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HUMAN MEMBRANE CHANNEL PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human
membrane
S channel proteins and to the use of these sequences in the diagnosis,
treatment, and prevention of
cell proliferative, immune/inflammatory, transport/secretory, osmoregulatory,
muscular,
cardiovascular, and neurological disorders.
BACKGROUND OF THE INVENTION
Channel proteins facilitate the transport of hydrophilic molecules across
membranes by
forming aqueous pores that can perforate a lipid bilayer. Many channels
consist of protein
complexes formed by the assembly of multiple subunits, at least one of which
is an integral
membrane protein that contributes to formation of the pore. In some cases, the
pore is constructed
to selectively allow passage of only one or a few molecular species. Distinct
types of membrane
t S channels that differ greatly in their distribution and selectiivity
include: ( 1 ) aquaporins, which
transport water; (2) protein-conducting channels, which transport proteins
across the endoplasmic
reticulum membrane; (3) gap junctions, which facilitate diiffusion of ions and
small organic
molecules between neighboring cells; and {4) ion channels, which regulate ion
flux through
various membranes.
Aquaporins
Aquaporins (AQP) are channels that transport water and, in some cases,
nonionic small
solutes such as urea and glycerol. Water movement is important for a number of
physiological
processes including renal fluid filtration, aqueous humor generation in the
eye, cerebrospinal fluid
production in the brain, and appropriate hydration of the lung. A variety of
aquaporins have been
found in higher animals, plants and microorganisms. The .mammalian aquaporins
appear to have
selective expression in particular tissues, with AQPO localized to lens
epithelium; AQPl localized
to many tissues including red blood cells, kidney, eye, lunl;, choroid plexus,
bile duct, and
vascular epithelium; AQP2 localized to the apical membrane of kidney
collecting duct cells; AQP3
localized to kidney, colon, trachea, urinary bladder, skin, and sclera of eye;
AQP4 localized to
kidney, colon, trachea, stomach, skeletal muscle, spinal cord, brain, and
retina; AQPS localized to
the apical membranes of exocrine tissues; AQP6 localized to kidney; and AQP7
localized to testis
(King, L.S. and P. Agre (1996) Annu. Rev. Physiol. 58:619-648; Ishibashi, K.
et al. (1997) J. Biol.
Chem. 272:20782-20786). AQP9 is expressed in peripheral leukocytes, less
abundantly in liver,
even less in lung and spleen, and not at al) in thymus tissue (Ishibashi, K.
et al. ( 1998) Biochem.
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Biophys. Res. Commun. 244:268-274).
Aquaporins are members of the major intrinsic protein (MIP) family of membrane
transporters. MIP family proteins are composed of four s~ubunits, each of
which may span the
membrane six times, and have their N-and C-termini facing the cell cytoplasm.
Proteins from
bacteria, yeast, plants, and animals have been shown to bt: members of the MIP
family {Reizer, J.
et al. {I993) Crit. Rev. Biochem. 28:235-257). Aquaporin subunits are integral
membrane
proteins with six transmembrane regions and two conserved Asn-Pro-Ala (NPA)
boxes (which are
sometimes found as Asn-Pro-Ser) found in loop regions between the
transmembrane regions
{King, supra; Ishibashi, ( 1997) supra). The, study of aquaporins may have
relevance to
understanding edema formation and fluid balance in both normal physiological
and disease states
(King, supra). Mutations in AQP2 cause autosomal recessive nephrogenic
diabetes insipidus
(Online Mendelian Inheritance in Man (OM1M) *107777 Aquaporin 2; AQP2).
Reduced AQP4
expression in skeletal muscle may be associated with Duchenne muscular
dystrophy (Frigeri, A. et
al. (1998) J. Clin. Invest. I02:695-703). Mutations in AQPO cause autosomal
dominant cataracts
in mice (OMIM * 154050 Major Intrinsic Protein of Lens :Fiber; MIP}.
Protein-Conducting Channels
Secreted and integral membrane proteins are transported from the cytoplasm to
the
endoplasmic reticuium (ER) through protein-conducting channels in the ER
membrane. The
channel is used for both co- and post-translational translocation. In the co-
translational process,
transport is initiated by the action of a cytopiasmic signal .recognition
particle (SRP) which
recognizes a signal sequence on a growing, nascent polype;ptide and binds the
polypeptide and its
ribosome complex to.the ER membrane through an SRP re;ceptor located on the
membrane. The
ribosome complex, together with the attached polypeptide" becomes membrane
bound. As the
nascent chain emerges from the ribosome, it is fed into the channel and across
the ER membrane.
The post-translational process also requires a signal sequence on the protein
to be translocated, but
does not require an SRP. The protein enters the channel and is driven across
the ER membrane by
the hydrolysis of adenosine triphosphate (ATP) by BiP, an ATPase and molecular
chaperone in
the ER lumen.
The protein-conducting channel, termed the Secd l p complex, is composed of
multiple,
probably two, heterotrimers of three membrane proteins, the alpha, beta, and
gamma subunits of
Sec6lp. The Sec6lp complex forms a ring structure visible by electron
microscopy (EM). EM
and quenching experiments indicate a channel diameter of 20 to 60 A.
Association of the Sec61 p
complex with the ribosome and with the proteins Secb2p, Sec63p, Sec71 p,
Sec72p, BiP, and
TRAM (translocating chain-associating membrane protein;l is required for some
of the channel's
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functions. The Sect 1 p alpha subunit contains ten membrane-spanning segments
and has been
found to line the path of the translocating polypeptide chain from one side of
the membrane to the
other. The sequences of dog and rat Sec61 p alpha genes have been determined.
Homologs of the
mammalian Sec6lp alpha are found in the yeast Saccharomvces cerevisiae
(Sec6lp) and in
bacteria (SecYp). (See Giirlich, D. et al. ( 1992) Cell 71:489-503; Matlack,
K.E.S. et al. ( 1998)
CeII 92:381-390.)
Defects in protein trafficking to organelles or to 'the cell surface are
involved in numerous
human diseases and disorders including cystic fibrosis, glucose-galactose
malabsorption
syndrome, hypercholesterolemia, diabetes mellitus, diabc;tes insipidus, hyper-
and hypoglycemia,
Grave's disease, goiter, Cushing's disease, and Addison';s disease. Cancer
cells secrete excessive
amounts of hormones or other biologically active peptides.
Gap Junctions
Gap junctions (also called eonnexons} are channels that function chemically
and
electrically to couple the cytoplasms of neighboring cells in many tissues.
Gap junctions function
as electrical synapses for intercellular propagation of actuon potentials in
excitable tissues. In
nonexcitable tissues, gap junctions have roles in tissue homeostasis,
coordinated physiological
response, metabolic cooperation, growth control, and the regulation of
development and
differentiation. Gap junctions help to synchronize heart and smooth muscle
contraction, speed
neural transmission, and propagate extracellular signals. Gap junctions can
open and close in
response to particular stimuli (e.g., pH, Ca+Z, and cAMP). The effective pore
size of a gap
junction is approximately 1.5 nm, which enables small molecules (e.g., those
under 1000 daltons)
to diffuse freely through the pore. Transported molecules include ions, small
metabolites, and
second messengers (e.g., Ca+2 and cAMP).
Each connexon is composed of six identical subunits called connexins. At least
thirteen
distinct connexin proteins exist, with each having similar structures but
differing tissue
distributions. Structurally, the connexins are integral membrane proteins with
four putative
membrane spanning regions and N- and C-termini oriented towards the cell
cytoplasm. Conserved
regions include the membrane spanning regions and two e:xtracellular loops.
The variable regions,
which are two cytoplasmic loops and the C-terminal region, may be responsible
for the regulation
of different connexins. (See Hennemann, H. et ai. ( 1992) .1. Biol. Chem.
267:17225-17233;
PRINTS PR00206 connexin signature.)
Connexins have many disease associations. Female mice lacking connexin 37
(Cx37) are
infertile due to the absence of the oocyte-granuIosa cell signaling pathway.
Mice lacking Cx43 die
shortfy after birth and show cardiac defects reminiscent of some forms of
stenosis of the
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pulmonary artery in humans. Mutations in Cx32 are associated with the X-linked
form of
Charcot-Marie-Tooth disease, a motor and sensory neuropathy of the peripheral
nervous system.
Cx26 is expressed in the placenta, and Cx26-deficient mi<;e show decreased
transplacental
transport of a glucose analog from the maternal to the fetal circulation. In
humans, Cx26 has been
identified as the first susceptibility gene for non-syndromic sensorineurai
autosomal deafness.
Cx46 is expressed in lens fiber cells, and Cx46-deficient mice develop early-
onset cataracts that
resemble human nuclear cataracts. (See Nicholson, S.M. and R. Bruzzone (1997)
Curr. Biol.
7:8340-8344.)
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. There are two basic types of ion
channel: ion
transporters and gated ion channels. Ian transporters utili:ae 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.
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 constists of six potential transmemlbrane segments (Sl,
S2, S3, S4, S5, and
S6). The center ofthe barrel forms a pore lined by a-helices or (3-strands.
The side chains ofthe
amino acid residues comprising the a-helices or p-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.
Ion Transporters
Ion transporters generate and maintain the resting electrical potential of a
cell. Utilizing
the energy derived from ATP hydrolysis, they transport ions against the ion's
concentration
gradient. These transmembrane ATPases are divided into three families. The
vacuolar (V) class
of ion transporters includes 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 Hr pumps in the
mitochondria. F-class ion transporters utilize a proton gradient to generate
ATP from ADP and
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inorganic phosphate (P;). The phosphorylated (P) class ion transporters,
including Na+-K+
ATPase, Ca+2-ATPase, and H+-ATPase, are activated by a phosphorylation event.
P-class ion
transporters are responsible for maintaining resting potential distributions
such that cytosolic
concentrations ofNa~ and Ca+2 are low and cytosolic concentration of K+ is
high. The resting
potential of the cell is utilized in many processes involving carrier proteins
and gated ion channels.
Carrier proteins utilize the resting potential to transport molecules into and
out of the cell. Amino
acid and glucose transport into many cells is linked to sodium ion co-
transport (symport) so that
the movement of Na+ down an electrochemical gradient drives transport of the
other molecule up a
concentration gradient. Similarly, cardiac muscle links transfer of Ca+= out
of the cell with
transport of Na+ into the cell (antiport).
Gated Ion Channels
Gated ion channels control ion flow by regulating the opening and closing of
pores. The
ability to control ion flux through various gating mechanisms allows ion
channels to mediate such
diverse signaling and homeostatic functions as neuronal and endocrine
signaling, muscle
contraction, fertilization; and regulation of ion and pH balance. Gated ion
channels are
categorized according to the manner of regulating the gating function.
Mechanically-gated
channels open their pores in response to mechanical stress.; voltage-gated
channels (e.g., Na+, K+,
Ca+2, and Cf channels) open their pores in response to changes in membrane
potential; and ligand-
gated channels (e.g., acetyicholine-, serotonin-, and glutamate- gated cation
channels, and GABA-
and glycine- gated chloride channels) open their pores in tl'ne presence of a
specific ion, nucleotide,
or neurotransmitter. The gating properties of a particular ion channel ( i.e.,
its threshold forand
duration of opening and closing) are sometimes modulated! by association with
auxiliary channel
proteins and/or post translational modifications, such as phospharylation. The
pore forming
subunits of voltage-gated and transmitter-gated cation channels form two
distinct superfamilies of
conserved multipass membrane proteins.
Voltage-gated Na+ and K+ channels are necessary for the function of
electrically excitable
cells such as nerve, endocrine, and muscle cells. Action potentials, which
lead to neurotransmitter
release and muscle contraction, arise from large, transient changes in the
permeability of the
membrane to Na+ and K+ ions. Depolarization of the membrane beyond the
threshold level opens
voltage-gated Na+ channels. Sodium ions flow into the cell, further
depolarizing the membrane
and opening more voltage-gated Na+ channels, thus propagating the
depolarization down the
length of the cell. Depolarization also opens voltage-gated K+ channels.
Consequently, potassium
ions flow outward, leading to repolarization of the membrane. Voltage-gated
channels utilize
charged residues in the fourth transmembrane segment (S4) to sense voltage
change. The open
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state lasts only about 1 millisecond, at which time the channel spontaneously
converts into an
inactive state that cannot be opened irrespective of the membrane potential.
Inactivation is
mediated by the channel's N-terminus, which acts as a plug that closes the
pore. The transition
from an inactive to a closed state requires a return to restiing potential.
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, pl and ~i2.
The [32 subunit is an integral membrane glycoprotein that: contains an
extracellular Ig domain, and
its association with a and X31 subunits correlates with increased function of
the channel, a change
in the channel's gating properties, as well as an increase iin whole cell
capacitance (Isom, L.L. et
al. (1995) Cell 83:433-442).
K+ channels are located in a!l cell types, and may be regulated by voltage,
ATP
concentration, or second messengers such as Ca'~'' and cA.MP. 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
IS 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 contains a Na+-K+ pump and ion channel; that provide the
redistribution ofNa+,
K+, and Cf. 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 Cl' to flow by passive
diffusion. Because of
the high negative charge within the cytosol, C1' flows out of the cell. The
flow of K+ is balanced
by an electromotive force pulling K+ into the cell, and a 1C+ 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).
K+ pore-forming subunits generally have six transrnembrane-spanning domains
with a
short region between the ffth 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 a ssociated 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 amyioid or A beta, also blocks voltage-gated potassium channels
in hippocampal
neurons. {See 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.
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( 1996) Biophys. J. 70:296-304.)
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 l:heir functions
differ dramatically, they
have similar subunit compositions. The channels are composed of three
subunits. The a, subunit
forms the membrane pore and voltage sensor, while the oN& and ~i 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 ~ genes. A fourth subunit, y,
has been identified in
skeletal muscle. (See Walker, D. et al. ( 1998) J. Biol. Chem. 273:2361-2367;
Say, S.D. et al.
( 1990) Science 248:490-492.)
Chloride channels are necessary in endocrine secretion and in regulation of
cytosolic and
organelle pH. In secretory epithelial cells, Cl' enters the. cell across a
basolateral membrane
through an Na+, K+/Cl' cotransporter, accumulating in the cell above its
electrochemical
equilibrium concentration. Secretion ofCl' from the apical surface, in
response to hormonal
stimulation; leads to flow of Na'~ and water into the secretory lumen. The
cystic fibrosis
transmembrane conductance regulator (CFTR) is a chloriide channel.encoded by
the gene for
cystic fibrosis, a common fatal genetic disorder in humans. Loss of CFTR
function decreases
transepithelial water secretion and, as a result, the layers of mucus that
coat the respiratory tree,
pancreatic ducts, and intestine are dehydrated and difficult to clear. The
resulting blockage of
~ these sites leads to pancreatic insufficiency, "meconium ileus," and
devastating "chronic
obstructive pulmonary disease" (AI-Awqatii, Q. et al. ( 1992) J. Exp. Biol.
172:245-266).
Many intracellular organelles contain H+-ATPase pumps that generate
transmembrane pH
and electrochemical differences by moving protons from the cytosol to the
organelle lumen. If the
membrane of the organelle is permeable to other ions, then the electrochemical
gradient can be
abrogated without affecting the pH differential. In fact, removal of the
electrochemical barrier
allows more H+ to be pumped across the membrane, increasing the pH
differential. Cl' is the sole
counterion of H+ translocation in a number of organelles,, including
chromaffin granules, Golgi
vesicles, lysosomes, and endosomes. Functions that require a low vacuolar pH
include uptake of
small molecules such as biogenic amines in chromaffin I;ranules, processing of
vacuolar
constituents such as pro-hormones by proteolytic enrym~es, and protein
degradation in lysosomes
(Al-Awqati, supra).
Liigand-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 extraceilular domain. These channels exist in
the postsynaptiic
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membrane of nerve or muscle cells. There are two types of neurotransmitter-
gated channels.
Sodium channels open in response to excitatory neurotransmitters, such as
acetylchoiine,
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 hyperpofarization of the membrane and the subsequent
generation of an
action potential.
Ligand-gated channels can be regulated by intracellular second messengers.
Calcium-
activated K+ channels are gated by internat calcium ions.. In nerve cells, an
influx of calcium
during depolarization opens K+ channels to modulate the. magnitude of the
action potential (Ishi,
T.M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:11651-11656). Cyclic
nucleotide-gated (CNG)
channels are gated by cytosoiic cyclic nucleotides. The best examples of these
are the cAMP-
gated Nat channels involved in olfaction and the cGMP-gated cation channels
involved in vision.
Both systems involve iigand-activation of a G-protein coupled receptor which
then alters the level
of cyclic nucleotide within the cell. In olfaction, binding; of an odorant to
the receptor activates
adenylate cyclase, leading to a rise in cytosolic cAMP. 'The cAMP binds to the
cAMP-gated Na+
channel causing an influx of Na+, depolarization of the nnembrane, and
initiation of a nerve
impulse that travels along the axon to the brain. In vision, light activation
of rhodopsin leads to
activation of cGMP phosphodiesterase, which hydrolyzes cGMP. As a result,
cytosolic cGMP
levels drop, cGMP dissociates from cGMP-gated cation channels, and the
channels close, resulting
in hyperpolarization of the membrane. (See Zagotta, W.M. and S.A. Siegelbaum (
1996) Annu.
Rev. Neurosci. 19:235-263; Molday, R.S. and L.L. Molday (1998) Vision Res.
38:1315-1323.)
The subunits or monomers of an ion channel may be identical or different. CNG
channels, for example, consist of a and p subunits that differ from each other
at the N-terminal
cytoplasmic tail. The central pore formed by the barrel arrangement is lined
by an antiparallel (3-
sheet, the pore (P) region, contained within each subunit. This region also
contains information
specifying the ion selectivity for the channel. In the case of K~ channels, a
GYG tripeptide is
involved in this selectivity (Ishi et al., supra). In voltage-gated channels;
one of the
transmembrane domains contains regularly spaced, positively charged amino
acids that act as a
voltage-sensor. In CNG channels, a region in the C-terminal cytoplasmic domain
acts as a cyclic
nucleotide binding site (Zagotta and Siegelbaum, supra). Ion channels also
have a domain that
functions in inactivation of the channel. In CNG K+ channels, the inactivation
domain is on the N-
terminal cytoplasmic tail of the (i-subunit. This domain acts as a tethered
ball to block ion flow
through the pore. This domain is also expressed as a separate protein, a
glutamic acid-rich protein
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(GARP), by alternative splicing and may act as an independent regulator of
pore activity (Sautter,
A. et ai. ( 1997) Molec. Brain Res. 48:171-175).
Ion channels are essential to a wide range of physiological functions
including neuronal
signaling, muscle contraction, cardiac pacemaking, hormone secretion, and cell
proliferation. 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 et
al., supra). 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).
The discovery of new human membrane channell 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 cell proliferative,
immune/inflammatory,
transpartJsecretory, osmoregulatory, muscular, cardiovascular, and
neurological disorders.
SUMMARY OF THE II~fVENTION
The invention features substantially purified polypeptides, human membrane
channel
proteins, referred to collectively as "MECHP" and individually as "MECHP-1,"
"MECHP-2,"
"MECHP-3," "MECHP-4," "MECHP-5," "MECHP-6," "MECHP-7," "MECHP-8," "MECHP-9,"
"MECHP-10," "MECHP-11," "MECHP-12," "MECHP-~13," "MECHP-14," "MECHP-15,"
"MECHP-16", "MECHP-17", and "MECHP-18." In one aspect, the invention provides
a
substantially purified polypeptide comprising an amino .acid sequence selected
from the group
consisting of SEQ ID NO:1-18, and fragments thereof.
The invention further provides a substantially purifed variant having at least
95% amino
acid sequence identity to at least one of the amino acid sequences selected
from the group
consisting of SEQ ID NO:I-I 8 and fragments thereof. 'lflhe invention also
provides an isolated and
purified polynucleotide encoding the polypeptide comprising an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-18 and fragments thereof. The
invention also includes
an isolated and purified polynucleotide variant having at least 95%
polynucleotide sequence
-g-
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identity to the polynucieotide encoding the poiypeptide comprising the amino
acid sequence
selected from the group consisting of SEQ ID NO:1-18 and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide
which hybridizes
under stringent conditions to the polynucleotide encoding the polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-18 and fragments
thereof. The invention
also provides an isolated and purified polynucleotide having a sequence which
is complementary to the
polynucleotide encoding the poiypeptiide comprising the amino acid sequence
selected from the group
consisting of SEQ ID NO: I-18 and fragments thereof.
The invention also provides a method for detectiing a polynucleotide in a
sample
containing nucleic acids, the method comprising the steps of (a) hybridizing
the complement of the
polynucleotide sequence to at least one of the polynucleotides of the sample,
thereby forming a
hybridization complex; and {b) detecting the hybridization complex, wherein
the presence of the
hybridization complex correlates with the presence of a poiynucleotide in the
sample. In one
aspect, the method further comprises amplifying the poiynucleotide prior to
hybridization.
The invention also provides an isolated and puriified polynucieotide
comprising a
polynucleotide sequence selected from the group consisting of SEQ ID N0:19-36,
and fragments
thereof. The invention further provides an isolated and purified
polynucleotide variant having at
least 95% polynucleotide sequence identity to the polyn~ucieotide sequence
selected from the
group consisting of SEQ ID N0:19-36 and fragments thereof. The invention also
provides an
isolated and purified polynucleotide having a sequence which is complementary
to the
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ
ID N0:19-36 and fragments thereof.
The invention further provides an expression vector containing at least a
fragment of the
polynucieotide encoding the polypeptide comprising an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-18 and fragments thereof. In another aspect,
the expression
vector is contained within a host cell.
The invention also provides a method for prodn~cing a polypeptide, the method
comprising
the steps of-. (a} culturing the host cell containing an expression vector
containing at least a
fragment of a polynucleotide under conditions suitable for the expression of
the polypeptide; and
(b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a
substantially
purified polypeptide having the amino acid sequence selected from the group
consisting of SEQ
ID NO:I-18 and fragments thereof, in conjunction widh a suitable
pharmaceutical carrier.
The invention further includes a purified antibody which binds to a
polypeptide selected
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from the group consisting of SEQ iD NO:1-18 and fragments thereof. The
invention also provides
a purified agonist and a purified antagonist to the polypeptide.
'The invention also provides a method for treating or preventing a disorder
associated with
decreased expression or activity of MECHP, the method comprising administering
to a subject in
need of such treatment an effective amount of a pharmaceutical composition
comprising a
substantially purified polypeptide having the amino acid sequence selected
from the group
consisting of SEQ ID NO:1-18 and fragments thereof, in conjunction with a
suitable
pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder
associated with
increased expression or activity of MECHP, the method comprising administering
to a subject in
need of such treatment an effective amount of an antagonist of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-I8 and fragments
thereof.
BRIEF DESCRIPTION OF THE FIGURES AND TABLES
IS Figure 1 shows the amino acid sequence alignment between MECHP-1 (1568324;
SEQ ID
NO:1 ) and rat glutamic acid-rich protein (GI 2924369; S~EQ ID N0:37),
produced using the
BLAST search tool.
Figure 2 shows the amino acid sequence alignment among MECHP-2 {4094907; SEQ
ID
N0:2), Drosophila voltage-gated potassium channel (GI 116443; SEQ ID N0:38),
and P.
penicillatus potassium channel a-subunit (GI 1763619; SEQ ID N0:39), produced
using the
multisequence alignment program of LASERGENE software (DNASTAR, Madison WI).
Figures 3A and 3B show the amino acid sequence alignment between MECHP-3
(518158;
SEQ ID N0:3) and rat calcium-activated potassium channel rSK3 (GI 2564072; SEQ
ID N0:40),
produced using the multisequence alignment program o1F LASERGENE software.
Figures 4A, 4B, and 4C show the amino acid sequence alignment among MECHP-4
(602926; SEQ ID N0:4), Droso~hila voltage-gated potassium channel (GI 116443;
SEQ ID
N0:38) and P. penicillatus potassium channel a-subunit (Gi 1763619; SEQ ID
N0:39), produced
using the multisequence alignment program of LASERCiENE software.
Figures SA and SB show the amino acid sequence alignment between MECHP-5
{922119;
SEQ ID NO:S) and rat aquaporin 7 (GI 2350843; SEQ ID N0:41 ), produced using
the
multisequence alignment program of LASERGENE software.
Figures 6A and 6B show the amino acid sequence alignment between MECHP-7
(2731369; SEQ ID N0:7) and mouse connexin 30.3 (GI 192647; SEQ ID N0:42),
produced using
the muitisequence alignment program of LASERGENE software.
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Figure 7 shows the amino acid sequence alignment between MECHP-16 (2069907;
SEQ
ID N0:16) and human beta subunit of Ca' activated K+ channel (GI 1055345; SEQ
ID N0:43);
produced using the multisequence alignment program of LASERGENE software.
Figures 8A and 8B show the amino acid sequence alignment between MECHP-17
(2243917; SEQ iD N0:17) and a homolog of Caenorhabditis elesans K+ channel
protein (GI
3292929; SEQ ID N0:44), produced using the multiseque~nce alignment program of
LASERGENE software.
Figures 9A and 9B show the amino acid sequence alignment between MECHP-18
(2597476; SEQ ID N0:18) and human aquaporin 9 {GI 2887407; SEQ ID N0:45),
produced using
the multisequence alignment program of LASERGENE software.
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 MECHP.
Table 2 shows features of each polypeptide sequence, including potential
motifs,
homologous sequences, and methods and algorithms used for identification of
MECHP.
Table 3 shows 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 MECHP were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze MECHP, along
with
applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE II'JVENTION
Before the present proteins, nucleotide sequences" and methods are described,
it is
understood that this invention is not limited to the particullar machines,
materials and methods
described, as these may vary. It is also to be understood tlhat the
terminology used herein is for the
purpose of describing particular embodiments only, and i<.; 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.
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Unless deftned otherwise, all technical and scientifc 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 method:. 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, reagemts 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
"MECHP" refers to the amino acid sequences of substantially purified MECHP
obtained
from any species, particularly a mammalian species, including bovine, ovine,
porcine, murine,
equine, and preferably the human species, from any source, whether natural,
synthetic,
semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which, when bound to MECHP, increases
or
prolongs the duration of the effect of MECHP. Agonists may include proteins,
nucleic acids,
carbohydrates, or any other molecules which bind to and modulate the effect of
MECHP.
An "allelic variant" is an alternative fonm of the gene encoding MECHP.
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. Any given
natural or recombinant gene may have none, one, or many allelic forms. Common
mutational
changes which give rise to allelic variants are generally ascribed to natural
deletions, additions, or
substitutions of nucleotides. Each of these types of changes may occur alone,
or in combination
with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding MECI';IP include those sequences
with
deletions, insertions, or substitutions of different nucleotidles, resulting
in a polypeptide the same
as MECHP or a polypeptide with at least one functional characteristic of
MECHP. Tncluded
within this definition are polymorphisms which may or may not be readily
detectable using a
particular oligonucleotide probe of the polynucleotide encoding MECHP, and
improper or
unexpected hybridization to allelic variants, with a locus other than the
normal chromosomal locus
for the polynucleotide sequence encoding MECHP. 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 MECHP. Deliberate amino
acid substitutions
may be made on the basis of similarity in polarity, charge, solubility,
hydrophobicity,
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hydrophilicity, and/or the amphipathic nature of the residues. as long as the
biological or
immunological activity of MECHP is retained. For example, negatively charged
amino acids may
include aspartic acid and glutamic acid, positively charged amino acids may
include lysine and
arginine, and amino acids with uncharged polar head groups having similar
hydrophilicity values
may include leucine, isoleucine, and valine; glycine and allanine; asparagine
and glutamine; seriile
and threonine; 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. In this context, "fragments," "immun~ogenic fragments,"
or "antigenic
fragments" refer to fragments of MECHP which are preferably at least 5 to
about I S amino acids
in length, most preferably at least 14 amino acids, and which retain some
biological activity or
immunological activity of MECHP. Where "amino acid sequence" is recited to
refer to an amino
acid sequence of a naturally occurring protein molecule, "aunino acid
sequence" and like terms are
not meant to limit the amino acid sequence to the complete; native amino acid
sequence associated
with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which, when bound to MECHP,
decreases the
amount or the duration of the effect of the biological or immunological
activity of MECHP.
Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or
any other molecules
which decrease the effect of MECHP.
The term "antibody" refers to intact molecules as well as' to fragments
thereof, such as
Fab, F(ab')2, and Fv fragments, which are capable of binding the epitopic
determinant. Antibodies
that bind MECHP polypeptides can be prepared using intact polypeptides or
using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oiigopeptide
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 (ICLHI). The coupled peptide is then used to
immunize the animal.
The term "antigenic determinant" refers to that fragment 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 {given regions or three-
dimensional structures on
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the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition containing a nucleic acid
sequence which
is complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules
may be produced by any method including synthesis or transcription. Once
introduced into a cell,
the complementary nucleotides combine with natural sequences produced by the
cell to form
duplexes and to block either transcription or translation. The designation
"negative" can refer to
the antisense strand, and the designation "positive" can refer to the sense
strand.
The term "biologically active" refers to a protein having structural,
regulatory, or
biochemical functions of a naturally occurring molecule. Likewise,
"immunologically active"
refers to the capability of the natural, recombinant, or synthetic MECHP, or
of any oligopeptide
thereof, to induce a specific immune response in appropriate animals or cells
and to bind with
specific antibodies.
The terms "complementary" and "complementarily" refer to the natural binding
of
polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds
to the
complementary sequence "3' T-C-A 5'." Complementarit~,~ between two single-
stranded molecules
may be "partial," such that only some of the nucleic acids bind, or it may be
"complete," such that
total complementarity exists between the single stranded molecules. The degree
of
complementarity between nucleic acid strands has significant effects on the
efficiency and strength
of the hybridization between the nucleic acid strands. This is of particular
importance in
amplification reactions, which depend upon binding between nucleic acids
strands, and in the
design and use of peptide nucleic acid {PNA) molecules.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising a given amino acid sequence" refer broadly to any composition
containing the given
polynucleotide or amino acid sequence. The composition may comprise a dry
formulation or an
aqueous solution. Compositions comprising polynucleotide sequences encoding
MECHP or
fragments of MECHP 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 dodecyi 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
resequenced to
resolve uncalled bases, extended using the XL-PCR kit (Pe:rkin-Elmer, Norwalk
CT) in the 5'
and/or the 3' direction, and resequenced, or which has been assembled from the
overlapping
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sequences of more than one Incyte Clone using a computer program for fragment
assembly, such
as the GELVIEW fragment assembly system (GCG, Madison WI). Some sequences have
been
both extended and assembled to produce the consensus sequence.
The term "correlates with expression of a polynuc:leotide" indicates that the
detection of
the presence of nucleic acids, the same or related to a nucleic acid sequence
encoding MECHP, by
northern analysis is indicative of the presence of nucleic acids encoding
MECHP in a sample, and
thereby correlates with expression of the transcript from the polynucteotide
encoding MECHP.
A "deietion" 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 the chemical modification of a polypeptide
sequence, yr a
polynucleotide sequence. Chemical modifications of a polynucleotide sequence
can include, for
example, replacement of hydrogen by an alkyl, acyl, or amino group. A
derivative pvlynucleotide
encodes a polypeptide which retains at least one biological or immunvlogical
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.
The term "similarity" refers to a degree of complementarity. There may be
partial
similarity or complete similarity. The word "identity" maw substitute for the
word "similarity." A
partially complementary sequence that at least partially inhibits an identical
sequence from
hybridizing to a target nucleic acid is referred to as "substantially
similar." The inhibition of
hybridization ofthe completely complementary sequence to the target sequence
may be examined
using a hybridization assay (Southern or northern blot, solution
hybridization, and the like) under
conditions of reduced stringency. A substantially similar sequence or
hybridization probe will
compete for and inhibit the binding of a completely similar (identical)
sequence to the target
sequence under conditions of reduced stringency. This is mot to say that
conditions of reduced
stringency are such that non-specific binding is permitted, as reduced
stringency conditions
require that the binding of two sequences to one another be a specific (i.e.,
a selective) interaction.
The absence of non-specific binding may be tested by the u.se of a second
target sequence which
lacks even a partial degree of complementarity (e.g., less than about 30%
similarity or identity).
In the absence of non-specific binding, the substantially similar sequence or
probe will not
hybridize to the second non-complementary target sequence.
The phrases "percent identity" and "% identity" refi~r to the percentage of
sequence
similarity found in a comparison of two or more amino acidl or nucleic acid
sequences. Percent
identity can be determined electronically, e.g., by using the MEGALIGN program
(DNASTAR)
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which creates alignments between two or more sequences according to methods
selected by the
user, e.g., the clustal method. (See, e.g., Higgins, D.G. arrd P.M. Sharp (
1988) Gene 73:237-244.)
Parameters for each method may be the default parameters provided by MEGALIGN
or may be
specified by the user. The clustal algorithm groups sequences into clusters by
examining the
distances between all pairs. The clusters are aligned painwise and then in
groups. The percentage
similarity between two amino acid sequences, e.g., sequence A and sequence B,
is calculated by
dividing the length of sequence A, minus the number of g,ap residues in
sequence A, minus the
number of gap residues in sequence B, into the sum of the residue matches
between sequence A
and sequence B, times one hundred. Gaps of low or of no similarity between the
two amino acid
sequences are not included in determining percentage similarity. Percent
identity between nucleic
acid sequences can also be counted or calculated by other methods known in the
art, e.g., the Jotun
Hein method. (See, e.g., Hein, J. ( 1990) Methods Enzymol. 183:626-645.)
Identity between
sequences can also be determined by other methods know~a in the art, e.g., by
varying
hybridization conditions.
"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 stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules 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 blinding ability.
"Hybridization" refers to any process by which a strand of nucleic acid binds
with a
complementary strand through base pairing.
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, flters, 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,
to the sequence found in the naturally occurring molecule.
"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, wind other
signaling molecules, which
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may affect cellular and systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotides on
a substrate.
The terms "element" and "array element" in a microarray context, refer to
hybridizable
polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of MECHP. For example,
modulation may cause an increase or a decrease in proteiin activity, binding
characteristics, or any
other biological, functional, or irnrnunological properties of MECHP.
The phrases "nucleic acid" or "nucleic acid seque;nce," as used herein. refer
to a
nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These
phrases also refer to
DNA or RNA of genomic or synthetic origin which may Ibe single-stranded or
double-stranded
and may represent the sense or the antisense strand, to pelptide nucleic acid
(PNA), or to any
DNA-like or RNA-like material. In this context; "fragments" refers to those
nucleic acid
sequences which comprise a region of unique polynucleotide sequence that
specifically identifies
SEQ ID N0:19-36, for example, as distinct from any other sequence in the same
genome. For
IS example, a fragment of SEQ ID N0:19-36 is useful in hybridization and
amplification
technologies and in analogous methods that distinguish S1:Q ID N0:19-36 from
related
polynucleotide sequences. A fragment of SEQ ID N0:19-36 is at least about 15-
20 nucleotides in
length. The precise length of the fragment of SEQ ID NO~:19-36 and the region
of SEQ ID
N0:19-36 to which the fragment corresponds are routinely determinable by one
of ordinary skill
in the art based on the intended purpose for the fragment. In some cases, a
fragment, when
translated, would produce polypeptides retaining some functional
characteristic, e.g., antigenicity,
or structural domain characteristic, e.g., ATP-binding site, of the full-
length polypeptide.
The terms "operably associated" and "operably linked" refer to functionally
related nucleic
acid sequences. A promoter is operably associated or operably linked with a
coding sequence if
the promoter controls the translation of the encoded polyp~eptide. While
operably associated or
operably linked nucleic acid sequences can be contiguous and in the same
reading frame, certain
genetic elements, e.g., repressor genes, are not contiguousily linked to the
sequence encoding the
polypeptide but still bind to operator sequences that contrcd expression of
the polypeptide.
The term "oligonucleotide" refers to a nucleic acidl sequence of at least
about 6 nucleotides
to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably
about 20 to 25
nucleotides, which can be used in PCR amplification or in a hybridization
assay or microarray.
"Oligonucleotide" is substantially equivalent to the terms "'amplimer,"
"primer," "oligomer," and
"probe," as these terms are commonly defined in the art.
"Peptide nucleic acid" (PNA) refers to an antisense; molecule or anti-gene
agent which
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comprises an oligonucleotide of at least about S 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.
The term "sample" is used in its broadest sense. ,A sample suspected of
containing nucleic
acids encoding MECHP, or fragments thereof, or MECHI' 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, or an antagonist. 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 containing 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
1S to the antibody.
The term "stringent conditions" refers to conditions which permit
hybridization between
polynucleotides and the claimed polynucleotides. Stringent conditions can be
defined by salt
concentration, the concentration of organic solvent, e.g., forrnamide,
temperature, and other
conditions well known in the art. In particular, stringency can be increased
by reducing the
concentration of salt, increasing the concentration of form~amide, or raising
the hybridization
temperature.
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 about 60%
free, preferably about 7S% free, and most preferably aboul: 90% free from
other components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or
nucleotides by
different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beadis, 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 polynucleotide;s or polypeptides
are bound.
"Transformation" describes a process by which exogenous DNA enters and changes
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
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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, 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 oftime.
A "variant" of MECHP polypeptides refers to an. amino acid sequence that is
altered by
one or more amino acid residues. The variant may have "conservative" changes,
wherein a
I0 substituted amino acid has similar structural or chemical properties (e.g.,
replacement of leucine
with isoleucine). More rarely, a variant may have "nonconservative" changes
(e.g., replacement of
glycine with tryptophan). Analogous minor variations m,ay also include amino
acid deletions or
insertions, or both. Guidance in determining which amino acid residues may be
substituted,
inserted, or deleted without abolishing biological or immunological activity
may be found using
15 computer programs well known in the art, for example, L,ASERGENE software
(DNASTAR).
The term "variant," when used in the context of a~ po(ynucleotide sequence,
may
encompass a polynucleotide sequence related to MECHP. This defnition may also
include, for
example, "allelic" (as defined above}, "splice," "species," or "polymorphic"
variants. A splice
variant may have significant identity to a reference molecule, but will
generally have a greater or
20 lesser number of polynucleotides due to alternate splicing; of exons during
mRNA processing. The
corresponding polypeptide may possess additional functional domains or an
absence of domains.
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
25 individuals of a given species. Polymorphic variants also may encompass
"single nucleotide
polymorphisms" (SNPs) in which the poiynucleotide sequence varies by one base.
The presence
of SNPs may be indicative of, for example, a certain population, a disease
state, or a propensity for
a disease state.
THE INVENTION
30 The invention is based on the discovery of new human membrane channel
proteins
(MECHP), the polynucleotides encoding MECHP, and the. use of these
compositions for the
diagnosis, treatment, or prevention of cell proliferative,
im~munelinflammatory, transport/secretory,
osmoregulatory, muscular, cardiovascular, and neurological disorders.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
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MECHP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs}
of the
polypeptide and nucleotide sequences, respectively. Column 3 shows the clone
IDs of the Incyte
clones in which nucleic acids encoding each MECHP were identified, and column
4 shows the
cDNA libraries from which these clones were isolated. Column 5 shows Incyte
clones and their
corresponding cDNA libraries. Clones for which cDNA libraries are not
indicated were derived
from pooled cDNA libraries. The clones in column S were used to assemble the
consensus
nucleotide sequence of each MECHP 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 the identity of each polypeptide; and
column 7 shows
analytical methods used to identify each polypeptide through sequence homology
and protein
motifs.
IS MECHP-1 has chemical and structural similarity with rat glutamic acid-rich
protein (GI
2924369; SEQ ID N0:37). In particular, MECHP-1 and rat glutamic acid-rich
protein share 15%
overall identity. As shown in Figure 1, BLAST analysis identifies regions of
MECHP-1 and rat
glutamic acid-rich protein which share 27-30% identity. 'These regions extend
from residue V12
through T163, P266 through 6344, P461 through E548, and E653 through 6709 in
MECHP-i.
As shown in Figure 2, MECHP-2 has chemical and structural similarity with
Drosonhila
voltage-gated potassium channel (GI 116443; SEQ ID NO:38) and P, penicillatus
potassium
channel a-subunit (GI 1763619; SEQ ID N0:39). In particular, MECHP-2 shares
18% identity
with Drosophila voltage-gated K+ channel, and 17% identity with P.
penicillatus K+ channel a-
subunit. In particular, MECHP-2 shares 27% identity with Drosophila voltage-
gated potassium
channel and P. penicillatus potassium channel a-subunit over the first 133
residues, from M1
through T 133 in MECHP-2.
As shown in Figures 3A and 3B, MECHP-3 has chemical and structural similarity
with rat
calcium-activated potassium channel rSK3 (GI 2564072; SEQ ID N0:40). In
particular, MECHP-
3 and rat rSk;3 share 40% identity. MECHP-3 and rat rSk;3 also share a
canonical ion pore (P)
region, including a GYG potassium ion selectivity sequence, from residue W192
through 6213 in
MECHP-3.
As shown in Figures 4A, 4B, and 4C, MECHP-4 h;as chemical and structural
similarity
with Drosophila voltage-gated potassium channel (GI 116f.43; SEQ ID N0:38) and
P. penicillatus
potassium channel a-subunit (GI 1763619; SEQ ID N0:39). In particular, MECHP-4
shares 28%
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identity with Drosophila voltage-gated K* channel, and 2Ei% identity with
P.,penicillatus K*
channel a-subunit, respectively. MECHP-4, Droso~hila voltage-gated K* channel,
and_P.
penicillatus K+ channel a-subunit also share a GYG potassium ion selectivity
sequence from
residue 6372 through 6374 in MECHP-4.
As shown in Figures SA and SB, MECHP-5 has chemical and structural similarity
with
rat aquaporin 7 (Gi 2350843; SEQ ID N0:41). In particular, MECHP-5 and rat
aquaporin 7 share
74% identity.
As shown in Figures 6A arid 6B, MECHP-7 has chemical and structural similarity
with
mouse connexin 30.3 (GI 192647; SEQ ID N0:42). In particular, MECHP-7 and
mouse connexin
30.3 (GI 192647) share 84% identity.
As shown in Figure 7, MECHP-16 has chemical and structural similarity with
human beta
subunit of Ca* activated K* channel (GI 1055345; SEQ ID N0:43). In particular,
MECHP-16 and
human beta subunit of Ca* activated K+ channel share 40%. identity.
As shown in Figures 8A and 8B, MECHP-17 has chemical and structural similarity
with a
homolog of C. elegans K* channel protein (GI 3292929; SEQ ID N0:44). In
particular, MECHP-
17 and the specified homolog of C. eleQans K* channel protein share 47%
identity.
As shown in Figures 9A and 9B, MECHP-I8 has chemical and structural similarity
with
human aquaporin 9 (GI 2887407; SEQ ID N0:45). In particular, MECHP-18 and
human
aquaporin 9 share 46% identity.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding MECHP. The first column of Table
3 lists the
nucleotide SEQ ID NOs. Column 2 lists tissue categories which express MECHP as
a fraction of
total tissue categories expressing MECHP. Column 3 lists diseases, disorders,
or conditions
associated with those tissues expressing MECHP. Column 4 lists the vectors
used to subclone the
cDNA library. Northern analysis shows the expression of SEQ ID N0:34 in only 7
libraries, of
which 6 (86%} are associated with cell proliferation. Two of these libraries
are associated with
brain tissue, one with pancreatic islet cells, one with kidney tissue, one
with fetal lung tissue, one
with ovarian tissue, and one with adrenal tissue. Northern analysis shows the
expression of SEQ
ID N0:36 in only 3 libraries, one of which is associated with ovarian tumor
tissue, one with
developing lung tissue, and one with gastrointestinal tissue associated with
inflammation. Of
particular note is the enriched expression of MECHP in neural and
neuroendocrine tissue, most
prominently the neural tissue-specific expression of SEQ IL) N0:30.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA
libraries from which cDNA clones encoding MECHP were ;isolated. Column 1
references the
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nucleotide SEQ ID NOs, column 2 shows the cDNA libraries from which these
clones were
isolated, and column 3 shows the tissue origins and other descriptive
information relevant to the
cDNA libraries in column 2.
The following fragments of the nucleotide sequences encoding MECHP are useful,
for
example, in hybridization or amplification technologies to identify SEQ ID
N0:19-36, and to
distinguish between SEQ ID NO: f 9-36 and related polynucleotide sequences.
The useful
fragments include the fragment of SEQ ID N0:19 from about nucleotide 764 to
about nucleotide
808; the fragment of SEQ ID N0:20 from about nucleotide 523 to about
nucleotide 582; the
fragment of SEQ ID N0:21 from about nucleotide 628 to .about nucleotide 669;
the fragment of
SEQ ID N0:22 from about nucleotide 779 to about nucleotide 826; the fragment
of SEQ ID
N0:23 from about nucleotide 64 to about nucleotide 108; 'the fragment of SEQ
ID N0:24 from
about nucleotide 1133 to about nucleotide 1180; the fragment of SEQ ID N0:25
from about
nucleotide 656 to about nucleotide 700; the fragment of SEQ ID N0:26 from
about nucleotide 153
to about nucleotide 197; the fragment of SEQ ID N0:27 from about nucleotide
2160 to about
nucleotide 22 i 9; the fragment of SEQ ID N0:28 from about nucleotide 1275 to
about nucleotide
1322; the fragment of SEQ ID N0:29 from about nucleotide 313 to about
nucleotide 348; the
fragment of SEQ ID N0:30 from about nucleotide 994 to about nucleotide 1041;
the fragment of
SEQ ID N0:31 from about nucleotide 443 to about nucleotide 478; the fragment
of SEQ ID
N0:32 from about nucleotide l I75 to about nucleotide 12Ct7; the fragment of
SEQ ID N0:34 from
about nucleotide 381 to about nucleotide 425; the fragment: of SEQ ID N0:35
from about
nucleotide 17 to about nucleotide 61; and the fragment of SEQ ID N0:36 from
about nucleotide
54 to about nucleotide 98. The polypeptides encoded by th.e fragments of SEQ
ID N0:19, SEQ ID
N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:25,
SEQ
ID N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID N0:2~>, SEQ ID N0:30, SEQ ID
N0:34,
SEQ ID N0:35, AND SEQ ID N0:36 are useful, for example, as immunogenic
peptides.
The invention also encompasses MECHP variants. A preferred MECHP variant is
one
which has at least about 80%, more preferably at least abou~,t 90%, and most
preferably at least
about 95% amino acid sequence identity to the MECHP amino acid sequence, and
which contains
at least one functional or structural characteristic of MECHP.
The invention also encompasses polynucleotides which encode MECHP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence
selected from the group consisting of SEQ ID N0:19-36, which encodes MECHP.
The invention also encompasses a variant,of a polynucleotide sequence encoding
MECHP. In particular, such a variant polynucleotide sequence will have at
least about 70%, more
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preferably at least about 85%. and most preferably at iea;st about 95%
polynucleotide sequence
identity to the polynucleotide sequence encoding MECHf. A particular aspect of
the invention
encompasses a variant of a sequence selected from the group consisting of SEQ
ID N0:19-36
which has at least about 70%, more preferably at least about 85%, and most
preferably at least
about 95% poiynucleotide sequence identity to a sequence selected from the
group consisting of
SEQ ID N0:19-36. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of MECHP.
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~MECHP, 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 polynucieotide
sequence that could be made by selecting combinations biased 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 MECHP, ;and ail such variations
are to be
considered as being specifically disclosed.
Although nucleotide sequences which encode ME',CHP and its variants are
preferably
capable of hybridizing to the nucleotide sequence of the naturally occurring
MECHP under
appropriately selected conditions of stringency, it may be advantageous to
produce nucleotide
sequences encoding MECHP 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 prol<:aryotic 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 M:ECHP 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 MECHP
and MECHP 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 MECHP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, iin particular, to
those shown in SEQ ID
NO: I9-36, or to a fragment of SEQ ID N0:19-36, under various conditions of
stringency. (See,
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e.g., Wahi, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel,
A.R. (1987)
Methods Enzymol. 152:507-51 I .) For example, stringent: salt concentration
will ordinarily be less
than about 750 mM NaCI and 75 mM trisodium citrate, preferably less than about
500 mM NaCI
and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCI
and 25 mM
trisodium citrate. Low stringency hybridization can be obtained in the absence
of organic solvent,
e.g., formamide, while high stringency hybridization can Ibe obtained in the
presence of at least
about 35% formamide, and most preferably at least about SO% formamide.
Stringent temperature
conditions will ordinarily include temperatures of at least about 30°C,
more preferably of at least
about 37°C, and most preferably of at least about 42°C. Varying
additional parameters, such as
i0 hybridization time, the concentration of detergent, e.g:, sodium dodecyl
sulfate {SDS), and the
inclusion or exclusion of carrier DNA, are well known to chose skilled in the
art. Various levels of
stringency are accomplished by combining these various conditions as needed.
In a preferred
embodiment, hybridization will occur at 30°C in 750 mM NaCI, 75 mM
trisodium citrate, and I%
SDS. In a more preferred embodiment, hybridization will occur at 37°C
in 500 mM NaCI, S0 mM
IS trisodium citrate, l% SDS, 35% formamide, and 100 p.g/rn~l denatured salmon
sperm DNA
(ssDNA). In a most preferred embodiment, hybridization will occur at
42°C in 250 mM NaCI, 25
mM trisodium citrate, 1% SDS, 50 % formamide, and 200 pg/ml ssDNA. Useful
variations on
these conditions will be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash
20 stringency conditions can be defined by salt concentration and by
temperature. As above, wash
stringency can be increased by decreasing salt concentration or by increasing
temperature. For
example, stringent salt concentration for the wash steps willl preferably be
less than about 30 mM
NaCI and 3 mM trisodium citrate, and most preferably less than about 15 mM
NaCI and 1.5 mM
trisodium citrate. Stringent temperature conditions for the wash steps will
ordinarily include
25 temperature of at least about 25°C, more preferably of at least
about 42°C, and most preferably of
at least about 68°C. In a preferred embodiment, wash steps. will occur
at 25°C in 30 mM NaCI, 3
mM trisodium citrate, and 0. I % SDS. In a more preferred embodiment, wash
steps will occur at
42°C in 1 S mM NaCI, 1.5 mM trisodium citrate, and 0.1 % SDS. In a most
preferred embodiment,
wash steps will occur at 68°C in 15 mM NaCI, 1.5 mM trisodium citrate,
and 0.1 % SDS.
30 Additional variations on these conditions will be readily apparent to those
skilled in the art.
Methods for DNA sequencing are well known in the art arid may be used to
practice any
of the embodiments of the invention. The methods may employ such enzymes as
the Klenow
fragment of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq
polymerise
(Perkin-Elmer), thetrnostable T7 polymerise (Amersham Pharmacia Biotech,
Piscataway NJ), or
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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 Robbins Hydra
microdispenser (Robbins
Scientific, Sunnyvale CA), Hamilton MICROLAB 2200 (Hamilton, Reno NV), Pettier
Thermal
S Cycler 200 (PTC200; MJ Research, Watertown MA) and the ABI CATALYST 800
(Perkin-
Elmer). Sequencing is then carried out using either ABI a73 or 377 DNA
sequencing systems
(Perkin-Eimer), 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. ( I 997) Short
Protocols in Molecular Biolo v, John Wiley & Sons, New York NY, unit 7.7;
Meyers, R.A.
( 1995) Molecular Biology and Biotechnolo~v, Wiley VCH, New York NY, pp. 856-
853.)
The nucleic acid sequences encoding MECHP 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
IS may be employed, restriction-site PCR, uses universal and nested primers to
amplify unknown
sequence from genomic DNA within a cloning vector. (Se:e, 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: i 11-119.) 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.
2S Other methods which may be used to retrieve unknown sequences are known in
the art. (See, e.g.,
Parker, J.D. et al. (I991) Nucleic Acids Res. 19:30SS-3060). Additionally, one
may use PCR,
nested primers, and PROMOTERFINDER libraries (Clonte,ch, Palo Alto CA) to walk
genomic
DNA. This procedure avoids the need to screen libraries arid is useful in
finding intron/exon
junctions. For ail 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 SO% 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
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sequences containing the 5' regions of genes, are preferable for situations in
which an oiigo 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 SEQIJENCE NAVIGATOR, Perkin-
Elmer),
IO 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 MECHP may be cloned in recombinant DNA molecules that direct
expression of
MECHP, 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
MECHP.
The nucleotide sequences of the present invention can be engineered using
methods
generally known in the art in order to alter MECHP-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.
In another embodiment, sequences encoding MECFtP 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. (1f80) Nucleic Acids
Symp. Ser.
7:225-232.) Alternatively, MECHP itself or a fragment thereof may be
synthesized using
chemical methods. For example, peptide synthesis can be performed using
various solid-phase
techniques. (See, e.g., Roberge, J.Y. et al. (1995) Science 26.9:202-204.)
Automated synthesis
may be achieved using the ABI 431A peptide synthesizer (Perkin-Elmer).
Additionally, the amino
acid sequence of MECHP, or any part thereof, may be altered during direct
synthesis and/or
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combined with sequences from other proteins, or any part thereof, to produce a
variant
poiypeptide.
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, T. ( 1984) Proteins. Structures and
Molecular Properties, WH
Freeman, New York NY.)
In order to express a biologically active MECHP, the nucleotide sequences
encoding
MECHP or derivatives thereof may be inserted into an appropriate expression
vector, i.e., a vector
which contains the necessary elements for transcriptional and translationai
control of the inserted
coding sequence in a suitable host. These elements inclucle regulatory
sequences, such as
enhancers, constitutive and inducible promoters, and 5' and 3' untranslated
regions in the vector
and in pofynucleotide sequences encoding MECHP. Such elements may vary in
their strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding MECHP. Such signals include the A'CG initiation codon and
adjacent
sequences, e.g. the Kozak sequence. In cases where sequences encoding MECHP
and its initiation
codon and upstream regulatory sequences are inserted into. the appropriate
expression vector, no
additional transcriptional or translational control signals m;ay 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 Nhe art may be used to
construct
expression vectors containing sequences encoding MECHF' 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, Flainview NY,
ch. 4, 8, and
16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolosv,
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 MECHP. These include, but are not limited to,
microorganisms such as
bacteria transformed with recombinant bacteriophage, plasrnid, or cosmid DNA
expression
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vectors; yeast transformed with yeast expression vectors.; insect cell systems
infected with viral
expression vectors (e.g., bacuiovirus}; plantceli 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'piasmids); or animal cell systems. 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 MECHP.
For example,
routine cloning, subcloning, and propagation of polynucleotide sequences
encoding MECHP can
be achieved using a multifunctional E. coli.vector such a;s PBLUESCRIPT
(Stratagene, La Jolla
CA) or pSPORTI plasmid (Life Technologies). Ligation of sequences encoding
MECHP into the
vector's multiple cloning site disrupts the IacZ 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.;1 When large quantities
of MECHP are
needed, e:g. for the production of antibodies, vectors which direct high level
expression of
MECHP 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 MECHP. 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, G.A. et al. ( 1987) Methods Enzymol. i 53:516-544; and
Scorer, C.A. et al.
(1994) Bio/Technology 12:181-184.}
Plant systems may also be used for expression of 1VIECHP. Transcription of
sequences
encoding MECHP may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV
used alone or in combination with the omega leader sequence from TMV
(Takamatsu, N. (1987)
EMBO J. 6:307-311 ). Alternatively, plant promoters such as the small subunit
of RUBISCO or
heat shock promoters may be used. (See, e:g., Coruzzi, G. et aL (1984) EMBO J.
3:1671-1680;
Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991)
Results Probl. Cell
Differ. 17:85-105.) These constructs can be introduced into plant cells by
direct DNA
transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill
Yearbook of
Science and Tech~ nolo~y (1992) McGraw Hill, New York rdY, pp. 191-196.)
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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 MECHP
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 MECHP 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 hiigh-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.3.
et al. ( 1997) Nat.
Genet. 15:345-355.)
For long term production of recombinant proteins iin mammalian systems, stable
expression of MECHP in cell lines is preferred. For example, sequences
encoding MECHP can be
transformed into cell lines using expression vectors which :may contain viral
origins of replication
and/or endogenous expression element's and a selectable marker gene on the
same or on a separate
vector. Following the introduction of the vector, cells may be al lowed 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 apI>ropriate 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 or apP cells, respectively.
(See, e.g., Wigler, M. et
al. ( 1977) Cell 1 I :223-232; Lowy, I. et al. ( I980) 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 or pat confer resistance to chlorsulfuron and phasphinotricin
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), (3
glucuronidase and its
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substrate f3-glucuronide, or luciferase and its substrate luciferin may be
used. These markers can
be used not only to identify transformants, but also to quantify the amount of
transient or stable
protein expression attributable to a specific vector system. (See, e.g.,
Rhodes, C.A. (1995)
Methods Mol. Biol. 55:121-131.)
Although the presencelabsence 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 MECHP is inserted within a marker gene
sequence,
transformed cells containing sequences encoding MECHP' can be identified by
the absence of
marker gene function. Alternatively, a marker gene can be placed in tandem
with a sequence
encoding MECHP 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 aequence encoding MECHP
and that
express MECHP may be identifed by a variety of procedures known to those of
skill in the art.
These procedures include, but are not limited to, DNA-DN'A or DNA-RNA
hybridizations, PCR
amplification, and protein bioassay or immunoassay techniiques which include
membrane,
solution, or chip based technologies for the detection and/or quantification
of nucleic acid or
protein sequences.
immunological methods for detecting and measuring the expression of MECHP
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
MECHP is preferred,
but a competitive binding assay may be employed. These a.nd other assays are
well known in the
art. (See, e.g., Hampton, R. et al. ( 1990) Serological Methods. a Laboratory
Manual, APS Press,
St. Paul MN, Sect. IV; Coligan, J.E. et ai. (1997) Current Protocols in
Immunolo y, Greene Pub.
Associates and Wiley-Interscience, New York NY; and Pound, J.D. ( 1998)
Immunochemical
Protocols, Humana Press, Totowa NJ.)
A wide variety of labels and conjugation techniques. are known by those
skilled in the art
and may be used in various nucleic acid and amino acid assays. Means for
producing labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding MECHP
include oligolabeling, nick translation, end-labeling, or PCR. amplification
using a labeled
nucleotide. Alternatively, the sequences encoding MECHP, 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
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appropriate RNA potymerase such as T7, T3, or SP6 and labeled nucleotides.
These procedures
may be conducted using a variety of commercially availalble 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 tike.
Host cells transformed with nucleotide sequences encoding MECHP maybe 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 intracel
lularly 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 MECHP' may be designed to
contain signal
sequences which direct secretion of MECHP 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
IS ofthe polypeptide include, but are not limited to, acetylation,
carboxylation, glycosyiation,
phosphorylation, lipidation, and acylation. Post-transtational processing
which cleaves a "prepro"
form of the protein may also be used to specify protein tarl;eting, folding,
and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HE:K293, and WI38), are
available from
the American Type Culture Collection (ATCC, Manassas 'JA) and may be chosen to
ensure the
correct modification and processing of the foreign protein.
In another embodiment ofthe invention, natural, modified, or recombinant
nucleic acid
sequences encoding MECHP 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 MECHP
protein containing a heterologous moiety that can be recognized by a
commercially available
antibody may facilitate the screening of peptide libraries for inhibitors of
MECHP activity.
Heterologous protein and peptide moieties may also facilitate purification of
fusion proteins using
commercially available amity matrices. Such moieties include, but are not
limited to, glutathione
S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx),
calmodufin binding
peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (H,A). 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 polyclonai antibodies that specifically recognize
these epitope tags. A
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fusion protein may also be engineered to contain a proteolytic cleavage site
located between the
MECHP encoding sequence and the heterologous protein sequence, so that MECHP
may be
cleaved away from the heterologous moiety following purification. Methods for
fusion protein
expression and purification are discussed in Ausubel ( 1955, 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 MECHP may
be
achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ
extract systems
(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, preferably 'SS-methionine.
Fragments of MECHP may be produced not only by recombinant production, but
also by
direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton,
supra pp. 55-60.)
Protein synthesis may be performed by manual techniques. or by automation.
Automated synthesis
1 S may be achieved, for example, using the ABI 43 I A Peptife Synthesizer
(Perkin-Elmer). Various
fragments of MECHP may be synthesized separately and then combined to produce
the full length
molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of MECHP and human membrane channel proteins. In addition, the
expression
of MECHP is closely associated with nervous, reproductive, and
gastrointestinal tissues; fetal
development; and neurological, immune/inflammatory, and cell proliferative
disorders, including
cancer. Therefore, MECHP appears to play a role in cell proliferative,
immune/inflammatory,
transportlsecretory, osmoregulatory, muscular, cardiovascular, and
neurological disorders. In the
treatment of disorders associated with increased MECHP expression or activity,
it is desirable to
decrease the expression or activity of MECHP. In the treatment of disorders
associated with
decreased MECHP expression or activity, it is desirable to increase the
expression or activity of
MECHP.
Therefore, in one embodiment, MECHP 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 MECHP. Examples of such disorders include, but are not limited to,
a cell proliferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis,
mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria,
polycythemia vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma,
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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;
an
immune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS), Addison's
disease, adult respiratory distress syndrome, ankylosing spondylitis,
amyloidosis, anemia, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmmne thyroiditis,
autoimmune
polyenodocrinopathy-candidiasis-ectodermal dystrophy (:APECED), bronchitis,
choiecystitis,
contact dermatitis, Crohn's disease, atopie dermatitis, derrnatomyositis,
diabetes mellitus,
emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis
fetaiis, erythema
nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout,
Graves' disease,
Hashimoto's thyroiditis, hypereosinophiIia, irritable bowel syndrome, multiple
sclerosis,
myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid
arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and
helminthic infections, and trauma; a transport/secretory diisorder such as
akinesia, amyotrophic
lateral sclerosis, ataxia telangiectasia, cystic fibrosis, Becker's muscular
dystrophy, Bell's palsy,
Charcot-Marie Tooth disease, Chediak-Higashi syndrome., diabetes mellitus,
diabetes insipidus,
diabetic neuropathy, hyperkalemic periodic paralysis, nonnokalemic periodic
paralysis, malignant
hyperthermia, multidrug resistance, myotonic dystrophy, <;atatonia, dystonias,
peripheral
neuropathy, neuroftbromatosis, postherpetic neuralgia, tril;eminal neuropathy,
sarcoidosis, sickle
cell anemia, toxic shock syndrome, Wilson's disease, cataracts, infertility,
pulmonary artery
stenosis, sensorineural autosomal deafness, hyperglycemia, hypoglycemia,
goiter, Cushing's
disease, glucose-galactose malabsorption syndrome, hypercholesterolemia, and
allergies,
including hay fever, asthma, and urticaria (hives); an osmoregulatory disorder
such as diabetes
insipidus, diarrhea, peritonitis, chronic renal failure, Addison's disease,
SIADH,
hypoaldosteronism, hyponatremia, adrenal insufficiency, hypothyroidism,
hypernatremia,
hypokalemia, Barter's syndrome, metabolic acidosis, metabolic alkalosis,
encephalopathy, edema,
hypotension, and hypertension; a muscular disorder such ass cardiomyopathy,
myocarditis,
Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic
dystrophy, central core
disease, nemaline myopathy, centronuclear myopathy, lipid myopathy,
mitochondrial myopathy,
infectious myositis, polymyositis, dermatornyositis, inclusion body myositis,
thyrotoxic myopathy,
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and ethanol myopathy; 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
S heart failure, ischemic heart disease, angina pectoris, myocardial
infaretian, hypertensive heart
disease, degenerative valvular heart disease, calcific aortic valve stenosis,
congenitally bicuspid
aortic valve, mitral annular calcification, rnitral valve prolapse, rheumatic
fever and rheumatic
heart disease, infective endocarditis, nonbacterial thromb~otic endocarditis,
endocarditis of
systemic lupus erythematosus, carcinoid heart disease, ca~rdiomyopathy,
myocarditis, pericarditis,
neoplastic heart disease, congenital heart disease, cornpiications of cardiac
transplantation;
congenital lung anomalies, atelectasis, pulmonary congestion and edema,
pulmonary embolism,
pulmonary hemorrhage, pulmonary infarction, pulmonan~ hypertension, vascular
sclerosis,
obstructive pulmonary disease, restrictive pulmonary disease, chronic
obstructive pulmonary
disease, emphysema, chronic bronchitis, bronchial asthma, bronchiectasis,
bacterial pneumonia,
IS viral and mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis,
diffuse interstitial
diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis,
desquarnative 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, pneuntothorax, pleural tumors, drug-induced lung disease,
radiation-induced
lung disease, and complications of lung transplantation; and a neurological
disorder such as
epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms,
Alzheimer's disease,
Pick's disease, Down syndrome, Huntington's disease, dementia, Parkinson's
disease and other
extrapyramidal disorders, amyotrophic lateral sclerosis arnd 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,
epidura) abscess, suppurative intracraniaI thrombophlebitis, myelitis and
radiculitis, viral central
nervous system disease; prion diseases including kuru, Cre;utzfeldt-Jakob
disease, and Gerstmann-
Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neuroflbromatosis, tuberous sclerosis, cer~ebelloretinal
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; neuromuscular
disorders including spinal
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muscular atrophy, carpal tunnel syndrome, monomeuriti:~ multiplex; muscular
dystrophies such as
Duchenne's, myotonic facioscapulohumeral, oculopharyngeal, scapuloperoneal,
congenital. distal,
and ocular; congenital and metabolic myopathies, myotonia, peripheral nervous
system disorders,
dermatomyositis and polymyositis; inherited, metabolic, endocrine, and toxic
myopathies;
myasthenia gravis, periodic paralysis; mental disorders including depression
and bipolar disorder,
and mood, anxiety, and schizophrenic disorders; seasonal affective disorder
(SAD); akathesia,
amnesia, catatonia, diabetic neuropathy, tardive dyskinesi.a, dystonias,
paranoid psychoses,
postherpetic neuralgia, and Tourette's disorder; abnormalities in electrolytes
such as calcium,
phosphate, magnesium, and potasium; hypo- and hyperfunction of the thyroid,
adrenal,
parathyroid, and pituitary; and primary and metastatic neaplasms.
In another embodiment, a vector capable of expressing MECHP 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 MECHP including, but: not limited to,
those described above.
In a further embodiment, a pharmaceutical composition comprising a
substantially
purified MECHP 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 MECHP
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of MECHP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of MECHP including, but not limited to, those Iiste~d above.
In a further embodiment, an antagonist of MECHP may be administered to a
subject to
treat or prevent a disorder associated with increased expression or activity
of MECHP: Such
disorders may include, but are not limited to, those discussed above. In one
aspect, an antibody
which specifically binds MECHP may be used directly as an antagonist or
indirectly as a targeting
or delivery mechanism for bringing a pharmaceutical agent to cells or tissue
which express
MECHP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding MECHP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of MECHP 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 Ibe administered in
combination with
other appropriate therapeutic agents. Selection of the appropriate agents for
use in combination
therapy may be made by one of ordinary skill in the art, according to
conventional pharmaceutical
principles. The combination of therapeutic agents may act synergistically to
effect the treatment
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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 MECHP may be produced using methods which are generally known
in
the art. In particular, purified MECHP may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind MECHP.
Antibodies to MECHP
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, a:nd single chain
antibodies, Fab
fragments, and fragments produced by a Fab, expression library. Neutralizing
antibodies (i.e.,
those which inhibit dimer formation) are especially 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 MECHP 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
IS 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 CaImette-Guerin) and
Corvnebacterium parvum
are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
MECHP have an amino acid sequence consisting of at least: about 5 amino acids,
and, more
preferably, 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 and
contain the entire amino acid sequence of a small, naturally occurring
molecule. Short stretches of
MECHP 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 MECHP 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) Moi. Cell Biol. 6:2:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes 1:0 obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See;, e.g.,
Morrison, S.L. et al. (1984)
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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 3 I4:452-454.) Alternativeoiy, techniques
described for the
production.of single chain antibodies may be adapted, using methods known in
the art, to produce
MECHP-specific single chain antibodies. Antibodies with related specificity,
but of distinct
idiotypic composition, may be generated by chain shuffling from random
combinatorial
immunoglobulin libraries. (See, e.g., Burton D.R. (1991) Proc. Natl. Acad.
Sci. USA 88:10134-
10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin.libraries or panels of highly
specific binding reagents
t0 as disclosed in the literature. (See, e.g., (?rlandi, 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 MECHP 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 speeifcity. Numerous protocols for competitive blinding or
immunoradiometric assays
using either polyclonal or monoclonal antibodies with estalblished
specificities are well known in
the art. Such immunoassays typically involve the measurement of complex
formation between
MECHP and its specific antibody. A two-site, monoclonal-based immunoassay
utilizing
monoclonal antibodies reactive to two non-interfering MEt:HP epitopes is
preferred, but a
competitive binding assay may also be employed (Pound,_sa~).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for MECHP.
Affinity is expressed as
an association constant, Ke, which is defined as the molar concentration of
MECHP-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 MECHP epito~pes, represents the
average affnity, or
avidity, of the antibodies for MECHP. The Ka determined i°or a
preparation of monoclonal
antibodies, which are monospeci$c for a particular MECHf epitope, represents a
true measure of
affinity. High-affinity antibody preparations with Ka ranging from about 109
to 10'2 I/mole are
preferred for use in immunoassays in which the MECHP-antibody complex must
withstand
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rigorous manipulations. Low-affinity antibody preparations with Ka ranging
from about 106 to 10'
I/mole are preferred for use in immunopurification and similar procedures
which ultimately
require dissociation of MECHP, preferably in active forrri, from the antibody
(Catty, D. (1988)
Antibodies. Volume I: A Practical Approach, IRL Press, Washington DC; Liddell,
J.E. and Cryer,
A. ( 1991 ) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New
York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example; a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml;
preferably 5-10 mg specific antibody/ml, is.preferred for use in procedures
requiring precipitation
of MECHP-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 MECHP, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, the
complement of the polynucleotide encoding MECHP may be used in situations in
which it would
be desirable to block the transcription of the mRNA. In particular, cells may
be transformed with
sequences complementary to polynucleotides encoding MIECHP. Thus,
complementary molecules
or fragments may be used to modulate MECHP activity, or to achieve regulation
of gene function.
Such technology is now well known in the art, and sense or antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding MECHP.
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. Methods which are well known to
those skilled in the art
can be used to construct vectors to express nucleic acid seqiuences
complementary to the
polynucieotides encoding MECHP. (See, e.g., Sambrook, ;~unra; Ausubel, 1995,
supra.)
Genes encoding MECHP can be turned off by transforming a cell or tissue with
expression vectors which express high levels of a polynucleotide, or fragment
thereof, encoding
MECHP. Such constructs may be used to introduce untranslatable sense or
antisense sequences
into a cell. Even in the absence of integration into the DNA, such vectors may
continue to
transcribe RNA molecules until they are disabled by endogenous nucleases.
Transient expression
may last for a month or more with a non-replicating vector, and may last even
longer if
appropriate replication elements are part of the vector system.
As mentioned above, modifications of gene expression can be obtained by
designing
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complementary sequences or antisense molecules (DNA, RNA, or PNA) to the
control, 5', or
regulatory regions ofthe gene encoding MECHP. Oligonucleotides derived from
the transcription
initiation site, e.g., between about positions -10 and +10 :from the start
site, are preferred.
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 polymerises, transcription factors, or rE:gulatory
molecules. Recent therapeutic
advances using triplex DNA have been described in the literature. (See, e.g.,
Gee, J.E. et al.
(1994) in Huber, B.E. and B.I. Carr, Molecular and Immuinologic 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 seduence-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 endonucleoiytic cleavage of sequences encoding MECHP.
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 tauge;t gene containing
the cleavage site, may
be evaluated for secondary structural features which may render the
oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing
accessibility to hybridization
with complementary oligonucieotides 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 oligonucIeotides such as solid
phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules may be
generated by in vitro
and in vivo transcription of DNA sequences encoding MEC;HP. Such DNA sequences
may be
incorporated into a wide variety of vectors with suitable Rr~fA polymerise
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' andlor 3'
ends of the molecule, or the use of phosphorothioate or 2' 0~-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
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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 endonucIeases.
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 vivp therapy, vectors
may be introduced into
stem cells taken from the patient and clonally propagated for autologous
transplant back into that
same patient. Delivery by transfection, by liposome injections, or by
polycationic amino polymers
may be.achieved using methods which are well known in the art. (See, e.g.,
Goldman, C.K. et aI.
( 1997) Nat. Biotech. 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 dogs, cats, cows,
horses, rabbits,
monkeys, and most preferably, humans.
An additional embodiment of the invention relates to the administration of a
pharmaceutical or sterile composition, in conjunction with a pharmaceutically
acceptable carrier,
for any of the therapeutic effects discussed above. Such pharmaceutical
compositions may consist
of MECHP, antibodies to MECHP, and mimetics, agonist:>, antagonists, or
inhibitors of MECHP.
The compositions may be administered alone or in combination with at least one
other agent, such
as a stabilizing compound, which may be administered in any sterile,
biocompatible
pharmaceutical carrier including, but not limited to, saline, buffered saline,
dextrose, and water.
The compositions may be administered to a patient alone, or in combination
with other agents,
drugs, or hormones.
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; transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain
suitable pharmaceutically-acceptable carriers comprising e:~ccipients and
auxiliaries which
facilitate processing of the active compounds into preparations which can be
used
pharmaceutically. Further details on techniques for formulation and
administration may be found
in the latest edition of Remin~ton's Pharmaceutical Sciences (Maack
Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using
pharmaceutically acceptable carriers well known in the art in dosages suitable
for oral
administration. Such carriers enable the pharmaceutical compositions to be
formulated as tablets,
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pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and
the like, far ingestion by
the patient:
Pharmaceutical preparations for oral use can be obtained through combining
active
compounds with solid excipient and processing the resultant mixture of
granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be
added, if desired. Suitable
excipients include carbohydrate or protein fillers, such as sugars, including
lactose, sucrose,
mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other
plants; cellulose, such as
methyl cellulose, hydroxypropylmethyf-cellulose, or sodium
carboxymethylcellulose; gums,
including arabic and tragacanth; and proteins, such as gelatin and collagen.
If desired,
disintegrating or solubilizing agents may be added, such as the cross-linked
polyvinyl pyrrolidone,
agar, and alginic acid or a salt thereof, such as sodium alginate.
Dragee cores may be used in conjunction with suiitable coatings, such as
concentrated
sugar solutions, which may also contain gum arabic, talc,
polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable
organic solvents or
solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee
coatings for
product identification or to characterize the quantity of active compound,
i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of
gelatin, as well as soft, sealed capsules made of gelatin and a coating, such
as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or
binders, such as lactose or
starches, lubricants, such as talc or magnesium stearate, and, optionally,
stabilizers. In soft
capsules, the active compounds maybe dissolved or suspended in suitable
liquids, such as fatty
oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be
formulated in
aqueous solutions, preferably in physiologically compatible buffers such as
Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqu<;ous injection
suspensions may contain
substances which increase the viscosity of the suspension, such as sodium
carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions o~f the active
compounds may be
prepared as appropriate oily injection suspensions. Suitable lipophilic
solvents or vehicles include
fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl
oleate, triglycerides, or
liposomes. Non-lipid polycationic amino polymers may also be used for
delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to increase the
solubility of the
compounds and allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular
barrier to be
permeated are used in the formulation. Such penetrants are; generally known in
the art.
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The pharmaceutical compositions of the present invention may be manufactured
in a
manner that is known in the art, e.g., by means of conventional mixing,
dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping, or
lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed
with many
acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic,
tartaric, malic, and
succinic acids. Salts tend to be more soluble in aqueous or other pratonic
solvents than are the
corresponding free base forms. In other cases, the prefewed preparation may be
a lyophilized
powder which may contain any or all of the following: I mM to 50 mM histidine,
O.I% to 2%
sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined
with buffer prior to
use.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate container and labeled for treatment of an indicated condition. For
administration of
MECHP, such labeling would include amount, frequency, and method of
administration.
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.
For any compound, the therapeutically effective dose can be estimated
initially either in
cell culture assays, e.g., of neoplastic cells or in animal models such as
mice, rats, rabbits, dogs, 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
MECHP or fragments thereof, antibodies of MECHP, and ;~gonists, antagonists or
inhibitors of
MECHP, 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 EDS° (the dose therapeutically effective in
50% of the population) or
LDs° (the dose lethal to 50% of the population) statistics. 7.'he dose
ratio of toxic to therapeutic
effects is the therapeutic index, which can be expressed as the
LDS°/EDS° 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 EDS° 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.
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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 healtlh 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 phar~r~aceutical
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 pg to 100,000 ug, up to a total
dose of
about I 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 polyheptides will be
specific to particular
cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind MECHP may be used
for the
diagnosis of disorders characterized by expression of MEC~HP, or in assays to
monitor patients
being treated with MECHP or agonists, antagonists, or inhibitors of MECHP.
Antibodies useful
for diagnostic purposes may be prepared in the same manner as described above
for therapeutics.
Diagnostic assays for MECHP include methods which utilize the antibody and a
label to detect
MECHP in human body fluids or in extracts of cells or tissues. The antibodies
may be used with
or without modification, and may be labeled by covalent o:r 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 MECHP, including ELISAs, RIAs, and FACS,
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of MECHP
expression. Normal or standard values for MECHP expression are established by
combining body
fluids or cell extracts taken from normal mammalian subjects, preferably
human, with antibody to
MECHP under conditions suitable for complex formation. The amount of standard
complex
formation may be quantitated by various methods, preferably by photometric
means. Quantities of
MECHP 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 polynu.cieotides encoding MECHP
may be
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used for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide
sequences, complementary RNA and DNA molecules, anal PNAs. The polynucleotides
may be
used to detect and quantitate gene expression in biopsied tissues in which
expression of MECHP
may be correlated with disease. The diagnostic assay ma;y be used to determine
absence,
presence, and excess expression of MECHP, and to monitor regulation of MECHP
levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide sequences, including genomic sequences, encoding MECHP or
closely related
molecules may be used to identify nucleic acid sequences which encode MECHP.
The specificity
1 a of the probe, whether it is made from a highly specific re8;ion, e.g., the
5' regulatory region, or
from a less specific region, e.g., a conserved motif, and the stringency of
the hybridization or
axnpiification (maximal, high, intermediate, or low), will determine whether
the probe identifies
only naturally occurring sequences encoding MECHP, allelic variants, or
related sequences.
Probes may also be used for the detection of related sequences, and should
preferably
have at least 50% sequence identity to any of the MECHP encoding sequences.
The hybridization
probes of the subject invention may be DNA or RNA and may be derived from the
sequence of
SEQ ID N0:19-36 or from genomic sequences including promoters, enhancers, and
introns of the
gene encoding MECHP.
Means for producing specific hybridization probes for DNAs encoding MECHP
include
the cloning of polynueleotide sequences encoding MECHP' or MECHP 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 radionuclidles such as'ZP or
355, or by enzymatic
labels, such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and
the like.
Polynucleotide sequences encoding MECHP may be used for the diagnosis of
disorders
associated with expression of MECHP. Examples of such disorders include, but
are not limited to,
a cell proliferative disorder such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed connective tissue disease (MCTD~), myelofibrosis,
paroxysmal nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myelom,a, 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, hung, muscle,
ovary, pancreas,
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parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus; an
immune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS), Addison's
disease, adult respiratory distress syndrome, ankylosing spondyiitis,
amyloidosis, anemia, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis,
contact dermatitis, Crohn's disease, atopic dermatitis, de~matomyositis,
diabetes mellitus,
emphysema, episodic lymphopenia with tymphocytotoxins, erythroblastosis
fetalis, erythema
nodosum, atrophic gastritis, glomerulonephritis, Goodpa,sture's syndrome,
gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple
sclerosis,
myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid
arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis;
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and
helminthic infections, and trauma; a transportlsecretory disorder such as
akinesia, amyotrophic
lateral sclerosis, ataxia telangiectasia, cystic fibrosis, Becker's muscular
dystrophy, Bell's palsy,
Charcot-Marie Tooth disease, Chediak-Higashi syndrome, diabetes mellitus,
diabetes insipidus,
diabetic neuropathy, hyperkalemic periodic paralysis, normokalemic periodic
paralysis, malignant
hyperthermia, multidrug resistance, myotonic dystrophy, catatonia, dystonias,
peripheral
neuropathy, neurofibromatosis, postherpetic neuralgia, tri,geminal neuropathy,
sarcoidosis, sickle
cell anemia, toxic shock syndrome, Wilson's disease, cataracts, infertility,
pulmonary artery
stenosis, sensorineural autosomal deafness, hyperglycemia, hypoglycemia,
goiter, Cushing's
disease, glucose-gaIactose malabsorption syndrome, hype:rcholesterolemia, and
allergies,
including hay fever, asthma, and urticaria {hives); an osmoregulatory disorder
such as diabetes
insipidus, diarrhea, peritonitis, chronic renal failure, Addison's disease,
SIADH,
hypoaldosteronism, hyponatremia, adrenal insufficiency, hypothyroidism,
hypernatremia,
hypokalemia, Barter's syndrome, metabolic acidosis, metabolic alkalosis,
encephalopathy, edema,
hypotension, and hypertension; a muscular disorder such ass cardiomyopathy,
myocarditis,
Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic
dystrophy, central core
disease, nemaline myopathy, centronuclear myopathy, lipid myopathy,
mitochondrial myopathy,
infectious myositis, polymyositis, dermatomyositis, inclusion body myositis,
thyrotoxic myopathy,
and ethanol myopathy; a cardiovascular disorder such as alrteriovenous
fistula, atherosclerosis,
hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections,
varicose veins,
thrombophlebitis and phlebothrombosis, vascular tumors, and complications of
thrombolysis,
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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,
neopiastic heart disease, congenital heart disease, 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 frbrosis,
desquamative interstitial
pneumonitis, hypersensitivity pneumonitis, pulmonary eo;>inophilia
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; and a neurological
disorder such as
epilepsy, ischemic cerebrovascular disease, stroke, cerebra.i neopiasms,
Alzheimer's disease,
Pick's disease, Down syndrome, 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 kunz, Creutzfeldt-Jakob
disease, and Gerstmann-
Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cere;belloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the
central nervous system, cerebral palsy, neuroskeletal disordlers, autonomic
nervous system
disorders, cranial nerve disorders, spinal cord diseases; neuromuscular
disorders including spinal
muscular atrophy, carpal tunnel syndrome, monomeuritis multiplex; muscular
dystrophies such as
Duchenne's, myotonic facioscapulohumeral, oculopharyngc;al, scapuloperoneal,
congenital, distal,
and ocular; congenital and metabolic myopathies, myotonia., peripheral nervous
system disorders,
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dermatomyositis and polymyositis; inherited, metabolic, endocrine, and toxic
myopathies;
myasthenia gravis, periodic paralysis; mental disorders including depression
arid bipolar disorder,
arid mood,. anxiety, and schizophrenic disorders; seasonal affective disorder
(SAD); akathesia,
amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias,
paranoid psychoses,
postherpetic neuralgia, and Tourette's disorder; abnormalities in electrolytes
such as calcium,
phosphate, magnesium, and potasium; hypo- and hyperfu.nction of the thyroid,
adrenal,
parathyroid, and pituitary; and primary and metastatic ne~oplasms. The
polynucleotide sequences
encoding MECHP 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
i0 assays; and in microarrays utilizing fluids or tissues from patients to
detect altered MECHP
expression. Such qualitative or quantitative methods are well known in the
art.
In a particular aspect, the nucleotide sequences encoding MECHP may be useful
in assays
that detect the presence of associated disorders, particularly those mentioned
above. The
nucleotide sequences encoding MECHP may be labeled by standard methods and
added to a fluid
t 5 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 quantitated
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 MECHP in the sample indicates the presence of the
associated disorder. Such
20 assays may also be used to evaluate the efficacy of a particular
therapeutic treatment regimen in
animal studies, in clinical trials, or to monitor the treatment of an
individual patient.
In order to provide a basis for the diagnosis of a diisorder associated with
expression of
MECHP, 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
25 sequence, or a fragment thereof, encoding MECHP, 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 sympl:omatic for a
disorder. Deviation from
30 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
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ranging fram several days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
over-expressed) in biopsied tissue from an individual mar indicate a
predisposition for the
development of the disease, or may provide a means for dietecting 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 ar further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding
MECHP 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 MECHP, or a fragment of a polynucieotide complementary
to the
polynucleotide encoding MECHP, and will be employed under optimized conditions
for
identi$cation of a specific gene or condition. Oligomers rnay also be employed
under less
stringent conditions for detection or quantitation of closely related DNA or
RNA sequences.
Methods which may also be used to quantitate the expression of MECHP include
radiolabeIing or biotinylating nucleotides, coampIification 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. Bioclhem. 212:229-236.)
The speed of
quantitation of multiple samples may be accelerated by running the assay in an
ELISA format
where the oligomer of interest is presented in various dilutions and a
spectraphotometric or
colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as targets in a
microarray. The
mieroarray can be used to monitor the expression level of large numbers of
genes simultaneously
and 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, and
to develop and monitor the activities of therapeutic agents.
Microarrays may be prepared, used, and analyzed wising 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. 93:10614-10619; Baldeschweiler et al. ( 1995) PCT application
W0951251116; Shalon,
D. et al. (1995) PCT application W095135505; Heller, R.A. et al. (1997) Proc.
Natl. Acad. Sci.
USA 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Pate:nt No. 5,605,662.)
In another embodiment of the invention; nucleic aciid sequences encoding MECHP
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic
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sequence. 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
P 1 constructions, or single chromosome cDNA libraries,. (See, e.g.,
Harrington, J.J. et al. ( 1997)
Nat. Genet. i 5:345-355; Price, C.M. ( 1993) Blood Rev. 7:127-134; and Trask,
B.J. ( 1991 ) Trends
Genet. 7: I 49-154.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome mapping techniques 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) site.
Correlation between the
location of the gene encoding MECHP on a physical chromosomal map and a
specific disorder, or
a predisposition to a specific disorder, may help define the region of DNA
associated with that
disorder. The nucleotide sequences of the invention may be used to detect
differences in gene
sequences among normal, carrier, and affected individuals.
IS 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 number or arm of a particular
human
chromosome is not known. New sequences can be assigned to chromosomal arms by
physical
24 mapping. This provides valuable information to investigators searching for
disease genes using
positional cloning or other gene discovery techniques. Once the disease or
syndrome has been
crudely localized by genetic linkage to a particular genomie region, e.g.,
ataxia-teiangiectasia to
I 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) rJature 336:577-
580.) The nucleotide
2S sequence ofthe subject invention may also be used to detect differences in
the chromosomal
location due to translocation, inversion, ete., among normal, carrier, or
affected individuals.
In another embodiment of the invention, MECHP, its cataiyt'tc or immunagenic
fragments,
or oligopeptides thereof can be used for screening libraries of compounds in
any of a variety of
drug screening techniques. The fragment employed in such screening may be free
in solution,
30 affixed to a solid support, borne on a cell surface, or located
intracellularly. The formation of
binding complexes between MECHP 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
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compounds are synthesized on a solid substrate. The test compounds are reacted
with MECHP, or
fragments thereof, and washed. Bound MECHP is then detected by methods well
known in the
art. Purified MECHP 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 MECHP specifically compete with a
test compound for
binding MECHP. In this manner, antibodies can be used to detect the presence
of any peptide
which shares one or mare antigenic determinants with MECHP.
In additional embodiments, the nucleotide sequences which encode MECHP may be
used
in any molecular biology techniques that have yet to be dE;veloped, 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
i5 preceding description, utilize the present invention to its fiullest
extent. The following preferred
specific embodiments are, therefore, to be construed as m<~rely illustrative,
and not iimitative of
the remainder of the disclosure in any way whatsoever.
The disclosures of ail patents, applications, and publications mentioned above
and below,
in particular U.S. Ser. No. [Attorney Docket No. PF-0589 P, filed September 2,
1998], U.S. Ser.
ZO No. [Attorney Docket No. PF-0632 P, filed November 12, 1998], U.S. Ser. No.
[Attorney Docket
No. PF-0648 P, filed December 9, 199$), U.S. Ser. No. [Attorney Docket No. PF-
0664 P, filed
January 26, 1999], and U.S. Ser. No. [Attorney Docket No. PF-0671 P, filed
February 10, 1999],
are hereby expressly incorporated by reference.
25 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
30 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
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purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was
isolated using oligo d(Trcoupled 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 o(igonucieotide adapters were iigatedl 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 1 iD00, SEPHAROSE CL2B, or
SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or
preparative
agarose gel electrophoresis. cDNAs were iigated into compatible restriction
enzyme sites of the
polylinker of a suitable plasmid, e.g., PBL;UESCR1PT plasmid (Stratagene),
pSPORTI piasmid
(Life Technologies), or pINCY (Incyte Pharmaceuticals, Palo Alto CA).
Recombinant plasmids
were transformed into competent E. coli cells including ~:L1-Blue, XLl-
BIueMRF, or SOLR from
Stratagene or DHSa, DH10B, or ElectroMAX DHI OB from Life Technologies.
II. Isola#ion of cDNA Clones
Plasmids were recovered from host cells by in vivo excision, using the UNIZAP
vector
system (Stratagene) or cell lysis. Plasmids were purified using at feast 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
purifcation 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
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cDNA sequencing reactions were processed using; standard methods or high-
throughput
instrumentation such as the AB1 CATALYST 800 (Perkin-Elmer) thermal cycler or
the PTC-200
thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser
(Robbins
Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA
sequencing
S 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 (Perkin-Elmer). 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 PRISPrI 373 or 377 sequencing
systems
(Perkin-Elmer) 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 V.
The polynucleotide sequences derived from cDNA, sequencing were assembled and
analyzed using a combination of software programs which utilize algorithms
well known to those
skilled in the art. Table 5 summarizes the tools, programs, and algorithms
used and provides
applicable descriptions, references, and threshold parameters. The first
column of Table 5 shows
the tools, programs, and algorithms used, the second column provides brief
descriptions thereof,
the third column presents appropriate references, all of which are
incorporated by reference herein
in their entirety, and the fourth column presents, where app~iicable, 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). 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 GienBank primate,
rodent, mammalian,
vertebrate, and eukaryote databases, and BLOCKS 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
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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 subsequE;ntly analyzed by
querying against
databases such as the GenBank databases (described above), SwissProt, BLOCKS,
PRINTS,
S Prosite, and Hidden Markov Model (HMM)-based proteim family databases such
as PFAM.
HMM is a probabilistic approach which analyzes consensus primary structures of
gene families.
(See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly a.nd analysis of full length
polynucleotide
and amino acid sequences were also used to. identify polynucieotide sequence
fragments from
SEQ ID N0:19-36. Fragments from about 20 to about 4040 nucleotides which are
useful in
hybridization and amplification technologies were described in The Invention
section above.
IV. Northern Analysis
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
1S RNAs from a particular cell type or tissue have been bound. (See, e.g.,
Sambrook, suura, ch. 7;
Ausubel, 1995, supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or
related molecules in nucleotide databases such as GenBanlk or LIFESEQ (Incyte
Pharmaceuticals,
Palo Alto CA). 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:
se4uence identity x % maXimurn BLAST score
100
2S The product score takes into account both the degree of similarity between
two sequences and the
length of the sequence match. For example, with a produce: score of 40, the
match will be exact
within a 1 % to 2% error, and, with a product score of 70, the match will be
exact. Similar
molecules are usually identified by selecting those which show product scores
between 1S and 40,
although lower scores may identify related molecules.
The results of northern analyses are reported as a percentage distribution of
libraries in
which the transcript encoding MECHP occurred. Analysis involved the
categorization of cDNA
libraries by.organ/tissue and disease. The organ/tissue categories included
cardiovascular,
dermatologic, developmental, endocrine, gastrointestinal,
h~ematopoietic/immune, musculoskeletal,
nervous, reproductive, and urologic. The disease/condition categories included
cancer,
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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 the description of the invention.
V. Extension of MECHP Encoding Polynucleotidles
The full length nucleic acid sequences of SEQ II) N0:19-36 was produced by
extension of
an appropriate fragment of the full length molecule using oligonucleotide
primers designed from
this fragment. One primer was synthesized to initiate 5' extension of the
known fragment, and the
other primer, to initiate 3' extension of the known fragment. The initial
primers were designed
using OLIGO 4.06 software (National Biasciences), or another appropriate
program, to be about
22 to 30 nucleotides in length, to have a GC content of abut 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 dimf:rizations was
avoided.
Selected human eDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art.
PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ
Research, Inc.). The
reaction mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mgz+,
(NH4)zSO4, and ~i-mercaptoethanol, Taq DNA polymerise; (Amersham Pharmacia
Biotech),
ELONGASE enzyme (Life Technologies), and Pfu DNA polymerise (Stratagene), with
the
following parameters for primer pair PCI A and PCI B: Step 1: 94 °C, 3
min; Step 2: 94°C, 1 S 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 I: 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; Steg 6: 6.8°C, S min;
Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p.l
PICO
GREEN quantitation reagent (0.25% (v/v) PICO GREEN; Molecular Probes, Eugene
OR)
dissolved in IX TE and 0.5 pl 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 S ul to 10,u1 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,
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digested with CviJI cholera virus endonucfease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to relegation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were relegated using T4 ligase {New England Biolabs, Beverly MA) into
pUC 18 vector
(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase {Stratagene) to
fill-in
restriction site overhangs, and transfected into competent E. coli cells.
Transformed cells were
selected on antibiotic-containing media, individual colonies were picked and
cultured overnight at
37°C in 384-well plates in LB/2x carb liquid media.
I0 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 I: 94°C, 3 min; Step 2: 94°C, i5 sec; Step 3:
60°C, I min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, S min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) a.s described above.
Samples with low
IS DNA recoveries were reamplified using the same conditions as described
above. Samples were
diluted with 20% dimethysulphoxide (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 (Perkin-Elmer).
In like manner, the nucleotide sequence of SEQ 1fD N0:19-36 is used to obtain
5'
20 regulatory sequences using the procedure above, oligonueleotides designed
for such extension,
and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:19-36 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of olig;onucieotides, consisting
of about 20
25 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 prnol of each
oligomer, 250 p.Ci of
tszp]-adenosine triphosphate (Amersham Pharmacia Biotec;h), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
30 SEPI-IADEX G-25 supe~ne size exclusion dextran bead column (Amersham
Pharmacia Biotech).
An aliquot containing 10'counts per minute ofthe 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 1, or Pvu iI (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
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membranes (Nytran Plus, Schleicher & Schueli, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are ;sequentially
washed at room temperature
under increasingly stringent conditions up to 0.1 x saline sodium citrate and
0.5% sodium dodecyl
sulfate. Hybridization patterns are visualized using autoradiography and
compared.
VII. Microarrays
A chemical coupling procedure and an ink jet device can be used to synthesize
array
elements on the surface of a substrate. (See, e.g., Baldesc:hweiler, supra.)
An array analogous to a
dot or slot blot may also be used to arrange and link elements to the surface
of a substrate using
thermal, L1V, chemical, or mechanical bonding procedures. A typical array may
be produced by
l0 hand or using available methods and machines and contain any appropriate
number of elements.
After hybridization, nonhybridized probes are removed and a scanner used to
determine the levels
and patterns of fluorescence. The degree of complementarity and the relative
abundance of each
probe which hybridizes to an element on the microarray may be assessed through
analysis of the
scanned images.
Full-length cDNAs, Expressed Sequence Tags (E;STs), or fragments thereof may
comprise the elements of the micraarray. Fragments suitable for hybridization
can be selected
using software well known in the art such as LASERGEN1E software (DNASTAR).
Full-length
cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide
sequences of the
present invention, or selected at random from a cDNA library relevant to the
present invention, are
arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed
to the slide using, e.g.,
W cross-linking followed by thermal and chemical treatments and subsequent
drying. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome
Res. 6:639-645.)
Fluorescent probes are prepared and used for hybridization. to the elements on
the substrate. The
substrate is analyzed by procedures described above.
VIII. Complementary Polynucleotides
Sequences complementary to the MECHP-encoding sequences, or any parts thereof,
are
used to detect, decrease, or inhibit expression of naturally occurring MECHP.
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
MECHP. To inhibit transcription, a complementary oligonucieotide is designed
from the most
unique 5' sequence and used to prevent promoter binding toy the coding
sequence. To inhibit
translation, a complementary oligonucieotide is designed to~ prevent ribosomal
binding to the
MECHP-encoding transcript.
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IX. Expression of MECHP
Expression and purification of MECHP are achieved using bacterial or virus-
based
expression systems. Far expression of MECHP 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 T5 or T7 bacterioplhage 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 MECHP upon induction
with isopropyl
beta-D-thiogalactopyranoside {1PTG). Expression of MECHP in eukaryotic cells
is achieved by
infecting insect or mammalian cell lines with recombinant Auto~raphica
catifornica nuclear
polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential
polyhedrin
gene of bacutovirus is replaced with cDNA encoding MECHP 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 bacuiovirus is used to infect,Spodoptera fruaiperda
(Sf~) insect cells
in most cases, or human hepatocytes, in some cases. Infection ofthe 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, MECHP is synthesiized as a fusion protein with,
e.g.,
giutathione 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 iaponicu:m, 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 MECHP at specifically engineered sites. FLAG, an
8-amino acid
peptide, enables immunoaffinity purification using commercially available
monoclonal and
polyclonai anti-FLAG antibodies (Eastman Kodak). 6-Hiis, a stretch of six
consecutive histidine
residues, enables purification on metal-chelate resins (QLAGEN). Methods for
protein expression
and purification are discussed in Ausubel (1995, supra, ch 10 and 16).
Purified MECHP obtained
by these methods can be used directly in the following activity assay.
X. Demonstration of MECHP Activity
Aquaporin Activit~of MECHP
Aquaporin activity of MECHP is demonstrated .as the ability to induce osmotic
water
permeability in Xenopus laevis oocytes injected with MECHP cRNA (Ishibashi, K.
et al. (1994)
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Proc. Natl. Acad. Sci. USA 91:6269-6273). Oocytes injiected with water are
used as the control.
Injected oocytes are given a hypotonic shock by being transferred from 200
mosM to 70 mosM
modified Barth's buffer. The increase in osmotic volume of the oocytes,
observed at 24 °C by
videomicroscopy, is proportional to the MECHP aquaporin activity in the
injected oocytes.
Protein Transport Activity of MECHP
Protein transport activity of MECHP is demonstrated by its ability to catalyze
the
translocation of newly synthesized preprolactin into proteoliposomes in an in
vitro system
(Gorlich, D. and T.A. Rapoport (1993) Cell 75:615-630;1. Proteoliposomes are
prepared
containing purified MECHP, purified dog Sec61 p beta and gamma, purified dog
SRP receptor,
and a mixture of phospholipids (phosphatidylcholine, phosphatidylethanolamine,
phosphatidylserine, and phosphatidylinositol) corresponding approximately to
those found in
native microsomes. The proteoliposomes are incubated in a wheat germ in vitro
translation system
in which a secretory protein (preprolactin) is synthesized in the presence of
SRP and radioactive
amino acids. After translation and synthesis of preprolactin, half of the
sample is treated with S00
IS llg/ml proteinase K while the other half remains untreated. Any
translocated preprolactin will be
inaccessible to proteinase K while any untranslocated preprolactin will be
degraded. The amount
of preprolactin in the samples with and without proteinase K treatment is
determined by sodium
dodecyl sulfate polyacrylamide gel electrophoresis followed by phosphor image
analysis. The
amount of preprolactin protected from proteinase K digestion in the proteinase
K-treated sample is
proportional to the protein transport activity of MECHP.
Gap Junction Activity of MECHP
Gap junction activity of MECHP is demonstrated as the ability to induce the
formation of
intercellular channels between paired Xenopus laevis oocytes injected with
MECHP cRNA
(Hennemann, supra). One week prior to the experimental injection with MECHP
cRNA, oocytes
are injected with antisense oligonucleotide to MECHP to reduce background.
MECHP cRNA-
injected oocytes are incubated overnight, stripped of viteelline membranes,
and paired for recording
of functional currents by dual cell voltage clamp. The measured conductances
are proportional to
gap junction activity of MECHP.
ton Channel Activity of MECHP
Ion channel activity of MECHP is demonstrated using an electrophysiological
assay for
ion conductance. MECHP can be expressed by transfornning a mammalian cell line
such as
COS7, HeLa or CHO with a eukaryotic expression vector encoding MECHP.
Eukaryotic
expression vectors are commercially available, and the techniques to introduce
them into cells are
well known to those skilled in the art. A second plasmid which expresses any
one of a number of
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marker genes, such as I3-galactosidase, is co-transformec! into the cells to
allow rapid identification
of those cells which have taken up and expressed the foreign DNA. The cells
are incubated for
48-72 hours after transformation under conditions appropriate for the cell
line to allow expression
and accumulation of MECHP and Q-galactosidase.
Transformed cells expressing I3-galactosidase are stained blue when a suitable
colorimetric substrate is added to the culture media under conditions that are
well known in the art.
Stained cells are tested for differences in membrane conductance due to
potassium ions by
electrophysiological techniques that are well known in the art. Untransformed
cells, andlor cells
transformed with either vector sequences alone or 13-gala~ctosidase sequences
alone, are used as
controls and tested in parallel. Cells expressing MECHP will have higher
cation conductance
relative to control cells. The contribution of MECHP to conductance can be
confirmed by
incubating the cells using antibodies specific for MECH.'P. The antibodies
will bind to the
extracellular side of MECHP, thereby blocking the pore in the ion channel, and
the associated
conductance.
IS Ion channel activity of MECHP is also measured as current flow across a
MECHP-
containing Xenopus oocyte membrane using the two-electrode voltage-clamp
technique (Ishi et
al., supra; Jegla, T. and L. Salkoff ( 1997) J. Neurosci. 1 T:32-44}. MECHP is
subcloned into an
appropriate Xenopus oocyte expression vector, such as p~BF, and 0.5-5 ng of
mRNA is injected
into mature stage IV oocytes. Injected oocytes are incubated at 18°C
for 1-5 days. Inside-out
macropatches are excised into an intracellular solution containing 116 mM K-
gluconate, 4 mM
KCI, and 10 mM Hepes (pH 7.2). The intracellular solution is supplemented with
varying
concentrations of the MECHP mediator, such as cAMP, cGMP, or Ca+Z (in the form
of CaCh},
where appropriate. Electrode resistance is set at 2-5 MSS! and electrodes are
filled with the
intracellular solution lacking mediator. Experiments are performed at room
temperature from a
holding potential of 0 mV. Voltage ramps (2.5 s) from -100 to 100 mV are
acquired at a sampling
frequency of 500 Hz. Current measured is proportional fro the activity of
MECHP in the assay.
XI. Functional Assays
MECHP function is assessed by expressing the sequences encoding MECHP 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 (L,ife Technologies) and
pCR3.1
(Invitrogen, Carlsbad CA), both of which contain the cyt:omegalovirus
promoter. 5-10 pg of
recombinant vector are transiently transfected into a hurr~an cell line,
preferably of endothelial or
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CA 02341148 2001-03-O1
WO 00112711 PCT/US99120468
hematopoietic origin, using either liposome formulations or electroporation. 1-
2 pg 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
trans~ected 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 bromodeo:Kyuridine uptake;
alterations in
expression of cell surface and intracellular proteins as measured by
reactivity with specific
antibodies; and alterations in plasma membrane composition as measured by the
binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. ( 1994) Flow Cytometry, Oxford, New York NY.
The influence of MECHP on gene expression can be assessed using highly
purified
populations of cells transfected with sequences encoding; MECHP and either
CD64 or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of tra~nsfected cells and bind
to conserved
regions of human immunoglobulin G (IgG). Transfected cells are efficiently
separated from
nontransfected cells using magnetic beads coated with eiither 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 MECHP and other
genes of interest
can be analyzed by northern analysis or rnicroarray techniques.
XII. Production of MECHP Specific Antibodies
MECHP 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 u;>ing standard
protocols.
Alternatively, the MECHP amino acid sequence is analyzed using LASERGENE
software (DNASTAR) to determine regions of high imrrmnogenicity, and a
corresponding
oligopeptide is synthesized and used to raise antibodies 'by means known to
those of skill in the
art. Methods for selection of appropriate epitopes, such as those near the C-
terminus or in
hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995,
su ra, ch. i l.)
Typically, oligopeptides 15 residues in length are synthesized using an ABI
431A peptide
-61-
CA 02341148 2001-03-O1
WO 00112711 PCTIUS99120468
synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to ICLH (Sigma-
Aldrich, St. Louis
MO} by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to
increase
immunogenicity. {See, e.g., Ausubel, 1995, su ra.) Rabbits are immunized with
the oligopeptide-
ICLH complex in complete Freund's adjuvant. Resulting autisera are tested for
antipeptide activity
by, for example, binding the peptide to plastic, blocking with 1% BSA,
reacting with rabbit
antisera, washing, and reacting with radio-iodinated goat anti-rabbit igG.
XIII. Purification of Naturally Occurring MECHP Using Specific Antibodies
Naturally occurring or recombinant MECHP is substantially purified by
immunoaffinity
chromatography using antibodies specific for MECHP. A.n immunoa~nity column is
constructed
by covalently coupling anti-MECHP 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 MECHP are passed over the irnmunoaffinity column, and the
column
is washed under conditions that allow the preferential abs~orbance of MECHP
(e.g., high ionic
IS strength buffers in the presence of detergent). The column is eluted under
conditions that disrupt
antibodyIMECHP 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 MECHP is collected.
XIV. Identification of Molecules Which Interact with MECHP
MECHP, or biologically active fragments thereof, are labeled with''-SI Bolton-
Hunter
reagent. {See, e.g., Bolton, A.E. and W.M. Hunter (1973;1 Biochem. J. 133:529-
539.) Candidate
molecules previously arrayed in the wells of a multi-well plate are incubated
with the labeled
MECHP, washed, and any wells with labeled MECHP complex are assayed. Data
obtained using
different concentrations of MECHP are used to calculate values for the number,
affinity, and
association of MECHP with the candidate molecules.
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
specific preferred
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.
-62-
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
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s
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_76_
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
o ~
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_77_
CA 02341148 2001-03-O1
WO 00/12711 PCT/IJS99120468
SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, TNC.
AU-YOUNG, Janice
SANDMAN, Olga
TANG, Y. Tam
REDDY, Roopa
HILLMAN, Jennifer L.
YUE, Henry
T~AT,, Preeti
CORLEY, Neil C.
GUEGLER, Kari J.
GORGONE, Gina
BAUGHN, Mariah R.
AZIMZAI, Yalda
<120> HUMAN MEMBRANE CHANNEL PROTEINS
<130> PF-OS89 PCT
<140> To Be Assigned
<141> Herewith
<150> 09/145,815; unassigned; 09/191,283; unas:aigned; 09/208,821; unassigned
09/237,506; unassigned; 09/247;891; unas:aigned
<151> 1998-09-02; 1998-09-02; 1998-hl-12; 199 8-11-12; 1998-12-09; 1998-12-09
1999-01-26; 1999-O1-26; 1999-02-10; 199 9-02-10
<160> 45
<170> PERL Program
<210> 1
<211> 724
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1568324CD1
<400> 1
Met Ser Phe Glu Ser Ile Ser Ser Leu Pro Glu Val Glu Pro Asp
1 5 10 1S
Pro Glu Ala Gly Ser Glu Gln Glu Val Phe Ser Ala Val Glu Giy
20 2S 30
Pro Ser Ala Glu Glu Thr Pro Ser Asp Thr Glu Ser Pro GIu Val
35 40 45
Leu Glu Thr Gln Leu Asp Ala His Gln Gly Leu Leu Gly Met Asp
50 5S 60
Pro Pro G1y Asp Met Va1 Asp Phe Val Ala Ala Glu Ser Thr Glu
6S 70 75
Asp Leu Lys Ala Leu Ser Ser Glu Glu Glu Glu Glu Met Gly Gly
80 85 90
Ala Ala Gln Glu Pro Glu Ser Leu Leu Pra Pro Ser Val Leu Asp
1/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
95 100 105
Gln Ala Ser Val Ile Ala Glu Arg Phe Val Ser S'~er Phe Ser Arg
110 115 120
Arg Ser Ser Val AIa Gln Glu Asp Ser Lys Ser Ser Gly Fhe Gly
125 I30 135
Ser Pro Arg Leu Val Ser Arg Ser Ser Ser Val Leu Ser Leu Glu
140 145 150
Gly Ser GIu Lys Gly Leu Ala Arg His Gly Ser Ala Thr Asp Ser
155 160 165
Leu Ser Cys Gln Leu Ser Pro Glu Val Asp Ile Ser Val Gly VaI
170 I75 180
Ala Thr Glu Asp Ser Pro Ser Val Asn Gly Met G:lu Pro Pro Ser
185 190 195
Pro Gly Cys Pro Val Glu Pro Asp Arg Ser Ser Cys Lys Lys Lys
200 205 210
Glu Ser Ala Leu Sex Thr Arg Asp Arg Leu Leu L<au Asp Lys IIe
215 220 225
Lys Ser Tyr Tyr Glu Asn AIa GIu His His Asp AIa Gly Phe Sex
230 235 240
Val Arg Arg Arg Glu Ser Leu Ser Tyr IIe Pro Lys Gly Leu Val
245 250 255
Arg Asn Ser Ile Ser Arg Phe Asn Ser Leu Pro Arg Pro Asp Pro
260 265 270
Glu Pro Val Pro Pro Val Gly Ser Lys Arg Gln Va:l Gly Ser Arg
275 280 285
Pro Thr Ser Trp Ala Leu Phe Glu Leu Pro Gly Pro Ser Gln Ala
290 295 300
Val Lys Gly Asp Pro Pro Pro Ile Ser Asp AIa Glu Phe Arg Pro
305 310 315
Ser Ser Glu Ile Val Lys Ile Trp Glu GIy Met GIu Ser Ser Gly
320 325 330
Gly Ser Pro G1y Lys GIy Pro Gly Gln GIy Gln Ala Asn Gly Phe
335 340 345
Asp Leu His Glu Pro Leu Phe Ile Leu Glu GIu Hi,s Glu Leu Gly
350 355 360
Ala Ile Thr Glu Glu Ser Ala Thr Ala Ser Pro Glu Ser Ser Ser
365 370 375
Pro Thr Glu Gly Arg Ser Pro Ala His Leu Ala Ar<fi Glu Leu Lys
380 385 390
Glu Leu VaI Lys Glu Leu Ser Ser Ser Thr Gln Gly Glu Leu Val
395 400 405
Ala Pro Leu His Pro Arg Ile Val Gln Leu Ser His Val Met Asp
410 415 420
Ser His Val Ser Glu Arg Val Lys Asn Lys Val Tyx- Gln Leu AIa
425 430 435
Arg Gln Tyr Ser Leu Arg Ile Lys Ser Asn Lys Pro Val Met Ala
440 445
450
Arg Pro Pro Leu Gln Trp Glu Lys Val Ala Pro Glu; Arg Asp Gly
455 460 465
Lys Ser Pro Thr Val Pro Cys Leu G1n Glu Glu Ala Gly Glu Pra
470 475 480
Leu Gly Gly Lys Gly Lys Arg Lys Pro Val Leu Ser Leu Phe Asp
485 490 495
Tyr Glu Gln Leu Met Ala Gln Glu His Ser Pro Pro Lys Pro Ser
500 SOS 510
Ser AIa Gly Glu Met Ser Pro Gln Arg Phe Phe Phe Asn Pro Pro
2/~3
CA 02341148 2001-03-O1
WO 40/12711 PCT/US99/20468
515 520 52S
Ala Val Ser Gln Arg Thr Thr Ser Pro Gly Gly Arg Pro Ser Ala
530 535 540
Arg Ser Pro Leu Ser Pro Thr Glu Thr Phe Sex Trp Pro Asp Val
545 550 555
Arg Glu Leu Cys Ser Lys Tyr Ala Ser Arg Asp G:Lu Ala Arg Arg
560 565 570
Ala Gly Gly Gly Arg Pro Arg Gly Pro Pro Val A:an Arg Ser His
575 580 585
Ser Val Pro Glu Asn Met Val Glu Pro Pro Leu Se:r Gly Arg Val
590 595 600
Gly Arg Cys Arg Ser Leu Ser Thr Lys Arg Gly Ai:g Gly Gly Gly
605 610 615
Glu Ala Ala Gln Ser Pro Gly Pro Leu Pro Gln Se:r Lys Pro Asp
620 625 630
Gly Gly Glu Thr Leu Tyr Val Thr Ala Asp Leu Thr Leu Glu Asp
635 640 645
Asn Arg Arg Val Ile Val Met Glu Lys Gly Pro Le:u Pro Ser Pro
650 655 660
Thr Ala Gly Leu Glu Glu Ser Ser Gly Gln Gly Pro Ser Ser Pro
665 670 675
Vai Ala Leu Leu Gly Gln Val Gln Asp Phe Gln G1n Ser Ala Glu
680 685 690
Cys Gln Pro Lys Glu Glu Gly Ser Arg Asp Pro Ala Asp Pro Ser
695 700 705
Gln Gln Gly Arg Val Arg Asn Leu Arg Glu Lys Phe Gln Ala Leu
710 715 720
Asn Ser Val Gly
<210> 2
<211> 257
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4094907CD1
<400> 2
Met Ser Arg Pro Leu Ile Thr Arg Ser Pro Ala Ser Pro Leu Asn
1 5 10 15
Asn Gln Gly Ile Pro Thr Pro Ala Gln Leu Thr Lys Ser Asn Ala
20 25 30
Pro Val His Ile Asp Val Gly Gly His Met Tyr Thr Ser Ser Leu
35 40 45
Ala Thr Leu Thr Lys Tyr Pro Glu Ser Arg Ile Gly Arg Leu Phe
50 55 60
Asp Gly Thr Glu Pro Ile Val Leu Asp Ser Leu Lys~ Gln His Tyr
65 70 75
Phe Ile Asp Arg Asp Gly Gln Met Phe Arg Tyr Ile Leu Asn Phe
80 85 90
Leu Arg Thr Ser Lys Leu Leu Ile Pro Asp Asp Phe Lys Asp Tyr
95 100 IOS
Thr Leu Leu Tyr Glu Glu Ala Lys Tyr Phe Gln Leu Gln Pro Met
110 115 120
3/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99I20468
Leu Leu Glu Met Glu Arg Trp Lys Gln Asp Arg C~lu Thr Gly Arg
125 130 135
Phe Ser Arg Pro Cys Glu Cys Leu Val Val Arg Val AIa Pro Asp
140 145 150
Leu Gly Glu Arg Ile Thr Leu Ser Gly Asp Lys Ser Leu Ile Glu
155 160 165
Glu Val Phe Pro GIu Ile GIy Asp Val Met Cys A.sn Ser Val Asn
170 175 180
Ala Gly Trp Asn His Asp Ser Thr His Val Ile Arg Phe Pro Leu
185 190 195
Asn Gly Tyr Cys His Leu Asn Ser Val Gln Val Leu Glu Arg Leu
200 205 210
Gln Gln Arg Gly Phe Glu Ile Val Gly Ser Cys G.ly Gly Gly Val
215 220 225
Asp Ser Ser Gln Phe Ser Glu Tyr Vah Leu Arg A:rg Glu Leu Arg
230 235 240
Arg Thr Pro Arg Val Pro Ser Val Ile Arg Ile Lys Gln Glu Pro
245 250 ° 255
Leu Asp
<210> 3
<211> 377
<212 > PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 518158CD1
<400> 3
Met Gly Gly Asp Leu Gly Leu Gly Leu Arg Arg
Val Leu Ala Arg
1 5 10 15
Lys Arg Leu Leu Glu Lys Ser Leu Gly Trp Ala
Gln Glu Ala Leu
20 25 30
Val Leu Ala Gly Thr Gly Leu Met Leu His Ala
Gly Ile Val Glu
35 40 45
Met Leu Trp Phe Gly Ser Trp Ala Ty:r Leu Phe
Gly Cys Leu Leu
50 55 60
Val Lys Cys Thr Ile Ser Thr Phe Leu Leu Cys
Ser Ile Leu Leu
65 70 75
Ile Val Ala Phe His Glu Val Gln Phe= Met Thr
Ala Lys Leu Asp
80 85 90
Asn Gly Leu Arg Asp Val Ala Leu Gly Arg GIn
Trp Arg Thr Ala
95 100 105
Ala Gln Ile Val Leu Val Val Cys Leu Has Pro
Glu Leu Gly Ala
110 115 120
Pro Val Arg GIy Pro Val Gln Asp Gly Ala Pro
Pro Cys Leu Leu
125 130 135
Thr Ser Pro GIn Pre GIy Phe Leu Gln Gly Glu
Trp Pro Gly AIa
140 145 150
Leu Leu Ser Leu AIa Leu Leu Gly Thr Leu Gly
Met Leu Leu Leu
155 160 165
Txp Leu Thr Thr AIa Leu Ser Val Glu. Arg Gln
Trp Val, Ala AIa
170 175 180
Val Asn AIa Thr Gly Ser Asp Thr Txp Leu Ile
His Leu Leu Pro
4/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
185 190 195
Ile Thr Phe Leu Thr Ile Gly Tyr Gly Asp Val Val Pro Gly Thr
200 205 210
Met Trp Gly Lys Ile Val Cys Leu Cys Thr Gly teal Met Gly Val
215 220 225
Cys Cys Thr Ala Leu Leu.Val Ala Val Val Ala 1?xg Lys Leu Glu
230 235 240
Phe Asn Lys Ala GIu Lys His Val His Asn Phe Nfet Met Asp Ile
245 250 255
Gln Tyr Thr Lys Glu Met Lys Glu Ser Ala AIa A.rg Vai Leu Gln
260 265 270
Glu Ala Trp Met Phe Tyr Lys His Thr Arg Arg Lys Glu Sex His
275 280 285
Ala Ala Arg Arg His Gln Arg Lys Leu Leu Ala Ala I1e Asn Ala
290 295 300
Phe Arg Gln Val Arg Leu Lys His Arg Lys Leu Arg Glu Gln Val
305 310 315
Asn Ser Met Val Asp Ile Ser Lys Met His Met Lle Leu Tyr Asp
320 325 330
Leu Gln Gln Asn Leu Ser Ser Ser His Arg Ala Leu Glu Lys Gln
335 340 345
Ile Asp Thr Leu Ala Gly Lys Leu Asp Ala Leu Tlar Glu Leu Leu
350 355 360
Ser Thr Ala Leu GIy Pro Arg Gln Leu Pro Glu Pro Ser Gln Gln
365 370 375
Ser Lys
<220> 4
<211> 49I
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 602926CD1
<400> 4
Met Val Phe Gly Glu Phe Phe His Arg Pro Gly Gln Asp Glu Glu
1 5 10 15
Leu Val Asn Leu Asn Val Gly Gly Phe Lys Gln Ser Val Asp Gln
20 25 30
Ser Thr Leu Leu Arg Phe Pro His Thr Arg Leu Gly Lys Leu Leu
35 40 45
Thr Cys His Ser GIu Glu Ala Ile Leu Glu Leu Cys Asp Asp Tyr
50 55 60
Ser Val Ala Asp Lys Glu Tyr Tyr Phe Asp Arg Assn Pro Ser Ser
65 70 75
Phe Arg Tyr Val Leu Asn Phe Tyr Tyr Thr G1y Ly;s Leu His VaI
80 85 90
Met Glu Glu Leu Cys Val Phe Ser Phe Cys Gln Glu Tle Glu Tyr
95 100 105
Trp Gly Ile Asn Glu Leu Phe Ile Asp Ser Cys Cys Ser Asn Arg
110 115 120
Tyr Gln Glu Arg Lys Glu Glu Asn His Glu Lys Asp Trp Asp Gln
125 130 135
5/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99120468
Lys Ser His Asp Val Ser Thr Asp Ser Ser Phe Glu Glu Ser Ser
140 145 150
Leu Phe Glu Lys Glu Leu Glu Lys Phe Asp Thr Leu Arg Phe Gly
I55 160 I65
Gln Leu Arg Lys Lys Ile Trp Ile Arg Met GIu A,sn Pro Ala Tyr
170 175 180
Cys Leu Ser Ala Lys Leu Ile Ala Ile Ser Ser Leu Ser Val Val
I85 190 I95
Leu Ala Ser Ile Val Ala Met Cys Val His Ser Met Ser Glu Phe
200 205 2Ip
Gln Asn Glu Asp Gly Glu Val Asp Asp Pro Val Lesu Glu Gly Val
215 220 225
Glu Ile Ala Cys Ile Ala Trp Phe Thr Gly Glu Le:u Ala Val Arg
230 235 240
Leu Ala Ala Ala Pro Cys Gln Lys Lys Phe Trp Lys Asn Pro Leu
245 250 255
Asn Ile Ile Asp Phe Val Ser Ile Ile Pro Phe Tyr Ala Thr Leu
260 265 270
Ala Val Asp Thr Lys Glu Glu Glu Ser Glu Asp Il.e Glu Asn Met
275 280 285
Gly Lys Val Val Gln Ile Leu Arg Leu Met Arg Il.e Phe Arg Ile
290 295 300
Leu Lys Leu Ala Arg His Sex Val Gly Leu Arg Se.r Leu Gly Ala
305 310 315
Thr Leu Arg His Ser Tyr His G1u Va1 Gly Leu Leu Leu Leu Phe
320 325 330
Leu Ser Val GIy Ile Ser Ile Phe Ser Val Leu Ile Tyr Ser Val
335 340 345
Glu Lys Asp Asp His Thr Ser Ser Leu Thr Ser Ile Pro Ile Cys
350 355 360
Trp Trp Trp Ala Thr Tle Ser Met Thr Thr Val Gly Tyr Gly Asp
3.65 370 375
Thr His Pro Val Thr Leu Ala Gly Lys Leu Ile Al;a Ser Thr Cys
380 385 390
Ile Ile Cys Gly Ile Leu Val Val AIa Leu Pro Ile Thr Ile Ile
395 400 405
Phe Asn Lys Phe Ser Lys Tyr Tyr Gln Lys Gln Lya Asp Ile Asp
410 415 420
Val Asp Gln Cys Ser Glu Asp Ala Pro Glu Lys Cy:a His Glu Leu
42S 430 435
Pro Tyr Phe Asn IIe Arg Asp IIe Tyr Ala Gln Arch Met His Ala
440 445 450
Phe Ile Thr Ser Leu Ser Ser Val Gly Ile Val Va7L Ser Asp Pro
455 460 465
Asp Ser Thr Asp Ala Ser Ser Ile Glu Asp Asn Glu Asp Ile Cys
470 475 480
Asn Thr Thr Ser Leu Glu Asn Cys Thr Ala Lys
485 490
<210> 5
<211> 341
<212> PRT
<213> Homo sapiens
<220>
6/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99120468
<221> misc_feature
<223> Incyte ID No: 922119CD1
<400> 5
Met Gly Ser Gly His Cys Leu Arg Ser Thr Arg G:Ly Ser Lys Met
1 5 10 15
Val Ser Trp Ser Val Ile Ala Lys Ile Gln Glu I7Le Leu Gln Arg
20 25 30
Lys Met Val Arg Glu Phe Leu Ala Glu Phe Met Se:r Thr Tyr Val
35 40 45
Met Met Val Phe Gly Leu Gly Ser Val Ala His Me;t Val Leu Asn
50 55 60
Lys Lys Tyr Gly Ser Tyr Leu Gly VaI Asn Leu Gl.y Phe Gly Phe
65 70 75
Gly Vai Thr Met Gly Val His Val Ala~Gly Arg Ile Ser Gly Ala
80 85 90
His Met Asn Ala Ala Val Thr Phe Ala Asn Cys Ala Leu Gly Arg
g5 100 105
Val Pro Trp Arg Lys Phe Pro Val Tyr Val Leu Gly Gln Phe Leu
110 115 120
Gly Ser Phe Leu Ala Ala Ala Thr Ile Tyr Ser Leu Phe Tyr Thr
125 130 135
Ala Ile Leu His Phe Ser Gly Gly Gln Leu Met Val Thr Gly Pro
140 145 150
Val Ala Thr Ala Gly Ile Phe Ala Thr Tyr Leu Pro Asp His Met
155 160 165
Thr Leu Trp Arg Gly Phe Leu Asn Glu Ala Trp Leu Thr Gly Met
170 175 180
Leu G1n Leu Cys Leu Phe Ala Ile Thr Asp Gln Glu Asn Asn Pro
185 190 195
Ala Leu Pro Gly Thr Glu Ala Leu Val Ile Gly Ilea Leu Val Val
2.00 205
210
Ile Ile Gly Val Ser Leu Gly Met Asn Thr Gly Tyr Ala Ile Asn
215 220 225
Pro Ser Arg Asp Leu Pro Pro Arg Tle Phe Thr Phe: Ile Ala Gly
230 235 240
Trp GIy Lys Gln Val Phe Ser Asn Gly Glu Asn Trp Trp Trp Val
245 250 255
Pro Val Val Ala Pro Leu Leu Gly Ala Tyr Leu Gly Gly Ile Ile
260 265
270 .
Tyr Leu Val Phe Ile Gly Ser Thr Ile Pro Arg Glue Pro Leu Lys
275 280 285
Leu Glu Asp Ser Val Ala Tyr Glu Asp His Gly Ile Thr Va1 Leu
290 295 300
Pro Lys Met Gly Ser His Giu Pro Thr Ile Ser Pra Leu Thr Pro
305 310 315
Val Ser Val Ser Pro Ala Asn Arg Ser Ser Val His Pro Ala Pro
320 325 330
Pro Leu His G1u Ser Met Ala Leu Glu His Phe
335 340
<210> 6
<211> 476
<212> PRT
<213> Homo sapiens
7143
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99I20468
<220>
<22I> misc_feature
<223> Incyte ID No: 2666782CD1
<400> 6
Met Gly Ile Lys Phe Leu Glu Val Ile Lys Pro Phe Cys Ala Val
1 5 10 15
Leu Pro Glu Ile Gln Lys pro GIu Arg Lys Ile Gln Phe Arg Glu
20 25 30
Lys Val Leu Trp Thr Ala Ile Thr Leu Phe Ile Phe Leu Val Cys
35 40 45
Cys Gln Ile Pro Leu Phe Gly Ile Met Ser Ser Asp Ser AIa Asp
50 55 60
Pro Phe Tyr Trp Met Arg Val IIe Leu Ala Ser Aan Arg Gly Thr
65 70 75
Leu Met Glu Leu Gly Ile Ser Pro Ile Val Thr Se=r Gly Leu IIe
80 85 90
Met GIn Leu Leu Ala Gly Ala Lys Ile Ile Glu Val Gly Asp Thr
95 100 105
Pro Lys Asp Arg Ala Leu Phe Asn Gly Ala Gln Lys Leu Phe Gly
110 115 120
Met Ile Ile Thr Ile Gly Gln Ala Ile Val Tyr Val Met Thr Gly
125 130 135
Met Tyr Gly Asp Pro Ala Glu Met Gly Ala Gly Il.e Cys Leu Leu
140 145 150
Ile Ile Ile Gln Leu phe Val Thr Ser Leu Ile Va.l Leu Leu Leu
I55 160 165
Asp Glu Leu Leu Gln Thr Gly Tyr Ser Leu Gly Ser Gly Ile Ser
170 175 180
Leu Val Ile Ala Thr Asn Ile Cys Glu Thr Ile Val Trp Lys Ala
185 190 195
Phe Ser Pro Thr Thr Ile Asn Thr Gly Arg Gly Thr Glu Phe Glu
200 205 210
Gly Ala Val Ile AIa Leu Phe His Leu Leu AIa Th:r Arg Thr Asp
215 220 225
Lys Val Arg Ala Leu Arg Glu Ala Phe Tyr Arg Gln Asn Leu Pro
230 235 240
Asn Leu Met Asrl Leu Ile Ala Thr Val Phe Val Phe A1a Val Val
245 250 255
Ile Tyr Phe Gln Gly Phe Arg Val Asp Leu Pro Ilea Lys Ser Ala
260 265 270
Arg Tyr Arg Gly Gln Tyr Ser Ser Tyr Pro Ile Ly.; Leu Phe Tyr
275 280 285
Thr Ser Asn Ile Pro Ile Ile Leu Gln Ser Ala Leu Val Ser Asn
290 295 300
Leu Tyr Val Ile Ser G1n Met Leu Ser Val Arg PhE: Ser Gly Asn
305 310 315
Phe Leu Val Asn Leu Leu Gly Gln Trp Ala Asp Val. Ser Gly Gly
320 325 330
Gly pro Ala Arg Ser Tyr Pro Va1 Gly Gly Leu Cys Tyr Tyr Leu
335 340 345
Ser Pro pro Glu Ser Met Gly Ala Ile Phe Glu Asp Pro Val His
350 355 360
VaI Val Val Tyr Ile Ile Phe Met Leu Gly Ser Cys Ala Phe Phe
365 370 375
Ser Lys Thr Trp Ile Glu Val Sex Gly Ser Ser Ala Lys Asp Val
8/43
CA 02341148 2001-03-O1
WO 00/127I I PCT/US99/20468
380 385 390
Ala Lys Gln Leu Lys Glu Gln Gln Met Val Met Ax~g Gly His Arg
395 400 405
Asp Thr Ser Met Val His Glu Leu Asn Arg Tyr Ile Pro Thr Ala
410 415 420
Aia Ala Phe Gly Gly Leu Cys Ile Gly Ala Leu Ser Val Leu Ala
425 430 435
Asp Phe Leu Gly Ala Ile Gly Ser Gly Thr Gly Ile Leu Leu Ala
440 445 450
Val Thr Ile Ile Tyr Gln Tyr Phe Glu Ile Phe Val Lys Glu Gln
455 460 465
Ala Glu Val Gly Gly Met Gly Ala Leu Phe Phe
470 475
<210> 7
<211> 266
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2731369CD1
<400> 7
Met Asn Ala Phe Leu Gln Leu Gly Asn
Trp Gly Leu Ser Val Lys
1 5 20 15
Tyr Ser Val Leu Ser Arg Leu Va7. Phe
Thr Ile Trp Ser Val Ile
20 25 30
Phe Arg Leu Val Tyr Val Ala Glu Trp
Val Val Ala Glu Val Asp
35 40 45
Asp Glu Lys Asp Phe Asp Thr Gln Gly
Gln Cys Asn Lys Pro Cys
50 55 60
Thr Asn Cys Tyr Asp Asn Pro Sex' Ile
Val Tyr Phe Ile Asn Arg
65 70 75
Leu Trp Leu Gln Leu Ile Thr Pro Leu
Ala Leu Val Cys Ser Leu
80 85 90
Val Val His Val Ala Tyr Glu Glu. Lys
Met Arg Glu Arg Arg His
95 100 105
His Leu His Gly Pro Asn Ser Tyr Asn
Lys Ala Pro Leu Asp Lew
110 115 120
Ser Lys Arg Gly Gly Leu Thr Leu Ser
Lys Trp Trp Tyr Leu Leu
125 I3 0 3.3
5
Ile Phe Ala Ala Val Asp Phe Tyr Phe
Lys Ala Gly Leu Ile His
140 145 150
Arg Leu Lys Asp Tyr Asp Arg Val Cys
Tyr Met Pro Val Ala Ser
155 160 165
Val Glu Cys Pro His Thr Cys Ile Arg
Pro Val Asp Tyr Ser Pro
170 175 180
Thr Glu Lys Val Phe Thr Met Thr.ThrAla
Lys Tyr Phe Val Ala
185 190 195
Ile Cys Leu Leu Asn Leu Val Tyr Val
Ile Ser Glu Phe Leu Gly
200 205 210
Lys Arg Met G1u Ile Phe Arg Arg Pro
Cys Gly Pro His Arg Arg
215 220 225
Cys Arg Cys Leu Pro Asp Pro Tyr Leu
Glu Thr Cys Pro Val Ser
9143
CA 02341148 2001-03-O1
WO 00//2711 PCT/US99/204b8
230 235 240
Gln Gly Gly His Pro Glu Asp Gly Asn Ser Val L~~u Met Lys Ala
245 250 255
Gly Ber Ala Pro Val Asp Ala Gly Gly Tyr Pro
260 265
<210> 8
<211> 182
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1375415CD1
<400> 8
Met Ala Glu Phe Pro Ser Lys Val Ser Thr Arg Thr Ser Ser Pro
1 5 10 15
Ala Gln Gly Ala Glu Ala Ser Val Ser Ala Leu Arg Pro Asp Leu
20 25 30
Gly Phe Val Arg Ser Arg Leu Gly Ala Leu Met Leu Leu Gln Leu
35 40 45
Val Leu Gly Leu Leu Val Trp Ala Leu Ile Ala Asp Thr Pro Tyr
50 55 60
His Leu Tyr Pro Ala Tyr Gly Trp Val Met Phe Val Ala Val Phe
65 70 75
Leu Trp Leu Val Thr Ile Val Leu Phe Asn Leu Tyr Leu Phe Gln
80 85 90
Leu His Met Lys Leu Tyr Met Val Pro Trp Pro Leu Val Leu Met
95 100 105
Ile Phe Asn Ile Ser Ala Thr Val Leu Tyr Ile Th:r Ala Phe Ile
110 115 120
Ala Cys Ser Ala Ala Val Asp Leu Thr Ser Leu Ar<1 Gly Thr Arg
125 130 , 135
Pro Tyr Asn Gln Arg Ala Ala Ala Ser Phe Phe Al:~ Cys Leu Val
140 145 150
Met Ile Ala Tyr Gly Va3 Ser Ala Phe Phe Ser Tyr Gln Ala Trp
155 160 165
Arg Gly Val Gly Ser Asn Ala Ala Thr Ser Gln Met: Ala Gly Giy
170 175 180
Tyr Ala
<210> 9
<211> 942
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2733282CD1
<400> 9
Met Thr Gln Arg Ser Ile Ala Gly Pro Ile Cys Asn Leu Lys Phe
1 5 10 15
10/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99120468
Val Thr Leu Leu Val Ala Leu Ser Ser GIu Leu P:ro Phe Leu Gly
20 25 30
AIa Gly Val Gln Leu GIn Asp Asn Gly Tyr Asn G:Ly Leu Leu Ile
35 40 45
Ala Ile Asn Pro Gln Val Pro GIu Asn Gln Asn Leu IIe Ser Asn
50 55 60
Ile Lys Glu Met IIe Thr Glu Ala Ser Phe Tyr Le:u Phe Asn Ala
65 70 75
Thr Lys Arg Arg Val Phe Phe Arg Asn IIe Lys I:Le Leu Ile Pro
80 85 90
Ala Thr Trp Lys AIa Asn Asn Asn Ser Lys Ile Lys Gln Glu Ser
95 100 105
Tyr GIu Lys Ala Asn Val Ile Val Thr Asp Trp Tyr Gly Ala His
110 115 120
Gly Asp Asp Pro Tyr Thr Leu Gln Tyr~Arg Gly C~~s Gly Lys Glu
125 130 135
Gly Lys Tyr Ile His Phe Thr Pro Asn Phe Leu Le:u Asn Asp Asn
3.40 145 150
Leu Thr Ala Gly Tyr Gly Ser Arg Gly Arg Val Phe Val His Glu
155 160 165
Trg Ala His Leu Arg Trp Gly Val Phe Asp Glu Tyr Asn Asn Asp
170 175 180
Lys Pro Phe Tyr Ile Asn Gly Gln Asn Gln Ile Lys Val Thr Arg
185 190 195
Cys Ser Ser Asp Ile Thr Gly Ile Phe VaI Cys Glu Lys Gly Pro
200 205 210
Cys Pro Gln Glu Asn Cys IIe Iie Ser Lys Leu Phe Lys Glu Gly
215 220 225
Cys Thr Phe Ile Tyr Asn Ser Thr Gln Asn Ala Thr Ala Ser Ile
230 235 240
Met Phe Met Gln Ser Tyr Leu Cys Gly Glu IIe Cy,s Asn Ala Ser
245 250 255
Thr His Asn Gln Glu Ala Pro Asn Leu Gln Asn GIn Met Cys Ser
260 265 270
Leu Arg Ser Ala Trp Asp Val Ile Thr Asp Ser Ala Asp Phe His
275 280 285
His Ser Phe Pro Met Asn Gly Thr Glu Leu Pro Pro Pro Pro Thr
290 295 300
Phe Ser Leu VaI Glu Ala Gly Asp Lys Val Val Cy:a Leu Val Leu
305 310 315
Asp Val Ser Ser Lys Met Ala Glu Ala Asp Arg Leu Leu Gln Leu
320 325 330
Gln Gln AIa Ala Glu Phe Tyr Leu Met Gln Ile Va7L Glu Ile His
335 340 345
Thr Phe Val Gly Ile Ala Ser Phe Asp Ser Lys Gly Glu Ile Arg
350 . 355 360
Ala Gln Leu His GIn Ile Asn Ser Asn Asp Asp Arch Lys Leu Leu
365 370 375
Val Ser Tyr Leu Pro Thr Thr Val Ser Ala Lys Thr Asp Ile Ser
380 3B5 390
Ile Cys Ser Gly Leu Lys Lys Gly Phe GIu Val Val. Glu Lys Leu
395 400 405
Asn Gly Lys Ala Tyr GIy Ser VaI Met Ile Leu Val Thr Ser Gly
410 415 420
Asp Asp Lys Leu Leu Gly Asn Cys Leu Pro Thr Val Leu Ser Ser
425 430 435
Z 1/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
Gly Ser Thr Ile His Ser Ile Ala Leu Gly Ser S'~er Ala Ala Pro
440 445 450
Asn Leu Glu Glu Leu Ser Arg Leu Thr Gly Gly Leu Lys Phe Phe
455 460 465
VaI Pro Asp Ile Ser Asn Ser Asn Ser Met Ile Asp Ala Phe Ser
470 475 480
Arg Ile Ser Ser Gly Thr Gly Asp IIe Phe Gln Gln His Ile Glri
485 490 495
Leu Glu Ser Thr Gly Glu Asn Val Lys Pro His His Gln Leu Lys
500 505 510
Asn Thr Val Thr Val Asp Asn Thr Val Gly Asn A;sp Thr Met Phe
515 520 525
Leu Val Thr Trp Gln Ala Ser Gly Fro Pro Glu I:Le Ile Leu Phe
S30 535 540
Asp Pro Asp Gly Arg Lys Tyr Tyr Thr Asn Asn Plze Ile Thr Asn
545 550 555
Leu Thr Phe Arg Thr Ala Ser Leu Trp Ile Pro G:Ly Thr Ala Lys
560 565 570
Pro Gly His Trp Thr Tyr Thr Leu Asn Asn Thr His His Ser Leu
57S 580 585
Gln Ala Leu Lys Val Thr Val Thr Ser Arg Ala Se:r Asn Ser Ala
S90 595 600
Val Pro Pro Ala Thr Val Glu Ala Phe Val Glu Az~g Asp Ser Leu
605 610 615
His Phe Pro His Pro Val Met Ile Tyr Ala Asn Va~.l Lys Gln Gly
620 625 630
Phe Tyr Pro Ile Leu Asn Ala Thr Val Thr Ala Th.r Val Glu Pro
635 640 645
Glu Thr Gly Asp Pro Val Thr Leu Arg Leu Leu Asp Asp Gly AIa
650 655 660
Gly AIa Asp Val Ile Lys Asn Asp Gly Ile Tyr Ser Arg Tyr Phe
665 670 675
Phe Ser Phe Ala Ala Asn Gly Arg Tyr Ser Leu Lys Val His Val
680 685 690
Asn His Ser Pro Ser Ile Ser Thr Pro Ala His Ser Ile Pro Gly
695 700 705
Ser His Ala Met Tyr Val Pro Gly Tyr Thr Ala Assn Gly Asn Ile
710 715 720
Gln Met Asn Ala Pro Arg Lys Ser Val Gly Arg Asn Glu Glu Glu
725 730 735
Arg Lys Tzp Gly Phe Ser Arg Val Ser Ser Gly Gly Ser Phe Ser
740 745 750
Val Leu Gly Val Pro Ala Gly Pro His Fro Asp Va:l Phe Pro Pro
755 760 765
Cys Lys Ile IIe Asp Leu Glu Ala Val Lys VaI Glu Glu GIu Leu
770 ?75 780
Thr Leu Ser Trp Thr Ala Pro Gly Glu Asp Phe Asp Gln Gly Gln
785 790 795
Ala Thr Ser Tyr Glu Ile Arg Met Ser Lys Ser Leu Gln Asn Ile
800 805 810
Gln Asp Asp Phe Asn Asn Ala I1e Leu VaI Asn Thzv Ser Lys Arg
815 820 825
Asn Pro Gln Gln Ala Gly Ile Arg Glu Ile Phe Thr Phe Ser Fro
830 $3S 840
G1n Ile Ser Thr Asn Gly Pro Glu His Gln Pro Asn, Gly Glu Thr
845 850 855
12/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
His Glu Ser His Arg Ile Tyr Val Ala Ile Arg Ala Met Asp Arg
860 865 870
Asn Ser Leu Gln Ser Ala Val Ser Asn Ile Ala Gln Ala Pro Leu.
875 880 88S
Phe Ile Pro Pro Asn Ser Asp Pro Val Pro Ala A.rg Asp Tyr Leu
890 895 900
Ile Leu Lys Gly Val Leu Thr Ala Met Gly Leu Ile Gly Ile Ile
905 910 915
Cys Leu Ile Ile Val Val Thr His His Thr Leu Ser Arg Lys Lys
920 925 930
Arg Ala Asp Lys Lys Glu Asn Gly Thr Lys Leu L~eu
935 940
<210> 10
<211> 519
<212> PI2T
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3148427CDI
<400> 10
Met Glu Glu Met Phe His Lys Lys Ser Glu Ala Va,l Arg Arg Leu
1 5 10 15
VaI Glu Ala AIa Glu Glu Ala His Leu Lys His Glu Phe Asp Ala
20 25 30
Asp Leu Gln Tyr Glu Tyr Phe Asn Ala Val Leu Ile Asn Glu Arg
35 40 45
Asp Lys Asp Gly Asn Phe Leu Glu Leu Gly Lys Glu Phe IIe Leu
50 55 60
AIa Pro Asn Asp His Phe Asn Asn Leu Pro Val Assn Ile Ser Leu
65 70 75
Ser Asp Val Gln Val Pro Thr Asn Met Tyr Asn Ly,s Asp Pro Ala
80 85 90
Ile Val Asn Gly Val Tyr Trp Ser Glu Sex Leu Asn Lys Val Phe
95 100 105
Val Asp Asn Phe Asp Arg Asp Pro Ser Leu Ile Trp Gln Tyr Phe
110 115 120
Gly Ser Ala Lys Gly Phe Phe Arg Gln Tyr Pro Gly Ile Lys Trp
125 130 135
Glu Pro Asp Glu Asn Gly Val Ile Ala Phe Asp Cys Arg Asn Arg
140 145 150
Lys Trp Tyr Ile Gln Ala Ala Thr Ser Pro Lys Asp Val Val Ile
155 160 165
Leu Val Asp Val Ser Gly Ser Met Lys Gly Leu Arg~ Leu Thr Ile
170 175 180
Ala Lys Gln Thr Val Ser Ser Ile Leu Asp Thr Leu~. Gly Asp Asp
185 190 195
Asp Phe Phe Asn Ile Ile Ala Tyr Asn Glu Glu Leu. His Tyr Val
200 205 210
Glu Pro Cys Leu Asn Gly Thr Leu Val G1n Ala Asp Arg Thr Asn
215 220 225
Lys Glu His Phe Arg GIu His Leu Asp Lys Leu Phe Ala Lys Gly
230 235 240
13/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
Ile Gly Met Leu Asp Ile Ala Leu Asn Glu Ala F?he Asn Tle Leu
245 250 255
Ser Asp Phe Asn His Thr Gly Gln Gly Ser Ile C'ys Ser Gln Ala
260 265 270
Ile Met Leu Ile Thr Asp Gly Ala Val Asp Thr Tyr Asp Thr Ile
275 280 285
Phe Ala Lys Tyr Asn Trp Pro Asp Arg Lys Val A.rg Ile Phe Thr
290 295 300
Tyr Leu Ile Gly Arg Glu Ala Ala Phe Ala Asp A,sn Leu Lys Trp
305 310 315
Met Ala Cys Ala Asn Lys Gly Phe Phe Thr Gln Ile Ser Thr Leu
320 325 330
Ala Asp Val Gln Glu Asn Val Met Glu Tyr Leu His Val Leu Ser
335 340 345
Arg Pro Lys Val Ile Asp Gln Glu His~Asp Val Vai Trp Thr Glu
350 355 360
Ala Tyr Ile Asp Ser Thr Leu Pro Gln Ala Gln Lvys Leu Thr Asp
365 370 375
Asp Gln Gly Pro Val Leu Met Thr Thr Val Ala Met Pro Val Phe
380 385 390
Ser Lys Gln Asn Glu Thr Arg Ser Lys Gly Tle Le~u Leu Gly Val
395 400 405
Val Gly Thr Asp Val Pro Val Lys Glu Leu Leu L;rs Thr Tle Pro
410 415 420
Lys Tyr Lys Leu Gly Ile His Gly Tyr Ala Phe Ala Ile Thr Asn
425 430 435
Asn Gly Tyr Ile Leu Thr His Pro Glu Leu Arg Le:u Leu Tyr Glu
440 445 450
Glu Gly Lys Lys Arg Arg Lys Pro Asn Tyr Ser Se:r Val Asp Leu
455 460 465
Ser Glu Val Glu Trp Glu Asp Arg Asp Asp Val Le:u Arg Asn Ala
470 475 480
Met Val Asn Arg Lys Thr Gly Lys Phe Ser Met Glu Val Lys Lys
485 490 495
Thr Val Asp Lys Gly Val His Phe Ser Gln Thr Ph.e Leu Leu Leu
500 505 510
Asn Leu Lys Gln Thr Thr Val Lys Asn
515
<210> 11
<211> 251
<212> PRT
<213> Homo sapiens
<220>
<221> miso_feature
<223> Incyte ID p'o: 3342358CD1
<400> 11
Met Thr Asp Ser Ala Thr Ala Asn Gly Asp Asp Arch Asp pra Glu
1 5 10 ~ 15
Ile Glu Leu Phe VaI Lys Ala Gly Ile Asp Gly Glu Ser Ile Gly
20 25 30
Asn Cys Pro Phe Ser Gln Arg Leu Phe Met Ile Leu Trp Leu Lys
35 40 4S
14/43
CA 02341148 2001-03-O1
WO 00/1271 i PCT/US99I20468
Gly Val Val Phe Asn Val Thr Thr Val Asp Leu L~rs Arg Lys Pro
50 55 60
Ala Asp Leu His Asn Leu Ala Pro Gly Thr His Px-o Pro Phe Leu
65 70 75
Thr Phe Asn Gly Asp Val Lys Thr Asp Val Asn Lys Ile Glu Glu
80 85 90
Phe Leu Glu Glu Thr Leu Thr Pro Glu Lys Tyr Pro Lys Leu Ala
95 100 105
Ala Lys His Arg Glu Ser Asn Thr Ala Gly Ile Asp Ile Phe Ser
110 115 120
Lys Phe Ser Ala Tyr Ile Lys Asn Thr Lys Gln Gln Asn Asn A1a
125 130 135
Ala Leu Glu Arg Gly Leu Thr Lys AIa Leu Lys Lys Leu Asp Asp
140 145 150
Tyr Leu Asn Thr Pro Leu Pro Glu Glu~Ile Asp Ala Asn Thr Cys
155 160 165
Gly Glu Asp Lys Gly Ser Arg Arg Lys Phe Leu As;p Gly Asp Glu
170 175 180
Leu Thr Leu Ala Asp Cys Asn Leu Leu Pro Lys Leu His Val Val
185 190 195
Lys Ile Val Ala Lys Lys Tyr Arg Asn Tyr Asp Ile Pro Ala Glu
200 205 210
Met Thr Gly Leu Trp Arg Tyr Leu Lys Asn Ala Tyr Ala Arg Asp
215 220 225
Glu Phe Thr Asn Thr Cys Ala Ala Asp Ser Glu Ile: Glu Leu Ala
230 235 240
Tyr Ala Asp Val Ala Lys Arg Leu Ser Arg Ser
245 250
<210> 12
<211> 323
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1267774CD1
<400> 12
Met Gly Leu Phe Asp Arg Gly Val Gln Met Leu Leu Thr Thr Val
1 5 10 1S
Gly Ala Phe Ala Ala Phe Ser Leu Met Thr Ile Ala Val Gly Thr
20 25 30
Asp Tyr Trp Leu Tyr Ser Arg Gly Val Cys Lys Thr Lys 5er Val
3S 40
Ser Glu Asn Glu Thr Ser Lys Lys Asn Glu Glu Val Met Thr His
S5 60
Ser Gly Leu Trp Arg Thr Cys Cys Leu Glu Gly Asn Ser Lys Gly
65 70 75
Leu Cys Lys Gln Ile Asp His Phe Pro Glu Asp Ala Asp Tyr GIu
80 85 90
Ala Asp Thr Ala Glu Tyr Phe Leu Arg Ala Va1 Arg Ala Ser Ser
95 100 105
Ile Phe Pro Ile Leu Ser Val Ile Leu Leu Phe Met Gly Gly Leu
110 115 120
15143
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/2046$
Cys Ile Ala Ala Ser Phe Tyr Lys Thr Arg Ile
Glu Hi.s Asn Ile
125 130 135
Leu Ser Ala Gly Ile Phe Val Ser Ala Gly Asn
Phe Le:u Ser Ile
140 145 1S0
Ile Gly Ile Ile Val Ile Ser Ala Asn Ala Pro
Tyr Gly Asp Ser
155 160 165
Lys Ser Asp Ser Lys Asn Ser Tyr Ser Tyr Ser
Lys Gly Trp Phe
170 175 180
Tyr Phe Gly Ala Leu Phe Ile Ile Ala Glu Gly
Ser Met Val Val
185 190 195
Leu Ala Val His Met IIe Asp Arg His Lys Arg
Phe Gl:n Leu Ala
200 205 210
Thr Ala Arg Ala Thr Tyr Leu Gln Ala Ser Thr
Asp Al,a Ile Arg
215 220 225
IIe Pro Ser Tyr Arg Arg Tyr Gln Arg Arg Ser
Tyr Se:r Arg Ser
230 235 240
Ser Arg Ser Thr Glu Ser His Ser Arg Asp Pro
Pro A1<~ Ser Val
245 250 255
Gly Ile Lys Gly Phe Thr Leu Pro Ser Thr Ser
Asn Glu Ile Met
260 265 270
Tyr Thr Leu Ser Arg Pro Leu Lys Ala Ala Pro
Asp Thr Thr Thr
275 280 285
Ala Thr Tyr Asn Ser Arg Asp Asn Ser Phe Val
Asp Leu Gln His
290 295 300
Asn Cys Ile Gln Lys
Glu Asn Lys Asp
Ser Leu His. Ser
Asn Thr
305 310 315
Ala Asn Arg Arg Thr Pro Val
Thr
320
<210> 13
<211> 51
<212> PRT
<213> Homa Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: I8I7329CD1
<400> 13
Met Asn Gln Gly Ser Gly Leu Asp Leu Leu Lys Ile Ser Tyr Gly
1 5 10 I5
Lys Gly Ala Arg Arg Lys Asn Arg Phe Lys Gly Ser Asp Gly Ser
20 25 30
Thr Ser Ser Asp Thr Thr Ser Asn Ser Phe Val Arg Gln Val Arg
35 40 45
Val Leu Ser Ser Trp Phe
<210> 14
<211> 113
<212> PRT
<213> Homo Sapiens
<220>
16/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
<221> misc_feature
<223> Incyte ID No: 3273307CD1
<400> 14
Met Glu Gln Arg Lys Leu Asn Asp Gln Ala Asn Thr Leu Val Asp
Z 5 10 15
Leu Ala Lys Thr Gln Asn Ile Met Tyr Asp Met Il~e Ser Asp Leu
20 25 30
Asn Glu Arg Ser Glu Asp Phe Glu Lys Arg Ile Va.l Thr Leu Glu
35 40 45
Thr Lys Leu Glu Thr Leu Ile Gly Ser Ile His Ala Leu Pro Gly
50 55 60
Leu Ile Ser Gln Thr Ile Arg Gin Gln Gln Arg Asp Phe Ile Glu
65 70 75
Ala Gln Met Glu Ser Tyr Asp Lys His Val Thr Tyr Asn Ala Glu
80 85 90
Arg Sex Arg Ser Ser Ser Arg Arg Arg Arg Ser Ser Ser Thr Ala
95 100 105
Pro Pro Thr Ser Ser Glu Ser Ser
110
<210> 15
c211> 215
< 212 > PTtT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3824833CD1
<400> 15
Met His Arg Asp Ala Trp Leu Pro Arg Pro Ala Phe Ser Leu Thr
1 5 10 15
Gly Leu Ser Leu Phe Phe Ser Leu Val Pro Pro Gly Arg Ser Met
20 25 30
Glu Val Thr Val Pro Ala Thr Leu Asn Val Leu Asn Gly Ser Asp
35 40 45
Ala Arg Leu Pro Cys Thr Phe Asn Ser Cys Tyr Thr Val Asn His
50 55 60
Lys Gln Phe Ser Leu Asn Trp Thr Tyr Gln Glu Cys Asn Asn Cys
65 70 75
Ser Glu GIu Met Phe Leu Gln Phe Arg Met Lys Ile Ile Asn Leu
80 85 90
Lys Leu Glu Arg Phe Gln Asp Arg Val Glu Phe Ser Gly Asn Pro
95 100 105
Ser Lys Tyr Asp Val Ser Val Met Leu Arg Asn Val Gln Pro Glu
110 115 120
Asp Glu Gly Ile Tyr Asn Cys Tyr Ile Met Asn Pro Pro Asp Arg
125 130 135
His Arg Gly His Gly Lys Ile His Leu Gln Val Leu Met Glu Glu
140 145 150
Pro Pro Glu Arg Asp Ser Thr Val Ala Val Ile Val Gly Ala Ser
155 160 165
Val Gly Gly Phe Leu Ala Val Val Ile Leu Val Leu Met Val Val
170 175 180
i ~r4~
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
Lys Cys Val Arg Arg Lys Lys GIu Gln Lys Leu Se:r Thr Asp Asp
185 190 195
Leu Lys Thr Glu Glu GIu Gly Lys Thr Asp GIy Glu Gly Asn Pro
200 205 210
Asp Asp Gly Ala Lys
2 3.5
<210> 16
<211> 235
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2069907CD1
<400> 16
Met Phe Ile Trp Thr Ser Gly Arg Thr Ser Ser Sexy Tyr Arg His
10 15
Asp Glu Lys Arg Asn Ile Tyr Gln Lys Ile Arg Asp His Asp Leu
20 25 30
Leu Asp Lys Arg Lys Thr Val Thr AIa Leu Lys Ala. Gly Glu Asp
35 40 45
Arg Ala Ile Leu Leu Gly Leu Ala Met Met Val Cys Ser IIe Met
50 55 60
Met Tyr Phe Leu Leu Gly Ile Thr Leu Leu Arg Ser Tyr Met Gln
65 70 75
Ser Val Trp Thr Glu Glu Ser Gln Cys Thr Leu Leu Asn Ala Ser
80 85 90
Ile Thr Glu Thr Phe Asn Cys Ser Phe Ser Cys Gly Pro Asp Cys
95 100 105
Trp Lys Leu Ser Gln Tyr pro Cys Pro Gln Val Tyr Va1 Asn Leu
110 115 120
Thr Ser Ser Gly Glu Lys Leu Leu Leu Tyr His Thr Glu Glu Thr
125 130 I35
Ile Lys Ile Asn Gln Lys Cys Ser Tyr Ile Pro Lys Cys Gly Lys
140 145 150
Asn Phe Glu Glu Ser Met Ser Leu Val Asn Val Val Met Glu Asn
155 160 165
Phe Arg Lys Tyr Gln His Phe Ser Cys Tyr Ser Asp Pro Glu Gly
170 175 1$0
Asn Gln Lys Ser Val Ile Leu Thr Lys Leu Tyr Ser Ser Asn Val
185 190 195
Leu Phe His Ser Leu Phe Trp Pro Thr Cys Met Met Ala Gly Gly
200 205 210
Val Ala Ile Val Ala Met Val Lys Leu Thr Gln Tyr Leu Ser Leu
215 220 225
Leu Cys Glu Arg Ile Gln Arg Ile Asn Arg
230 235
<210> 17
<211> 234
<212> PRT
<213> Homo Sapiens
18/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
<220>
<22i> misc_feature
<223> Incyte ID No: 2243917CDI
<400> 17
Met Ala Glu Asn His Cys Glu Leu Leu Ser Pro A.la Arg Gly Gly
1 5 10 15
Ile Gly Ala Gly Leu Gly Gly Gly Leu Cys Arg A.rg Cys Ser Ala
20 25 30
Gly Leu Gly Ala Leu Ala Gln Arg Pro Gly Ser Val Ser Lys Trp
35 40 45
VaI Arg Leu Asn Val Gly Gly Thr Tyr Phe Leu T.hr Thr Arg Gln
50 55 60
Thr Leu Cys Arg Asp Pro Lys Ser Phe.Leu Tyr A:rg Leu Cys Gln
65 70 75
Ala Asp Pro Asp Leu Asp Ser Asp Lys Asp Glu Thr Gly Ala Tyr
80 85 90
Leu IIe Asp Arg Asp Pro Thr Tyr Phe Gly Pro V<~1 Leu Asn Tyr
95 100 105
Leu Arg His Gly Lys Leu Vai Ile Asn Lys Asp Le~u Ala Glu G1u
110 115 120
Gly Val Leu Glu Glu Ala Glu Phe Tyr Asn Ile Thr Ser Leu Ile
125 130 135
Lys Leu Val Lys Asp Lys Ile Arg Glu Arg Asp Se:r Lys Thr Ser
140 145 150
Gln Val Pro Val Lys His Val Tyr Arg Val Leu Gl.n Cys Gln Glu
155 160 165
Glu Glu Leu Thr Gln Met Val Ser Thr Met Ser As;p Gly Trp Lys
170 175 180
Phe Glu Gln Leu Val Ser Ile Gly Ser Ser Tyr Asn Tyr Gly Asn
185 190 195
Glu Asp G1n Ala Glu Phe Leu Cys VaI Val Ser Lys Glu Leu His
200 205 210
Asn Thr Pro Tyr Gly Thr Ala Ser Glu Pro Ser Glu Lys Ala Lys
225 220 225
Ile Leu Gln Glu Arg Gly Ser Arg Met
230
<210> 18
<211> 301
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2597476CD1
<400> 18
Met Val Phe Thr Gln Ala Pro Ala Glu Ile Met Gly His Leu Arg
1 5 i0 15
Ile Arg Sex Leu Leu Ala Arg Gln Cys Leu Ala Glu Phe Leu Gly
20 25 30
Val Phe Val Leu Met Leu Leu Thr Gln Gly Ala Val Ala Gln Ala
35 40 45
Va1 Thr Ser Gly Glu Thr Lys Gly Asn Phe Phe Thr Met Phe Leu
19143
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
50 55 60
Ala Gly Ser Leu Ala Val Thr.Ile Ala Ile Tyr Val Gly Gly Asn
65 70 75
Val Ser G1y Ala His Leu Asn Pro Ala Phe Ser Leu Ala Met Cys
SQ 85 90
Ile Val Gly Arg Leu Pro Trp Val Lys Leu Pro Il,e Tyr Ile Leu
95 100 105
Val Gln Leu Leu Ser Ala Phe Cys Ala Ser Gly Ala Thr Tyr Val
110 115 120
Leu Tyr His Asp Ala Leu Gln Asn Tyr Thr Gly Gly Asn Leu Thr
125 130 135
Val Thr Gly Pro Lys Glu Thr Ala Ser Ile Phe Ala Thr Tyr Pro
140 145 150
Ala Pro Tyr Leu Ser Leu Asn Asn Gly.Phe Leu Asp Gln Val Leu
155 160 165
GIy Thr Gly Met Leu Ile Val Gly Leu Leu Ala Il~e Leu Asp Arg
170 175 180
Arg Asn Lys Gly Val Pro Ala Gly Leu Glu Pro Va:l Val Val Gly
185 190 195
Met Leu Ile Leu Ala Leu Gly Leu Ser Met Gly A1;~ Asn Cys Gly
200 205 210
Ile Pro Leu Asn Pro Ala Arg Asp Leu Gly Pro Arg Leu Phe Thr
215 220 225
Tyr Val Ala Gly Trp Gly Pro Glu Val Phe Ser Ala Gly Asn Gly
230 235 240
Trp Trp Trp Val Pro Val Val Ala Pro Leu Val Gly Ala Thr Val
245 250 255
Gly Thr Ala Thr Tyr Gln Leu Leu Val Ala Leu Hi.o His Pro Glu
260 265 270
Gly Pro Glu Pro Ala Gln Asp Leu Val Ser Ala Glr.~ His Lys A1a
275 280 285
Sex Glu Leu Glu Thr Pro Ala Ser Ala Gln Met Leu Glu Cys Lys
290 295 300
Leu
<210> 19
<211> 2994
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1568324CB1
<400> 19
gggaggcact gcccgtgttg ggatgcagaa gggatcagct tcc<~gttgtc ttggagttga 60
tgactgacct ctacctctgc agggaaaggg gcgcagggag tctc;aaagct ccaggagcag 120
cagaaggccc 'agtggccggt ctccaaccag tactgagaag cgca~tgagct tcgagtccat 180
ttcttccctg ccagaggttg agccggaccc tgaggctggg agtc~agcaag aggtattttc 240
tgctgtggaa gggcccagtg ccgaggagac gccttcagac acac~aatctc cagaagtcct 300
ggagacacag 'cttgatgccc accagggcct tctggggatg gaccccccag gtgacatggt 360
ggacttcgtg gcagctgaga gcactgagga ccttaaggcc ctgagcagcg aggaggaaga 420
agaaatggga ggtgccgccc aggagcctga gagccttctg ccac;cctctg tgctggacca 480
ggccagcgtc attgcggagc gatttgtcag cagcttctct cggc:ggagca gcgtggcaca 540
ggaggacagc aagtccagtg gctttgggag cccgcggctg gtcaegecgga gcagcagcgt 600
20/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
gctcagcctg gagggcagcg agaagggcct ggcccggcat gc~cagtgcca cagactccct 660
cagctgtcag ctctccccag aagtggacat cagtgtgggg gt~ggccacag aggacagccc 720
ttctgtcaat gggatggagc ccccaagccc aggctgccca gt~ggagcctg accggtcttc 780
ctgcaagaag aaggaatcag cactctccac ccgagaccgg ct:gttgctag acaagattaa 840
gagctattat gaaaatgcag aacaccatga tgcaggcttc ac~cgtccgtc gccgggagag 900
cctctcctac atccccaaag gactggtaag aaactccatc tc:caggttca acagccttcc 960
ccggccagac ccagagccag tacctccagt ggggagcaag ac~acaggtgg gctcccggcc 1020
gacttcgtgg gccctgtttg agctcccagg accaagccag gc:agtcaaag gggacccacc 1080
tcccatctca gatgctgagt tccgcccatc ttcagaaatt gtgaagatct gggagggaat 1140
ggagtcttcc ggagggagcc ctgggaaggg gccaggccag gcrccaggcca atggctttga 1200
cctgcatgag ccactcttca tcctggagga gcatgagctg ggragccatca cagaggagtc 1260
ggccactgcc tccccggaaa gctcctctcc cactgagggg cg;cagcccgg cccacctggc 1320
ccgggagctg aaagagctgg tgaaggagct gagcagcagt acccaggggg agctggtggc 1380
cccactgcac ccccgcatcg tgcagctctc ccacgtaatg ga.cagccacg tgagcgagcg 1440
cgtcaagaac aaggtctacc agctggcccg ccagtacagc ctccggatca agagcaacaa 1500
gccagtgatg gccaggccac cactgcagtg ggaaaaggtg gcccctgaga gggatgggaa 1560
gagccccact gtgccctgtc tacaggaaga ggctggagag ccattaggtg gcaaaggtaa 1620
gaggaagccg gtgctgtctc tatttgacta tgagcagctg atggcccagg agcacagccc 1680
tcccaagccc tcctcggctg gggagatgtc accacagcgt ttcttcttca acccgcctgc 1740
tgtcagccag aggaccacct cgcctggggg ccggccctcc gcccggagcc ccctcagccc 1800
cacagagacc ttcagctggc ccgacgtccg tgagctctgc tccaagtatg cctcccgcga 1860
tgaggcacgc cgagcagggg gcggccggcc ccgcggccca cccgtcaaca ggagccactc 1920
ggtgccggag aacatggtag agccacctct gtcgggcagg gtgggccgct gccgcagcct 1980
gagcaccaag aggggccggg gaggcggaga ggctgcccaa tcccctgggc ctctgcccca 2040
gagcaagccg gatggaggcg agaccctgta tgtcactgca gacctcaccc tggaggacaa 2100
ccggcgggtg attgtcatgg agaagggacc ccttcccagc cccactgcag ggctggagga 2160
gagcagtggc cagggaccaa gctcaccggt ggccctgctg gggcaggttc aggacttcca 2220
gcagtctgca gagtgccagc cgaaggaaga gggttccagg gacccggcag acccgagcca 2280
gcagggcaga gtgagaaacc ttagagagaa gttccaggcc ttgaactctg tcggttgatg 2340
ctgactcctg ggggagggag gagtcatgtt ggaggttggg gaagaacctg ggcatccttc 2400
ccctcaagcc tgggctcatg gagcccctgc ccagggccct caggtgggcg gaaagtccat 2460
cccctccgcc cttcaggaag gatgctcccg tgtgcagggg tct~cctgcct gtgccatcca 2520
ctggggctcg agacaatttc ccactcacct gtgaggccgg tgt:ggctgct tcccttgtaa 2580
atagttgttc tctggtaaga agccaaatat ttaagctcac ttcatcccag agagaggaag 2640
ctctgctcag gcctccagcg ttggctggcc atggccacag ccagatggag gagcccatcc 2700
ccaggagact caggcagtgg cctggagagg ctttgttctg taacggtgcc ttttcttagg 2760
gtccaggcag gaatgaagcc aataatttat tgctttccat tct:gtggtat gatgtgcgtg 2820
tgcgtgagtg tgtggcccct gtttattccc ctcctgtcaa gaatgaagtg gattcagttc 2880
aggtactttt gagggttgtt gtgctgaccc tgtggttgtc gct:gatgtac acacatttca 2940
ttatttgcca atggtgcaat aaccaatgct gacaaaccca aaa~aaaaaaa aaaa 2994
<210> 20
<211> 1298
<2I2> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4094907081
<400> 20
cacgaccccc tgcctcttct tccagaagcc aatgggcaca gaagcaccaa ttctcccaca 60
atagtttcac ctgctattgt ttcccccacc caggacagtc ggcccaatat gtcaagacct 120
ctgatcacta gatcccctgc atctccactg aacaaccaag gcatccctac tccagcacaa 180
ctcacaaaat ccaatgcgcc tgtccacatt gatgtgggcg gccacatgta caccagcagc 240
ctggccaccc tcaccaaata ccctgaatcc agaatcggaa gac~tttttga tggtacagag 300
21/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
cccattgttt tggacagtct caaacagcac tatttcattg acagagatgg acagatgttc 360
agatatatct tgaattttct acgaacatcc aaactcetca ttcctgatga tttcaaggac 420
tacactttgt tatatgaaga ggcaaaatat tttcagcttc agcccatgtt gttggagatg 480
gaaagatgga 'agcaggacag agaaactggt cgattttcaa ggccctgtga gtgcctcgtc 540
gtgcgtgtgg ccccagacct cggagaaagg atcacgctaa gc:ggtgacaa atccttgata 600
gaagaagtat ttccagagat cggcgacgtg atgtgtaact ct:gtcaatgc aggctggaat 660
cacgactcga cgcacgtcat caggtttcca ctaaatggct acagtcacct caactcagtc 720
caggtcctcg agaggttgca gcaaagagga tttgaaatcg tgggctcctg tgggggagga 780
gtagactcgt cccagttcag cgaatacgtc cttcggcggg aactgaggcg gacgccccgt 840
gtaccctccg tcatccggat aaagcaagag cctctggact aaatggacat atttcttatg 900
caaaaaggaa aacacacaca accaataact caaacaaaaa ac~ggacattt atgtgcagtt 960
gggacagcaa accaagtcct ggacgtaaaa tcgaataaaa ga~cacattta tatccaatag 1020
agaccacacc tgtattcata tgggaacaat tggaatagtg at:atcctcaa ggtgtaaaaa 1080
atatataaat atatatatat atgtcaaaag gtaggaaatg ca.aaaaagaa aaaaaaaaaa 1140
aggtgacagc cgcagttggt gctgtgatgg ccgtgaagtg tcctgggcct cccgaggcct 1200
ctgacaaata aacaagccat gagtggtgag gacacagtct ccttacagtt tccattgcca 1260
acaacagcca tccatatttc ttttttcctt tgtctttc 1298
<210> 21
<2i1> 1877
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte TD No: 518158CB1
<400> 21
gcctggccgt caccactccc cagagggcac agggctctgc tgtgcctcag agcaaaagtc 60
ccagagccag-cagagcaggc tgacgacctg caagccacag tggctgccct gtgcgtgctg 120
cgaggtgggg gaccctgggc aggaagctgg ctgagcccca agaccccggg ggccatgggc 180
ggggatctgg tgcttggcct gggggccttg agacgccgaa agc:gcttgct ggagcaggag 240
aagtctctgg ccggctgggc actggtgctg gcaggaactg gcattggact catggtgctg 300
catgcagaga tgctgtggtt cggggggtgc tcgtgggcgc tct;acctgtt cctggttaaa 360
tgcacgatca gcatttccac cttcttactc ctctgcctca tcc~tggcctt tcatgccaaa 420
gaggtccagc tgttcatgac cgacaacggg ctgcgggact ggc;gcgtggc gctgaccggg 480
cggcaggcgg cgcagatcgt gctggagctg gtggtgtgtg ggc;tgcaccc ggcgcccgtg 540
cggggcccgc cgtgcgtgca ggatttaggg gcgccgctga cct:ccccgca gccctggccg 600
ggattcctgg gccaagggga agcgctgctg tccctggcca tgctgctgct cggcctcacg 660
cttggcctct ggctgaccac cgcctgggtg ctgtccgtgg ccg~agaggca ggctgttaat 720
gccactgggc acctttcaga cacactttgg ctgatcccca tca.cattcct gaccatcggc 780
tatggtgacg tggtgccggg caccatgtgg ggcaagatcg tctgcctgtg cactggagtc 840
atgggtgtct gctgcacagc cctgctggtg gccgtggtgg cccggaagct ggagtttaac 900
aaggcagaga agcacgtgca caacttcatg atggatatcc agtataccaa agagatgaag 960
gagtccgctg cccgagtgct acaagaagcc tggatgttct acaaacatac tcgcaggaag 1020
gagtctcatg ctgcccgcag gcatcagcgc aagctgctgg ccgccatcaa cgcgttccgc 1080
caggtgcggc tgaaacaccg gaagctccgg gaacaagtga actccatggt ggacatctcc 1140
aagatgcaca tgatcctgta tgacctgcag cagaatctga gca~gctcaca ccgggccctg 1200
gagaaacaga ttgacacgct ggcggggaag ctggatgccc tgactgagct gcttagcact 1260
gccctggggc cgaggcagct tccagaaccc agccagcagt cca~agtagct ggacccacga 1320
ggaggaacca ggctactttc cccagtactg aggtggtgga catcgtctct gccactcctg 1380
acccagccct gaacaaagca cctcaagtgc aaggaccaaa ggg<3gccctg gcttggagtg 1440
ggttggcttg 'ctgatggctg ctggagggga cgctggctaa agtgggtagg ccttggccca 1500
cctgaggccc caggtgggaa catggtcacc cccactctgc ata<:cctcat caaaaacact 1560
ctcactatgc tgctatggac gacctccagc tctcagttac aagt;gcaggc gactggaggc 1620
aggactcctg ggtccctggg aaagagggta ctaggggccc ggat:ccagga ttctgggagg 1680
22/43
CA 02341148 2001-03-O1
WO 00/1271 I PCT/US99/2046$
cttcagttac cgctggccga gctgaagaac tgggtatgag gcaggggcgg ggctggaggt 1740
ggcgccccct ggtgggacaa caaagaggac accatttttc cagagctgca gagagcacct 1800
ggtggggagg aagaagtgta actcaccagc ctctgctctt at:ctttgtaa taaatgttaa 1860
agccagaaaa aaaaaaa 1877
<210> 22
c211> 2517
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 602926CB1
<400> 22
gccaccccgc tctcctcgcc gccgcggcgg caggcgcggg gccgcgccgc gaggcctgat 60
ccctgcagcg cgggcaggcg gcgtcgcaga gcggagctca gctggatgcg tccggactcc 120
tgcaggtgag ag'tgattttc cagtgattgc tttggcctgt acaaccagag aacaggattc 180
ttcccttctt tttggccacc aaatgcctat gtgcaccaca cattccagtg tgctgagaag 240
ggcagagctt cttggatgat gatggacgtc ccaccgggca gg<~tgaaggc agagcgtgtg 300
gcatctccac ctcaagggtg cagcctgatc ttcctcttct cccttgccag ccagcactct 360
gccttctgta tccaccatgg tgtttggtga gtttttccat cgccctggac aagacgagga 420
acttgtcaac ctgaatgtgg ggggctttaa gcagtctgtt gaccaaagca ccctcctgcg 480
gtttcctcac accagactgg ggaagctgct tacttgccat tct:gaagagg ccattctgga 540
gctgtgtgat gattacagtg tggccgataa ggaatactac ttt:gatcgga atccctcctc 600
gttcagatat gttttgaatt tttattacac ggggaagctg cat:gtcatgg aggagctgtg 660
cgtattctca ttctgccagg agatcgagta ctggggcatc aac:gagctct tcattgattc 720
ttgctgcagc aatcgctacc aggaacgcaa ggaggaaaac cac:gagaagg actgggacca 780
gaaaagccat gatgtgagta ccgactcctc gtttgaagag tcc~tctctgt ttgagaaaga B40
gctggagaag tttgacacac tgcgatttgg tcagctccgg aagraaaatct ggattagaat 900
ggagaatcca gcgtactgcc tgtccgctaa gcttatcgct atctcctcct tgagcgtggt 960
gctggcctcc atcgtggcca tgtgcgttca cagcatgtcg gag~ttccaga atgaggatgg 1020
agaagtggat gatccggtgc tggaaggagt ggagatcgcg tgcattgcct ggttcaccgg 1080
ggagcttgcc gtccgcctgg ctgccgctcc ttgrtcaaaag aaattctgga aaaaccctct 1140
gaacatcatt gactttgtct ctattattcc cttctatgcc acgttggctg tagacaccaa 1200
ggaggaagag agtgaggata ttgagaacat gggcaaggtg gtccagatcc tacggcttat 1260
gaggattttc cgaattctaa agcttgcccg gcactcggta ggacttcggt ctctaggtgc 1320
cacactgaga cacagctacc atgaagttgg gcttctgctt ctcttcctct ctgtgggcat 1380
ttccattttc tctgtgctta tctactccgt ggagaaagat gaccacacat ccagcctcac 1440
cagcatcccc atctgctggt ggtgggccac catcagcatg aca~actgtgg gctatggaga 1500
cacccacccg gtcaccttgg cgggaaagct catcgccagc acatgcatca tctgtggcat 1560
cttggtggtg gcccttccca tcaccatcat cttcaacaag ttttccaagt actaccagaa 1620
gcaaaaggac attgatgtgg accagtgcag tgaggatgca ccagagaagt gtcatgagct 1680
accttacttt aacattaggg atatatatgc acagcggatg cac<~ccttca ttaccagtct 1740
ctcttctgta 'ggcattgtgg tgagcgatcc tgactccaca gat<Icttcaa gcattgaaga 1800
caatgaggac atttgtaaca ccacctcctt ggagaattgc acadcaaaat gagcgggggt 1860
gtttgtgcct gtttctctta tcctttcccg acattaggtt aacacagctt tataaacctc 1920
agtgggttcg ttaaaatcat ttaattctca gggtgtacct ttcagccata gttggacatt 1980
cattgctgaa ttctgaaatg atagaattgt ctttattttt ctct:gtgagg tcaattaaat 2040
gccttgttct gaaatttatt ttttacaaga gagagttgtg atat:agtttg gaatataaga 2100
taaatggtat tgggtggggt ttgtggctac agcttatgca tcatactgtg tttgtcattt 2160
actcacattg agctaacttt aaattactga caagtagaat caaa~ggtgca gctgactgag 2220
acgacatgca tgtaagatcc acaaaatgag acaatgcatg taaa~tccatg ctcatgttct 2280
aaacatggaa actaggagcc taataaactt cctaattcag tatgrgtatac caaaaaatcc 2340
gggcggcctg cgactagcga gctcgtctga ccgggaatcc attccgcgac ggtacctgcc 2400
ggcgtaccag cctttcccat agtgagtcgg attagagctt ggcg~gaatca tggtcatagc 2460
23/43
CA 02341148 2001-03-O1
WO 00/12T11 PCT/US99/20468
cggttcccgt gggaaactgt tatccggcca caattccata caacactcga gccggga 2517
<210> 23
<211> 1154
<212> DNA
<213> Iiomo Sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 922119CB1
<400> 23
ttaactcagc tgggagttga agagccgatg ggcagcaggc ag~acttgagt ctcctttctg 60
tccatgggct cgggccactg tctcaggtcc acccgtggct ccaaaatggt ctcctggtcc 120
gtgatagcaa agatccagga aatactgcag aggaagatgg tgcgagagtt cctggccgag 180
ttcatgagca catatgtcat gatggtattc ggccttggtt ccgtggccca tatggttcta 240
aataaaaaat atgggagcta ccttggtgtc aacttgggtt ttggcttcgg agtcaccatg 300
ggagtgcacg tggcaggccg catctctgga gcccacatga acgcagctgt gacctttgct 360
aactgtgcgc tgggccgcgt gccctggagg aagtttccgg tctatgtgct ggggcagttc 420
ctgggctcct tcctggcggc tgccaccatc tacagtctct tctacacggc cattctccac 480
ttttcgggtg gacagctgat ggtgaccggt cccgtcgcta ca~gctggcat ttttgccacc 540
taccttcctg atcacatgac attgtggcgg ggcttcctga atgaggcgtg gctgaccggg 600
atgctccagc tgtgtctctt cgccatcacg gaccaggaga ac;aacccagc actgccagga 660
acagaggcgc tggtgatagg catcctcgtg gtcatcatcg gggtgtccct tggcatgaac 720
acaggatatg ccatcaaccc gtcccgggac ctgccccccc gc~atcttcac cttcattgct 780
ggttggggca aacaggtctt cagcaatggg gagaactggt ggtgggtgec agtggtggca 840
ccacttctgg gtgcctatct aggtggcatc atctacctgg tcttcattgg ctccaccatc 900
ccacgggagc ccctgaaatt ggaggattct gtggcgtatg aagaccacgg gataaccgta 960
ttgcccaaga tgggatctca tgaacccacg atctctcccc tcacccccgt ctctgtgagc 1020
cctgccaaca gatcttcagt ccaccctgcc ccacccttac at<~aatccat ggccctagag 1080
cacttctaag 'cagagattat ttgtgatccc atccattccc caataaagca aggcttgtec 1140
gacaaaaaaa aaaa 1154
<210> 24
<211> 1879
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2666782CB1
<400> 24
ggtaccgagg cccgagccgc gggagtcgag cgaaggcagc gccgaggccg cggtttcccc 60
ctgggcctcc ccagcagcag ccatgggcat caaattttta gaagttatca aaccattctg 120
tgcagttcta ccagaaattc agaaaccgga aaggaaaatc cagtttagag agaaggttct 180
gtggactgct ataacgctct tcattttctt agtgtgttgt cag~atcccac tgtttggaat 240
catgtcatca gattctgcag atcctttcta ctggatgaga gtt;attctgg cttccaatag 300
aggaacttta atggaattgg gtatctcccc aattgtaaca tctggtttga ttatgcagtt 360
gttagctgga gccaaaatca ttgaagttgg agatacaccg aaagatagag ctttattcaa 420
tggagcccag aaactgtttg gtatgatcat taccattggg caagccattg tgtatgtcat 480
gacggggatg tatggggacc ctgcagaaat gggtgccgga atc~tgtctcc tgatcatcat 540
tcagttgttt gttactagtt tgattgtgct actgttagat gag<~tactac agacaggtta 600
cagcttgggg tctgggattt ccctcgttat tgccaccaac atctgtgaga ccattgtctg 660
gaaggccttt agtcccacta ccattaacac tggcagaggt actgagtttg agggtgcagt 720
catagctctg ttccatttgt tggccaccag gacggacaaa gtcc:gagctt tacgggaggc 780
24/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
tttttatcgg cagaacttac ccaatctcat gaacctcatt gctacagttt ttgtgtttgc 840
tgttgttata tatttccaag gatttcgcgt tgatctgccc at taagtcgg cccgttaccg 900
aggacagtac agcagctacc ccatcaaact cttctacacc tccaacatcc ccatcatcct 960
ccagtcggcc ctggtgtcca acctgtatgt tatttcccag atgctgtctg ttcgatttag 1020
tggcaacttt ttagtaaatt tactaggaca gtgggccgat gt:cagtgggg gaggacccgc 1080
acgttcttac ccagttggag gcctttgtta ctatctttct ccacctgagt ccatgggcgc 1140
catctttgag gatcctgtcc atgtcgttgt ttatatcatc tt:catgttgg ggtcatgtgc 1200
attcttctct aagacatgga ttgaagtgtc tggttcctca gc:caaagatg tagctaaaca 1260
gctgaaagaa cagcagatgg taatgagggg ccaccgagat ac:ctctatgg ttcatgagct 1320
taataggtac atccccaccg cagctgagtt tggcggtttg tc~cattggcg ccctgtcagt 1380
gctggctgac ttcctggggg ccattggatc tggcactgga atactgctag cagtcactat 1440
tatttaccag tattttgaaa tatttgttaa agaacaggcc gaiagttggtg ggatgggtgc 1500
tttgtttttc taaatgttca aatatttcat tttgtgcgtg tg~aaagggaa aacactttga 1560
cggatcgttt ttgtcagatg acactggtgg ctccectttt ct.cccctcac agtttcttgt 1620
ttcgagtgct gactgacccg tttctgaaat gggcaccgag ctaagtctgt gtgcagcatt 1680
agtacccgct gccttaaaac tcaagtttac attattcatt aa.aaaaagta catctagtgt 1740
tgcctgtaat gctggaaacc agtgtatcta ccttgctgtg ttaaatcatg acagtgagac 1800
ggtgagatgg attcgttttg cacacaacat tcaaaacact tcatattgcc cccacttgtt 1860
gaaaaataaa tgtagttca 1879
<210> 25
<211> 1537
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2731369CB1
<400> 25
ctggcccagc cctggcagct gatgggagag cagatcgtcc aatacaagcc ttcctacggt 60
ctctttagaa gacatgggct gaaacttggg gtaatagagg ttc~gctggca tatccatgca 120
ggatgattgt cttcacatgt atctattacc ttgtagaata agc~tagaccc tgattttgga 180
acctgaagac 'caaagtgcaa gattagctct gctacttcca tct:gtggacc attgggcagg 240
tatctctggg ccttcactta ctctttgtga aatgaggaca ggc~gcaatcc ctaccctacc 300
aagtcattgg gagtgaagac atgatgacac ggtgattgtg aaa~agatttt gtcaatcgca 360
ccagcattaa gggtgcccat ctccaggttc ccccaggcct caaiggctccc aaggcctgag 420
tgggcaggta gcacccaggt atagaccttc cacgtgcagc acc:caggaca cagccagcat 480
gaactgggca tttctgcagg gcctgctgag tggcgtgaac aagtactcca cagtgctgag 540
ecgcatctgg ctgtctgtgg tgttcatctt tcgtgtgctg gtg~tacgtgg tggcagcgga 600
ggaagtgtgg gacgatgagc agaaggactt tgactgcaac accaagcagc ccggctgcac 660
caacgtctgc tacgacaact acttccccat ctecaacatc cgcctctggg ccctacagct 720
catcctggtc acgtgcccct cactgctcgt ggtcatgcac gtggcctacc gcgaggaacg 780
cgagcgcaag caccacctga aacacgggcc caatgccccg tccctgtacg acaacctgag 840
caagaagcgg ggcggactgt ggtggacgta cttgctgagc ctcatcttca aggccgccgt 900
ggatgctggc ttcctctata tcttccaccg cctctacaag gattatgaca tgccccgcgt 960
ggtggcctgc tccgtggagc cttgccccca cactgtggac tgttacatct cccggcccac 1020
ggagaagaag gtcttcacct acttcatggt gaccacagct gcc~atctgca tcctgctcaa 1080
cctcagtgaa gtcttctacc tggtgggcaa gaggtgcatg gag;atcttcg gccccaggca 1140
ccggcggcct cggtgccggg aatgcctacc cgatacgtgc ccaccatatg tcctctccca 1200
gggagggcac cctgaggatg ggaactctgt cctaatgaag get<~ggtcgg ccecagtgga 1260
tgcaggtggg tatccataac ctgcgagatc agcagataag atcaacaggt cccccccaca 1320
tgaggccacc caggaaaaaa ggcaggggca gtggcatcct tgccgtagca gggtggtgag 1380
gagggtggct gtgggggctc aggaagctcg cccaggggcc aatgtgggag gttgggggta 1440
gtttggtccc tgggtcctga gcctcagggg agggaggttg atagctactg gggattttgt 1500
atatggcaac 'agtatatgtc aaacctctta ataaatt 1537
25/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
<210> 26
<21I> 884
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 790, 827, 860
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte ID No: 1375415CB1
<400> 26
gcgcctggag ccacacaggg atccggagcc tgggggaaaa gcggcgcggg agccggcacc 60
caccgctgga ggggcggcga cggcggccgt agcgacctcg ggaggcaagc ggagccgcca 120
tggccgagtt cccgtcgaaa gttagcacgc ggaccagcag tcctgcgcag ggcgccgaag 180
cctcggtgtc ggcgctgcgc ccggacctgg gcttcgtgcg ctcccgcctc ggggcgctca 240
tgctgctgca gctggtgctg gggctgctgg tgtgggcgct gattgcggac accccgtacc 300
acctgtatcc ggcctatggc tgggtgatgt tcgtcgctgt cttcctctgg ctggtgacaa 360
tcgtcctctt caacctctac ctgtttcagc tgcacatgaa gttgtacatg gttccctggc 420
cactggtgtt aatgatcttt aacatcagcg ccaccgttct ct~acatcacc gccttcatcg 480
cctgctctgc ggcagttgac ctgacatccc tgaggggcac ccggccttat aaccagcgcg 540
cggctgcctc gttctttgcg tgtttggtga tgatcgccta tggagtgagt gccttcttca 600
gctaccaggc ctggcgagga gtaggcagca atgcggccac ca~3tcagatg gctggcggct 660
atgcctaaac cacctgtgcc acggccccct ctggggctga agccgccgct gggtcacaga 720
gcagggtcac cctgcaagcc tgaagctggg gagccctgcg tg<~agtcagc ccaacaggga 780
ctgcatttgn 'ctcctctctg cccgtcagac ataagctctc acagcgntaa ggaagcaggc 840
ccaggctggc agacatctcn gcttgcagga ggccaactgt ga<~a gg4
<210> 27
<211> 3156
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2733282CB1
<400> 27
gttcaggaag aaaccatctg catccatatt gaaaacctga cacaatgtat gcagcaggct 60
cagtgtgagt gaactggagg cttctctaca acatgaccca aaggagcatt gcaggtccta 120
tttgcaacct gaagtttgtg actctcctgg ttgccttaag ttcagaactc ccattcctgg 180
gagctggagt acagcttcaa gacaatgggt ataatggatt gctcattgca attaatcctc 240
aggtacctga gaatcagaac ctcatctcaa acattaagga aat~gataact gaagcttcat 300
tttacctatt taatgctacc aagagaagag tatttttcag aaatataaag attttaatac 360
ctgccacatg gaaagctaat aataacagca aaataaaaca aga;atcatat gaaaaggcaa 420
atgtcatagt gactgactgg tatggggcac atggagatga tccatacacc ctacaataca 480
gagggtgtgg aaaagaggga aaatacattc atttcacacc taaitttccta ctgaatgata 540
acttaacagc tggctacgga tcacgaggcc gagtgtttgt ccatgaatgg gcccacctcc 600
gttggggtgt gttcgatgag tataacaatg acaaaccttt cta<:ataaat gggcaaaatc 660
aaattaaagt gacaaggtgt tcatctgaca tcacaggcat ttttgtgtgt gaaaaaggtc 720
cttgccccca agaaaactgt attattagta agctttttaa agaaggatgc acctttatct 780
acaatagcac ccaaaatgca actgcatcaa taatgttcat gcaaagttat ctctgtggtg 840
aaatttgtaa tgccagtacc cacaaccaag aagcaccaaa cctacagaac cagatgtgca 900
26/43
CA 02341148 2001-03-O1
WO 00/12711 PCTNS99/20468
gcctcagaag tgcatgggat gtaatcacag actctgctga ctttcaccac agctttccca 960
tgaacgggac tgagcttcca cctcctccca cattctcgct t.gtagaggct ggtgacaaag 1020
tggtctgttt agtgctggat gtgtccagca agatggcaga ggctgacaga ctccttcaac 1080
tacaacaagc cgcagaattt tatttgatgc agattgttga aattcatacc ttcgtgggca 1140
ttgccagttt cgacagcaaa ggagagatca gagcccagct acaccaaatt aacagcaatg 1200
atgatcgaaa gttgctggtt tcatatctgc ccaccactgt atcagctaaa acagacatca 1260
gcatttgttc agggcttaag aaaggatttg aggtggttga aaaactgaat ggaaaagctt 1320
atggctctgt gatgatatta gtgaccagcg gagatgataa gcttcttggc aattgcttac 1380
ccactgtgct cagcagtggt tcaacaattc actccattgc cctgggttca tctgcagccc 1440
caaatctgga ggaattatca cgtcttacag gaggttaaaa gttctttgtt ccagatatat 1500
caaactccaa tagcatgatt gatgctttca gtagaatttc ctctggaact ggagacattt 1560
tccagcaaca tattcagctt gaaagtacag gtgaaaatgt caaacctcac catcaattga 1620
aaaacacagt gactgtggat aatactgtgg gcaacgacac t<~tgtttcta gttacgtggc 1680
aggccagtgg tcctcctgag attatattat ttgatcctga tggacgaaaa tactacacaa 1740
ataattttat caccaatcta acttttcgga cagctagtct ttggattcca ggaacagcta 1800
agcctgggca ctggacttac accctgaaca atacccatca tt:ctctgcaa gccctgaaag 1860
tgacagtgac ctctcgcgcc tccaactcag ctgtgccccc ac~ccactgtg gaagcctttg 1920
tggaaagaga 'cagcctccat tttcctcatc ctgtgatgat tt:atgccaat gtgaaacagg 1980
gattttatcc cattcttaat gccactgtca ctgccacagt tgagccagag actggagatc 2040
ctgttacgct gagactcctt gatgatggag caggtgctga tcrttataaaa aatgatggaa 2100
tttactcgag gtattttttc tcctttgctg caaatggtag at:atagcttg aaagtgcatg 2160
tcaatcactc tcccagcata agcaccccag cccactctat tc:cagggagt catgctatgt 2220
atgtaccagg ttacacagca aacggtaata ttcagatgaa tgctecaagg aaatcagtag 2280
gcagaaatga ggaggagcga aagtggggct ttagccgagt ca,gctcagga ggctcctttt 2340
cagtgctggg agttccagct ggcccccacc ctgatgtgtt tccaccatgc aaaattattg 2400
acctggaagc tgtaaaagta gaagaggaat tgaccctatc ttggacagca cctggagaag 2460
actttgatca gggccaggct acaagctatg aaataagaat gagtaaaagt ctacagaata 2520
tccaagatga ctttaacaat gctattttag taaatacatc aaagcgaaat cctcagcaag 2580
ctggcatcag ggagatattt acgttctcac cccaaatttc cacgaatgga cctgaacatc 2640
agccaaatgg agaaacacat gaaagccaca gaatttatgt tgcaatacga gcaatggata 2700
ggaactcctt acagtctgct gtatctaaca ttgcccaggc gcctctgttt attcccccca 2760
attctgatcc tgtacctgcc agagattatc ttatattgaa aggagtttta acagcaatgg 2820
gtttgatagg aatcatttgc cttattatag ttgtgacaca tc~atacttta agcaggaaaa 2880
agagagcaga caagaaagag aatggaacaa aattattata aataaatatc caaagtgtct 2940
tccttcttag atataagacc catggccttc gactacaaaa acatactaac aaagtcaaat 3000
taacatcaaa actgtattaa aatgcattga gtttttgtac aatacagata agatttttac 3060
atggtagatc aacaaattct ttttgggggt agattagaaa acccttacac tttggctatg 3120
aacaaataat aaaaattatt ctttaaaaaa aaaaaa 3156
<210> 28
<211> 1774
<212> DNA
<213> Homo sapieas
<220>
<221> misc_feature
<223> Incyte ID No: 3148427081
<400> 28
cgctgctata ccgtgcaccc gcgtgctcgt gagggaactc gggctcgctt tggtggggag 60
aaaaatccat gcgctaatac tcggttccca gcttctgcaa aagaaataca aagagtatga 120
gaaagacgtt gccatagaag aacatcgatg gcctccaact ggtaaagaag ctggcaaaga 180
acatggaaga gatgtttcac aagaagtctg aggccgtcag gcgtctggtg gaggctgcag.240
aagaagcaca cctgaaacat gaatttgatg cagacttaca gtatgaatac ttcaatgctg 300
tgctgataaa tgaaagggac aaagacggga attttttgga gct~gggaaag gaattcatct 360
tagccccaaa tgaccatttt aataatttgc ctgtgaacat cagtctaagt gacgtccaag 420
27/3
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99I20468
taccaacgaa catgtacaac aaagaccctg caattgtcaa tc~gggtttat tggtctgaat 480
ctctaaacaa agtttttgta gataactttg accgtgaccc at:ctctcata tggcagtact 540
ttggaagtgc aaagggcttt tttaggcagt atccggggat taaatgggaa ccagatgaga 600
atggagtcat tgccttcgac tgcaggaacc gaaaatggta catccaggca gcaacttctc 660
cgaaagacgt ggtcatttta gttgacgtca gtggcagcat gs~aaggactc cgtctgacta 720
tcgcgaagca aacagtctca tccattttgg atacacttgg gg~atgatgac ttcttcaaca 780
taattgctta taatgaggag cttcactatg tggaaccttg cctgaatgga actttggtgc 840
aagccgacag gacaaacaaa gagcacttca gggagcatct gg~acaaactt ttcgccaaag 900
gaattggaat gttggatata gctctgaatg aggccttcaa ca.ttctgagt gatttcaacc 960
acacgggaca aggaagtatc tgcagtcagg ccatcatgct ca.taactgat ggggcggtgg 1020
acacctatga tacaatcttt gcaaaataca attggccaga tegaaaggtt cgcatcttca 1080
catacctcat tggacgagag gctgcgtttg cagacaatct aaagtggatg gcctgtgcca 1140
acaaaggatt ttttacccaa atctccacct tggctgatgt gcaggagaat gtcatggaat 1200
accttcacgt gcttagccgg cccaaagtca tcgaccagga gcatgatgtg gtgtggaccg 1260
aagcttacat tgacagcact ctccctcagg cacaaaagct gactgatgat cagggccccg 1320
tcctgatgac cactgtagcc atgcctgtgt ttagtaagca gaacgaaacc agatcgaagg 1380
gcattcttct gggagtggtt ggcacagatg tcccagtgaa agaacttctg aagaccatcc 1440
ccaaatacaa gttagggatt cacggttatg cctttgcaat cacaaataat ggatatatcc 1500
tgacgcatcc ggaactcagg ctgetgtacg aagaaggaaa aa~agcgaagg aaacctaact 1560
atagtagcgt tgacctctct gaggtggagt gggaagaccg ag~atgacgtg ttgagaaatg 1620
ctatggtgaa tcgaaagacg gggaagtttt ccatggaggt gaagaagaca gtggacaaag 1680
gggtacattt ttctcaaaca tttttgctgc ttaatttaaa ac~aaaccact gtgaaaaatt 1740
agctttgaaa gctatatctg gaataaatga cttc 1774
<210> 29
<211> 1505
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3342358CB1
<400> 29
cgcggatcct gtgacacctc cgggcagccc ggcacttgtt gct:cccacga cctgttgtca 60
ttcccttaac ccggctttcc ccgtggcccc ccgcctcctc ccg~gcttcgc tccttttcat 120
gtgagcatct gggacactga tctctcagac cccgctgctc ggg~ctggaga atagatggtt 180
ttgtgaaaaa ttaaacaccg ccctgaagag gagccccgct gggcagcggc aggagcgcag 240
agtgctggcc caggtgctgc agaggtggcg cctccccggc ccg~ggacggt agccccgggc 300
gccaacggca tgacagactc ggcgacagct aacggggacg aca.gggaccc cgagatcgag 360
ctctttgtga aggctggaat cgatggagaa agcatcggca actgtccttt ctctcagcgc 420
ctcttcatga tcctctggct gaaaggagtc gtgttcaatg tcaccactgt ggatctgaaa 480
agaaagccag ctgacctgca caacctagcc cccggcacgc acccgccctt cctgaccttc 540
aacggggacg tgaagacaga cgtcaataag atcgaggagt tcctggagga gaccttgacc 600
cctgaaaagt accccaaact ggctgcaaaa caccgggaat ccaacacagc gggcatcgac 660
atcttttcca agttttctgc ctacatcaaa aataccaagc agcagaacaa tgctgctctt 720
gaaagaggcc taaccaaggc tctaaagaaa ttggatgact acctgaacac ccctctacca 780
gaggagattg acgccaacac ttgtggggaa gacaaggggt cccggcgcaa gttcctggat 840
gggrgatgagc tgaccctggc tgactgcaat ctgttgccca agctccatgt ggtcaagatt 900
gtggccaaga aataccgcaa ctatgatatc ccggctgaga tga~aaggcct gtggcggtac 960
ctcaagaacg cctatgcccg tgatgagttc accaacacct gtgcagctga cagtgagatc 1020
gagttggcct acgctgatgt cgccaaacgc ctcagccgat cctgagcaca gccattttgc 1080
cccatccccg ctgcagaagg actcaaccac tcccctaaga ctccagcttc atagactcct 1140
ctgtatcact gccttgaggc gcacttttta taatcaagcc tcatcttgct ggtatcatgg 1200
gaactccagc ctgctatctt tcatgaaggt cagcaccatc ctgggcctcc tcacataggg 1260
atctagcaga aatgatagac acagtccacc tttcggccgg ccacficctgat ctgggrgctca 1320
28/43
CA 02341148 2001-03-O1
WO 00112711 PCT/US99120468
gcatgtttgg gggtcagtca gtgttgggag agcccacata tc~gggattgc cattaggttt 1380
tttttgccat tatcaaaaat tacttttcaa gaggctttag gc~aacatggc aacaacactt 1440
ctttttctaa cctccttttc ggcctatacc acaaggggca gc~ggcaaacg gcattttcat 1500
tcttt 1505
<210> 30
<211> 1478
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1267774CB1
<400> 30
gaaactggag accagaattt tagaaaaaga gattaaggca tctcacttgg tggggtgggg 60
ggtgtctttt tatttttttt ttccttttct ttttaaaaaa aaaacactgc aactggaaca 120
gtttctgatc tcaaaaggca agcctcttcc cgtgtgatct ttataattta cactcttttc 180
cgtgagcttt cttacctccc tttbtttata actctccata ttctctattc atacatatat 240.
ccattatatt agtagtggaa taatttttat ttttatttat tttttttggc tttagtactt 300
gcaccctcac acacactctc ccgagaacca gaagtcggtt gggtgtttat ataatgaaga 360
attatggggc tgtttgatcg aggtgttcaa atgcttttaa cc~accgttgg tgctttcgct 420
gccttcagcc tgatgaccat agctgtggga accgactatt ggctctactc cagaggggtt 480
tgcaagacca aaagtgtcag tgagaatgaa accagcaaaa ag,aacgagga agttatgacc 540
cattccggat tatggagaac ctgctgccta gaagggaatt ca;aaaggtct gtgcaagcaa 600
attgatcact tcccagagga tgcagattac gaagctgaca cagcagaata tttcctccgg 660
gccgtgaggg cctccagcat tttcccaatc ctgagtgtga ttctgctttt catgggtggc 720
ctctgcatcg cagccagcga gttctacaaa actcgacaca acatcatcct gagtgccggc 780
atcttcttcg tgtctgcagg tctgagtaac atcattggca tcatagtgta catatctgcc 840
aatgccggag acccctccaa gagcgactcc aaaaagaata gttactcata cggctggtcc 900
ttctacttcg gggccctgtc cttcatcatc gccgagatgg tcggggtgct ggcggtgcac 960
atgtttatcg accggcacaa acagctgcgg gccacggccc gcc~ccacgga ctacctccag 1020
gcctctgcca tcacccgcat ccccagctac cgctaccgct accagcgccg cagccgctcc 1080
agctcgcgct ccacggagcc ctcacactcc agggacgcct cc<:ccgtggg catcaagggc 1140
ttcaacaccc tgccgtccac ggagatctcc atgtacacgc tcagcaggga ccccctgaag 1200
gccgccacca cgcccaccgc cacctacaac tccgacaggg ataacagctt cctccaggtt 1260
cacaactgta tccagaagga gaacaaggac tctctccact ccaacacagc caaccgccgg 1320
accacccccg tataaagacc gcgggcctcg ccagaagacc gcc~ggaggag ggcgcggtcc 1380
ccgggggcgg ggcggggcgg ggagacccag accctccgct ggc~agacctt ccaaaagcaa 1440
aaacaaaaaa caaaaaaaac aagtatacag gagagaga 1478
<210> 31
<211> 1971
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1817329CB1
<400> 31
cgggcggggg agacctgcca gtcctcccag acttctcccg ggttgctcca gctggccctc 60
ctcgcccctt cccgggagag gcacatggag agacatgaat cag~gggagtg gactggacct 120
gctgaagatc tcatatggaa aaggagccag aaggaaaaac agatttaaag gatctgatgg 180
aagcacgtca tctgatacta cctcaaatag ttttgttcgc caggtaagag ttttaagttc 240
atggttttga taagtacctt aaaatgactt tagattttta aaggtgggtt tcactctttt 300
29/43
CA 02341148 2001-03-O1
WO OU112711 PCT/US99/20468
ccttaagatt tatgtagtat gattgtactt acttttaatt ga;agtgaaga cggtgctgtt 360
taatgtcata attaaaatat tttaatctta agtgagacta agtaataatt acactctttc 420
ctggcaagtt gaggaaagag aagtgtggca ttcattacag aggatttctt tgaaacagtg 480
agagaactcc agcagaaatg attatggatt tggggggatt gtitttttttt ttttttttca 540
gtgggacaga gagctgatgt gcatct:gtat ccctacctgt gacfiatactgg ggttcttcta 600
gttgagcttt cttcttctca cttgggcttg cattgaaaac ta<3aaaatca ttcttggtct 660
tgaaatggtt gaggctatcg aggttaaaca gaacatgcag tatcactgag acataattta 720
aaccatttct gcctttggag acatagcctt cgtctcattt aatgtgtagt atcttcctgc 780
caagcagcct ggaacagtcc cctaaacaga cagtgccagg ctccctaaca tataaacacg 840
attatatatg gtaacagaaa ggaattccat taccagagga agagcataga ggcagctgga 900
cctctgaggg ttgtggttat gtgttgagaa tgaagtctca tce~ggaatga gtgagcttgt 960
ttcttcctct aacccctctc catggggtgg aaagtagggg caclagcatgc agcagagaat 1020
ctgttcttgt ggcccaggga tgtccagtgt ctgatcagta ctca catcat ctcttgtcaa 1080
tgacagctca ctctacggaa cagtggtagt caactggaaa agcagcctcg tgtcatttgg 1140
atgccacttt ccccagtgcc gcttgagtcc atcatcatgg ccatagattc cacccctgcc 1200
ccctgtttgt gtggcaggac tgtttcctat aaatcctata tgaccttttg ggttttattt 1260
ttattgggga cagttacatt tccctagctg tctaccctta ttc~gctcctt gtggcactcc 1320
ccaagtgcct ccctcatgtt tccttcccac agttagctgc agtagaactt gaacttgtcc 1380
acctgcagca tcggtgggga ttttgatctt ggctggttgc tgca tctttg cactgtcctt 1440
agacgaagga tgatctgtcc ccagccatca gtccccgctt gcaacatttg agtatgccag 1500
tggtacttcc aagtaacttg gcaactggaa aaaaactggt cct:ggtccgt gccaattggg 1560
aattgttgtg gttgcaggaa gtgagaaaaa gagttactta aga~aggggca aagctttttt 1620
ggttagaaac atttagaaag aaaaaagatg aagccacgtg aggctcaacc ctagagctat 1680
gttctaatgg tgtcagcagg agctaggaag gctgtcagag gagtggaagg tgtatgcgtt 1740
ggcctcatgt tttcgtgggc agaatccagg catcatggtc ctgrctgatga agggagggcc 1800
ttgggacata gtgacttggg agagtcagtt aaaggaaagt tac~ccatcgc cttttccatt 1860
tcaagctatt tattctgcct ctgagacaaa gagattgaac ctg~gagagct agagttagaa 1920
tctacaactc tgagtcatca tgcaggacct gaaacagacc aaa.aaaaaaa a 1971
<210> 32
<211> 1424
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3273307CB1
<400> 32
cccttctcat ttgtcccatc catttgaaat acagtgtact taaatttcca acaaaatcat 60
aacgtagact ctgtcagtta attaaagtat actaacaagc atacaaattg ctaagtcatt 120
ctgggaggct atcagtgctg gatgtactta tacattcagc act~ggaaaca tacccttcca 180
gtagtgcaca gccagaaata ccaagcaggc ttgcaaagga caa~gattaac cttggcctca 240
tggtattttg tctttctgca gggaaaggaa gatcatggag aca,acaattg caatacacgg 300
tggagaactc aacacccctt gattaaagaa aaaaaatagt tga~ataaaaa agaagtttcc 360
tgaaggagta agatagaatt taccacagaa agaaagtggt agtatctccc tatttcaatt 420
taagtgttgt tcccaggcag gtacaagtac ttctaaaaag gcatagatta aagaaaaatg 480
gtatttggag tcatggttac taaatgaagc tacagctcac catgatcctt gggatgaaga 540
ctatgacccc cagcaaagtt acaaaggatt ctgctctgga atttatcaac tgctttgttt 600
gttctcttaa caggtaaaaa atgcagctgc caatgtactc agggaaacat ggctaattta 660
caaaaataca aagctagtga aaaagataga tcatgcaaaa gta~agaaaac atcaacgaaa 720
attcctgcaa gctattcatc aattaagaag tgtaaaaatg gagcagagga aactgaatga 780
ccaagcaaac actttggtgg acttggcaaa gacccagaac atcatgtatg atatgatttc 840
tgacttaaac gaaaggagtg aagacttcga gaagaggatt gttaccctgg aaacaaaact 900
agagactttg attggtagca tccacgccct ccctgggctc ata<~gccaga ccatcaggca 960
gcagcagaga gatttcattg aggctcagat ggagagctac gacaagcacg tcacttacaa 1020
30/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/z0468
tgctgagcgg tcccggtcct cgtccaggag gcggcggtcc tcttccacag caccaccaac 1080
ttcatcagag agtagctaga agagaataag ttaaccacaa aataagactt tttgccatca 1140
tatggtcaat attttagctt ttattgtaaa gcccctatgg ttctaatcag cgttatccgg 1200
gttctgatgt cagaatcctg ggaacctgaa cactaagttt t,aggccaaaa tgagtgaaaa 1260
ctcttttttt ttctttcaga tgcacaggga atgcacctat t;attgctata tagattgttc 1320
ctcctgtaat ttcactaact ttttattcat gcacttcaaa caaactttac tactacatta 1380
tatgatatat aataaaaaaa gttaatttct gcacaaaaaa aaaa 1424
<210> 33
<211> 1224
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3824833CB1
<400> 33
cggccatatt agagagatgg aaataaagct tccttaatgt tc~tatatgtc tttgaagtac 60
atccgtgcat ttttttttag catccaacca ttcctccctt gt:agttctcg ccccctcaaa 120
tcaccctctc ccgtagccca cccgactaac atctcagtct ct:gaaaatgc acagagatgc 180
ctggctacct cgccctgcct tcagcctcac ggggctcagt ctctttttct ctttggtgcc 240
accaggacgg agcatggagg tcacagtacc tgccaccctc aa.cgtcctca atggctctga 300
cgcccgcctg ccctgcacct tcaactcctg ctacacagtg aa.ccacaaac agttctccct 360
gaactggact taccaggagt gcaacaactg ctctgaggag atgttcctcc agttccgcat 420
gaagatcatt aacctgaagc tggagcggtt tcaagaccgc gtggagttct cagggaaccc 480
cagcaagtac gatgtgtcgg tgatgctgag aaacgtgcag ccggaggatg aggggattta 540
caactgctac atcatgaacc cccctgaccg ccaccgtggt catggcaaga tccatctgca 600
ggtcctcatg gaagagcccc ctgagcggga ctccacggtg gccgtgattg tgggtgcctc 660
cgtcgggggc ttcctggctg tggtcatctt ggtgctgatg gtggtcaagt gtgtgaggag 720
aaaaaaagag cagaagctga gcacagatga cctgaagacc gaggaggagg gcaagacgga 780
cggtgaaggc aacccggatg atggcgccaa gtagtgggtg gccggcctgc agcctcctct 840
aggggttgca cccagcgctc cctcaggagg gccttggcct ggcacggctg tgctcctccc 900
ctgctcccag cccagagcag ccatcaggct ggaggtgacg at~gagttcct gaaacttgga 960
ggggcatgtt aaagggatga ctgtgcattc cagggcactg acggaaagcc agggctgcag 1020
gcaaagctgg acatgtgccc tggcccagga ggccatgttg gg~ccctcgtt tccattgcta 1080
gtggcctcct tggggctcct gttggctcct aatcccttag ga~~tgtggat gaggccagac 1140
tggaagagca gctccaggta gggggccatg tttcccagcg ggc~acccacc aacagaggcc 1200
agtttcaaag tcagctgagg gget 1224
<210> 34
<211> 1300
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2069907CB1
<400> 34
gctgtttacc ttgcatggtt gactgctctc ttctcacatt gtc~tgccagg aagggcactg 60
caccttggta taaatgctgc tgggcaccgt tctgttttct ttcatttctt aatcctatcc 120
aagtatgcag tacgctcttg ggtcgtctca tgagacccag gggcatgttg gaaagaactg 180
agagaaagag caacaaagcg gcgagtggtg tgagagggca gca,cgcgctg tggggccctt 240
ccagagaaat gtactgaaaa agtctacgca atgtctggga ttt.gctaaac aatacctgga 300
aagcagacag gtctttttgc cattcctcca ggacatccac cat.aaggaaa ggagaccctg 360
31/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99120468
gaccaacatt ctctaagatg tttatatgga ccagtggccg gacctcttca tcttatagac 420
atgatgaaaa aagaaatatt taccagaaaa tcagggacca tgacctcctg gacaaaagga 480
aaacagtcac agcactgaag gcaggagagg accgagctat tcacctggga ctggctatga 540
tggtgtgctc catcatgatg tattttctgc tgggaatcac acacctgcgc tcatacatgc 600
agagcgtgtg gaccgaagag tctcaatgca ccttgctgaa tc~cgtccatc acggaaacat 660
ttaactgctc cttcagctgt ggtccagact gctggaaact tt;ctcagtac ccctgccccc 720
aggtgtacgt taacctgact tcttccgggg aaaagctcct cctctaccac acagaagaga 780
caataaaaat caatcagaag tgctcctata tacctaaatg tc~gaaaaaat tttgaagaat 840
ccatgtccct ggtgaatgtt gtcatggaaa acttcaggaa gt:atcaacac ttctcctgct 900
attctgaccc agaaggaaac cagaagagtg ttatcctaac ca~aactctac agttccaacg 960
tgctgttcca ttcactcttc tggccaacct gtatgatggc tg~ggggtgt:g gcaattgttg 1020
ccatggtgaa acttacacag tacctctccc tactatgtga ga.ggatccaa cggatcaata 1080
gataaatgca aaaatggata aaataatttt tgttaaagct ca.aatactgt tttctttcat 1140
tcttcaccaa agaaccttaa gtttgtaacg tgcagtctgt tatgagttcc ctaatatatt 1200
cttatatgta gagcaataat gcaaaagctg ttctatatgc aaacatgatg tctttattat 1260
tcaggagaat aaataactgt tttgtgttaa aaaaaaaaaa 1300
<210> 35
<211> 1060
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No; 2243917CB1
<400> 35
gcttgctggg atcatggcgg agaatcactg cgagctcctg tcgecggccc ggggcggcat 60
cggggcgggg ctggggggcg gcctgtgccg ccgctgcagc gctgggctcg gcgccctggc 120
ccagcgccct ggcagcgtgt ccaagtgggt ccgactcaac gtc:ggcggca cctacttcct 180
caccactcgg cagaccctgt gccgggaccc gaaatccttc ctdtaccgct tatgccaggc 240
cgatcccgac ctggactcag acaaggatga aacaggcgcc tatataatcg acagagaccc 300
cacctacttt gggcctgtgc tgaactacct gagacacggc aac~ctggtga ttaacaaaga 360
cctcgcggag gaaggagtgt tggaggaagc agaattttac aat:atcacct cattaataaa 420
acttgtaaag gacaaaatta gagaacgaga cagcaaaaca tcc~caggtgc ctgtgaagca 480
tgtgtaccgt gtgctgcagt gccaggagga ggagctcacg cac~atggtgt ccaccatgtc 540
cgacggctgg aagttcgagc agttggtcag catcggctcc tctaacaact atgggaacga 600
agaccaagcc gagttcctct gtgtggtgtc caaggagctg cac:aacaccc cgtacggtac 660
ggccagcgag cccagcgaga aggccaagat tttgcaagaa cga~ggctcaa ggatgtgagg 720
gacacagtat tgacagctga agaaatgatt tacgttttcc cga;gatgtaa tgaactgcca 780
tgtccaggaa gcttggctgt gagaagaaac ctgcttttga tca.tttttct agagatctgg 840
gtgtgaatcc ttttttgcct ctgaggtggg tggtgagaga cgg~gcccagc tgtccaaggc 900
cagacgtccc caagttgggg gagcacggcg gccgggtggg cgctgcctct tgggggggcc 960
tcgctctgtt ttttccaagt gccacgtggg actgaggcag acactcccag tcagcccgct 1020
cgatcctgaa gatcgtgtga aggaagcgtt cttggtgcta 1060
<210> 36
<211> 1815
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2597476C'B1
<400> 36
32/43
CA 02341148 2001-03-O1
WO ObII2711 PCT/US99I20468
gtgcctatgc agacagaggg agcagtgaat agcaataggg tgtttccacc atggtcttca 60
ctcaggcccc ggctgaaatc atgggccacc tccggatacg cagcctcctg gcccggcagt 120
gcctggcaga gtttctgggt gtgtttgtac tcatgctcct cacccaagga gctgtggccc 3.80
aggctgtcac cagtggagaa accaaaggca acttcttcac catgtttctg gctggctctc 240
tggccgttac gatagccatc tacgtgggtg gtaacgtctc a<~gggcccac ctgaatccag 300
ccttctccct ggccatgtgc atcgttggac gcctcccctg g<fitcaagctc cccatttaca 360
tcttggtgca gttgctgtct gctttctgtg cttcgggagc cacctatgtt ctctaccatg 420
atgccctaca gaactataca ggtgggaacc tgacagtgac t<;gccccaag gagacagcct 480
ccatttttgc cacctatcct gccccctatc tgtccctgaa caatggcttc ctggatcagg 540
ttctgggcac tgggatgctg attgtggggc tcttggccat ccaggacaga cggaacaagg 600
gagtccctgc gggtctggag cctgtggtgg tggggatgct gatcctggcc ctcgggttat 660
ccatgggtgc caactgcggg attccactca accctgcccg gc~acctgggc ccacgtctct 720
tcacctacgt ggctggctgg ggtcctgaag tcttcagtgc tc~gtaatggc tggtggtggg 780
tgcctgtggt ggcccctctg gtgggggcca ccgttggcac acfccacttac cagctgttgg 840
tggctctgca ccaccctgag ggcccagagc cagctcagga tcaggtgtct gctcaacaca 900
aagcctcaga gttggaaact cctgcctcag ctcagatgct gc~agtgtaag ctatgattag 960
gacaaccctc acttcactca tggaccctgg agccagccac ta~accccgcc tgggaacaac 1020
agtcattctt cctctttgtt aatgtgccag aacctgggag gcttctctgt ttatctgttt 1080
ggcatccctt cctcctaaac taagaaggat cctggacagg ga.gaagtgga ggaggataag 1140
gtaccaggac tcaggcttct catcccctcc tcccgcaaag cg~gttttctg accctcaggg 1200
cctctcggaa tgtagttgct cgaggtaacc gctagagggt gcgcacctgg atgctggatg 1260
gggacggctg cgggcatctg cagggtggag ggggccacca tccagtgtag ggcacaaccc 1320
tggggactgc cctccatagc ctgtcccgac tgccgactcc tagctctcat cgcctcggcg 1380
cctcccacct tcaccctctc ggggatgcct ccccaagagg gtagttaggg gtggggaagc 1440
cgcctccacc cagggggcgt ggtgggggcg gagggaagga gggcggcggg gcacagagac 1500
agagagcaag gctgtgaaac tgaggcaccg ttcctagaca tctcggtgct gtgtcgttca 1560
ttcaaggaga gttgagatac agtgaaatga gccagggcga ggagggaggg tgaaggaacg 1620
gagggcgggc ggctccgagg agcgagagtc gggctgaggg caacctggcg ccagggaaaa 1680
ttctggttat tcaccacttc tacagctctc ctgccgctcc cbgcagagga tgctcgtttt 1740
gcagagaagg cagtgttcct ctattccctt cttccgaatt aa~aaataccc cctcagagcg 1800
aaaaaaaaaa aaaaa 1815
<210> 37
<211> 315
<212> PRT
<213> Rattus norvegicus
<220>
<221> misc_feature
<223> GenBank ID No: 82924369
<400> 37
Met Leu Gly Trp Val Gln Arg Val Leu Pro Gln Pro Pro Gly Thr
1 5 10 15
Pro Gln Lys Thr Glu GIu GIy Ala Gly Pro Gln Pro Glu Thr Glu
20 25 30
Ser.Lys Pro GIu Ala Asn Pro Gln Pro Glu Pro Glu Val Gln Pro
35 40 45
Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro G1u Pro Ala
50 55 60
Pro Glu Glu Ala Ala Pro Glu Val Gln Thr Leu Pro Pro Glu Glu
65 70 75
Pro Val Glu GIy Glu Asp Val Ala Glu Ala Gly Pro~ Ser Leu Gln
80 85 90
Glu Thr Gln Glu Ala Asp Pro Pro Gln Pro Thr Ser Gln Ala Gln
95 100 105
33143
CA 02341148 2001-03-O1
WO 00/1271 Z PCT/IJS99/20468
Val Ala Val Val Lys Val Asn Arg Pro Ser Ser Trp Met Leu Ser
I10 115 120
Trp Phe Trp Lys Giy Met Glu Lys Val Val Pro Gln Pro Val Tyr
125 130 135
Ser Ser Ser Gly Gly Gln Asn Leu Ala Ala Gly G.lu Gly Gly Pro
140 . 145 150
Asp Gln Asp Gly Ala Gln Thr Leu Glu Pro Cys G:ly Thr Gly Asp
155 160 165
Pro Gly Ser Giu Asp Gly Ser Asp Lys Thr Ser L~~rs Thr Gin Asp
170 175 180
Thr Glu Pro Ser Leu Trp Leu Leu Arg Trp Leu Glu Leu Asn Leu
185 190 195
Glu Lys Val Leu Pro Gln Pro Pro Thr Pro Ser G:Ln Ala Trp Lys
200 205 210
Val Glu Pro Glu Gly Ala Val Leu Glu~Pro Asp Pro Pro Gly Thr
215 220 225
Pro Met Glu Val Glu Pro Thr Glu Asn Pro Ser Gl.n Pro Asn Pro
230 235 240
Gly Pro Val Glu Pro Glu Glu Glu Pro Ala Ala Gl.u Pro Gln Pro
245 250 255
Gly Phe Gln.Ala Ser Ser Leu Pro Pro Pro Gly A~~p Pro Val Arg
260 265 270
Leu Ile Glu Trp Leu Leu His Arg Leu Glu Met Ai.a Leu Pro Gln
275 280 285
Pro Val Leu His Gly Lys Ala Ala Glu Gln Glu Pro Ser Cys Pro
290 295 300
Gly Thr Cys Asp Val Gin Thr Arg Ala Thr Ala Ala Giy Giy Leu
305 3I0 315
<210> 38
<211> 490
<212> PRT
<213> Drosophila melanogaster
<220>
<221> misc_feature
<223> GenBank ID No: g116443
<400> 38
Met Ala Ser Ala Trp Leu Pro Phe Ala Ala
Val Ala Arch p~.7.a Aia
~
1 5 10 15
Ile Gly Trp Pro Ala Thr His Pro Leu Pro
Val Ile Pro Pro Pro
20 25 30
Met Pro Lys Arg Lys Thr Asp Asp Glu Leu
Asp Arg Lys Leu Ile
35 40 45
Asn Val Ser Arg Phe Glu Thr Txp Arg Leu
Gly Arg Asn Thr Glu
50 55 60
Lys Tyr Pro Thr Leu Gly Ser Asn Giu Phe
Asp Leu Arch Glu Phe
65 70 75
Tyr Asp Glu Cys Glu Tyr Phe Phe Asp Pro
Asp Lys Arch Asp Asp
BO 85 90
Iie Phe Arg Ile Asn Tyr Tyr Arg Thr Leu
His Leu Gly Lys His
95 100 205
Tyr Pro Lys Giu Leu Thr Ser Tyr Asp Leu
His Cys Glu Glu Ala
110 115 120
34/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/204b8
Phe Phe Gly Ile Met Pro Asp Cys Tyr
Val Ile G1y Asp Cya Glu
125 130 135
Asp Tyr Arg Asp Arg Lys Arg Leu Met
Glu Asn Ala Glu Ar<1 Asp
140 145 150
Asp Lys Leu Ser Glu Asn Gly Gln Leu
Asp Gln Asn Leu Gln Thr
155 160 165
Asn Met Arg Gln Lys Met Trp Pro His
Arg Ala Phe Glu Asn Thr
170 175 180
Ser Thr Ser Ala Leu Val Phe Phe Phe
Tyr Tyr Val Thr Gly Ile
185 190 195
Ala Val Ser Val Met Ala Asn Glu Thr Pro Cys
Val Val VaI. Gly
200 205 210
His Arg Pro Gly Arg Ala Gly Glu Arg
Thr Leu Pro Cys Gly Tyr
215 220 225
Lys Ile Val Phe Phe Cys Leu Ala Cys Met Ile
Asp Thr Val Phe
230 235 240
Thr Ala Glu Tyr Leu Leu Arg Ala Ala Asp Arg
Leu Phe Pro Cys
245 250 255
Lys Phe Val Arg Ser VaI Met Ile Asp Val Ala
Ser Ile Val Ile
260 265 270
Met Pro Tyr Tyr Ile Gly Leu Thr Asp Asp Asp
Gly Ile Asn Val
275 280 285
Ser Gly Ala Phe Val Thr Leu Phe Arg Phe Arg
Arg Val Val Ile
290 295 300
Phe Lys Phe Ser Arg His Ser Leu Arg Leu Gly
Gln Gly Ile Tyr
305 310 315
Thr Leu Lys Ser Cys Ala Ser Gly Phe Val Phe
Glu Leu Leu Ser
320 325 330
Leu Ala Met AIa Ile Ile Ile Thr Val Phe Tyr
Phe Ala Met Ala
335 340 345
Glu Lys Asn VaI Asn Gly Thr Thr Ser Pro Ala
Asn Phe Ile Ala
350 355 360
Phe Trp Tyr Thr Ile Val Thr Thr Leu Tyr Gly
Met Thr Gly Asp
365 370 375
Met Val Pro Glu Thr Ile Ala Ile Val Gly Val
Gly Lys Gly Cys
380 385 390
Ser Leu Ser Gly Val Leu Val Leu Pro Pro Val
Ile Ala Val Ile
395 400 405
Val Ser Asn Phe Ser Arg Ile Gln Asn
Tyr His Gln Arg
Ala Asp
410 415 420
Lys Arg Lys Ala Gln Arg Lys Leu Ala Ile Arg
Ala Arg Arg Ile
425 430 435
AIa Lys Ala Ser Ser Gly Ala Val Ser
Ala Phe Lys Lys
Lys Ala
440 445 450
Ala Glu Ala Arg Trp Ala Ala Ser Gly
Gln Glu Ile Glu
Leu Asp
455 460 46S
Asp Asn Tyr Arg Asp Glu Asp Glu Leu
Ile Phe Gln His
His His
470 475 480
Leu Leu Arg Cys Leu Glu Lys
Thr Thr Met
485 490
<210> 39
<211> 478
<212> PRT
<213> Polyorchis penicillatus
35/43
CA 02341148 2001-03-O1
WO 001127I1 PCT/US99120468
<220>
<221> misc_feature
<223> GenBank ID No: g1763619
<400> 39
Met Asn Gly Asp Ile
Gly Ala Trp Ile Ser
Cys Ala Arg Thr Ala
1 5 10 15
Gly Ile Gly Trp Val Glu Pro Ser Ala
Pro Ile Ser Ser Lys Tyr
20 25 30
Leu Asn Lys Gln Val
Cys Asn Glu Asn GIu
Lys Asn Asn Ala Lys
35 40 45
Leu Thr Ile Asn Val Gln Th:r Tyr Ser
Ser Gly Arg Arg Tyr His
50 55 60
Thr Leu Arg Lys Phe Gly Ser Gln Glu
Lys Glu Thr Leu Leu Arg
65 70 75
Asp Tyr Phe Tyr Asp Leu Glu Tyr Ty~.~ Phe
Glu Ser Glu Asp Arg
80 85 90
Asp Pro Asp Leu Phe Ile Leu Tyr Tyr Arg Thr
Arg His Asn Gly
95 100 105
Lys Leu His Phe Pro Glu Cys Ser Ser Phe Glu
Lys Asn Val Asp
110 115 120
Glu Leu Thr Phe Phe Lys Gly Asn Ile: Asn Asn
Gly Ile Phe Cys
125 130 135
Cys Trp Asp Asp Tyr Lys Lys Glu Cye~ Thr Glu
His Asp Arg Arg
140 145 150
Leu Asn Glu Ser Asp Leu Thr Ser Glu Ile Asn
Val Met Ser Glu
155 160 165
Lys Ser Asp Thr Met Asp Val Met Asn Asn His
Gly Ile Gln Gln
170 175 180
Ala Lys Asn Phe Arg Val His Leu Phe. Glu Asn
Gln Lys Gly Pro
185 ~ 190 195
Gln Ser Thr Phe Leu Ile Leu Tyr Ile Thr Gly
Ala Arg Tyr Phe
200 205 210
Phe Ile Ala Val Ser Ser Thr Ile Glu Thr Ile
Val Gly Ile Asp
215 220 225
Cys Ser Ala Asn Arg Gly Glu Tyr Asn Lys Ile
Pro Cys Val Phe
230 235 240
Phe Asn Ile Glu Ala Val Val Phe Thr Ile Glu
Val Cys Val Tyr
245 250 255
Leu Ala Arg Leu Tyr Pro Cys Phe Arg His Ala
Ser Ala Arg Arg
260 265 270
Ile Ser Leu Ser Ile Val Ile Ile Leu Pro Phe
Ile Asp Ala Tyr
275 280 285
Ile Gly Leu Ala Met Thr Ser Ser Gly Ala Phe
Thr Lys Ile Val
290 295 300
Ser Leu Arg Val Phe Phe Arg Phe Lys Phe Ser
Arg Val Ile Arg
305 310 315
His Ser Lys Gly Leu Leu Giy Thr Leu Thr Ser
Arg Ile Ser Cys
320 325 330
Ala Ser Glu Leu Gly Leu Phe Leu Ser Met Ala
Phe Leu Ser Ile
335 340 345
Ile Ile Phe Ala Thr Phe Tyr
Val Val Val Glu
Lys Asp
Val Asn
350 355 360
Asp Ser Asp Phe Thr Pro Ala
Ser Ile Ser Phe
Trp Tyr
Thr Ile
365 370 375
Val Thr Met Thr Thr
Leu Gly Tyr Gly Asp
Met Val Pro Lys Thr
36/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99120468
380 385 390
Ile Pro Gly Lys Leu Va1 Giy Ser Ile Cys Ser Le:u Ser Gly Val
395 400 405
Leu Val Ile.Ala Leu Pro Val Pro Val Ile Val Ser Asn Phe Ser
410 415 420
Arg Iie Tyr Leu GIn Asn Gln Arg Ala Asp Lys Arg Arg Ala Asn
425 430 435
Gln Lys Leu Arg Asn Lys Cys Glu Glu Lys Giu Glu Lys Lys Lys
440 445 450
Glu Ser Ser Ser Glu Thr Val Thr Arg Phe Ile Ile Ser Asn Gln
455 460 465
Met Tyr Thr Ile Phe Ser Met Lys Phe Ala Leu Thr Arg
470 475
<210> 40
<211> 732
<212> PRT
<213> Rattus norvegicus
<220>
<221> misc_feature
<223> GenBank ID No: 82564072
<400> 40
Met Asp Thr Ser Gly Phe His G1u Ser Gly Gly Asp
His Va:L Leu
1 5 10 15
Asp Glu Asp Pro Lys Pro Cys Pro Ser Ser Asp Glu
Cys Gl;l Gln
20 25 30
Gln Gln Gln Gln Gln Pro Pro Pro Ser Ala Pro Ala
Pro Pro Val
35 40 45
Pro Gln Gln Pro Pro Pro Leu Leu Gln Pro Pro Pro
Gly Gln Gln
50 55 60
Leu Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
Gln Gln Gln
65 70 75
Gln Gln Gln Gln Gln Pro Leu His Pro Leu Gln Leu
Ala Pro Ala
80 85 90
Gln Leu Gln Ser Gln Val His Pro Gly Leu His Ser
Val Leu Ser
100 105
Pro Thr Ala Phe Arg Pro Asn Ser Ala Asn Thr Ala
Ala Sex' Ile
110 115 120
Leu His Pro Ser Ser Gln GIy Ser Gln Leu Leu Asn
Arg Asn Asp
125 130 135
His Leu Val Gly His Pro Ser Ser Thr Ala Ser Gly
Ser Thr Pro
140 145 3.50
Gly Gly Gly Ser Arg Arg Gln Ala Ser Pro Val His
His Val Arg
I55 160 165
Arg Asp Ser Asn Pro Thr Glu Ile Ala Met Ser Cys
Phe Ser Lys
170 175 180
Tyr Ser Gly Gly Val Lys Pro Leu Asn Arg Ser Ala
Met Leu Ser
185 190 195
Arg Arg Asn Leu Ile Ala Glu Pro Glu Gly Pro Leu
Glu Gln Gln
200 205 210
Leu Phe Ser Pro Ser Pro Pro Glu Ile ile Ser Ser
Asn Ile Arg
215 220 225
Glu Asp Asn His Ala Gln Thr Leu Leu His Pro Asn
His His Ala
37/43
CA 02341148 2001-03-O1
WO 00112711 PCT/US99/20468
230 235 240
Thr His Asn His.Gln His Ala Gly Thr Thr Ala G:Ly Ser Thr Thr
245 250 255
Phe Pro Lys.Ala Asn Lys Arg Lys Asn Gln Asn I7Le Gly Tyr Lys
260 265 270
Leu Gly His Arg Arg Ala Leu Phe Glu Lys Arg Lys Arg Leu Ser
275 280 285
Asp Tyr Ala Leu Ile Phe Gly Met Phe Gly Ile Val Val Met Val
290 295 300
Ile Glu Thr Glu Leu Ser Trp Gly Leu Tyr Ser Lys Asp Ser Met
305 310 315
Phe Ser Leu Ala Leu Lys Cys Leu Ile Ser Leu Se:r Thr Ile Ile
320 325 330
Leu Leu Gly Leu Ile Ile Ala Tyr His Thr Arg Glu Val Gln Leu
335 340 345
Phe Val Ile Asp Asn Gly Ala Asp Asp Trp Arg Ile Ala Met Thr
350 355 360
Tyr Glu Arg Ile Leu Tyr Ile Ser Leu Glu Met Leu Val Cys Ala
365 370 375
Ile His Pro Ile Pro Gly Glu Tyr Lys Phe Phe Tr;p Thr Ala Arg
380 385 390
Leu Ala Phe Ser Tyr Thr Pro Ser Arg Ala Glu Aha Asp Val Asp
395 400 405
Ile Ile Leu Ser Ile Pro Met Phe Leu Arg Leu Ty:r Leu Ile Ala
410 415 420
Arg Val Met Leu Leu His Ser Lys Leu Phe Thr Asp Ala Ser Ser
425 430 435
Arg Ser Ile Gly Ala Leu Asn Lys Ile Asn Phe Asn Thr Arg Phe
440 445 450
Val Met Lys Thr Leu Met Thr Ile Cys Pro Gly Thr Val Leu Leu
455 460 465
Val Phe Ser Ile Ser Leu Trp Ile Ile Ala Ala Trl> Thr Val Arg
470 475 480
Val Cys.Glu Arg Tyr His Asp Gln Gln Asp Val Thr Sex Asn Phe
485 490 495
Leu Gly Ala Met Trp Leu Ile Ser IIe Thr Phe Leu Ser Ile Gly
500 505 51p
Tyr Gly Asp Met Val Pro His Thr Tyr Cys Gly Lys~ Gly Val Cys
515 520 525
Leu Leu Thr Gly Tle Met Gly A1a Gly Cys Thr Ala, Leu Val Val
530 535 540
Ala Val Val Ala Arg Lys Leu Glu Leu Thr Lys Ala. Glu Lys His
545 550 555
Val His Asn Phe Met Met Asp Thr Gln Leu Thr Lys Arg Ile Lys
560 565 570
Asn Ala Ala Ala Asn VaI Leu Arg Glu Thr Trp Leu Ile Tyr Lys
575 580 585
His Thr Lys Leu Leu Lyst Lys Ile Asp His Ala Lys Val Arg Lys
590 595 600
His Gln Arg Lys Phe Leu Gln Ala Ile His Gln Leu Arg Gly Val
605 6I0 615
Lys Met Glu Gln Arg Lys Leu Ser Asp Gln Ala Asn Thr Leu Val
620 625 630
Asp Leu Ser Lys Met Gln Asn Val Met Tyr Asp Leu Ile Thr Glu
635 640 645
Leu Asn Asp Arg Ser Glu Asp Leu Glu Lys Gln Ile Gly Ser Leu
38/43
CA 02341148 2001-03-O1
WO OO/i2713 PCT/US99/20468
650 655 660
Glu Ser Lys Leu Glu His Leu Ser A:an Leu
Thr Ala Phe Ser Pro
665 670 675
Leu Leu Ile Ala Asp Thr Leu Gln GAn Leu
Arg Gln Gln Gln Leu
680 685 690
Thr Ala Phe Val GIu Ala Arg Ser A7.a Gly
Gly Ile VaI Val Thr
695 700 705
Ser His Ala Pro Pro Ser Asp Ile Il.e Ser
Ser Pro Gly Ser Thr
710 715 720
Ser Phe Pro Thr Pro Tyr Thr Ser Cys
Ser Ser Ser
725 730
<210> 41
<211> 269
<212> PRT
<213> Rattus norvegicus
<220>
<221> misc_feature
<223> GenHank ID No: 82350843
<400> 41
Met Ala Gly Ser Val Leu Glu Asn Ile Gln Ser Va:1 Leu G1n Lys
1 5 20 15
Thr Trp Val Arg Glu Phe Leu Ala Glu Phe Leu Asn Thr Tyr Val
20 25 30
Leu Met Val Phe Gly Leu GIy Ser Val Ala His Met: Val Leu Gly
35 40 45
Glu Arg Leu Gly Sex Tyr Leu Gly Val Asn Leu Gly Phe Gly Phe
50 55 60
Gly VaI Thr Met Gly Ile His Val Ala Gly Gly Ile; Ser Gly Ala
65 70 75
His Met Asn Pro Ala Val Thr Phe Thr Asn Cys Alai Leu Gly Arg
80 85 90
Met Ala Gly Arg Lys Phe Pro Ile Tyr Val Leu Gly Gln Phe Leu
95 100 105
Gly Ser Phe Leu Ala Ala Ala Thr Thr Tyr Leu Ile Phe Tyr Gly
110 115 120
Ala IIe Asn His Tyr Ala Gly Glu Thr Leu Leu Val Thr Gly Pro
125 130 135
Lys Ser Thr Ala Asn Ile Phe Ala Thr Tyr Leu Pro Glu His Met
140 145 150
Thr Leu Tzp Arg Gly Phe Val Asp Glu Val Phe Val Thr Gly Met'
15S 160 165
Leu GIn Leu Cys Ile Phe Ala Ile Thr Asp Lys Leu Asn Ser Pro
1?0 I75 180
Ala Leu Gln Gly Thr Glu Pro Leu Met Ile Gly Ile Leu Val Cys
185 190 195
Val Leu Gly VaI Ser Leu Gly Met Asn Thr Gly Tyr Ala Ile Asn
200 205 210
Pro Ser Arg Asp Leu Pro Pro Arg Phe Phe Thr Phe Ile Ala Gly
215 220 225
Trp Gly Lys Lys Val Phe Ser Ala Gly Asn Asn Trp Trp Trp Val
230 235 240
Pro Val Val AIa Pro Leu Leu Gly Ala Tyr Leu Gly Gly Ile Val
39/43
CA 02341148 2001-03-O1
WO 00/12711 PCTIUS99/20468
245 250 255
Tyr Leu Gly Leu Ile His Ala Gly Ile Pro Pro Gln Gly Ser
260 265
<210> 42
<211> 266
<212> P12T
<213> Mus musculus
<220>
<221> misc_feature
<223> GenBank ID No: 8192647
<400> 42
Met Asn Trp Gly Phe Leu Ile Leu Gly Val Lys
Gln Gly Ser Asn
1 5 10 15
Tyr Ser Thr Ala Leu Gly Trp Leu Ile
Arg Ile Ser Val
Val Phe
20 25 30
Phe Arg Val Leu Val Tyr Ala Ala GIu Val Asp
Val Val Glu Trp
35 40 45
Asp Asp Gln Lys Asp Phe Asn Thr Gln Pro Cys
Ile Cys Lys Gly
50 55 60
Pro Asn Vai Cys Tyr Asp Phe Pro Ser His Arg
Glu Phe Val VaI
65 70 75
Leu Trp Ala Leu Gln Leu Val Thr Pro Ser Leu
Ile Leu Cys Leu
g5 90
Val Val Met His Val Ala Glu Glu Glu Arg His
Tyr Arg Arg Lys
95 100 105
Arg Leu Lys His Gly Pro Pro Ala Tyr Ser Leu
Asn Ala Leu Asn
110 115 120
Ser Lys Lys Arg Gly Gly Trp Thr Leu Leu Leu
Leu Trp Tyr Ser
125 130 135
Tle Phe Lys Ala Ala Val Gly Phe Tyr Ile His
Asp Ser Leu Phe
140 145 150
Cys Ile Tyr Lys Asp Tyr Pro Arg Val Ala Ser
Asp Met Val Cys
155 160 165
Val Thr Pro Cys Pro His Asp Cys Ile Ala Pro
Thr Val Tyr Arg
170 175 180
Thr Glu Lys Lys Val Phe Phe Met Val Thr Ala
Thr Tyr Val Ala
1$5 190 195
Ile Cys Ile Leu Leu Asn Glu Va1 Tyr Leu Gly
Leu Ser Val Val
200 205 210
Lys Arg Cys Met Glu Val Pro Arg Arg Lys Ser
Phe Arg Arg Ala
215 220 225
Arg Arg His Gln Leu Pro Cys Pro Tyr Val Ser
Asp Thr Pro Ile
230 235 240
Lys Gly Gly His Pro Gln heu Thr Ala
Asp Glu Ser Val Ile Lys
245 250 255
Gly Met Ala Thr Val Asp
Ala Gly Val Tyr Pro
260 265
<210> 43
<21I> 191
< 212 > PitT
40/43
CA 02341148 2001-03-O1
WO 00/12711 PCT/US99/20468
<213> Homo sapiens
<220>
<221> misc_feature
<223> GenBank ID No: 81055345
<400> 43
Met Val Lys Lys Leu Va1 Met Ala Gln Lys Arg Gl.y Glu Thr Arg
1 5 10 15
Ala Leu Cys Leu Gly Val Thr Met Val Val Cys Ala Val Ile Thr
20 25 30
Tyr Tyr Ile Leu VaI Thr Thr Val Leu Pro Leu Tyr Gln Lys Ser
35 40 45
Val Trp Thr Gln Glu Ser Lys Cys His Leu Ile Glu Thr Asn Ile
50 55 60
Arg Asp Gln Glu Glu Leu Lys Giy Lys Lys Val Pro Gln Tyr Pro
65 70 75
Cys Leu Trp Val Asn Val Ser Ala Ala Gly Arg Trp Ala Val Leu
80 ' 85 90
Tyr His Thr Glu Asp Thr Arg Asp GIn Asn Gln Gln Cys Ser Tyr
95 100 105
Ile Pro Gly Ser Val Asp Asn Tyr Gln Thr Ala Ar<3 Ala Asp Val
110 115 ' 120
Glu Lys Val Arg Ala Lys Phe Gln Glu Gln Gln Va:C Phe Tyr Cys
125 130 135
Phe Ser Ala Pro Arg GIy Asn Glu Thr Ser Val Leu Phe Gln Arg
140 145 150
Leu Tyr Gly Pro Gln Aia Leu Leu Phe Ser Leu Phe: Trp Pro Thr
155 160 165
Phe Leu Leu Thr Gly Gly Leu Leu Ile Ile Ala Met: Val Lys Ser
170 175 180
Asn Gln Tyr Leu Ser Ile Leu Ala Ala Gln Lys
185 190
<210> 44
<211> 308
<212> PRT
<213> Caenorhabditis elegans
<220>
<221> misc_feature
<223> GenBank ID No: 83292929
<400> 44
Met Ser Thr Val Phe Ile Asn Ser Arg Lys Ser Pro Asn Val Leu
1 5 10 15
Lys Lys Gln Gly Thr Asp Gln Trp Val Lys Leu Asn Val Gly Gly
20 25 30
Thr Tyr Phe Leu Thr Thr Lys Thr Thr Leu Ser Arg Asp Pro Asn
35 40 45
Ser Phe Leu Ser Arg Leu Ile Gln Glu Asp Cys Asp Leu Ile Ser
50 55 60
Asp Arg Asp Glu Thr Gly Ala Tyr Leu ile Asp Arg Asp Pro Lys
65 70 75
Tyr Phe Ala Pro Val Leu Asn Tyr Leu Arg His Gly Lys Leu Val
41/43
CA 02341148 2001-03-O1
WO 00/12711 PCTIUS99/20468
80 85 90
Leu Asp Gly Val Ser Glu Glu Gly Val Leu Glu G~.u Ala Glu Phe
95 100 105
Tyr Asn Val.Thr Gln Leu Ile Ala Leu Leu Lys G~.u Cys Ile Leu
I10 115 120
His Arg Asp Gln Arg Pro Gln Thr Asp Lys Lys Ax~g Val Tyr Arg
125 I30 135
Val Leu Gln Cys Arg Glu Gln Glu Leu Thr Gln Mea Ile Ser Thr
140 145 150
Leu Ser Asp Gly Trp Arg Phe Glu Gln Leu Ile Ser Met Gln Tyr
155 160 165
Thr Asn Tyr Gly Pro Phe Glu Asn Asn Glu Phe Leu Cys Val Val
170 175 I80
Ser Lys Glu Cys Gly Thr Thr Ala Gly Arg Glu Leu GIu Leu Asn
185 190 195
Asp Arg Ala Lys Val Leu Gln Gln Lys G1y Ser Arg Ile Asn Thr
200 205 210
Ile Ser His Ser Ala Thr Pro Thr Gln His Gln Leu Asp Ala Ala
21S 220 225
Lys Glu Ala Arg Ala Thr Ala Thr Ala Thr Ser Asn Thr Thr Asn
230 235 240
His Thr Arg Ser Asp Gln Thr Gln Pro Gln Ala Gln Ile Thr His
245 250 255
Gln Asp Gln Pro Glu Ser Pro Lys Gln Ser Pro Gln Gly Asp Tyr
260 265 270
AIa Ser Phe Ala Phe Glu Thr Lys Leu Thr Gly Thi- Thr Ala Ile
275 280 285
Arg Phe Ser Pro Leu Trp Pro Phe Cys Ala Leu Tyr Glu Val Cys
290 295 300
Ala Gly Val His Val Phe Asn Leu
305
<2I0> 45
<211> 295
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> GenBank ID No: 82887407
<400> 45
Met Gln Pro Glu Gly Ala Glu Lys Gly Lys Ser Phe Lys Gln Arg
1 5 10 15
Leu Val Leu Lys Ser Ser Leu Ala Lys Glu Thr Leu Ser Glu Phe
20 25 30
Leu GIy Thr Phe Ile Leu Ile Val Leu Gly Cys Gly Cys Val Ala
35 40 45
GIn Ala Ile Leu Ser Arg Gly Arg Phe Gly Gly Val Ile Thr Ile
50 55 60
Asn Val Gly Phe Ser Met Ala Val Ala Met Ala Ile Tyr VaI Ala
65 70 7S
Gly Gly Val Ser Gly Gly His Ile Asn Pro Ala Val Ser Leu Aia
80 85 90
Met Cys Leu Phe Gly Arg Met Lys Trp Phe Lys Leu Pro Phe Tyr
42/43
CA 02341148 2001-03-O1
WO 00/12711 PC"T/US99120a68
~5 100 105
Val Gly Ala Gln Phe Leu Gly Ala Phe Val Gly A7.a Ala Thr Val
110 115 120
Phe Gly Ile Tyr Tyr Asp Gly Leu Met Ser Phe Al.a Gly Gly Lys
125 130 135
Leu Leu Ile Val Gly Glu Asn AIa Thr Ala His Ile Phe Aia Thr
140 145 150
Tyr Pro Ala Pro Tyr Leu Ser Leu Ala Asn Ala Ph.e Ala Asp Gln
155 160 165
Val Val Ala Thr Met Ile Leu Leu Ile Ile Val Ph.e Ala Ile Phe
170 175 180
Asp Ser Arg Asn Leu Gly Ala Pro Arg Gly Leu GIu Pro Ile Ala
185 190 195
Ile Gly Leu Leu Ile IIe Val Ile Ala Ser Ser Leu Gly Leu Asn
200 205 210
5er Gly Cys Ala Met Asn Pro Ala Arg Asg Leu Ser Pra Arg Leu
215 220 225
Phe Thr Ala Leu A1a Gly Trp Gly Phe Glu Val Ph~e Arg Ala Gly
230 23S 240
Asn Asn Phe Trp Trp Ile Pro VaI Val Gly Pro Leu VaI Gly Ala
245 250 255
Val Ile Gly Gly Leu IIe Tyr Val Leu Val Ile Glu Ile His His
260 265 270
Pro Glu Pro Asp Ser Val Phe Lys A1a Glu Gln Ser Glu Asp Lys
2?5 280 28S
Pro Glu Lys Tyr Glu Leu Ser Val Ile Met
290 295
43/43
