ctgatggctg catacctgaa 1020
ggtgctgccc ctcttcataa tggtgttccc tgggatggtc agccgcatcc tcttcccaga 1080
tcaagtggcc tgtgcagatc cagagatctg ccagaagatc tgcagcaacc cctcaggctg 1140
ttcggacatc gcgtatccca aactcgtgct ggaactcctg cccacagggc tccgtgggct 1200
gatgatggct gtgatggtgg cggctctcat gtcctccctc acctccatct ttaacagtgc 1260
cagcaccatc ttcaccatgg acctctggaa tcacctccgg cctcgggcat ctgagaagga 1320
gctcatgatt gtgggcaggg tgtttgtgct gctgctggtc ctggtctcca tcctctggat 1380
ccctgtggtc caggccagcc agggcggcca gctcttcatc tatatccagt ccatcagctc 1440
ctacctgcag ccgcctgtgg cggtggtctt catcatggga tgtttctgga agaggaccaa 1500
tgaaaagggt gccttctggg gcctgatctc gggcctgctc ctgggcttgg ttaggctggt 1560
cctggacttt atttacgtgc agcctcgatg cgaccagcca gatgagcgcc cggtcctggt 1620
gaagagcatt cactacctct acttctccat gatcctgtcc acggtcaccc tcatcactgt 1680
ctccaccgtg agctggttca cagagccacc ctccaaggag atggtcagcc acctgacctg 1740
gtttactcgt cacgaccccg tggtccagaa ggaacaagca ccaccagcag ctcccttgtc 1800
tcttaccctc tctcagaacg ggatgccaga ggccagcagc agcagcagcg tccagttcga 1860
gatggttcaa gaaaacacgt ctaaaaccca cagctgtgac atgaccccaa agcagtccaa 1920
agtggtgaag gccatcctgt ggctctgtgg aatacaggag aagggcaagg aagagctccc 1980
ggccagagca gaagccatca tagtttccct ggaagaaaac cccttggtga agaccctcct 2040
ggacgtcaac ctcattttct gcgtgagctg cgccatcttt atctggggct attttgctta 2100
gtgtggggtg aacccagggg tccaaactct gtttctcttc agtgctccat ttttttaatg 2160
aaagaaaaaa taataaagct tttgtttacc aaaaaaaaaa 2200
<210> 57
<211> 2823
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2069971CB1
<400> 57
gaaagacata cacacttcat gtaatgctac ctgcaagtct ccctagaaaa gcagtttttg 60
taggtgaaaa caatgaagcc aggtaatatt gcaaggaggc tgtaatttta gcagacctac 120
caacaacact gatgtaggaa gctcattatt ttaatttctg gagcctttta attttttctt 180
tagaaagtgt ataaataatt gcagtgctgc tttgcttcca aaactgggca gtgagttcaa 240
caacaacgac aacaacagcc gcagctcatc ctggccgtca tggagtttct tgaaagaacg 300
tatcttgtga atgataaagc tgccaagatg tatgctttca cactagaaag aaggagctgc 360
aaatgaacac ttcatagcaa tgtggaactc caacagaaac cggtgaataa agatcagtgt 420
cccagagaga gaccagagga gctggagtca ggaggcatgt accactgcca cagtggctcc 480
aagcccacag aaaagggggc gaatgagtac gcctatgcca agtggaaact ctgttctgct 540
tcagcaatat gcttcatttt catgattgca gaggtcgtgg gtgggcacat tgctgggagt 600
cttgctgttg tcacagatgc tgcccacctc ttaattgacc tgaccagttt cctgctcagt 660
ctcttctccc tgtggttgtc atcgaagcct ccctctaagc ggctgacatt tggatggcac 720
cgagcagaga tccttggtgc cctgctctcc atcctgtgca tctgggtggt gactggcgtg 780
ctagtgtacc tggcatgtga gcgcctgctg tatcctgatt accagatcca ggcgactgtg 840
atgatcatcg tttccagctg cgcagtggcg gccaacattg tactaactgt ggttttgcac 900
cagagatgcc ttggccacaa tcacaaggaa gtacaagcca atgccagcgt cagagctgct 960
tttgtgcatg cccttggaga tctatttcag agtatcagtg tgctaattag tgcacttatt 1020
atctacttta agccagagta taaaatagcc gacccaatct gcacattcat cttttccatc 1080
ctggtcttgg ccagcaccat cactatctta aaggacttct ccatcttact catggaaggt 1140
gtgccaaaga gcctgaatta cagtggtgtg aaagagctta ttttagcagt cgacggggtg 1200
ctgtctgtgc acagcctgca catctggtct ctaacaatga atcaagtaat tctctcagct 1260
catgttgcta cagcagccag ccgggacagc caagtggttc ggagagaaat tgctaaagcc 1320
cttagcaaaa gctttacgat gcactcactc accattcaga tggaatctcc agttgaccag 1380
gaccccgact gccttttctg tgaagacccc tgtgactagc tcagtcacac cgtcagtttc 1440
ccaaatttga caggccacct tcaaacatgc tgctatgcag tttctgcatc atagaaaata 1500
aggaaccaaa ggaagaaatt catgtcatgg tgcaatgcac attttatcta tttatttagt 1560
tccattcacc atgaaggaag aggcactgag atccatcaat caattggatt atatactgat 1620
cagtagctgt gttcaattgc aggaatgtgt atatagatta ttcctgagtg gagccgaagt 1680
aacagctgtt tgtaactatc ggcaatacca aattcatctc ccttccaata atgcatcttg 1740
agaacacata ggtaaatttg aactcaggaa agtcttacta gaaatcagtg gaagggacaa 1800
43/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
atagtcacaa aattttacca aaacattaga aacaaaaaat aaggagagcc aagtcaggaa 1860
taaaagtgac tctgtatgct aacgccacat tagaacttgg ttctctcacc aagctgtaat 1920
gtgatttttt tttctactct gaattggaaa tatgtatgaa tatacagaga agtgcttaca 1980
actaattttt atttacttgt cacattttgg caataaatcc ctcttatttc taaattctaa 2040
cttgtttatt tcaaaacttt atataatcac tgttcaaaag gaaatatttt cacctaccag 2100
agtgcttaaa cactggcacc agccaaagaa tgtggttgta gagacccaga agtcttcaag 2160
aacagccgac aaaaacattc gagttgaccc caccaagttg ttgccacaga taatttagat 2220
atttacctgc aggaaggaat aaagcagatg caaccaattc attcagtcca cgagcatgat 2280
gtgagcactg ctttgtgcta gacattgggc ttagcattga aactataaag aggaatcaga 2340
cgcagcaagt gcttctgtgt tctggtagca actcaacact atctgtggag agtaaactga 2400
agatgtgcag gccaacattc tggaaatcct atgtcactgg gtttggtttg gaaacctgga 2460
cttctgcatt tttaaaagtt acccagagat gcttctaaag atgagccata gtctagaaga 2520
ttgtcaacca caggagttca ttgagtggga cagctagata catacattgg cagctacaat 2580
agtatcatga attgcaatga tgtagtgggg tataaaagga aagcgatgga tattgccgga 2640
tgggcatggc cagtgatgtt tcacgtcatt gaggtgacag ctctgctgga ctttgaatta 2700
catatggagg ctctccagga agacgaagaa gagaaggaca ttctaggcaa aaagaagact 2760
aggcacaagg cacacttatg tttgtctgtt agcttttagt tgaaaaagca agatacaggg 2820
tcg 2823
<210> 58
<211> 1491
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2329339CB1
<400> 58
cgcctccctc cagctgcgag tgcggcctcg gctggcggcg gcaccaggcc acagttgtaa 60
gggatcttgt ggctgtcagg atggcagagg agcaggagtt cacccagctc tgcaagttgc 120
ctgcacagcc ctcacaccca cactgcgtga acaacaccta ccgcagcgca cagcactccc 180
aggctctgct ccgagggctg ctggctctcc gggacagcgg aatcctcttc gatgttgtgc 240
tggtggtgga gggcagacac atcgaggccc atcgcatcct gctggctgcg tcctgcgatt 300
acttcagagg aatgtttgct gggggattga aggagatgga acaggaagag gtcctgatcc 360
acggtgtgtc ctacaatgct atgtgccaaa tcctacattt catatacacc tccgagctgg 420
agctcagcct gagcaatgta caagagacac tggtggctgc ctgccagctt cagatcccag 480
aaattatcca tttctgctgt gatttcctca tgtcctgggt ggacgaagag aacattctcg 540
atgtctaccg gctggcagag ctgtttgact tgagccgcct gactgagcaa ctggacacct 600
atatcctcaa aaactttgtg gccttctctc ggactgacaa gtaccgccag cttccattgg 660
agaaggtcta ctccctcctc agcagcaatc gcctggaggt ctcctgcgag accgaggtat 720
atgagggggc ccttctctac cattatagcc tggagcaggt gcaggctgac cagatctcgc 780
tgcacgagcc cccaaagctc cttgagacag tgcggtttcc gctgatggaa gctgaggtcc 840
tgcagcggct gcatgacaag ctggacccca gccctttgag ggacacagtg gccagcggcc 900
tcatgtacca ccggaacgag agcctacagc ccagcctgca gagcccgcaa acggagctgc 960
ggtcggactt ccagtgcgtt gtgggcttcg ggggcattca ctccacgccg tccactgtcc 1020
tcagcgacca ggccaagtat ctaaacccct tactgggaga gtggaagcac ttcactgcct 1080
ccctggcccc ccgcatgtcc aaccagggca tcgcggtgct caacaacttc gtatacttga 1140
ttggagggga caacaatgtc caaggatttc gagcagagtc ccgatgctgg aggtatgacc 1200
cacggcacaa ccgctggttc cagatccagt ccctgcagca ggagcacgcc gacctgtccg 1260
tgtgtgttgt aggcaggtac atctacgctg tggcgggccg tgactaccac aatgacctga 1320
atgctgtgga gcgctacgac cctgccacca actcctgggc atacgtggcc ccactcaaga 1380
gggaggtgta tgcccacgca ggcgcgacgc tggaggggaa gatgtatatc acctgcggac 1440
gcaagcttat tccctttagt gagggttaat tttagcttgc actggccgtc g 1491
<210> 59
<211> 986
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2540219CB1
<400> 59
ggacgccaac ctcggtttga agtccagggc agtggctcct gcggacagcc agcataccag 60
gggccagtgc actgcattac aaccattgtg aggaatgagg gcctgtgccg tgtggctggc 120
gggcggcatg gcaggagcaa tttcttgggg gacagcgact cctatggatg tcgtgaaaag 180
44/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
tcgactccaa gctgatgggg tttatttaaa caaatataaa ggtgtcctgg actgtatctc 240
ccagagttac cagaaggaag gtcttaaagt gtttttcaga ggcatcactg tgaacgcggt 300
gcggggcttc cccatgagtg cggccatgtt ccttgggtac gagctgtcgc tgcaggctat 360
ccgcggggac cacgcagtga cgagcccata agcgccagga ggtgaacaca ggatgactac 420
agtgttcccc tgggcctcat ctctgcatgt gaagccctga gagctgcaga tgtttgccct 480
ttggacctcc aagtggacat caattagcaa gcgtgggcta ggatggtgca gacactgacg 540
tggcccttct gatgcctggg atgcctcatg agtcactgat tcaagccctc caaggttctg 600
atccccaatg cccactctgc taggctggca tcaaagagct ttccaagaaa tgtttggtcc 660
agctgagaag tcctgaccat gagcaccagg gagccagaaa ccacccagag aaacgttgct 720
tcactcctct gtctgaggat ggggaggggc cagtgagctc tgggctcagc cactccctcc 780
agtctcaagt aacacgtccc cgtgcctcca gtctcctctc agcaccgacc aggtttttcc 840
ccgctcctgc acccgtggat cctgaggaca gcggtagcgc cttcctcacc gcacgctgag 900
tccagtgcgt gctcctcact gtgcacttat tagtgtctgt tgagtgatta aatcacatcc 960
tcaggtctgc agcaaataaa tgaaag 986
<210> 60
<211> 4023
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2722462CB1
<400> 60
gtccggggcg gcgcgtatgg cggcactatg ccatccacga ctcccaggcc cccagtctca 60
gctctggggg tgagagttcc ccctccagcc ccgcacacaa ctgggagatg aattaccaag 120
aggcagcaat ctacctccag gaaggcgaga acaacgacaa gttcttcacc caccccaagg 180
atgccaaggc gctggcggcc tacctctttg cacacaatca cctcttctac ctgatggagc 240
tggccacggc cctgctgctg ctgctgctct ccctgtgcga ggcccccgcc gtccccgcac 300
tccggcttgg catctatgtc cacgccaccc tggagctgtt tgccctgatg gtggtagtgt 360
ttgaactctg catgaagtta cgctggctgg gcctccacac cttcatccgg cacaagcgga 420
ccatggtcaa gacctcggtg ctggtggtgc agtttgtcga ggccatcgtg gtgttggtac 480
ggcagatgtc ccatgtgcgg gtgacccgag cactgcgctg cattttcctg gtggactgtc 540
ggtattgcgg tggcgtccgg cgcaacctgc ggcagatctt ccagtccctg ccgcccttca 600
tggacatcct cctgctgctg ctgttcttca tgatcatctt tgccatcctc ggtttctact 660
tgttctcccc taacccttca gacccctact tcagcaccct ggagaacagc atcgtcagtc 720
tgtttgtcct tctgaccaca gccaatttcc cagatgtgat gatgccctcc tactcccgga 780
acccctggtc ctgcgtcttc ttcatcgtgt acctctccat cgagctgtat ttcatcatga 840
acctgcttct ggctgtggtg ttcgacacct tcaatgacat tgagaaacgc aagttcaagt 900
ctttgctact gcacaagcga accgctatcc agcatgccta ccgcctgctc atcagccaga 960
ggaggcctgc cggcatctcc tacaggcagt ttgaaggcct catgcgcttc tacaagcccc 1020
ggatgagtgc cagggagcgc tatcttacct tcaaggccct gaatcagaac aacacacccc 1080
tgctcagcct aaaggacttt tacgatatct acgaagttgc tgctttgaag tggaaggcca 1140
agaaaaacag agagcactgg tttgatgagc ttcccaggac ggcgctcctc atcttcaaag 1200
gtattaatat ccttgtgaag tccaaggcct tccagtattt catgtacttg gtggtggcag 1260
tcaacggggt ctggatcctc gtggagacat ttatgctgaa aggtgggaac ttcttctcca 1320
agcacgtgcc ctggagttac ctcgtctttc taactatcta tggggtggag ctgttcctga 1380
aggttgccgg cctgggccct gtggagtact tgtcttccgg atggaacttg tttgacttct 1440
ccgtgacagt gttcgccttc ctgggactgc tggcgctggc cctcaacatg gagcccttct 1500
atttcatcgt ggtcctgcgc cccctccagc tgctgaggtt gtttaagttg aaggagcgct 1560
accgcaacgt gctggacacc atgttcgagc tgctgccccg gatggccagc ctgggcctca 1620
ccctgctcat cttttactac tccttcgcca tcgtgggcat ggagttcttc tgcgggatcg 1680
tcttccccaa ctgctgcaac acgagtacag tggcagatgc ctaccgctgg cgcaaccaca 1740
ccgtgggcaa caggaccgtg gtggaggaag gctactatta tctcaataat tttgacaaca 1800
tcctcaacag ctttgtgacc ctgtttgagc tcacagttgt caacaactgg tacatcatca 1860
tggaaggcgt cacctctcag acctcccact ggagccgcct ctacttcatg accttttaca 1920
ttgtgaccat ggtggtgatg acgatcattg tcgcctttat cctcgaggcc ttcgtcttcc 1980
gaatgaacta cagccgcaag aaccaggact cggaagttga tggtggcatc acccttgaga 2040
aggaaatctc caaagaagag ctggttgccg tcctggagct ctaccgggag gcacgggggg 2100
cctcctcgga tgtcaccagg ctgctggaga ccctctccca gatggagaga taccagcaac 2160
attccatggt gtttctggga cggcgatcaa ggaccaagag cgacctgagc ctgaagatgt 2220
accaggagga gatccaggag tggtatgagg agcatgccag ggagcaagag cagcagcgac 2280
aactcagcag cagtgcagcc cccgccgccc agcagccccc aggcagccgc cagcgctccc 2340
agaccgttac ctagcccagc gcccgaaagc cgtctcttct atgcaataac acaatagtat 2400
tactctactg cgatgtacgg aactgcggtg tgtgtacaca tactcacgta tatgcacata 2460
tttatataca ggaagaaaaa agacagacaa gatggggctt ggtttataac caccttgccc 2520
tgtcttcctt aactccagaa gccagtttgg tgaggggtgg gggtgcggcc accaggtctg 2580
45/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
agctcttcct actgtggaag gctccagaag gcccttcaca aggagacccc tcacctggat 2640
ccagtcgact gcggggcttg cccctcatgt gggctggcct ccatcggcca cgtccaaagc 2700
tgtcactgct actgcttcag gctcacatcc ccccgacctg atggcgtgcc cgccccctct 2760
ccctgcggcc catgccacag gtttctgtgt tttgctttag ggacagaacc acttaggaaa 2820
gaaagaactc ccggtctcca gggtggtatt tcagtgtctg tgataatgtc acgcaacacc 2880
tcttcgggga ccagtgccca ggatctaatg gaagcggaat tggggcaact gggcccgtgt 2940
ggccagagct cagttagcca gtgccgggcg gccacagatt acactgacca atctcctccc 3000
ttggctctgc aagcctccca cccagccttc tctggcttaa cccttgttgg cgaaaactct 3060
tccacagtgg cctccttggg gacccagaac ccggaggaag gggcatgagg caggaagtgg 3120
ggccgatgtc tgcaacccag accacttcgt ggaatgggct cttgaccaaa tccctttttt 3180
ttgcgattta cccgttcaag caaaacaacg ttttggttaa ctaaggattg tgctaaagcc 3240
gataccaggt ccttcacacg tgtgcactag gaacaggagc gaacagcaca gagagacgct 3300
ccctgtggga cgcagcagcc ccgtggcccc ggcccagttc ccagccaccc tccctggctc 3360
tgctcacacc agagatttcc atagcaggag cggttggtgc agaagtaggt tcagatgaac 3420
ctcagttaac gtcgccaccc ctcctcccac catggtaccc tgtaggagcc ctgtatgaca 3480
tctgagcgtg gtggaggtag gagggttgcc agctgcagtg accctgccac agaggcaggg 3540
tcagtgcaga ggtcgctttg gttccgcttc cctgggccac agaacggaac acagcatagg 3600
ttctgcagca ggagccgcag tggcaggatg gagggtgcga agggcaagga gtgcactgct 3660
gggcattcct ggccagcccc ggccctctgg tgcctgcttc ctgtgacttc agaaggcagg 3720
tggacagagc ctccctctgg ccttgtcctc ttcccagcca cagaacgggc agggtggcac 3780
ccgaccccag gggagcagta cctggtcccc caccccctcc tcccaaccac ctccaaggcc 3840
aagctgggtc ccatagccag cacggcatgg ttctcccctt ccccccttcc caggtcaggg 3900
gagttggaca agtagcaggt gtttgttttt aaagcacagc cctttgggaa agcaacacat 3960
tattgagact cactgtgatt cccccgggag tcagactggc tttgtctctt tctctctgga 4020
ggg 4023
<210> 61
<211> 2345
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2739264081
<400> 61
gaaaggaggc ggaactcggt gatctgactg gcggtttccc ggccggactg agaaggggag 60
cgcgctgcgc gtcgcaggag taacctactt ggtctcctgc tttcgcgaca tggccttcaa 120
ttttggggct ccctcgggca cctccggtac cgctgcagcc accgcggccc ccgcgggtgg 180
gtttggagga tttgggacaa catctacaac tgcaggttct gcattcagct tttctgcccc 240
aactaacaca ggcactactg gactctttgg tggtactcag aacaaaggtt ttggatttgg 300
tactggtttt ggcacaacaa cgggaactag tactggttta ggtactggtt tgggaactgg 360
actgggattt ggaggattta atacacagca gcagcagcaa actacattag gtggtctctt 420
cagtcagcct acacaagctc ctacccagtc caaccagctg ataaatactg cgagtgctct 480
ttctgctcca acgctgttgg gagatgagag agatgctatt ttggcaaaat ggaatcaact 540
gcaggccttt tggggaacag gaaaagggta tttcaacaat aatattccgc cagtggaatt 600
cacacaagaa aatccctttt gccgatttaa ggcagtaggt tatagttgca tgcccagtaa 660
taaagatgaa gatgggctag tggttttagt tttcaacaaa aaagaaacag agattcgaag 720
ccaacaacaa cagttggtag aatcattgca taaagttttg ggaggaaacc agacccttac 780
tgtaaatgta gagggcacta aaacattgcc agatgatcag acagaagttg ttatttatgt 840
tgttgagcgt tcgccaaatg gtacttcaag aagagttcca gctacaacgc tatatgccca 900
ttttgaacaa gccaatataa aaacacaatt gcagcaactt ggtgtaaccc tttctatgac 960
tagaacagaa ctttctcctg cacagatcaa acagctttta cagaatcctc ctgctggtgt 1020
tgatcctatt atctgggaac aggccaaggt agataaccct gattctgaaa agttaattcc 1080
tgtaccaatg gtgggtttta aggaacttct ccgaagactg aaggttcaag atcagatgac 1140
taagcagcat caaaccagat tagatatcat atctgaagat attagtgagc tacaaaagaa 1200
tcaaactaca tctgtagcca aaattgcaca atacaagagg aaactcatgg atctttccca 1260
tagaacttta caggtcctaa tcaaacagga aattcaaagg aagagtggtt atgccattca 1320
ggctgatgaa gagcagttgc gagttcagct ggatacgatt cagggtgaac taaatgcacc 1380
tactcagttc aagggccgac taaatgaatt gatgtctcaa atcaggatgc agaatcattt 1440
tggagcagtc agatctgaag aaaggtatta catagatgca gatctgttac gagaaatcaa 1500
gcagcatttg aaacaacaac aggaaggcct tagccatttg attagcatca ttaaagacga 1560
tctagaagat ataaagctgg tcgaacatgg attgaatgaa accatccaca tcagaggtgg 1620
tgtctttagt tgacagttca caaacttgtg taaaggtttg tgaaatgcat cttcttactg 1680
catcagacct tccttaagaa tgaaaccgac cacatggagg gaaaaagaaa acaattcttt 1740
cttggattgg ttttttgaga agtttactga caaattactg ttcatcaaat ctgaaatagt 1800
cacctcacag ctcttcaaag aaaacctttg aaagatttat atctaaaagc tgtatttact 1860
ttaaaagaag tgcataatta ccaaaattgt atgtactatt gtacattttt acaacagcat 1920
46/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
tttcttaaac ataatctgtg tttaatgatt attgtccatt gagcctgtac tctgctttcc 1980
ataccaagta aatatgaaat aatctacttt gcacataaca gaacaaacta taattacttg 2040
gctgttggag atttgtactt gagtataaat gtacaccagt ttttgtattt gtgaactcat 2100
ctgtgggagg agtaaagaaa atccaaaagc atttaatgtt ttgtttttgt tctataaaga 2160
tatgaaaatg tatttttata ttattttact tatttggaat ttacagagca cacctaagca 2220
attaggatat aacaaaacta cttaaccatt tttgcaacca ttttgttttt taagcctttt 2280
tatttctaaa aagatgaaaa cttataaata aattcttaat ttgtaattac ttttaaaaaa 2340
aaaaa 2345
<210> 62
<211> 2085
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2758310CB1
<400> 62
cggagatgtg gcgaccgttt ctggcatcat tctgagactc ggcagttgct tctcactgct 60
gcggccgggc ctgtctgtgg gagctgcatc ctcctcatct gcaggcgctg gaaaaccaga 120
cacgatcgga catgcatgtg gttctgcggc caaagcacgc cctttggttg tgaacttcat 180
gatacctgtg tgcagctctg tcatttccac tctgctctgc tgcacagaag gcagaagccc 240
tggccgtccc ctgctgtgtt cttcaggaga aacgtcaggg gccttcctcc aaggttctcc 300
agccccacac ccctgtggag gaaggtgctc tccaccgcgg tagtgggggc gcccctgctc 360
ctcggagccc gctatgtcat ggcagaggca cgggagaaga ggaggatgcg gctcgtggtg 420
gatggcatgg ggcgctttgg caggtctctg aaggtcggcc tgcagatctc cctggactac 480
tggtggtgca ccaatgttgt ccttcgaggg tggaagagcc caggctactt ggaggtgatg 540
tctgcgtgtc accagcgggc ggctgatgcc ctggtggcag gggccatcag caacgggggc 600
ctctacgtga agctgggcca ggggctgtgc tccttcaacc acctgcttcc ccccgagtat 660
acccggaccc tgcgcgtgct agaggacagg gccctcaagc ggggcttcca ggaggtggat 720
gagttgttcc ttgaggactt ccaggccctc ccccacgagc tcttccagga gtttgactac 780
cagccaattg ctgccgccag cctggcacag gtgcacagag ccaagctgca cgatggcacc 840
agcgtggctg tgaaggtgca gtacatcgac ctgcgggacc gctttgatgg ggacatccac 900
accctggagc tcctgctgcg gctcgttgag gtcatgcacc ccagctttgg cttcagctgg 960
gtcctccagg acctgaaggg gaccctggcc caggagctgg acttcgagaa tgagggccgc 1020
aacgcagagc gctgtgcgcg ggagctggcg cacttcccct acgtcgtggt gccccgcgtg 1080
cactgggaca agtccagcaa gcgcgtgctc actgccgact tctgcgccgg ctgcaaggtc 1140
aacgatgtgg aggccatcag gagccagggg ctggcagtgc atgacatagc agaaaagctc 1200
atcaaggcct ttgctgagca gatattttac accggcttca tccactcgga cccacatcct 1260
ggcaacgttc tggtgcggaa aggcccggac gggaaagcgg agctggtgct gctggaccac 1320
gggctctacc agttcctgga ggagaaggac cgcgcagccc tctgccagct gtggcgggcc 1380
atcatcctgc gggacgacgc cgccatgagg gcgcacgcag ccgcactggg ggtgcaagac 1440
tacctcctgt tcgccgagat gctcatgcag cgccccgtgc gcctggggca gctgtggggc 1500
tcgcacctac tgagccgcga agaggcggcc tacatggtgg acatggcccg cgagcgcttc 1560
gaggccgtca tggcggtgct cagggagctg ccgcggccca tgctgctggt gctgcgcaac 1620
atcaacaccg tgcgcgctat caacgtggcc ctcggcgccc ccgtggaccg ctacttcctt 1680
atggctaaaa gggctgtccg gggctggagc cgcctggcgg gcgccacgta tcggggtgtc 1740
tacggcacca gcctcctgcg ccacgccaag gtcgtctggg agatgctcaa gtttgaagtg 1800
gcgctcaggc tggagacctt ggccatgcgg ctgaccgccc tcctggctcg tgctctggtc 1860
cacctgagcc tcgtgccccc agcggaggag ctctaccagt acctggagac ctagggtgca 1920
gccgcccagg gccggcgggg cccttttcac cttgggctga cggaggtggc ggggctagag 1980
gtgtagacac cccgagcccc gtgggcactc gcactggggg gctgtgacag cagctgggcc 2040
aggaggccgt gtaatgacca cacactcctc tcaagcaaaa aaaaa 2085
<210> 63
<211> 3014
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2762348CB1
<400> 63
ggaggcaaag gccaggctga ggatcagggt ggcccgggtg gcagcgggga ggcgctgcat 60
gctggaggct gtgctgagtg cccggtgcag gtgagccggt cctgcggagt tgtgccgagt 120
gcctgctgca gaccgaggct gggccaagat ggcgtctgtg tttcgaagcg aggagatgtg 180
47/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
tttgtcacaa ctgtttctcc aggtggaagc tgcatattgc tgtgtggctg agctcggaga 240
gctcggattg gttcagttca aagatttaaa tatgaatgtg aacagctttc aaaggaaatt 300
tgtgaatgaa gtcagaaggt gtgaatcact ggagagaatc ctccgttttc tggaagacga 360
gatgcaaaat gagattgtag ttcagttgct cgagaaaagc ccactgaccc cgctcccacg 420
ggaaatgatt accctggaga ctgttctaga aaaactggaa ggagagttac aggaagccaa 480
ccagaaccag caggccttga aacaaagctt cctagaactg acagaactga aatacctcct 540
gaagaaaacc caagacttct ttgagacgga aaccaattta gctgatgatt tctttactga 600
ggacacttct ggcctcctgg agttgaaagc agtgcctgca tatatgaccg gaaagttggg 660
gttcatagcc gggtgtgatc caacagggaa gaggatggct tcctttgagc ggttactgtg 720
gcgagtctgc cgaggaaacg tgtacttgaa gttcagtgag atggacgccc ctctggagga 780
tcctgtgacg aaagaagaaa ttcagaagca catattcatc atattttacc aaggagagca 840
gctcaggcag aaaatcaaga agatctgtga tgggtttcga gccactgtct acccttgccc 900
agagcctgcg gtggagcgca gagagatgtt ggagagcgtc aatgtgaggc tggaagattt 960
aatcaccgtc ataacacaaa cagagtctca ccgccagcgc ctgctgcagg aagccgctgc 1020
caactggcac tcctggctca tcaaggtgca gaagatgaaa gctgtctacc acatcctgaa 1080
catgtgcaac atcgacgtca cccagcagtg tgtcatcgcc gagatctggt tcccggtggc 1140
agatgccaca cgtatcaaga gggcactgga gcaaggcatg gaactaagtg gctcctccat 1200
ggcccccatc atgaccacag tgcaatctaa aacagcccct cccacattta acaggaccaa 1260
taaattcaca gctggcttcc agaatattgt tgatgcctat ggtgtcggca gctaccggga 1320
gataaaccca gccccctaca ccatcatcac tttccccttc ctgttcgctg tgatgtttgg 1380
agactgtggt catggaaccg tgatgctcct ggctgcactt tggatgattc tgaatgagag 1440
acgcttgctc tcccagaaga cagacaatga gatttggaac accttcttcc acgggcgcta 1500
tctgatccta cttatgggca tcttctccat ctacacgggt ttgatctaca atgactgctt 1560
ctccaagtcc ttgaacatct ttggctcttc ttggagtgtc caacccatgt tcagaaacgg 1620
cacatggaat actcatgtaa tggaggaaag tctatatctg cagctggacc cagccatacc 1680
aggagtgtat tttggaaatc catacccgtt tgggattgat ccgatttgga acttggcttc 1740
aaacaaactc acatttctga actcgtataa aatgaagatg tcggtgatcc tgggaattgt 1800
ccagatggtt tttggtgtca tcctcagcct tttcaatcac atatacttca gaagaactct 1860
caacatcatt ctgcaattta tccctgagat gatttttatc ctgtgtctgt ttggatacct 1920
ggttttcatg atcattttca aatggtgctg ctttgacgtc cacgtatctc agcacgcccc 1980
cagcatcctc atccacttca tcaacatgtt tctgtttaac tacagtgact cttccaacgc 2040
acccctctac aaacatcagc aagaagtcca aagtttcttt gtggttatgg ctttgatttc 2100
tgtgccgtgg atgcttctga ttaagccgtt tattcttaga gccagtcatc ggaaatccca 2160
gctgcaggca tccaggatcc aagaagatgc cactgagaac attgaaggtg atagctccag 2220
cccttctagc cgttctggcc agaggacttc tgcagatacc cacggggctc tggacgacca 2280
tggagaagag ttcaactttg gagacgtctt tgtccaccaa gccatccaca ccatcgagta 2340
ctgcctgggc tgcatttcaa acacagcctc ctacctgcgg ctctgggccc tcagcctggc 2400
tcatgcacaa ctgtctgaag tgctctggac tatggtgatg aacagcggcc ttcagacgcg 2460
aggctgggga ggaatcgtcg gggtttttat tatttttgcc gtatttgctg tcctgacagt 2520
agccatcctt ctgatcatgg agggcctctc tgctttcctg cacgccctgc gactgcactg 2580
ggttgagttc cagaacaagt tctatgtcgg ggatggttac aagttttctc cattctcctt 2640
taaacacatc ctggatggca cagccgagga gtaggctgag ggctgcacct cccacggtgg 2700
tcaccatgcc aatgaaggaa gttcagtctt gtctttgata tcagcccctg caaggcgctc 2760
aatgggaagg ttgttcttgg ctcacctgaa gcatgaaact gtgtattatt tggacgtcag 2820
cctgtggatt tgatacgact taaccacgtc agaggaagga ctttggcaag tgatattgtc 2880
ttcatgtggg gtattaattc tcaaataata aagtaattga caaatgaggg gagaatgcta 2940
aacagatgtc ttcttgcaat attttaaata ttgtatttga gaaaataaac atctgagtca 3000
ttcaaaaaaa aaaa 3014
<210> 64
<211> 1726
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3715961CB1
<400> 64
tgaaaatgcc tgctgcgtac caaggtatgt actagggcat ctggggtaag taaaaacaaa 60
cacatagagc ctgcctggag aagctcatgg tctgatggaa agataagcaa gaagagttaa 120
tttctaatca atatgataaa aaggtcagag agcagtttct gaaaaacatg tttttgagtt 180
gagtcctgaa agacaaggag atgttagtaa agcagagaag ggagaattca ttctagaaag 240
atcagacagt gtgtgggaag ggcagagtct gaaaagagca tgccccattt ggagaagcat 300
caagaagccc acgtgttaga agcaccggcc ccatgagaca aagacacagc tagagagatt 360
gactaggcca tgtcggaatg tcctcttatt ttatacatac ataagcatat agatacatat 420
agccaaagtt acctttttaa tgatcttttt tacccagtgt attctggagg tcgaatggtc 480
acatatgaac atctccgaga ggttgtgttt ggcaaaagtg aagatgagca ttatcccctt 540
48/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
tggaaatcag tcattggagg gatgatggct ggtgttattg gccagttttt agccaatcca 600
actgacctag tgaaggttca gatgcaaatg gaaggaaaaa ggaaactgga aggaaaacca 660
ttgcgatttc gtggtgtaca tcatgcattt gcaaaaatct tagctgaagg aggaatacga 720
gggctttggg caggctgggt acccaatata caaagagcag cactggtgaa tatgggagat 780
ttaaccactt atgatacagt gaaacactac ttggtattga atacaccact tgaggacaat 840
atcatgactc acggtttatc aagtttatgt tctggactgg tagcttctat tctgggaaca 900
ccagccgatg tcatcaaaag cagaataatg aatcaaccac gagataaaca aggaagggga 960
cttttgtata aatcatcgac tgactgcttg attcaggctg ttcaaggtga aggattcatg 1020
agtctatata aaggcttttt accatcttgg ctgagaatga ccccttggtc aatggtgttc 1080
tggcttactt atgaaaaaat cagagagatg agtggagtca gtccatttta aacccctaaa 1140
gatgcaaccc ttaaagatac agtgttcagt attattgaaa tatgggcatc tgcaacacat 1200
accccctatt atttctacct ctttaggaag acacctattc cacagagact gatttatagg 1260
gggcagcact ttattttttt ctggaaaccc aagttctctt tgactcctct ttttgtccaa 1320
aagtgatctg gtcggatctc acaaggccat ccaatgagac cccgcacagc attttctaaa 1380
gaagaatcga agcctgacca ctttcacctt gggcaagaag gtttggcctt tgagttgcta 1440
ttctatgctg aagagcctgc ttagaggagg agtaccagga gggagccagc atttcagatc 1500
tgaagtagac gataggaatg tggaagaaca catacatagt gcttaagaaa tacatttaac 1560
ctgttatgtc agtatttatc aatgaagttt gataattcac ttttctgtca ttgttaaagc 1620
gtacatactg taaattaaag ggaggtgaat ggaaattaat gaataaacat tttgagtttc 1680
cctagtgttg aaggaaggtg tactttttct tgtcagaaag ataaaa 1726
<210> 65
<211> 899
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5108194CB1
<400> 65
gcggcggcga cgcgtccggg ccggtgaggg ggcgcggggg gcgccggggg ggcccaagcg 60
tcagcgcccg cgcctgtcgg gctgaactga ggaccgagtc tcctgccatt ccgagcaggc 120
ctggtatggg taatggtgtg aaggaaggcc cggtgcgatt gcatgaggat gctgaggctg 180
tcctgtcctc gtccgtctca tcaaagcgtg accacaggca agtgctcagc tccctgctgt 240
ctggggccct ggctggtgcc cttgccaaaa cagcggtagc tcccctggac cgaaccaaaa 300
tcatcttcca agtgtcttca aaaagatttt ctgccaagga ggccttccgg gtcctctact 360
acacctacct caacgaggga tttctcagct tgtggcgcgg gaactcggcc accatggtgc 420
gcgtggtgcc ctacgccgcc atccagttca gcgcacacga ggagtacaag cgcatcctgg 480
gcagctacta tggcttccgt ggagaagccc tgcccccttg gcctcgcctc ttcgccggcg 540
cactggctgg aacgacagcc gcttcactga cctaccccct ggacctggtc agagcgcgga 600
tggccgtaac cccgaaggaa atgtacagca acatctttca tgtcttcatc cgcatctcga 660
gagaagaggg gctgaagact ctctaccatg gatttatgcc caccgtgctg ggggtcattc 720
cctacgctgg cctgagcttc ttcacctatg agacgctcaa gagcttgcac agagagtaca 780
gcggccgcaa gcttattccc tttagtgagg gttaatttta gcttggcact ggccgtcgtt 840
ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcaca 899
<210> 66
<211> 643
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5503122CB1
<400> 66
ctttaagctg tagctgtggt ttctgcagca attttgtttt tgccttgaaa gaggtgctct 60
ggattatcac acctccatgt atgacaattt gtacctgcat ggaattgaag actcggaggc 120
tggttcagcg gattcctaca caagcaggcc gtctgactcc gatgtctctt tggaagagga 180
ccgggaagca attcgacagg agagagaaca gcaagcagct atccagcttg agagagcaaa 240
gtccaaacct gtagcatttg ccgtgaagac aaatgtgagc tactgcggcg ccctggacga 300
ggatgtgcct gttccaagca cagctatctc ctttgatgct aaagactttc tacatattaa 360
agagaaatat aacaatgatt ggtggatagg aaggctggtg aaagagggct gtgaaattgg 420
cttcattcca agtccactca gattggagaa catacggatc cagcaagaac aaaaaagagg 480
acgttttcac ggagggaaat caagtggaaa ttcttcttca agtcttggag aaatggtatc 540
tgggacattc cgagcaactc ccacatcaac aggtgagggt tgtagttaaa ctctttttca 600
tacactgtat tccttttaaa aatatttgaa cacacatgca agc 643
49/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
<210> 67
<211> 2574
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5517972CB1
<400> 67
gcgcctccca cgccggccgc cgccgacgtg atcgctcggg cgcgccgggc gtggttgggg 60
gaaggggttg tgccgcgcga cggtctgcgt gctgtgccca ctcaaaaggt tccgggcgcg 120
caggacggaa gacgcagtgc tcgccactcc cactgagatt gagagacgcg gcaaggaggc 180
agcctgtgga ggaactgggt aggatttagg aacgcaccgt gcacatgctt ggtggtcttg 240
ttaagtggaa actgctgctt tagagtttgt ttggaaggtc cgggtgactc atcccaacat 300
ttacatcctt aattgttaaa gcgctgcctc cgagcgcacg catcctgaga tcctgagcct 360
ttggttaaga ccgagctcta ttaagctgaa aagataaaaa ctctccagat gtcttccagt 420
aatgtcgaag tttttatccc agtgtcacaa ggaaacacca atggcttccc cgcgacagct 480
tccaatgacc tgaaggcatt tactgaagga gctgtgttaa gttttcataa catctgctat 540
cgagtaaaac tgaagagtgg ctttctacct tgtcgaaaac cagttgagaa agaaatatta 600
tcgaatatca atgggatcat gaaacctggt ctcaacgcca tcctgggacc cacaggtgga 660
ggcaaatctt cgttattaga tgtcttagct gcaaggaaag atccaagtgg attatctgga 720
gatgttctga taaatggagc accgcgacct gccaatttca aatgtaattc aggttacgtg 780
gtacaagatg atgttgtgat gggcactctg acggtgagag aaaacttaca gttctcagca 840
gctcttcggc ttgcaacaac tatgacgaat catgaaaaaa acgaacggat taacagggtc 900
attcaagagt taggtctgga taaagtggca gactccaagg ttggaactca gtttatccgt 960
ggtgtgtctg gaggagaaag aaaaaggact agtataggaa tggagcttat cactgatcct 1020
tccatcttgt tcttggatga gcctacaact ggcttagact caagcacagc aaatgctgtc 1080
cttttgctcc tgaaaaggat gtctaagcag ggacgaacaa tcatcttctc cattcatcag 1140
cctcgatatt ccatcttcaa gttgtttgat agcctcacct tattggcctc aggaagactt 1200
atgttccacg ggcctgctca ggaggccttg ggatactttg aatcagctgg ttatcactgt 1260
gaggcctata ataaccctgc agacttcttc ttggacatca ttaatggaga ttccactgct 1320
gtggcattaa acagagaaga agactttaaa gccacagaga tcatagagcc ttccaagcag 1380
gataagccac tcatagaaaa attagcggag atttatgtca actcctcctt ctacaaagag 1440
acaaaagctg aattacatca actttccggg ggtgagaaga agaagaagat cacagtcttc 1500
aaggagatca gctacaccac ctccttctgt catcaactca gatgggtttc caagcgttca 1560
ttcaaaaact tgctgggtaa tccccaggcc tctatagctc agatcattgt cacagtcgta 1620
ctgggactgg ttataggtgc catttacttt gggctaaaaa atgattctac tggaatccag 1680
aacagagctg gggttctctt cttcctgacg accaaccagt gtttcagcag tgtttcagcc 1740
gtggaactct ttgtggtaga gaagaagctc ttcatacatg aatacatcag cggatactac 1800
agagtgtcat cttatttcct tggaaaactg ttatctgatt tattacccat gaggatgtta 1860
ccaagtatta tatttacctg tatagtgtac ttcatgttag gattgaagcc aaaggcagat 1920
gccttcttcg ttatgatgtt tacccttatg atggtggctt attcagccag ttccatggca 1980
ctggccatag cagcaggtca gagtgtggtt tctgtagcaa cacttctcat gaccatctgt 2040
tttgtgttta tgatgatttt ttcaggtctg ttggtcaatc tcacaaccat tgcatcttgg 2100
ctgtcatggc ttcagtactt cagcattcca cgatatggat ttacggcttt gcagcataat 2160
gaatttttgg gacaaaactt ctgcccagga ctcaatgcaa caggaaacaa tccttgtaac 2220
tatgcaacat gtactggcga agaatatttg gtaaagcagg gcatcgatct ctcaccctgg 2280
ggcttgtgga agaatcacgt ggccttggct tgtatgattg ttattttcct cacaattgcc 2340
tacctgaaat tgttatttct taaaaaatat tcttaaattt ccccttaatt cagtatgatt 2400
tatcctcaca taaaaaagaa gcactttgat tgaagtattc aatcaagttt ttttgttgtt 2460
ttctgttccc ttgccatcac actgttgcac agcagcaatt gttttaaaga gatacatttt 2520
tagaaatcac aacaaactga attaaacatg aaagaaccca agaaaaaaaa aaaa 2574
<210> 68
<211> 1571
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5593114CB1
<400> 68
ggcgcggcga cagctagggt tcacggccac tggggcagag gagccgcgag aagatgtggg 60
tttttggtta cgggtccctg atctggaagg tggatttccc ctatcaggac aagctggtcg 120
gatacatcac caactacagc aggcgcttct ggcagggcag cacggaccac cgcggggtcc 180
ccggcaagcc tggaagagtt gtgactcttg ttgaagatcc tgcgggatgt gtatggggtg 240
50/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
ttgcttacag attgccagta ggaaaggaag aagaagtaaa agcatacctt gacttcagag 300
aaaaaggagg ctacagaacc acaacagtca ttttttatcc aaaagatccc acaacaaaac 360
cattcagtgt attgctatat attggaacat gtgataatcc tgattatctt ggtcctgcac 420
ctctggaaga cattgctgaa caaattttta atgcagctgg tccaagtgga agaaatacag 480
aatatctttt tgaacttgca aattctatta ggaaccttgt gccagaagaa gcagatgagc 540
atcttttcgc tttggaaaaa ttagtaaagg aacgtttaga agggaaacag aacctcaatt 600
gcatataatt tagtcttcag agaattaact tcagtgcaca atgacaatat gatttggaaa 660
tacgtttact taaagatctt atttttaatg tagtgaggat attatttaaa cttttatttt 720
aactggaaat gtcctgaaac acatatttaa aatattggga tacagtgaaa gaaaaattca 780
aattttaata acataaagat ttcctaactt tatgttattg aacacttact cactagaagt 840
gagttcttta gaaaaataca gtgaaggact cagttcagtc ttgtttttat cagagtgata 900
atcatcctgt ttcacatccc aatactattt tgaaattcta aacaattaaa ccaaaattcc 960
aataaatata aggttatgcc ttcaatatat tcctatacaa ttctgtaacc atggtttaaa 1020
atacacaagc ttaaaataac atgcttagaa atacacaata atatgaacag tatttcagcc 1080
ttaattgtga atttccttgt tattcaagta ttaaatgaaa tcttttgagt ttttagccaa 1140
aaattggcat ttttaaaata cgaaaatttc cttggaatta taatgtactg tacctcttct 1200
tttttaaata aaggcatttt actatatgga aaataactca ctaaagcata aattacatta 1260
tacaaatcat gatcactaat gatgtagtct gtcattcact ttgtattaat cttataccaa 1320
aactgaaaaa gatgggctga tactacaaat taatggcaca tataatgaaa atttagtttt 1380
taaaacagct tttggaattc tttgtctgtc actatctcaa tttgtgtgtg tgtgtgtgta 1440
tatacataaa tatacatata aaattttttt tttctttgca gcctgcgtct ggccatccca 1500
caggctggaa agtgtaacct ctggcagaag ccaagaacag gcacctcctg gaattataat 1560
tttgttttgt t 1571
<210> 69
<211> 1549
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 044775C81
<400> 69
cgcgcttagg caggcggtgg cgcggctgga gtgccgcggg gagggctgtg ccggttgctt 60
tctgcagccg catctcggcc agctctcctc gccgtccccg gggcgctgtg cgtctccagt 120
ccgggaccga agccgcctgc cgtagcgggc ggccagatcc gcgtcccgcc tcagcggccg 180
gaggacatgc gggagagaga atgagccaga gggacacgct ggtgcatctg tttgccggag 240
gatgtggtgg tacagtggga gctattctga catgtccact ggaagttgta aaaacacgac 300
tgcagtcatc ttctgtgacg ctttatattt ctgaagttca gctgaacacc atggctggag 360
ccagtgtcaa ccgagtagtg tctcccggac ctcttcattg cctaaaggtg atcttggaaa 420
aagaagggcc tcgttccttg tttagaggac taggccccaa tttagtgggg gtagcccctt 480
ccagagcaat atactttgct gcttattcaa actgcaagga aaagttgaat gatgtatttg 540
atcctgattc tacccaagta catatgattt cagctgcaat ggcaggcatt tactgtacat 600
ttctcccgag aaaagagtga gatcgtgtca tctcatgctc cccatccgca ggtcacttcc 660
tgtagaaata tggactaact taaacctcgt tttactgcaa tcacagcaac caaccccatt 720
tggcttataa agactcggtt acagcttgat gcaaggaacc gcggggaaag gcgaatgggt 780
gcttttgaat gtgttcgtaa agtgtatcag acagatggac taaaaggatt ttataggggc 840
atgtctgctt catatgctgg tatatcagag actgttatcc attttgttat ttatgaaagt 900
ataaaacaaa aactactgga atataagact gcttctacaa tggaaaatga tgaagagtct 960
gtgaaagaag catcagattt tgtgggaatg atgctagctg ctgccacctc aaaaacttgt 1020
gccacaacta tagcatatcc acatgaagtt gtaagaacaa gactacgtga agagggaaca 1080
aaatacagat ctttttttca gactctatct ttgcttgttc aagaagaagg ttatgggtct 1140
ctttatcgtg gtctgacaac tcatctagtg agacagattc caaacacagc cattatgatg 1200
gccacctatg aattggtggt ttacctactc aatggatagc agcacgagga ctgctgtact 1260
gcaaaaaaag aagaccaaaa gattacagtg gaccatggga tacagaagcc agcatggcag 1320
acagaagaaa aatagtttgg gaacatgtaa ctattctaag tggaagtttt gttgtaggaa 1380
ttatagtaat cacaccacat tacttggcct ttcggtaatg tgaaaaaaaa aaaaaaacct 1440
cagagcctcc aaggaaatgc ctttagaagc actcctctct caaaattgcc attttctcta 1500
ccatgtcccc cagacacagt tgggttttgt tgatttatgg cagtcttct 1549
<210> 70
<211> 2237
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
51 /60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
<223> Incyte ID No: 116588CB1
<400> 70
gtcactggtg ccacggggcc tcagcgcact tctgtcttaa gctcctgggc ctccttattt 60
tcccctttgc gatcgattcc agccacacct gtggatgttg ctagttactc cgcgtccgga 120
acgtggaggt cgagggactg agctgggcga gttttgtggc actcctttgc tcttcagcag 180
ctatttttgc tatgataatc ctgctgccct tcagactcaa gttaaacgag atatgcaagt 240
gaataccacg aaattcatgc tgctgtatgc ctggtattct tggcccaatg tagttttgtg 300
tttctttggt ggctttttga tagaccgagt atttggaata cgatggggca caatcatttt 360
tagctgcttt gtttgcattg gacaggttgt ttttgccctg ggtggaatat ttaatgcttt 420
ttggctgatg gaatttggaa gatttgtatt tgggattggt ggcgagtcct tagcagttgc 480
ccagaataca tatgctgtga gctggtttaa aggcaaagaa ttaaacctgg tgtttggact 540
tcaacttagc atggctagaa ttggaagtac agtaaacatg aacctcatgg gatggctgta 600
ttctaagatt gaagctttgt taggttctgc tggtcacaca accctcggga tcacacttat 660
gattgggggt gtaacgtgta ttctttcact aatctgtgcc ttggctcttg cctacttgga 720
tcagagagca gagagaatcc ttcataaaga acaaggaaaa acaggtgaag ttattaaatt 780
aactgatgta aaggacttct ccttacccct gtggcttata tttatcatct gtgtctgcta 840
ttatgttgct gtgttccctt ttattggact tgggaaagtt ttctttacag agaaatttgg 900
attttcttcc caggcagcaa gtgcaattaa cagtgttgta tatgtcatat cagctcccat 960
gtccccggtg tttgggctcc tggtggataa aacagggaag aacatcatct gggttctttg 1020
cgcagtagca gccactcttg tgtcccacat gatgctggcc tttacgatgt ggaacccttg 1080
gattgctatg tgtcttctgg gactctccta ctcattgctt gcctgtgcat tgtggccaat 1140
ggtggcattt gtagttcctg aacatcagct gggaactgca tatggcttca tgcagtccat 1200
tcagaatctt gggttggcca tcatttccat cattgctggt atgatactgg attctcgggg 1260
gtatttgttt ttggaagtgt tcttcattgc ctgtgtttct ttgtcacttt tatctgtggt 1320
cttactctat ttggtgaatc gtgcccaggg tgggaaccta aattattctg caagacaaag 1380
ggaagaaata aaattttccc atactgaatg agaagttaaa atgaatgtgt catgagaatg 1440
ggcttaacac atcgttggtt tgaaaacttc catttttaaa aatttagagt ttagtcatta 1500
gaaaaaataa tggactggaa agttatattt atatccaaat atacctattt caaagtgtat 1560
ttgtgaggcc tgttttagcc tgtgtctttt gtattgtgtg ttgctaaaga attctacttt 1620
tagtaggcta atcaacaatg aaagggttag aaaattgctg tggaacatcc aggtgaactt 1680
caggaaagac agtgaaaaat ggaaaacgtt ggagcttctg ttgagataat cttcattagg 1740
tatatatctt agggatacag ccttttcttt atcttatagc aggaaaaaaa aacttttgag 1800
ggaaatagaa gggctgcgtt acacaaaata aacaatggca ttgtcatagg ccttcctttt 1860
actagtaggg cataatgcta gggaatatgt gaagatgttt ttatgaagtc tctttctgat 1920
cacgaacaat agcttgcgct ctactctgta gttatgtgga ttgccgagca atgacccttt 1980
tcaatttctt atttctgtgt tactgaggac cctaatcact tagggatgta attttatagt 2040
ataaactttc tgtacagttt ttcttatagt ctaataagta aaaagtgtcc ttcaaattat 2100
gataattgcc tatgtacatg gataaattaa aacactgcac acggagtaaa aaaaaaaaaa 2160
aaaaaaaaaa aaaaaatgag cggccgcaag cttattccct ttagtgaggg ttaattttag 2220
cttggcactg gccgtcg 2237
<210> 71
<211> 1114
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 875369CB1
<400> 71
tgggaacgga gcagccccgg gggccccctt gaggcggcga ggccgcgaag ggcgcggggc 60
tggagggtag gagagcgcgg gaaagcgccc cagacgccac tcgcggcgga cggcggccag 120
ttcccagggg tttggagccg gcccgcggcg ccatggctca cgtcggctcc cgcaagcgct 180
cgaggagtcg cagccggtcc cggggacggg ggtcggaaaa gagaaagaag aagagcagga 240
aagacacctc gaggaactgc tcggcctcca catcccaagg tcgcaaggcc agcacggccc 300
ctggggcgga ggcctcacct tctccctgca tcacagagag aagcaagcag aaggcccgga 360
ggagaacaag atccagctcc tcctcctctt cttccagttc ttctagctcc tcttcttcct 420
cctcgtcctc ctcctcttcc tccagtgatg gccggaagaa gcgggggaag tacaaggaca 480
agaggaggaa gaagaagaag aagaggaaga agctgaagaa gaagggcaag gagaaggcgg 540
aagcacagca ggtggaggct ctgccgggcc cctcgctgga ccagtggcac cgatcagctg 600
gggaggaaga ggatggccca gtcctgacgg atgagcagaa gtcccgaatc caggccatga 660
agcccatgac caaggaggag tgggatgccc ggcagagcat catccgcaag gtggtggacc 720
ctgagacggg gcgcaccagg cttattaagg gagatggcga ggtcctagag gaaatcgtaa 780
ccaaagaacg acacagagag atcaacaagc aagccacccg aggggactgc ctggccttcc 840
agatgcgagc tgggttgctt ccctgagggc ccccgctggc caaggcctgt ggacgacgct 900
ggcggcccag cctgggcagg tttcagggtg ccagtgggaa gcctgatggg tgctggtggc 960
52/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
ctttcccccg tggattggtc tctggcccag cccagtctct tctcaggggc agggggtgga 1020
ggttggggtc accggcctgc ttggcacccc catctgaaag agcagcactt ctcagctatt 1080
aaaggccccc tggatagaca aaaaaaaaaa aaaa 1114
<210> 72
<211> 998
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1325518CB1
<400> 72
gactaaaaaa gccatgtatt ctttcgtttc tctctaaaag aagaaaaata taatttaaaa 60
atacattgcg tattttctaa aacaataaat ttatagtgtt aatattcata gggtcaatca 120
aaatgaagct tctcctttgg gcctgcattg tatgtgttgc ttttgcaagg aagagacggt 180
tccccttcat tggtgaggat gacaatgacg atggtcaccc acttcatcca tctctgaata 240
ttccttatgg catacggaat ttaccacctc ctctttatta tcgcccagtg aatacagtcc 300
ccagttaccc tgggaatact tacactgaca cagggttacc ttcgtatccc tggattctaa 360
cttctcctgg attcccctat gtctatcaca tccgtggttt tcccttagct actcagttga 420
atgttcctcc tctccctcct aggggtttcc cgtttgtccc tccttcaagg tttttttcag 480
cagctgcagc acccgctgcc ccacctattg cagctgagcc tgctgcagct gcacctctta 540
cagccacacc tgtagcagct gagcctgctg caggggcccc tgttgcagct gagcctgctg 600
cagaggcacc tgttggagct gagcctgctg cagaggcacc tgttgcagct gagcctgctg 660
cagaggcacc tgttggagtg gagccagctg cagaggaacc ttcaccagct gagcctgcta 720
cagccaagcc tgctgcccca gaacctcacc cttctccctc tcttgaacag gcaaatcagt 780
gaaattctct agaagagtac catgggttca tttctatact gatgcagaaa taagtgaaat 840
ctacaaaagt tttctttctt ttccaaagac tatttcattc tgttgtattc agagtattca 900
tctcactaca ttgatttgtt tgtggtagtt tttccttgga cttaatttat attgaaaaaa 960
cattgataat taaataaata aaatagataa tttagaca 998
<210> 73
<211> 2348
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2060987CB1
<400> 73
gggccagccg gctcgcccgg gggccatggc agcagcggct actgcagccg agggggtccc 60
cagtcggggg cctcccgggg aagtcatcca tctgaatgtg ggaggcaaga gattcagtac 120
ctctcgccag actctcacct ggatcccaga ctccttcttc tccagtcttc tgagcggacg 180
catctcgacg ctgaaagatg agaccggagc aatcttcatc gacagggacc ctacagtctt 240
cgcccccatc ctcaacttcc tgcgcaccaa agagttggat cccaggggtg tccacggttc 300
cagcctcctc catgaagccc agttctatgg gctcactcct ctggttcgtc gcctgcagct 360
tcgagaggag ttggatcgat cttcttgtgg aaacgtcctc ttcaatggtt acctgccgcc 420
accagtgttc ccagtgaagc ggcggaaccg gcacagccta gtggggcctc agcagctagg 480
aggacggcca gcccctgtcc gacggagcaa cacgatgccc cccaaccttg gcaatgcagg 540
gctgctgggc cgaatgctgg atgagaaaac ccctccctca ccctcaggac aacctgagga 600
gccggggatg gtgcgcctgg tgtgtggaca ccataattgg atcgctgtgg cctataccca 660
gtttctagtc tgctacaggt tgaaggaagc ctctggctgg cagctggtgt tttccagccc 720
ccgcctggac tggcccatcg aacgactggc gctcacagcc cgggtgcatg gtggggcttt 780
gggtgaacat gacaagatgg tggcagcagc caccggcagc gagatcctgc tatgggctct 840
gcaggcggaa ggcggtggct ccgagatagg ggtctttcat ctgggggtgc ctgtggaggc 900
cttgttcttc gtcgggaacc agctcattgc tacaagccac acagggcgca tcggggtgtg 960
gaatgccgtc accaagcact ggcaggtcca ggaggtgcag cccatcacca gttatgacgc 1020
ggcaggctcc ttcctcctcc tgggctgcaa caacggctcc atttactacg tggatgtgca 1080
gaagttcccc ttgcgcatga aagacaacga cctccttgtc agcgagctct atcgggaccc 1140
agcggaggat ggggtcaccg ccctcagtgt ctacctcacc cccaagacca gtgacagtgg 1200
gaactggatc gagatcgcct atggcaccag ctcagggggc gtgcgggtca tcgtgcagca 1260
cccggagact gtgggctcgg ggcctcagct cttccagacc ttcactgtgc accgcagccc 1320
tgtcaccaag atcatgctgt cggagaagca cctcatctca gtctgtgccg acaacaacca 1380
cgtgcggaca tggtctgtga ctcgcttccg cggcatgatt tccacccagc ccggctccac 1440
cccactcgct tcctttaaga tcctggctct ggagtcggca gatgggcatg gcggctgcag 1500
tgctggcaat gacattggcc cctacggtga gcgggacgac cagcaagtgt tcatccagaa 1560
53/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
ggtggtgccc agtgccagcc agctcttcgt gcgtctctca tctactgggc agcgggtgtg 1620
ctccgtgcgc tccgtggacg gctcacccac gacggccttc acagtgctgg agtgcgaggg 1680
ctcccggcgg ctcggctctc ggccccggcg ctacctgctc actggccagg ccaacggcag 1740
cttggccatg tgggacctaa ccaccgccat ggacggcctc ggccaggccc ctgcaggtgg 1800
cctgacggag caagagctga tggaacagct ggaacactgt gagctggccc cgccggctcc 1860
ttcagctccc tcatggggct gtctccccag cccctcaccc cgcatctccc tcaccagcct 1920
ccactcagcc tccagcaaca cctccttgtc tggccaccgt gggagcccaa gccccccgca 1980
ggctgaggcc cggcgccgtg gtgggggcag ctttgtggaa cgctgccagg aactggtgcg 2040
gagtgggcca gacctccgac ggccacccac accagccccg tggccctcca gcggtctcgg 2100
cactcccctc acacctccca agatgaagct caatgaaact tccttttgaa caacgcagct 2160
gccatgatgc cttgggatgc cctggtcctg ggggactcag gtgcctccct gattcctgtg 2220
ggaaccccgg gttcagggcc agggcctcct tggaataaat ggttattgtt actaggtccc 2280
caccttccct cttttctgga agccaaagtc accctcccca ataaagtcct cactgccaaa 2340
aaaaaaaa 2348
<210> 74
<211> 1139
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2172064CB1
<400> 74
ctcgagctgg gatgtgtggc aggttcctgc ggcggctgct ggcggaggag agccggcgct 60
ccacccccgt ggggcgcctc ttgcttcccg tgctcctggg attccgcctt gtgctgctgg 120
ctgccagtgg gcctggagtc tatggtgatg agcagagtga attcgtgtgt cacacccagc 180
agccgggctg caaggctgcc tgcttcgatg ccttccaccc cctctccccg ctgcgttcct 240
gggtcttcca ggtcatcttg gtggctgtac ccagcgccct ctatatgggt ttcactctgt 300
atcacgtgat ctggcactgg gaattatcag gaaaggggaa ggaggaggag accctgatcc 360
agggacggga gggcaacaca gatgtcccag gggctggaag cctcaggctg ctctgggctt 420
atgtggctca gctgggggct cggcttgtcc tggagggggc agccctgggg ttgcagtacc 480
acctgtatgg gttccagatg cccagctcct ttgcatgtcg ccgagaacct tgccttggta 540
gtataacctg caatctgtcc cgcccctctg agaagaccat tttcctaaag accatgtttg 600
gagtcagcgg tttctgtctc ttgtttactt ttttggagct tgtgcttctg ggtttgggga 660
gatggtggag gacctggaag cacaaatctt cctcttctaa atacttccta acttcagaga 720
gcaccagaag acacaagaaa gcaaccgata gcctcccagt ggtggaaacc aaagagcaat 780
ttcaagaagc agttccagga agaagcttag cccaggaaaa acaaagacca gttggaccca 840
gagatgcctg agttggagat gaactttggc caactttcct catcaccata cttaaaatcc 900
tgtccaagga ggagcttatt caccattttc tatacatgtg acatatgtag cagcataacc 960
gacaactggg actgcgctgc cttgactcca cctctacata caatgactca gctaaccaga 1020
ctaataaaag ccatgtttgc accattgctc agggaggcat tgctttgggg aattattccc 1080
agtgtcctcc ttacttatcg caagtaataa aaatcccctg ggaaatcctc aaaaaaaaa 1139
<210> 75
<211> 863
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2219267CB1
<400> 75
tcagagctag cttccgccgc agcctggcac ctcaggtgcc gaccttctac ctgccctact 60
tcctggaggc cggcctggag gcggcagcct tggtcttcct gctcctgacg gcagattgct 120
gtggacgccg ccccgtgctg ctgctgggca ccatggtcac aggcctggca tccctgctgc 180
tcctcgctgg ggcccagtat ctgccaggct ggactgtgct gttcctctct gtcctggggc 240
tcctggcctc ccgggctgtg tccgcactca gcagcctctt cgcggccgag gtcttcccca 300
cggtgatcag gggggccggg ctgggcctgg tgctgggggc cgggttcctg ggccaggcag 360
ccggccccct ggacaccctg cacggccggc agggcttctt cctgcaacaa gtcgtcttcg 420
cctcccttgc tgtccttgcc ctgctgtgtg tcctgctgct gcctgagagc cgaagccggg 480
ggctgcccca gtcactgcag gacgccgacc gcctgcgccg ctccccactc ctgcggggcc 540
gcccccgcca ggaccacctg cctctgctgc cgccctccaa ctcctactgg gccggccaca 600
cccccgagca gcactagtcc tgcctggtgg ccctgggagc caggatggga ccaaagtcaa 660
ggcctggggc atggctgagt accccagacg tctggtccag ggcagacaca ttcctctcag 720
aagcccgtgt ctcagtgcag gtggagccgt ggggacagcg tgaaggtgtc tccagccagg 780
54/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
ccccaggcac tgggaggccc tgggtctccc cccagccaca cccagtaggt gtggaggata 840
aaggcttctg tggaaaaaaa aaa 863
<210> 76
<211> 1322
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2308629CB1
<400> 76
ccggggggcc ggcgcgcggg gaggccgggg cctgcaggcc cccggtacga caagatccgg 60
actccggccc ggactacgag gcgctgccgg ctggagccac tgtcaccacg cacatggtgg 120
caggcgccgt ggcagggatc ctggagcact gcgtgatgta ccccatcgac tgcgtcaaga 180
cccggatgca gagtctacag cctgacccag ctgcccgcta tcgcaatgtg ttggaggccc 240
tctggaggat tataagaacg gagggcctat ggaggcccat gagggggctg aacgtcacag 300
caacaggcgc agggcctgcc cacgcccttt attttgcctg ctacgaaaag ttaaaaaaga 360
cattgagtga tgtaatccac cctgggggca atagccatat tgccaatggt gcggccgggt 420
gtgtggcaac attacttcat gatgcagcca tgaaccctgc ggaagtggtc aagcagagga 480
tgcagatgta caactcacca taccaccggg tgacagactg tgtacgggca gtgtggcaaa 540
atgaaggggc cggggccttt taccgcagct acaccaccca gctgaccatg aacgttcctt 600
tccaagccat tcacttcatg acctatgaat tcctgcagga gcactttaac ccccagagac 660
ggtacaaccc aagctcccac gtcctctctg gagcttgcgc aggagctgta gctgccgcag 720
ccacaacccc actggacgtt tgcaaaacac tgctcaacac ccaggagtcc ttggctttga 780
actcacacat tacaggacat atcacaggca tggctagtgc cttcaggacg gtatatcaag 840
taggtggggt gaccgcctat ttccgagggg tgcaggccag agtaatttac cagatcccct 900
ccacagccat cgcatggtct gtgtatgagt tcttcaaata cctaatcact aaaaggcaag 960
aagagtggag ggctggcaag tgaagtagca ctgaacgaag ccaggggttc agatgacact 1020
gctgcatcct ggtcacattc tctgtctcct ggaatgctcc cacctcaagt ggagttagaa 1080
ggaaggtaga ggggctctcc cccaggattt tggtgttttg actaacacca gttcctgcca 1140
acctctgttg ccaccacctt tccttccagg ccctaagcac gtgcagcaaa gcacaccaca 1200
gcacctttga taacctctct ccatcctggg cctgatgacc tgctctagac tgttatagag 1260
ggataagcag ctcattcccc tggttcctaa taaaaagcct ttaaattaaa aaaaaaaaaa 1320
as 1322
<210> 77
<211> 1869
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2660038CB1
<400> 77
cagggacgct cattcctggc cagagtcctg acttctttct cggatcgaga tctcgttgct 60
ggctcgagga aatcaccggc tcttctcccg gatccttttc ttctctcact tgctggcttt 120
cttcttcctg cctccctggc ttccatctcc caaccccgcg attccctcct ctactcccgt 180
gctcccgtcg cccgccatcc tgagccatcc cacctgcaac cttctgtctt ttgcccctcc 240
ttgacctcag agggtcctgc cttaagcttc tcaccagaat ctcctagatt tctatctctt 300
ccctgcttgc cagctcttga ctcccaaatt ccagctgacg tttgaccact tgacatttga 360
ccctgacacc cttgactgca aatctaaatt cttatcttct gcaacctgta ctgctgaaac 420
aggccttccc cctgtcttcc aaccccgctt tctgacaccc atttttactt tcttactctt 480
gggtcagttc ctgctacagc tatcccacca tggacttctt gatgagtggc ctggcagcct 540
gcggggcctg tgtattcacc aatcccctgg aggtggtgaa gaccaggatg cagttgcaag 600
gagaactgca ggcccctggc acataccagc ggcactaccg aaatgtcttc catgccttca 660
tcaccatcgg caaggtggat ggccttgctg ccctgcagaa aggcctggcc cccgccctct 720
tgtaccagtt cctgatgaat ggcatccgac tgggcaccta tgggctggct gaggctgggg 780
gctacctgca cacagccgaa gcgacccaca gtcctgcccg cagcgcagca gctggggcca 840
tggctggggt catgggagcc tacttgggga gccccatcta catggtgaag acacacctgc 900
aggcacaggc agcctcagaa attgctgtag ggcaccagta taagcatcag ggcatgtttc 960
aggcgctaac cgagattggc cagaaacatg gtctggtggg gttatggcgt ggggctctgg 1020
gcggcctgcc ccgagttatc gtcggttcct ccacccagct gtgcaccttc tcatccacca 1080
aggacctcct gagccagtgg gagatctttc ctccccagag ctggaagttg gcgctggtgg 1140
ctgccatgat gagtggcatt gcagttgtct tggccatggc accctttgat gtggcctgca 1200
caaggctcta caaccagccc acagatgcac agggcaaggg cctcatgtac cgggggatac 1260
55/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
tggacgctct gctgcagaca gctcggaccg agggcatttt tggcatgtac aagggtatag 1320
gtgcctccta cttccgcctc ggcccccaca ccatcctctc cctcttcttc tgggaccagc 1380
tgcgctccct ctactacaca gacactaaat aacagccgct ttcccagtct ccaccaaatg 1440
agcactcctt ggccacttgt gcctccacca ctatgtcctg gtgactactg attaggtgac 1500
ctttcatcca tccatggggg acagccaacc ccactcccca tctgttctca gggttgaatc 1560
actacaagag atgagtttcc cttctttcct tgggtgttgc tttaaacctt ccctacccat 1620
tccctgggta actcacaccc ctctctcagg gctgaacgag tcatcccaaa gtgtatttcc 1680
tcccactcac cactgccacc cttgagtccc tcctgctccc atgcacagtt ttaaactcct 1740
ccctccaaaa ccaaagggaa tcgagagacc caattcccag gcgtctggga cccaggtgtc 1800
ctgttagatt caaaggcaca gagattatat tgattataaa gcaagtttat tctgaaaaaa 1860
aaaaaaaaa 1869
<210> 78
<211> 1881
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2670745CB1
<400> 78
gaagaaccga gcttggctgt gtttatctcg ttggggacta aggcgtcggt tggcgcgcaa 60
cgggttctag gctgcaggca gctcgaggac ccgcggcccc gccccggctc ggcctggcag 120
atagcagagg cagcaggccg tgccgggggg gcatgttgct gtaaccagtg gcccagggga 180
tgttacggtg gacagtgcac ctggagggcg ggccccgcag ggtgaaccat gctgcagtgg 240
ctgtcgggca tcgggtatac tccttcgggg gttactgctc tggtgaagac tatgagacac 300
tgcgtcagat agatgtgcac attttcaatg cagtgtcctt gcgttggaca aagctgcccc 360
cggtgaagtc tgccatccgt gggcaagctc ctgtggtacc ctacatgcgc tatggacact 420
caaccgtcct catcgacgac acagtcctcc tttggggcgg gcggaatgac accgaagggg 480
cctgcaatgt gctctatgcc tttgacgtca atacgcacaa gtggttcaca ccccgagtgt 540
cagggacagt tcctggggcc cgggatggac attcagcctg tgtcctaggc aagatcatgt 600
acatttttgg gggctacgag cagcaggcgg actgtttttc caatgacatt cacaagctag 660
ataccagcac catgacatgg actcttatct gtacaaaggg cagccctgca cgctggaggg 720
acttccactc agccacaatg ctgggaagtc acatgtatgt ctttgggggc cgtgccgacc 780
gctttgggcc attccattcc aacaatgaga tttactgcaa ccgcattcga gtctttgaca 840
ccagaactga ggcttggctg gactgtcccc cgactccagt gctgcctgag gggcgccgga 900
gccactcggc ctttggctac aatggggagc tgtacatctt tggtggttat aatgcaaggc 960
tgaaccggca cttccatgac ctctggaagt ttaatcctgt gtcctttacc tggaaaaaga 1020
ttgaaccgaa ggggaagggg ccatgtcccc gccggcgcca gtgctgctgt attgttggtg 1080
acaagattgt cctctttggg ggtaccagtc catctcctga ggaaggcctg ggagatgaat 1140
ttgaccttat agatcattct gacttacaca ttttggactt tagccctagt ctgaagactc 1200
tgtgcaaact ggccgtgatt cagtataacc tagaccagtc ctgtttgcct catgatatca 1260
ggtgggagct gaatgccatg accaccaaca gcaatatcag tcgccccatc gtctcctccc 1320
atgggtagga ggaagtttct gccacctccc ctcctgagcc tgctgtcatc ttcactgccc 1380
ctgcccatct gtcacccacc tgctcctttg acccctggac ttggtatacc tccatgtgga 1440
gttgttgggc gagaggtgtt ctctgtgctg tgaattcagt ggggagctgt agcggggtgg 1500
gggctaggtt cctcccccct tgggccgagg gccccttccc cttggtgctc tgtccccatc 1560
cacctccttt cagctgctcc tgggcctcag ctctgcccag ggccagccag gttctgctgg 1620
gaagggaagg gaatggggag aagggagaag caagcagtgt ctgagcctca ggagcttccc 1680
cctccccctt tgcctatccc ctcccctctg cttgagcctt gagccttgac tgggagctga 1740
aaggagttgc agctgttggc atgagacctc cttctccccg tcttggggag gtggggacca 1800
gcagataaat cccacccttc cttgagctgt cgctgtactc tgaagttcag ccagctcaga 1860
ttttataaaa attaattaaa a 1881
<210> 79
<211> 2004
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2676443CB1
<400> 79
aatcgcaggg ggcgtggcct agcggtcccg cccccggggc gcgcgcgcgc attggctgtg 60
cggggtgcgg gcgcgcgggc ggcgctttga accgggcgcg gggcgcgggg cgcggggcgc 120
tgcggccggt acacgccggg gtagggccgg ggtcgggttg tggtcgggcc gggattgggc 180
56/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
tctcctgggc catggcagcc gaggcgcgcg tgtcgcgctg gtacttcggg gggctggcct 240
cctgcggggc cgcctgctgc acgcacccgc tggacctgct caaggtgcat ctgcagacgc 300
agcaggaggt gaagctgcgc atgacgggca tggcgctgcg ggtggtgcgt accgacggca 360
tcctggcact ctacagcggc ctgagcgcct cgctgtgcag acagatgacc tactccctga 420
ctcggttcgc catctacgag actgtgcggg accgcgtggc caagggcagc caggggcctc 480
tccccttcca cgagaaggtg ttgctgggct ccgtcagcgg tttagctgga ggcttcgtgg 540
ggacgcccgc agacttggtc aacgtcagga tgcagaacga cgtgaagctg ccccagggtc 600
agcggcgcaa ctacgcccat gcgctggatg gcctgtaccg cgtagctcgt gaagagggtc 660
tcaggagact gttctcgggt gcaaccatgg catccagccg aggggcctta gtcactgtgg 720
gccagctgtc ctgctacgac caggccaagc agctggtcct tagcaccggg tacctctctg 780
acaacatctt cactcacttt gtcgccagct ttattgcagg tggatgtgcc acgttcctgt 840
gccagcccct ggatgtgctg aagactcgcc tgatgaactc caagggggag tatcagggcg 900
ttttccactg cgccgtggag acagcgaagc tcgggcctct ggccttttac aagggcctcg 960
tcccagctgg catccgcctc atcccccaca ccgtgctcac ttttgtgttt ctggaacagc 1020
tacgcaaaaa ctttggcatc aaagtgccat cctgaccagc cgtgggaatg gctgggctgc 1080
caggccagac acgctaggtt cttccaaaga gtcccaagcc cagcacctgc tcctggggcc 1140
acgacctccc tggccgtggc cacccatcct ccgcagcagg cccctgctgt ccccccacct 1200
gctggctgag ctcctcctgg cctcgtcccc tctcagctgt agctgcacca cccccgctct 1260
ggctaccagg ctctcccggc tgggcactgc gtggccttgc ccctctcccg ctggcagctc 1320
ctcaggggaa caggggctac cagaggctga tttctcccct ctcctgggcc aggggagggg 1380
tattatccct gcctcctgcc cccgatgccc aaagcagcat cttccagcac tttccatcga 1440
ggacttgggt ggcagagtgt gggtgcagcc tggctgttgc tcacccaagt gctagctctg 1500
cacttcgtgt ctgctgagag caaccagacc ttccatgtcc tcgggcagct gcaactcccc 1560
gcgagacccc gcagctgggt gggatgaaca agcaacgcag accacaagcg agtgcctggg 1620
agggagtggc ccagggtggt tctggagcca ttgtgggtga gggtcgaggg ccaccgaggt 1680
cccgcgcacc gctgcctgcc ctgcagtggc tttaacagtt agttttgcca aagcctctcc 1740
actcaccagc aggcggtctc tgtcttcagg gattgtgcct gcgtccctcg ggcacctggg 1800
cccccccgct tggctccctg ggggaatggc ccaggcgggc cgcggttcct ccttagggcc 1860
ttctccccga caaggagtcc gacggggcgg atgctgcatc ctctgcctcc ctggtcgctg 1920
ggcttcaccc cacctgggaa gggcagtgtg ctctgtgggg gctgcaatca ataaatgccg 1980
ggagctgcca aaaaaaaaaa aaaa 2004
<210> 80
<211> 3555
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3295764CB1
<400> 80
gaccgtgggc gagtccagaa cgtcctggcc ttacagggag aaggcgtcac tcgcggttac 60
aagtgcctga ccctcactcc agttggcgga ggaggagaag gaaggggccg ggccgggtcc 120
cctcccctcg cgccccggat ggatgtgccc ggcccggtgt ctcggcgggc ggcggcggcg 180
gcggccactg tgctcctgcg gaccgctcgg gtccgtcgcg aatgctggtt cttgccgacc 240
gcgctgctct gcgcctacgg cttcttcgcc agcctcaggc cgtccgagcc cttcctgacc 300
ccgtacctgc tggggccgga caagaacctg accgagaggg aggtcttcaa tgaaatttat 360
ccagtatgga cttactctta cctggtgcta ctgtttcctg tgttccttgc cacagactac 420
ctccgttata aacctgttgt tctactgcag gggctcagcc ttattgttac atggtttatg 480
ctgctctatg cccagggact gctggccatt caatttctag aattttttta tggcatcgcc 540
acagccactg aaattgccta ttactcttat atctacagtg tggtggacct gggcatgtac 600
cagaaagtca caagttactg tcgaagtgcc actttggtgg gctttacagt gggctctgtc 660
ctagggcaaa tccttgtctc agtggcaggc tggtcgctgt tcagcctgaa tgtcatctct 720
cttacctgtg tttcagtggc ttttgctgtg gcctggtttt tacctatgcc acagaagagc 780
ctcttctttc accacattcc ttctacctgc cagagagtga atggcatcaa ggtacaaaat 840
ggtggcattg ttactgacac cccagcttct aaccaccttc ctggctggga ggacattgag 900
tcaaaaatcc ctctaaatat ggaggagcct cccgtggagg aaccggaacc caagccagac 960
cgtctccttg tattgaaagt actatggaat gatttcctga tgtgctactc ctctcgccct 1020
cttctctgct ggtctgtgtg gtgggccctc tctacctgtg gctattttca agttgtgaac 1080
tacacacagg gcctgtggga gaaagtgatg ccttctcgct atgctgctat ctataatggt 1140
ggcgtggagg ccgtttcaac cttactgggt gctgttgctg tgtttgcagt tggttatata 1200
aaaatatcct ggtcaacttg gggagaaatg acattatctc tcttttctct cctgattgct 1260
gctgcagtgt atatcatgga cactgtgggt aacatttggg tgtgctatgc atcctatgtt 1320
gtcttcagaa tcatctacat gttactcatc acgatagcaa cttttcaaat tgctgcaaac 1380
ctcagcatgg aacgctatgc cctagtattt ggtgtaaata ccttcattgc cctggcactg 1440
cagacgctgc tcactctaat tgtggtagat gccagtggcc ttggattaga aattaccact 1500
cagtttttga tctatgccag ttattttgca ctcatcgctg tggttttcct ggccagtggt 1560
57/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
gcagtcagtg ttatgaagaa atgtagaaag ctggaagatc cacaatcaag ttctcaagta 1620
accacttcat aatatactgc tgaagggctt cttcttatag caagaactct gcacagcaac 1680
tgcctggatg tatttgattt tttaaagcgt agacatatat ttatgaatgt gcatttcttg 1740
acttcacagc agccacttga ctaatacctt gtgttccggg aataacatga tactattcag 1800
aggagccaga agtaaagttt atttcatgga ttatttatga gagctaattt aaggatgact 1860
ttttttctga ttcaaaagtg aacttgattt taaaaaccag tcaagagcaa tcaaagcagc 1920
acatggtgtt gtatacttca ttagcaagtg agtttggtgt tttataggtc acatatgtct 1980
gtatctactt agccagatgc ttggcctggt gggaccaggg ctccacagag gccacaaaat 2040
gttgcaagtc atgatggatg gaaatatgtt ctaacagcat ctgcctctat tcaatttaat 2100
tcttatttct gtgttactca tgtacattgg tctttctaca tagttattct atcactggca 2160
atatttgttc tggtttagtg ttctgtattt taaggtgtac gtatcatttc taattttaag 2220
ttattttaaa aaaattcatc atatgaatgt tcttggttcc cattgtgacg attatttatt 2280
tctgtaaaat ttgtttagaa gtacgttttt gcattattca tatgcttccc agagaagctc 2340
atttagttag aaaataaggc aagttttgaa gcctgctaaa tgaagagact taagaaagct 2400
taaggtacgc ttgcttgtct ttaaatcttc aatatgaagg actattaatt ccaagattaa 2460
aagttcatat ataggctaaa gatgtaacta ggccatttgt atttgtattc ccttttattt 2520
ccaaaataaa atgaaaaatc tttttttaat aatttcatcc ctatttatag tttttatatt 2580
aatttgtttt tcttatccaa gtaaagatgt caataggaat tgcattagtc caaggccttt 2640
ttcataaact gagcctcttt tcaattattt caatgggaca ggaactagga tagatgtgat 2700
tcctgcattt ttttacctta aatctgcctt tgtttctaaa ggtagatcat cttgaatatt 2760
tgcttaaaat tgctagtgat ttcattacca agttacttga aaaaatgttc tatatgcatt 2820
taattctgaa atcagtctac caaggggctg ctagtatatg tcagacatga aaactatttt 2880
aaagctgact ttgttgcctt atcttgaaaa gaatctagat aggtgctttt aactggggta 2940
ttaacttttt tagaatgaca cagctgaaca gtgttaataa tagtgtgtca agattgcaaa 3000
gtcgacatac tcatttggtt taagcaggaa tcctagaagc aaatggatgg ggataagaat 3060
aggtcatttt ctattcacca tcctttacta ttaagggaaa ggaaaagaac actagctaag 3120
gaagggaaag ggaagtgata tcataaaagt agcaaccttc attttacatt ctgtctgttt 3180
ttcttttttt gctttgtttt gtttgtgcta atttgggaat tgtgtactcc gaaacaagta 3240
gaaaagtgct gtttgaggga ttttattaaa tcttttttta atggaatgtg gtacaaattg 3300
ttcatgttac caaagcaata tttccctgga atttaattca aagtttgtgg catacaacct 3360
gagccttttc ttatataaga caagaatatg ttcacatctt ggtatgtggc catatttata 3420
gaatgctgaa ctcaatgtgc aagttgtact gtatgcagtt ttgtaaataa gtgaaaataa 3480
tttgttgtac tttttattca attctgtata gattataaaa ttatttttat taaataaata 3540
ttttacagta tattt 3555
<210> 81
<211> 1293
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3438320CB1
<400> 81
atatgcccca cgcgggactc atactacgtt tcccgtgaac acgtgcagtc caaaccccgc 60
ccctgatatt tatctcagtg gacggtggcc ggaaaaggac aatggtttcc atgtcagcgg 120
ataaacgctc tcccctcggc tcccggacgc gacggaggtc gtagtagtag tgagtacgtg 180
ctgaggagca aaggagtaac caagagatcc agtgaccgac agagcaagag ccatgccgcg 240
ccggggcctg gtggctgggc cagacttgga gtattttcag cgtcgctatt tcacgccggc 300
ggaggtggcc caacataaca ggcccgaaga cctctgggta tcttacctgg gacgcgtgta 360
cgacctaacg tcattggcac aggaatacaa ggggaacctg ctgctgaaac ccatcgtgga 420
agttgcaggc caggatatca gccactggtt tgatccaaag accagagaca tccgcaagca 480
catagatccg ctgaccggct gcctgaggta ctgcaccccg cggggccgct ttgtgcacgt 540
tccgcctcag ctgccctgtt cggactgggc caacgatttt gggaagccct ggtggcaggg 600
gtcgtattat gaggtggggc ggctgtctgc caagacccgg agcatccgca tcattaacac 660
gctcacgtcg caggagcaca cactggaggt gggggttctg gagtccatat gggaaatcct 720
acaccgctat ctcccctata actcacatgc tgccagctac acgtggaaat atgaagggaa 780
gaacctgaac atggatttta ccctggaaga gaatgggatc cgggatgagg aggaagaatt 840
tgactatctc agtatggacg gtacacttca cacacctgca atacttctgt acttcaatga 900
tgatctcacg gagttgtagg caaggagatg tacactcgtg tagactcaag acgtatttcg 960
agtttggctt tttctgtgcc ttgaggaaaa gtggtggggc cgaggggtgc ctggacccag 1020
atctccactc ctctccagga gctagcctgt gcccttctga agtgtaaagg ccctattccc 1080
tgccttcatt acagtttgct ctgagaaaat tagtgaatta atctttggga atgatacaag 1140
aagatcaagt accttggttt agggagatgt agaagaggat agtcagagtt caggcagaac 1200
tgtttgatag ttaagagaga gtagttctac aggggtgagg gatggaagga cttttttggc 1260
aatgatggaa atgagatgtc tgcaggagat ggg 1293
58/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
<210> 82
<211> 1489
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3986488CB1
<400> 82
cggggctggc gggcggcgct cttctacggg acctgctcct tcctcatcgt gcttgtcaac 60
aaggcgctgc tgaccaccta cggtttcccg tcaccaattt tccttggaat tggacagatg 120
gcagccacca taatgatact atatgtgtcc aagctaaaca aaatcattca cttccctgat 180
tttgataaga aaattcctgt aaagctgttt cctctgcctc tcctctacgt tggaaaccac 240
ataagtggat tatcaagcac aagtaaatta agcctaccga tgttcaccgt gctcaggaaa 300
ttcaccattc cacttacctt acttctggaa accatcatac ttgggaagca gtattcactc 360.
aacatcatcc tcagtgtctt tgccattatt ctcggggctt tcatagcagc tgggtctgac 420
cttgctttta acttagaagg ctatattttt gtattcctga atgatatctt cacagcagca 480
aatggagttt ataccaaaca gaaaatggac ccaaaggagc tagggaaata cggagtactt 540
ttctacaatg cctgcttcat gattatccca actcttatta ttagtgtctc cactggagac 600
ctgcaacagg ctactgaatt caaccaatgg aagaatgttg tgtttatcct acagtttctt 660
ctttcctgtt ttttggggtt tctgctgatg tactccacgg ttctgtgcag ctattacaat 720
tcagccctga cgacagcagt ggttggagcc atcaagaatg tatccgttgc ctacattggg 780
atattaatcg gtggagacta cattttctct ttgttaaact ttgtagggtt aaatatttgc 840
atggcagggg gcttgagata ttccttttta acactgagca gccagttaaa acctaaacct 900
gtgggtgaag aaaacatctg tttggatttg aagagctaaa gagtctgcag caggattgga 960
gactgacttg tgactgcggg ctgggggggc attcccagta ggaatgtgaa gccagaggtt 1020
tcggattcgt gacatccacc ccctgggcaa gtgagagcat ctgcaaaatg caaagagaac 1080
tacctcatat gcaggatgag ccaatggcag tctcaagaaa tgtactcggg cgacacctta 1140
cctgtggaaa gcaaatcttt tcaaaataag ccactgggac tcggtaggtg gagccccagc 1200
tgctcttcta gggacctatg gggccttcgt ggcatctctg tgctgtgtgc tggggaggag 1260
gttgatgtaa tggtgactct tttctgatca gcaccttggc cgtgattccc aaggtcccag 1320
ccaaagcaaa gggccagttg tttcagttta aacagacatg tctttagtct aataaaatta 1380
gttaactgcc agtaaagtta tttgttagct ttgatgaaag ctatgttggt atctttccct 1440
aatcatcaaa gtaaataaaa aatcatttct atgtaaaaaa aaaaaaaaa 1489
<210> 83
<211> 927
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4378816CB1
<400> 83
ctccctgctt tcctctgccg catggtcctg ggccgttggc gtcggaagcc tgaagcatgg 60
gcgctgagtg ggagctgggg gccgaggctg gcggttcgct gctgctgtgc gccgcgctgc 120
tggcggcggg ctgcgccctg ggcctgcgcc tgggccgcgg gcagggggcg gcggaccgcg 180
gggcgctcat ctggctctgc tacgacgcgc tggtgcactt cgcgctggaa ggcccttttg 240
tctacttgtc tttagtagga aacgttgcaa attccgatgg cttgattgct tctttatgga 300
aagaatatgg caaagctgat gcaagatggg tttattttga tccaaccatt gtgtctgtgg 360
aaattctgac cgtcgccctg gatgggtctc tggcattgtt cctcatttat gccatagtca 420
aagaaaaata ttaccggcat ttcctgcaga tcaccctgtg cgtgtgcgag ctgtatggct 480
gctggatgac cttcctccca gagtggctca ccagaagccc caacctcaac accagcaact 540
ggctgtactg ttggctttac ctgttttttt ttaacggtgt gtgggttctg atcccaggac 600
tgctactgtg gcagtcatgg ctagaactca agaaaatgca tcagaaagaa accagttcag 660
tgaagaagtt tcagtgaact ttcaaaacca taaacaccat tatctaactt catgaaccag 720
aatgaatcaa atctttttgt ttggccaaaa tgtaatacat tccagtctac actttgtttt 780
tgtattgttg ctcctgaaca acctgtttca aattggtttt aaggcgacca gttttcgttg 840
tattgttgtt caattaaatg gtgatatagg gaaaagagaa caaatttgaa tttgtaataa 900
taaaatgttt aattatacaa aaaaaaa 927
<210> 84
<211> 970
<212> DNA
<213> Homo Sapiens
59/60
CA 02375493 2001-12-11
WO 00/78953 PCT/US00/16668
<220>
<221> misc_feature
<223> Incyte ID No: 4797137CB1
<400> 84
ggatgcagca gagaggagca gctggaagcc gtggctgcgc tctcttccct ctgctgggcg 60
tcctgttctt ccagggtgtt tatatcgtct tttccttgga gattcgtgca gatgcccatg 120
tccgaggtta tgttggagaa aagatcaagt tgaaatgcac tttcaagtca acttcagatg 180
tcactgacaa gcttactata gactggacat atcgccctcc cagcagcagc cacacagtat 240
caatatttca ttatcagtct ttccagtacc caaccacagc aggcacattt cgggatcgga 300
tttcctgggt tggaaatgta tacaaagggg atgcatctat aagtataagc aaccctacca 360
taaaggacaa tgggacattc agctgtgctg tgaagaatcc cccagatgtg caccataata 420
ttcccatgac agagctaaca gtcacagaaa ggggttttgg caccatgctt tcctctgtgg 480
cccttctttc catccttgtc tttgtgccct cagccgtggt ggttgctctg ctgctggtga 540
gaatggggag gaaggctgct gggctgaaga agaggagcag gtctggctat aagaagtcat 600
ctattgaggt ttccgatgac actgatcagg aggaggaaga ggcgtgtatg gcgaggcttt 660
gtgtccgttg cgctgagtgc ctggattcag actatgaaga gacatattga tgaaagtctg 720
tatgacacaa gaagagtcac ctaaagacag gaaacatccc attccactgg cagctaaagc 780
ctgtcagaga aagtggagct ggcctggacc atagcgatgg acaatcctgg agatcatcag 840
taaagacttt aggaaccact tatttattga ataaatgttc ttgttgtatt tataaactgt 900
tcaggaagtc tcataagaga ctcatgactt cccctttcaa tgaattatgc tgtaattgaa 960
tgaagaaatc 970
<210> 85
<211> 594
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5470806CB1
<400> 85
gacaggatgg cttcccttcg cctgttcctc ctctgcctcg ctgtactgat atttgcgtct 60
gaagctggcc ctgggggtgc tggagaatcc aagtgtcctc tgatggtcaa agtcctggat 120
gctgtccgag gcagccctgc tgttgatgtg gccgtgaaag tgttcaaaaa gactgcagac 180
ggaagctggg agccgtttgc ctctgggaag accgccgagt ctggagagct gcacgggctc 240
accacagatg agaagttcac ggaaggggtg tacagggtag aactggacac caaatcgtac 300
tggaaggctc ttggcatttc cccattccat gaatacgcag aggtggtttt cacagccaat 360
gactctggtc atcgccacta caccatcgca gccctgctca gcccgtactc ctacagcacc 420
actgctgtcg tcagtaaccc ccagaactga gggacccagc ccagtaggac caggatcttg 480
ccaaagcagt agcttcccat ttgtactgaa acagtgttct tgctctataa accgtgttag 540
caactcggga agatgccgtg aaacgatctt attaaaccac ctgtgatgcc aaaa 594
<210> 86
<211> 618
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5473242CB1
<400> 86
gtgttgactc gcaacctcag gaacagacac catggtgcac ctaactgatg ctgagaaggc 60
tactgttaat ggcctgtggg gaaaggtgaa ccctgttgaa attggcgctg agtcccttgc 120
cagtctgctg attgtctacc cttggaccca gaggtacttt tctaaatttg gggacctgtc 180
ctctgtctct gctatcatgg gtaaccccca ggtgaaggcc catggcgaaa aggtgataaa 240
cgccttcgat gatggcctga aacacttgga caacctcaag ggcacctttg ccagcctcag 300
tgaactccac tgtgacaagc tgcatgtgga tcctgagaac ttcaggctcc tgggcaatat 360
gattgtgatt atgatgggcc accacctggg caaggaattc accccgagtg cacaggctgc 420
cttccagaag gtggtggctg gagtggccag tgccctggct cacaagtacc actaaacctc 480
ttttcctgct cttgtctttg tgcaatggtc aattgttccc aagagagctt ctgtcagttg 540
ttgtcaaaat gacaaagacc tttgaaaatc tgtcctacta attaaagcat ttggttcaag 600
tgttctgttg agataccc 618
60/60
