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RECEPTORS AND MEMBRANE-ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of receptors
and membrane-
s associated proteins and to the use of these sequences in the diagnosis,
treatment, and prevention of
cell proliferative, autoimmune/inflammatory, neurological, metabolic,
developmental, and endocrine
disorders, and in the assessment of the effects of exogenous compounds on the
expression of nucleic
acid and amino acid sequences of receptors and membrane-associated proteins.
BACKGROUND OF THE INVENTION
Signal transduction is the general process by which cells respond to
extracellular signals.
Signal transduction across the plasma membrane begins with the binding of a
signal molecule, e.g., a
hormone, neurotransmitter, or growth factor, to a cell membrane receptor. The
receptor, thus
activated, triggers an intracellular biochemical cascade that ends with the
activation of an intracellular
target molecule, such as a transcription factor. This process of signal
transduction regulates all types
of cell functions including cell proliferation, differentiation, and gene
transcription.
Biological membranes surround organelles, vesicles, and the cell itself.
Membranes are
highly selective permeability barriers made up of lipid bilayer sheets
composed of phosphoglycerides,
fatty acids, cholesterol, phospholipids, glycolipids, proteoglycans, and
proteins. Membranes contain
ion pumps, ion channels, and specific receptors for external stimuli which
transmit biochemical
signals across the membranes. These membranes also contain second messenger
proteins which
interact with these pumps, channels, and receptors to amplify and regulate
transmission of these
signals.
Plasma Membrane Proteins
Plasma membrane proteins (MPs) are divided into two groups based upon methods
of protein
extraction from the membrane. Extrinsic or peripheral membrane proteins can be
released using
extremes of ionic strength or pH, urea, or other disruptors of protein
interactions. Intrinsic or integral
membrane proteins are released only when the lipid bilayer of the membrane is
dissolved by
detergent.
The majority of known integral membrane proteins are transmembrane proteins
(TM) which
are characterized by an extracellular, a transmembrane, and an intracellular
domain. TM domains are
typically comprised of 15 to 25 hydrophobic amino acids which are predicted to
adopt an a-helical
conformation. TM proteins are classified as bitopic (Types I and II) and
polytopic (Types III and IV)
(Singer, S.J. (1990) Annu. Rev. Cell Biol. 6:247-96). Bitopic proteins span
the membrane once while
polytopic proteins contain multiple membrane-spanning segments. TM proteins
carry out a variety of
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important cellular functions, including acting as cell-surface receptor
proteins involved in signal
transduction. These functions are represented by growth and differentiation
factor receptors, and
receptor-interacting proteins such as Drosophila pecanex and frizzled
proteins, LIV-1 protein, NF2
protein, and GNS 1/SUR4 eukaryotic integral membrane proteins. TM proteins
also act as
transporters of ions or metabolites, such as gap junction channels
(connexins), and ion channels, and
as cell anchoring proteins, such as lectins, integrins, and fibronectins. TM
proteins are found in
vesicle organelle-forming molecules, such as caveolins; or cell recognition
molecules, such as cluster
of differentiation (CD) antigens, glycoproteins, and mucins.
Many MPs contain amino acid sequence motifs that serve to localize proteins to
specific
subcellular sites. Examples of these motifs include PDZ domains, KDEL, RGD,
NGR, and GSL
sequence motifs, von Willebrand factor A (vWFA) domains, and EGF-like domains.
RGD, NGR,
and GSL motif containing peptides have been used as drug delivery agents in
targeted cancer
treatment of tumor vasculature (Arap, W. et al. (1998) Science, 279:377-380).
Furthermore, MPs
may also contain amino acid sequence motifs that serve to interact with
extracellular or intracellular
molecules, such as carbohydrate recognition domains (CRD).
Chemical modification of amino acid residue side chains alters the manner in
which MPs
interact with other molecules, for example, phospholipid membranes. Examples
of such chemical
modifications to amino acid residue side chains are covalent bond formation
with
glycosaminoglycans, oligosaccharides, phospholipids, acetyl and palmitoyl
moieties, ADP-ribose,
phosphate, and sulphate groups.
RNA encoding membrane proteins may have alternative splice sites which give
rise to
proteins encoded by the same gene but with different messenger RNA and amino
acid sequences.
Splice variant membrane proteins may interact with other ligand and protein
isoforms.
Receptors
The term receptor describes proteins that specifically recognize other
molecules. The
category is broad and includes proteins with a variety of functions. The bulk
of receptors are cell
surface proteins which bind extracellular ligands and produce cellular
responses in the areas of
growth, differentiation, endocytosis, and immune response. Other receptors
facilitate the selective
transport of proteins out of the endoplasmic reticulum and localize enzymes to
particular locations in
the cell. The team may also be applied to proteins which act as receptors for
ligands with known or
unknown chemical composition and which interact with other cellular
components. For example, the
steroid hormone receptors bind to and regulate transcription of DNA.
Cell surface receptors are typically integral plasma membrane proteins. These
receptors
recognize hormones such as catecholamines; peptide hormones; growth and
differentiation factors;
small peptide factors such as thyrotropin-releasing hormone; galanin,
somatostatin, and tachykinins;
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and circulatory system-borne signaling molecules. Cell surface receptors on
immune system cells
recognize antigens, antibodies, and major histocompatibility complex (MHC)-
bound peptides. Other
cell surface receptors bind ligands to be internalized by the cell. This
receptor-mediated endocytosis
functions in the uptake of low density lipoproteins (LDL), transferrin,
glucose- or mannose-terminal
glycoproteins, galactose-terminal glycoproteins, immunoglobulins,
phosphovitellogenins, fibrin,
proteinase-inhibitor complexes, plasminogen activators, and thrombospondin
(Lodish, H. et al. (1995)
Molecular Cell Biolo~y, Scientific American Books, New York NY, p. 723;
Mikhailenko, I. et al.
(1997) J. Biol. Chem. 272:6784-6791).
Receptor Protein I~inases
Many growth factor receptors, including receptors for epidermal growth factor,
platelet-derived growth factor, fibroblast growth factor, as well as the
growth modulator a-thrombin,
contain intrinsic protein kinase activities. When growth factor binds to the
receptor, it triggers the
autophosphorylation of a serine, threonine, or tyrosine residue on the
receptor. These phosphorylated
sites are recognition sites for the binding of other cytoplasmic signaling
proteins. These proteins
participate in signaling pathways that eventually link the initial receptor
activation at the cell surface
to the activation of a specific intracellular target molecule. In the case of
tyrosine residue
autophosphorylation, these signaling proteins contain a common domain referred
to as a Src
homology (SH) domain. SH2 domains and SH3 domains are found in phospholipase C-
y, PI-3-I~ p85
regulatory subunit, Ras-GTPase activating protein, and pp60G5'°
(Lowenstein, E.J. et al. (1992) Cell
70:431-442). The cytokine family of receptors share a different common binding
domain and include
transmembrane receptors for growth hormone (GH), interleukins, erythropoietin,
and prolactin.
Other receptors and second messenger-binding proteins have intrinsic
serine/threonine
protein kinase activity. These include activin/TGF-~3/BMP-superfamily
receptors, calcium- and
diacylglycerol-activated/phospholipid-dependant protein kinase (PK-C), and RNA-
dependant protein
kinase (PK-R). In addition, other serine/threonine protein kinases, including
nematode Twitchin,
have fibronectin-like, immunoglobulin C2-like domains.
G-protein coupled receptors
The G-protein coupled receptors (GPCRs), encoded by one of the largest
families of genes
yet identified, play a central role in the transduction of extracellular
signals across the plasma
membrane. GPCRs have a proven history of being successful therapeutic targets.
GPCRs are integral membrane proteins characterized by the presence of seven
hydrophobic
transmembrane domains which together form a bundle of antipaxallel alpha (a)
helices. GPCRs range
in size from under 400 to over 1000 amino acids (Strosberg, A.D. (1991) Eur.
J. Biochem. 196:1-10;
Coughlin, S.R. (1994) Curr. Opin. Cell Biol. 6:191-197). The amino-terminus of
a GPCR is
extracellular, is of variable length, and is often glycosylated. The carboxy-
terminus is cytoplasmic
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and generally phosphorylated. Extracellular loops alternate with intracellular
loops and link the
transmembrane domains. Cysteine disulfide bridges linking the second and third
extracellular loops
may interact with agonists and antagonists. The most conserved domains of
GPCRs are the
transmembrane domains and the first two cytoplasmic loops. The transmembrane
domains account,
in part, for structural and functional features of the receptor. In most
cases, the bundle of a helices
forms a ligand-binding pocket. The extracellular N-terminal segment, or one or
more of the three
extracellular loops, may also participate in ligand binding. Ligand binding
activates the receptor by
inducing a conformational change in intracellular portions of the receptor. In
turn, the large, third
intracellular loop of the activated receptor interacts with a heterotrimeric
guanine nucleotide binding
(G) protein complex which mediates further intracellular signaling activities,
including the activation
of second messengers such as cyclic AMP (cAMP), phospholipase C, and inositol
triphosphate, and
the interaction of the activated GPCR with ion channel proteins. (See, e.g.,
Watson, S. and S.
Arkinstall (1994) The G-protein Linked Receptor Facts Book, Academic Press,
San Diego CA, pp. 2-
6; Bolander, F.F. (1994) Molecular Endocrinolo~y, Academic Press, San Diego
CA, pp. 1G2-176;
Baldwin, J.M. (1994) Curr. Opin. Cell Biol. 6:180-190.)
GPCRs include receptors for sensory signal mediators (e.g., light and
olfactory stimulatory
molecules); adenosine, y-aminobutyric acid (GABA), hepatocyte growth factor,
melanocortins,
neuropeptide Y, opioid peptides, opsins, somatostatin, tachykinins, vasoactive
intestinal polypeptide
family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine and
norepinephrine, histamine,
glutamate (metabotropic effect), acetylcholine (muscarinic effect), and
serotonin); chemokines; lipid
mediators of inflammation (e.g., prostaglandins and prostanoids, platelet
activating factor, and
leukotrienes); and peptide hormones (e.g., bombesin, bradykinin, .calcitonin,
C5a anaphylatoxin,
endothelin, follicle-stimulating hormone (FSH), gonadotropic-releasing hormone
(GnRI~,
neurokinin, and thyrotropin-releasing hormone (TRH), and oxytocin). GPCRs
which act as receptors
for stimuli that have yet to be identified are known as orphan receptors.
The largest family of GPCRs consists of the rhodopsin-like GPCRs, wluch
transmit diverse
extracellular signals including hormones, neurotransmitters, and light.
Rhodopsin is a photosensitive
GPCR found in animal retinas. In vertebrates, rhodopsin molecules axe embedded
in membranous
stacks found in photoreceptor (rod) Bells. Each rhodopsin molecule responds to
a photon of light by
triggering a decrease in cGMP levels which leads to the closure of plasma
membrane sodium
channels. In this manner, a visual signal is converted to a neural impulse.
Other rhodopsin-like
GPCRs axe directly involved in responding to neurotransmitters. These GPCRs
include the receptors
for adrenaline (adrenergic receptors), acetylcholine (muscarinic receptors),
adenosine, galanin, and
glutamate (N-methyl-D-aspartate/NMDA receptors). (Reviewed in Watson, S. and
S. Arkinstall
(1994) The G-Protein Linked Receptor Facts Book, Academic Press, San Diego CA,
pp. 7-9, 19-22,
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32-35, 130-131, 214-216, 221-222; Habert-Ortoli, E. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:9780-9783.)
The largest subfamily of GPCRs, the olfactory receptors, are also members of
the rhodopsin-
like GPCR family. These receptors function by transducing odorant signals.
Numerous distinct
olfactory receptors are required to distinguish different odors. Each
olfactory sensory neuron
expresses only one type of olfactory receptor, and distinct spatial zones of
neurons expressing distinct
receptors are found in nasal passages. For example, the RAlc receptor which
was isolated from a rat
brain library, has been shown to be limited in expression to very distinct
regions of the brain and a
defined zone of the olfactory epithelium (Raining, K. et al. (1998) Receptors
Channels 6:141-151).
The olfactory mucosa also appears to possess an additional group of odorant-
binding proteins
which recognize and bind separate classes of odorants. For example, cDNA
clones from rat have
been isolated which correspond to mRNAs highly expressed in olfactory mucosa
but not detected in
other tissues. The proteins encoded by these clones are homologous to proteins
that bind
lipopolysaccharides or polychlorinated biphenyls, and the different proteins
appear to be expressed in
specific areas of the mucosal tissue. These proteins are believed to interact
with odorants before or
after specific recognition by odorant receptors, perhaps acting as selective
signal filters (Deax, T.N. et
al. (1991) EMBO J. 10:2813-2819; Vogt, R.G. et al. (1991) J. Neurobiol. 22:74-
84).
Members of the secretin-like GPCR subfamily have as their ligands peptide
hormones such as
secretin, calcitonin, glucagon, growth hormone-releasing hormone, parathyroid
hormone, and
vasoactive intestinal peptide. For example, the secretin receptor responds to
secretin, a peptide
hormone that stimulates the secretion of enzymes and ions in the pancreas and
small intestine
(Watson, supra, pp. 278-283). Secretin receptors are about 450 amino acids in
length and are found
in the plasma membrane of gastrointestinal cells. Binding of secretin to its
receptor stimulates the
production of cAMP.
Examples of secretin-like GPCRs implicated in inflammation and the immune
response
include the EGF module-containing, mucin-like hormone receptor (Emr1) and CD97
receptor
proteins. CD97 is predominantly expressed in leukocytes and is markedly
upregulated on activated B
and T cells (McKnight, A.J. and S. Gordon (1998) J. Leukoc. Biol. 63:271-280).
These GPCRs are
members of the recently characterized EGF-TM7 receptors subfamily. These seven
transmembrane
hormone receptors exist as heterodimers in vivo and contain between three and
seven potential
calcium-binding EGF-like motifs. The EGF motif is about forty amino acid
residues in length and
includes six conserved cysteine residues, and a calcium-binding site near the
N-terminus of the
signature sequence. Post-translational hydroxylation of aspartic acid or
asparagine residues has been
associated with EGF-like domains in several proteins (Prosite PDOC00010
Aspartic acid and
asparagine hydroxylation site).
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A number of proteins that contain calcium-binding EGF-like domain signature
sequences are
involved in growth and differentiation. Examples include bone morphogenic
protein 1, which
induces the formation of cartilage and bone; crumbs, which is a Drosonhila
epithelial development
protein; Notch and a number of its homologs, which are involved in neural
growth and differentiation,
and transforming growth factor beta-1 binding protein (Expasy PROSITE document
PDOC00913;
Soler, C. and Carpenter, G., in Nicola, N.A. (1994) The Cytokine Facts Book,
Oxford University
Press, Oxford, UK, pp 193-197). EGF-like domains mediate protein-protein
interactions for a variety
of proteins. For example, EGF-like domains in the ECM glycoprotein fibulin-1
have been shown to
mediate both self association and binding to fibronectin (Tram H. et al.
(1997) J. Biol. Chem.
272:22600-22606). Point mutations in the EGF-like domains of ECM proteins have
been identified
as the cause of human disorders such as Marfan syndrome and
pseudochondroplasia (Maurer, P. et al.
(1996) Curr. Opin. Cell Biol. 8:609-617).
GPCR mutations, which may cause loss of function or constitutive activation,
have been
associated with numerous human diseases (Coughlin, supra,). For instance,
retinitis pigmentosa may
arise from mutations in the rhodopsin gene. Furthermore, somatic activating
mutations in the
thyrotropin receptor have been reported to cause hyperfunctioning thyroid
adenomas, suggesting that
certain GPCRs susceptible to constitutive activation may behave as
protooncogenes (Parma, J. et al.
(1993) Nature 365:649-651). GPCR receptors for the following ligands also
contain mutations
associated with human disease: luteinizing hormone (precocious puberty);
vasopressin VZ (X-linked
nephrogenic diabetes); glucagon (diabetes and hypertension); calcium
(hyperparathyroidism,
hypocalcuria, hypercalcemia); parathyroid hormone (short limbed dwarfism); ~i3-
adrenoceptor
(obesity, non-insulin-dependent diabetes mellitus); growth hormone releasing
hormone (dwarfism);
and adrenocorticotropin (glucocorticoid deficiency) (Wilson, S. et al. (1998)
Br. J. Pharmocol.
125:1387-1392; Stadel, J.M. et al. (1997) Trends Phaxmacol. Sci. 18:430-437).
GPCRs are also
involved in depression, schizophrenia, sleeplessness, hypertension, anxiety,
stress, renal failure, and
several cardiovascular disorders (Horn, F. and G. Vriend (1998) J. Mol. Med.
76:464-468).
In addition, within the past 20 years several hundred new drugs have been
recognized that are
directed towards activating or inhibiting GPCRs. The therapeutic targets of
these drugs span a wide
range of diseases and disorders, including cardiovascular, gastrointestinal,
and central nervous system
disorders as well as cancer, osteoporosis and endometriosis (Wilson, supra;
Stadel, supra). For
example, the dopamine agonist L-dopa is used to treat Parkinson's disease,
while a dopamine
antagonist is used to treat schizophrenia and the early stages of Huntington's
disease. Agonists and
antagonists of adrenoceptors have been used for the treatment of asthma, high
blood pressure, other
cardiovascular disorders, and anxiety; muscarinic agonists are used in the
treatment of glaucoma and
tachycardia; serotonin 5HT1D antagonists are used against migraine; and
histamine Hl antagonists
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are used against allergic and anaphylactic reactions, hay fever, itching, and
motion sickness (Horn,
supra).
Nuclear Hormone Receptors
The nuclear hormone receptors, also known as the nuclear receptors or the
intracellular
receptors, constitute a protein superfamily whose members are both receptors
and transcriptional
regulators. Nuclear hormone receptors rely on both their receptor function and
their transcriptional
regulatory function to affect a broad array of biological processes, including
development,
homeostasis, cell proliferation, and cell differentiation. (Reviewed in
Mangelsdorf, D.J. et al. (1995)
Cell 83:835-840; Wen, D.X. and D.P. McDonnell (1995) Curr. Opin. Biotechnol.
6:582-589;
Perlmann, T. and R.M. Evans (1997) Cell 90:391-397; Tenbaum, S. and A.
Baniahmad (1997) Int. J.
Biochem. Cell Biol. 29:1325-1341; Moras, D. and H. Gronemeyer (1998) Curr.
Opin. Cell Biol.
10:384-391; Willy, P.J. and D.J. Mangelsdorf (1998) in: Hormones and Signaling
(ed: B.W.
O'Malley) vol. 1, Academic Press, San Diego CA, pp. 307-358; Weatherman, R.V.
et al. (1999)
Annu. Rev. Biochem. 68:559-581.)
Nuclear hormone receptors as receptors
Generally, the term receptor describes a protein that specifically recognizes
other molecules.
As receptors, nuclear hormone receptors specifically recognize and bind to
their cognate ligands.
Although nuclear hormone receptors are located intracellularly, many receptors
are extracellular cell
surface proteins which bind extracellular ligands. Such extracellular
receptors produce cellular
responses affecting growth, differentiation, endocytosis, and the immune
response. Other receptors
facilitate the selective transport of proteins out of the endoplasmic
reticulum and localize enzymes to
particular regions of the cell. Transcriptional regulation by nuclear hormone
receptors, propagation
of cellular signals by extracellular receptors, and transport and localization
of proteins by other
receptors, all rely upon specific interactions between the receptors and a
variety of cellular
components. In many cases, the identity of the cognate ligand to which a
receptor binds is unknown.
Such receptors are termed orphan receptors. This term also applies to those
nuclear hormone
receptors which carry out their transcriptional regulatory functions without
binding any ligands.
Nuclear hormone receptors as transcriptional re u1
Multicellular organisms are comprised of diverse cell types that differ
dramatically both in
structure and function. The identity of a cell is determined by its
characteristic pattern of gene
expression, and different cell types express overlapping but distinctive sets
of genes throughout
development. Spatial and temporal regulation of gene expression is critical
for the control of cell
proliferation, cell differentiation, apoptosis, and other processes that
contribute to organismal
development. As transcriptional regulators, nuclear hormone receptors play key
roles in controlling
these fundamental biological processes. Other transcriptional regulators
affect gene expression in
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response to extracellular signals that mediate cell-cell communication and
that coordinate the
activities of different cell types.
In general, transcriptional regulators such as nuclear hormone receptors
initiate, activate,
repress, or terminate gene transcription by binding to the promoter, enhancer,
and upstream
regulatory regions of a gene in a sequence-specific manner. However, some
transcriptional
regulators bind regulatory elements within or downstream of a gene's coding
region. Transcriptional
regulatory proteins may bind to a specific region of DNA singly, or in a
complex with other
accessory factors. (Reviewed in Lewin, B. (1990) in: Genes IV, Oxford
University Press, New York
NY, and Cell Press, Cambridge MA, pp. 554-570.)
Mechanism of nuclear hormone receptor function
In the unliganded state, a nuclear hormone receptor exists in association with
a multiprotein
complex of chaperones, including heat shock proteins such as hsp90 and
immunophilins such as
hsp56. These chaperones maintain the ligand-free receptor in an inactive state
which is amenable to
binding of free ligand, and prevent the ligand-free receptor from
translocating to the nucleus. Upon
activation by its cognate ligand, the receptor may form a homodimer or
heterodimer which
translocates to the nucleus, binds to specific DNA sequences, and exerts its
transcriptional regulatory
function. In order to effectively carry out its regulatory roles, an activated
nuclear hormone receptor
dissociates from a histone deacetylase-containing corepressor complex and
associates with a histone
acetyltransferase-containing coactivator complex (Xu, L. et al. (1999) Curr.
Opin. Genet. Dev. 9:140-
147). The association of the activated receptor with coactivator proteins
results in remodeling of
chromatin so that it adopts an open transcriptionally active state, providing
access to the
transcriptional regulatory elements of the activated nuclear receptor (Lemon,
B.D. and L.P. Freedman
(1999) Curr. Opin. Genet. Dev. 9:499-504).
Structure of nuclear hormone receptors
Nuclear hormone receptors function as signal transducers by converting
hormonal signals
into transcriptional responses. In general, nuclear hormone receptors consist
of a variable amino-
terminal domain, a highly conserved DNA-binding domain, and a conserved C-
terminal ligand-
binding domain. In the steroid-binding nuclear hormone receptors, the amino-
terminal domain
harbors a trans-activation element termed AF-1. Some nuclear hormone receptors
also contain a
trans-activation element in the ligand-binding domain termed AF-2. The DNA-
binding and ligand-
binding domains of nuclear hormone receptors may contain dimerization
elements, and the DNA-
binding domain may contain a nuclear localization signal (Weatherman, R.V. et
al. (1999) Annu. Rev.
Biochem. 68:559-581).
The DNA-binding domain of nuclear hormone receptors is composed of two zinc
finger
motifs which mediate recognition of specific DNA sequences. A zinc finger
motif contains
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periodically spaced cysteine and histidine residues which coordinate Zn+2.
Examples of this sequence
pattern include the C2H2-type, C4-type, and C3HC4-type ("RING" finger) zinc
fingers, and the PHD
domain (Lewin, supra; Aasland, R. et al. (1995) Trends Biochem. Sci. 20:56-
59). A zinc finger motif
contains an a helix and an antiparallel !3 sheet whose proximity and
conformation are maintained by
the zinc ion. Contact with DNA is made by the arginine preceding the oc helix
and by the second,
third, and sixth residues of the a helix. Zinc finger motifs may be repeated
in a tandem array within a
protein such that the a helix of each zinc finger in the protein makes contact
with the major groove of
the DNA double helix. This repeated contact between the protein and the DNA
produces a strong and
specific DNA-protein interaction. The strength and specificity of the
interaction can be regulated by
the number of zinc forger motifs within the protein. Although zinc fingers
were originally identified
in DNA-binding proteins as regions that interact directly with DNA, they have
since been found in
proteins that do not bind to DNA. (See, e.g., Lodish, H. et al. (1995)
Molecular Cell Biolo~y,
Scientific American Books, New York NY, pp. 447-451.)
The ligand-binding domain of nuclear hormone receptors is responsible for
binding to
ligands, coactivator proteins, and coreprescor proteins. This domain is
composed of three layers of a
helices, with the central layer consisting of two helices containing many
hydrophobic side chains
(Moms, D. and H. Gronemeyer (1998) Curr. Opin. Cell Biol. 10:384-391). These
two central a
helices thus create a hydrophobic pocket which is the site of ligand binding.
A ligand bound in this
hydrophobic ligand-binding site is completely buried inside the receptor
protein and is not exposed to
solvent. This suggests that large conformational changes in the ligand-binding
domain would
accompany binding of a ligand. One of the a helices of the ligand-binding
domain provides many of
the inter-subunit contacts in dimers of nuclear receptors. This a helix
contacts the ligand when it is
bound in the ligand-binding pocket, suggesting that ligand binding can affect
formation of receptor
dimers (Weatherman, R.V. et al. (1999) Annu. Rev. Biochem. 68:559-581).
Classes of nuclear hormone receptors
Nuclear hormone receptors can be grouped into three broad classes: the steroid
receptors, the
RXR-heterodimeric receptors, and the orphan nuclear hormone receptors. The
steroid receptors bind
to steroid hormones, and this class includes the androgen receptor,
mineralocorticoid receptor,
estrogen receptor, glucocorticoid receptor, and progesterone receptor. The RXR-
heterodimeric
receptors bind to nonsteroid ligands, and this class includes the thyroid
hormone receptor, retinoic
acid receptor, vitamin D receptor, ecdysone receptor, and peroxisome
proliferator activated receptor.
The orphan nuclear hormone receptors include steroidogenic factor 1, nerve
growth factor-induced
receptor, and X-linked orphan receptor DAX-1.
The steroid hormone receptors are activated upon binding to specific steroid
hormones. The
conformational change induced by ligand binding leads to dissociation of the
receptor from heat
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shock proteins and formation of receptor homodimers which recognize specific
palindromic DNA
sequences called hormone response elements (HREs). Upon binding to an HRE, a
steroid hormone
receptor homodimer can regulate the transcription of target genes.
For example, the progesterone receptor (PR) is a steroid hormone receptor
which is activated
by progesterone, a 4-pregnene-3,20-dione derived from cholesterol which is a
critical oscillating
component of the female reproductive cycle. These oscillations correlate with
anatomical and
morphological changes including menstruation and pregnancy. The activities of
progesterone are
mediated through PR. In the cytoplasm, PR associates with several other
proteins and factors known
as the PR heterocomplex. This heterocomplex includes heat shock proteins and
immunophilins such
as hsp70, hsp90, hsp27, p59 (hsp56), p48, and p23 (Johnson, J.L. et al. (1994)
Mol. Cell. Biol.
14:1956-1963). Upon binding progesterone, activated PR translocates to the
nucleus, binds to
canonical DNA transcriptional elements, and regulates progesterone-regulated
genes implicated in
differentiation and the cell cycle (Moutsatsou, P and C.E. Sekeris (1997) Ann.
N.Y. Acad. Sci.
816:99-115). The PR antagonist RU 486, which can be used to terminate a
pregnancy, is an example
of a commercial therapeutic targeted toward a steroid hormone receptor.
The RXR-heterodimeric nuclear receptors are distinguished from the steroid
hormone
receptors in that members of the former group bind to their target DNA
sequences upon formation of
heterodimers with retinoid X receptors (RXRs) (Mangelsdorf, D.J. and R.M.
Evans (1995) Cell
83:841-850). Three different isoforms of RXR have been identified (Minucci, S.
and I~. Ozato
(1996) Curr. Opin. Genet. Dev. 6:567-574). The retinoic acid receptors (RARs)
are examples of
RXR-heterodimeric nuclear receptors. Retinoic acid (RA) is a biologically
active metabolite of
vitamin A (retinol), a fat-soluble vitamin found mainly in fish liver oils,
liver, egg yolk, butter, and
cream. While 9-cis-RA binds to RARs and RXRs, all-trans-RA binds only to RXRs.
RAR/RXR
heterodimers bind with high affinity to specific DNA sequences known as
retinoic acid response
elements (RAREs), thus acting as regulators of RA-dependent transcription.
Peroxisome proliferator activated receptors (PPARs) are therapeutically
important RXR-
heterodimeric nuclear receptors which are induced by fatty acids and
eicosanoid~. There are three
known isotypes of PPAR, each with specific expression patterns, and these
PPARs are involved in the
regulation of genes involved in systemic homeostatic of glucose and lipids
(I~liewer, S.A. and T.M.
Willson (1998) Curr. Opin. Genet. Dev. 8:576-581; Michalik, L. and W. Wahli
(1999) Curr. Opin.
Biotechnol. 10:564-570). As such, PPARs are therapeutic targets for disorders
such as diabetes,
dyslipidemia, and obesity (Smith, S.A. (1996) Pharmacol. Rev. Commun. 8:57-64;
Willson, T.M. and
W. Wahli (1997) Curr. Opin. Chem. Biol. 1:235-241; Barroso, I. et al. (1999)
Nature 402:880-883).
The orphan nuclear receptors either have no known activating ligand, or can
exert their
transcriptional regulatory activities without benefit of ligand binding. For
example, in Caenorhabditis
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ele~ans, the X-chromosome encoded nuclear hormone receptor homologue SEX-1
regulates
transcription of the sex determination gene xol-1 (Carmi, I, et al. (1998)
Nature 396:168-173). Rather
than relying on ligand binding, SEX-1 acts as a transcriptional regulator in a
dose-dependent manner,
in effect controlling sexual differentiation through an X-chromosome-counting
mechanism.
Some nuclear hormone receptors lack the conventional DNA-binding domain
typically
associated with the nuclear hormone receptor family. DAX-1 is one such nuclear
hormone receptor
lacking the conventional DNA-binding domain, and mutations in DAX-1 have been
shown to cause
X-linked adrenal hypoplasia congenita (Zanaria, E.F. et al. (1994) Nature
372:635-641). DAX-1 is an
orphan nuclear receptor which interacts directly with steroidogenic factor 1
(SF-1) (Ito, M. et al.
(1997) Mol. Cell. Biol. 17:1476-1483), and DAX-1 is capable of modulating the
action of SF-1 in
sex-specific gene expression (Nachtigal, M.W. et al. (1998) Cell 445-454). SF-
1 is an orphan nuclear
receptor which acts as a transcription factor for several steroidogenic enzyme
genes in the adrenal
gland and gonads (Lala, D.S. et al. (1992) Mol. Endocrinol. 6:1249-1258;
Lynch, J.P. et al. (1993)
Mol. Endocrinol. 7:776-786; Clemens, J.W. et al. (1994) Endocrinology 134:1499-
1508), and can
also regulate several genes expressed in pituitary gonadotrope cells
(Barnhart, K.M. and P.L. Mellon
(1994) Mol. Endocrinol. 8:878-885; Ingraham, H.A. et al. (1994) Genes Dev.
8:2302-2312;
Halvorson, L.M. et al. (1996) J. Biol. Chem. 271:6645-6650; Keri, R.A. and
J.H. Nilson (1996) J.
Biol. Chem. 271:10782-10785).
SF-1 also acts as a potent transactivator of small heterodimer partner (SHP;
short heterodimer
partner) (Lee, Y.K. et al. (1999) J. Biol. Chem. 274:20869-20873). SHP is
another example of a
nuclear hormone receptor lacking the conventional DNA-binding domain (Seol, W.
et al. (1996)
Science 272:1336-1339; Lee, H.-K. et al. (1998) J. Biol. Chem. 273:14398-
14402). SHP interacts
with many members of the nuclear hormone receptor family, including retimoid
receptors, estrogen
receptor, thyroid hormone receptor, and the orphan receptor CAR. SHP acts as
an inhibitor of
estrogen receptor-mediated transcriptional activation by competing with
coactivators for binding to
estrogen receptor (Johansson, L. et al. (1999) J. Biol. Chem. 274:345-353).
SHP also inhibits
transactivation by the orphan receptor hepatocyte nuclear factor 4, and by
retinoid X receptor (Lee,
Y.K. et al. (2000) Mol. Cell. Biol. 20:187-195).
Consequences of defective transcription regulation
Many neoplastic disorders in humans can be attributed to inappropriate gene
expression.
Malignant cell growth may result from either excessive expression of tumor
promoting genes or
insufficient expression of tumor suppxessor genes (Cleary, M.L. (1992) Cancer
Surv. 15:89-104).
Chromosomal translocations may also produce chimeric loci which fuse the
coding sequence of one
gene with the regulatory regions of a second unrelated gene. Such an
arrangement likely results in
inappropriate gene transcription, potentially contributing to malignancy.
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In addition, the immune system responds to infection or trauma by activating a
cascade of
events that coordinate the progressive selection, amplification, and
mobilization of cellular defense
mechanisms. A complex and balanced program of gene activation and repression
is involved in this
process. However, hyperactivity of the immune system as a result of improper
or insufficient
regulation of gene expression may result in considerable tissue or organ
damage. This damage is
well documented in immunological responses associated with arthritis,
allergens, heart attack, stroke,
and infections. (See, e.g., Isselbacher et al. (1996) Harnson's Principles of
Internal Medicine, 13/e,
McGraw Hill, Inc. and Teton Data Systems Software.)
Furthermore, the growth of multicellular organisms is based upon the induction
and
coordination of cell differentiation at the appropriate stages of development.
Central to this process
is differential gene expression, which confers the distinct identities of
cells and tissues throughout the
body. Failure to regulate gene expression during development could result in
developmental
disorders.
Ligand-Gated Receptor Ion Channels
Ligand-gated receptor ion channels fall into two categories. The first
category, extracellular
ligand-gated receptor ion channels (ELGs), rapidly transduce neurotransmitter-
binding events into
electrical signals, such as fast synaptic neurotransmission. ELG function is
regulated by post-
translational modification. The second category, intracellular ligand-gated
receptor ion channels
(II,Gs), are activated by.many intracellular second messengers and do not
require post-translational
modifications) to effect a channel-opening response.
ELGs depolarize excitable cells to the threshold of action potential
generation.. In non-
excitable cells, ELGs permit a limited calcium ion-influx during the presence
of agonist. ELGs
include channels directly gated by neurotransmitters such as acetylcholine, L-
glutamate, glycine,
ATP, serotonin, GABA, and histamine. ELG genes encode proteins having strong
structural and
functional similarities. lLGs are encoded by distinct and unrelated gene
families and include
receptors for cAMP, cGMP, calcium ions, ATP, and metabolites of arachidonic
acid.
Macrophage Scavenger Receptors
Macrophage scavenger receptors with broad ligand specificity may participate
in the binding
of low density lipoproteins (LDL) and foreign antigens. Scavenger receptors
types I and II are
trimeric membrane proteins with each subunit containing a small N-terminal
intracellular domain, a
transmembrane domain, a large extracellular domain, and a C-terminal cysteine-
rich domain. The
extracellular domain contains a short spacer domain, an a-helical coiled-coil
domain, and a triple
helical collagenous domain. These receptors have been shown to bind a spectrum
of ligands,
including chemically modified lipoproteins and albumin, polyribonucleotides,
polysaccharides,
phospholipids, and asbestos (Matsumoto, A. et al. (1990) Proc. Natl. Acad.
Sci. USA 87:9133-9137;
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Elomaa, O. et al. (1995) Cell 80:603-609). The scavenger receptors are thought
to play a key role in
atherogenesis by mediating uptake of modified LDL in arterial walls, and in
host defense by binding
bacterial endotoxins, bacteria, and protozoa.
T-Cell Receptors
T cells play a dual role in the immune system as effectors and regulators,
coupling antigen
recognition with the transmission of signals that induce cell death in
infected cells and stimulate
proliferation of other immune cells. Although a population of T cells can
recognize a wide range of
different antigens, an individual T cell can only recognize a single antigen
and only when it is
presented to the T cell receptor (TCR) as a peptide complexed with a major
histocompatibility
molecule (MHC) on the surface of an antigen presenting cell. The TCR on most T
cells consists of
immunoglobulin-like integral membrane glycoproteins containing two polypeptide
subunits, a and (3,
of similar molecular weight. Both TCR subunits have an extracellular domain
containing both
variable and constant regions, a transmembrane domain that traverses the
membrane once, and a short
intracellular domain (Saito, H. et al. (1984) Nature 309:757-762). The genes
for the TCR subunits
are constructed through somatic rearrangement of different gene segments.
Interaction of antigen in
the proper MHC context with the TCR initiates signaling cascades that induce
the proliferation,
maturation, and function of cellular components of the immune system (Weiss,
A. (1991) Annu. Rev.
Genet. 25: 487-510). Rearrangements in TCR genes and alterations in TCR
expression have been
noted in lymphomas, leukemias, autoimmune disorders, and immunodeficiency
disorders (Aisenberg,
A.C. et al. (1985) N. Engl. J. Med. 313:529-533; Weiss, supra).
Selectins
Selectins, or LEC-CAMS, comprise a specialized lectin subfamily involved
primarily in
inflammation and leukocyte adhesion (reviewed in Lasky, L.A. (1991) J. Cell.
Biochem. 45:139-146).
Selectins mediate the recruitment of leukocytes from the circulation to sites
of acute inflammation
and are expressed on the surface of vascular endothelial cells in response to
cytokine signaling.
Selectins bind to specific ligands on the leukocyte cell membrane and enable
the leukocyte to adhere
to and migrate along the endothelial surface. Binding of selectin to its
ligand leads to polarized
rearrangement of the actin cytoskeleton and stimulates signal transduction
within the leukocyte .
(Brenner, B. et al. (1997) Biochem. Biophys. Res. Commun. 231:802-807; Hidari,
K.I. et al. (1997) J.
Biol. Chem. 272:28750-28756). Members of the selectin family possess three
characteristic motifs: a
lectin or carbohydrate recognition domain; an epidermal growth factor-like
domain; and a variable
number of short consensus repeats (scr or "sushi" repeats). Sushi domains,
also known as
complement control protein (CCP) modules, or short consensus repeats (SCR),
occur in a wide
variety of complement and adhesion proteins (Norman, D.G. et al. (1991) J.
Mol. Biol. 219:717-725).
Netrin Receptors
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The netrins are a family of molecules that function as diffusible attractants
and repellants to
guide migrating cells and axons to their targets within the developing nervous
system. The netrin
receptors include the C. elegans protein UNC-5, as well as homologues recently
identified in
vertebrates (Leonardo, E.D. et al. (1997) Nature 386:833-838). These receptors
are members of the
immunoglobulin superfamily, and also contain a characteristic domain called
the ZU5 domain.
Mutations in the mouse member of the netrin receptor family, Rcm (rostral
cerebellar malformation)
result in cerebellar and midbrain defects as an apparent result of abnormal
neuronal migration
(Ackerman, S.L. et al. (1997) Nature 386:838-842).
VPS 10 Domain Containing Receptors
The members of the VPS 10 domain containing receptor family all contain a
domain with
homology to the yeast vacuolar sorting protein 10 (VPS10) receptor. This
family includes the mosaic
receptor SorLA, the neurotensin receptor sortilin, and SorCS, which is
expressed during mouse
embryonal and early postnatal nervous system development (Hermey, G. et al.
(1999) Biochem.
Biophys. Res. Commun. 266:347-351; Hermey, G. et al. (2001) Neuroreport 12:29-
32). A recently
identified member of this family, SorCS2, is highly expressed in the
developing and mature mouse
central nervous system. Its main site of expression is the floor plate, and
high levels are also detected
transiently in brain regions including the dopaminergic brain nuclei and the
dorsal thalamus
(Rezgaoui, M. (2001) Mech. Dev. 100:335-338).
Membrane-Associated Proteins
Tetraspan Family Proteins
The transmembrane 4 superfamily (TM4SF) or tetraspan family is a multigene
family
encoding type III integral membrane proteins (Wright, M.D. and Tomlinson, M.G.
(1994) Immunol.
Today 15:588). The TM4SF is comprised of membrane proteins which traver$e the
cell membrane
four times. Members of the TM4SF include platelet and endothelial cell
membrane proteins,
melanoma-associated antigens, leukocyte surface glycoproteins, colonal
carcinoma antigens, tumor-
associated antigens, and surface proteins of the schistosome parasites
(Jankowski, S.A. (1994)
Oncogene 9:1205-1211). Members of the TM4SF share about 25-30% amino acid
sequence identity
with one another. A number of TM4SF members have been implicated in signal
transduction, control
of cell adhesion, regulation of cell growth and proliferation, including
development and oncogenesis,
and cell motility, including tumor cell metastasis. Expression of TM4SF
proteins is associated with a
variety of tumors and the level of expression may be altered when cells are
growing or activated.
Tumor Antigens
Tumor antigens are surface molecules that are differentially expressed in
tumor cells relative
to normal cells. Tumor antigens distinguish tumor cells immunologically from
normal cells and
provide diagnostic and therapeutic targets for human cancers (Takagi, S. et
al. (1995) Int. J. Cancer
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61:706-715; Liu, E. et al. (1992) Oncogene 7:1027-1032).
Ion Channels
Ion channels are found in the plasma membranes of virtually every cell in the
body. For
example, chloride channels mediate a variety of cellular functions including
regulation of membrane
potentials and absorption and secretion of ions across epithelial membranes.
When present in
intracellular membranes of the Golgi apparatus and endocytic vesicles,
chloride channels also
regulate organelle pH. (See, e.g., Greger, R. (1988) Annu. Rev. Physiol.
50:111-122.)
Electrophysiological and pharmacological properties of chloride channels,
including ion conductance,
current-voltage relationships, and sensitivity to modulators, suggest that
different chloride channels
exist in muscles, neurons, fibroblasts, epithelial cells, and lymphocytes.
Many channels have sites for
phosphorylation by one or more protein kinases including protein kinase A,
protein kinase C, tyrosine
kinase, and casein kinase II, all of which regulate ion channel activity in
cells. Inappropriate
phosphorylation of proteins in cells has been linked to changes in cell cycle
progression and cell
differentiation. Changes in the cell cycle have been linked to induction of
apoptosis or cancer.
Changes in cell differentiation have been linked to diseases and disorders of
the reproductive system,
immune system, and skeletal muscle.
Cerebellar granule neurons possess a non-inactivating potassium current which
modulates
firing frequency upon receptor 'stimulation by neurotransmitters and controls
the resting membrane
potential. Potassium channels that exhibit non-inactivating currents include
the ether a go-go (EAG)
channel. A membrane protein designated KCR1 specifically binds to rat EAG by
means of its C-
terminal region and regulates the cerebellar non-inactivating potassium
current. KCRl is predicted to
contain 12 transmembrane domains, with intracellular amino and carboxyl
termini. Structural
characteristics of these transmembrane regions appear to be similar to those
of the transporter
superfamily, but no homology between KCR1 and known transporters was found,
suggesting that
KCR1 belongs to a novel class of transporters. KCR1 appears to be the
regulatory component of
non-inactivating potassium channels (Hoshi, N. et al. (1998) J. Biol. Chem.
273:23080-23085).
ABC Transporters
ATP-binding cassette (ABC) transporters, also called the "traffic ATPases",
are a superfamily
of membrane proteins that mediate transport and channel functions in
prokaryotes and eukaryotes
(Higgins, C.F. (1992) Annu~ Rev. Cell Biol. 8:67-113). ABC proteins share a
similar overall
structure and significant sequence homology. All ABC proteins contain a
conserved domain of
approximately two hundred amino acid residues which includes one or more
nucleotide binding
domains. Mutations in ABC transporter genes are associated with various
disorders, such as
hyperbilirubinemia II/Dubin-Johnson syndrome, recessive Stargardt's disease, X-
linked
adrenoleukodystrophy, multidrug resistance, celiac disease, and cystic
fibrosis.
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Semaphorins and Neuropilins
Semaphorins are a large group of axonal guidance molecules consisting of at
least 30
different members and are found in vertebrates, invertebrates, and even
certain viruses. All
semaphorins contain the sema domain which is approximately 500 amino acids in
length. Neuropilin,
a semaphorin receptor, has been shown to promote neurite outgrowth in vitro.
The extracellular
region of neuropilins consists of three different domains: CUB, discoidin, and
MAM domains. The
CUB and the MAM motifs of neuropilin have been suggested to have roles in
protein-protein
interactions and are thought to be involved in the binding of semaphorins
through the sema and the
C-terminal domains (reviewed in Raper, J.A. (2000) Curr. Opin. Neurobiol.
10:88-94).
Membrane Proteins Associated with Intercellular Communication
Intercellular communication is essential for the development and survival of
multicellular
organisms. Cells communicate with one another through the secretion and uptake
of protein
signaling molecules. The uptake of proteins into the cell is achieved by
endocytosis, in which the
interaction of signaling molecules with the plasma membrane surface, often via
binding to specific
receptors, results in the formation of plasma membrane-derived vesicles that
enclose and transport
the molecules into the cytosol. The secretion of proteins from the cell is
achieved by exocytosis, in
which molecules inside of the cell are packaged into membrane-bound transport
vesicles derived
from the trar2s Golgi network. These vesicles fuse with the plasma membrane
and release their
contents into the surrounding extracellular space. Endocytosis and exocytosis
result in the removal
and addition of plasma membrane components, and the recycling of these
components is essential to
maintain the integrity, identity, and functionality of both the plasma
membrane and internal
membrane-bound compartments.
Nogo has been identified as a component of the central nervous system myelin
that prevents
axonal regeneration in adult vertebrates. Cleavage of the Nogo-66 receptor and
other
glycophosphatidylinositol-linked proteins from axonal surfaces renders neurons
insensitive to Nogo-
66, facilitating potential recovery from CNS damage (Fournier, A.E. et al.
(2001) Nature 409:341-
346).
The slit proteins are extracellulax matrix proteins expressed by cells at the
ventral midline of
the nervous system. Slit proteins are ligands for the repulsive guidance
receptor Roundabout (Robo)
and thus play a role in repulsive axon guidance (Brose, K. et al. (1999) Cell
96:795-806).
Lysosomes are the site of degradation of intracellular material during
autophagy and of
extracellular molecules following endocytosis. Lysosomal enzymes are packaged
into vesicles which
bud from the traps-Golgi network. These vesicles fuse with endosomes to form
the mature lysosome
in which hydrolytic digestion of endocytosed material occurs. Lysosomes can
fuse with
autophagosomes to form a unique compartment in which the degradation of
organelles and other
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intracellular components occurs.
Protein sorting by transport vesicles, such as the endosome, has important
consequences for a
variety of physiological processes including cell surface growth, the
biogenesis of distinct
intracellular organelles, endocytosis, and the controlled secretion of
hormones and neurotransmitters
(Rothman, J.E. and Wieland, F.T. (1996) Science 272:227-234). In particular,
neurodegenerative
disorders and other neuronal pathologies are associated with biochemical flaws
during endosomal
protein sorting or endosomal biogenesis (Mayer R.J. et al. (1996) Adv. Exp.
Med. Biol. 389:261-
269).
Peroxisomes are organelles independent from the secretory pathway. They are
the site of
many peroxide-generating. oxidative reactions in the cell. Peroxisomes are
unique among eukaryotic
organelles in that their size, number, and enzyme content vary depending upon
organism, cell type,
and metabolic needs (Waterham, H.R. and Cregg, J.M. (1997) BioEssays 19:57-
66). Genetic defects
in peroxisome proteins which result in peroxisomal deficiencies have been
linked to a number of
human pathologies, including Zellweger syndrome, rhizomelic chonrodysplasia
punctata, X-linked
adrenoleukodystrophy, aryl-CoA oxidase deficiency, bifunctional enzyme
deficiency, classical
Refsum's disease, DHAP alkyl transferase deficiency, and acatalasemia (Moser,
H.W. and Moser,
A.B. (1996) Ann. NY Acad. Sci. 804:427-441). In addition, Gartner, J. et al.
(1991; Pediatr. Res.
29:141-146) found a 22 kDa integral membrane protein associated with lower
density peroxisome-
like subcellular fractions in patients with Zellweger syndrome.
Normal embryonic development and control of germ cell maturation is modulated
by a
number of secretory proteins which interact with their respective membrane-
bound receptors. Cell
fate during embryonic development is determined by members of the activin/TGF-
(3 superfamily,
cadherins, IGF-2, and other morphogens. In addition, proliferation,
maturation, and redifferentiation
of germ cell and reproductive tissues are regulated, for example, by IGF-2,
inhibins, activins, and
follistatins (Petraglia, F. (1997) Placenta 18:3-8; Mather, J.P. et al. (1997)
Proc. Soc. Exp. Biol. Med.
215:209-222). Transforming growth factor beta (TGFbeta) signal transduction is
mediated by two
receptor Ser/Thr kinases acting in series, type II TGFbeta receptor and
(TbetaR-II) phosphorylating
type I TGFbeta receptor (TbetaR-I). TbetaR-I-associated protein-1 (TRECAP-1),
which distinguishes
between quiescent and activated forms of the type I transforming growth factor
beta receptor, has
been associated with TGFbeta signaling (Charng, M.J et al. (1998) J. Biol.
Chem. 273:9365-9368).
Retinoic acid receptor alpha (RAR alpha) mediates retinoic-acid induced
maturation and~has
been implicated in myeloid development. Genes induced by retinoic acid during
granulocytic
differentiation include E3, a hematopoietic-specific gene that is an immediate
target for the activated
RAR alpha during myelopoiesis (Scott, L.M. et al. (1996) Blood 88:2517-2530).
The ~.-opioid receptor (MOR) mediates the actions of analgesic agents
including morphine,
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codeine, methadone, and fentanyl as well as heroin. MOR is functionally
coupled to a G-protein-
activated potassium channel (Mestek A. et al. (1995) J. Neurosci. 15:2396-
2406). A variety of MOR
subtypes exist. Alternative splicing has been observed with MOR-1 as with a
number of G
protein-coupled receptors including somatostatin 2, dopamine D2, prostaglandin
EP3, and serotonin
receptor subtypes 5-hydroxytryptamine4 and 5-hydroxytryptamine7 (Pan, Y.X. et
al. ( 1999) Mol.
Pharm. 56:396-403).
Peripheral and Anchored Membrane Proteins
Some membrane proteins are not membrane-spanning but are attached to the
plasma
membrane via membrane anchors or interactions with integral membrane proteins.
Membrane
anchors are covalently joined to a protein post-translationally and include
such moieties as prenyl,
myristyl, and glycosylphosphatidyl inositol groups. Membrane localization of
peripheral and
anchored proteins is important for their function in processes such as
receptor-mediated signal
transduction. For example, prenylation of Ras is required for its localization
to the plasma membrane
and for its normal and oncogenic functions in signal transduction.
Extracellular Messengers
Intercellular communication is essential for the growth and survival of
multicellular
organisms, and in particular, for the function of the endocrine, nervous, and
immune systems. In
addition, intercellular communication is critical for developmental processes
such as tissue
construction and organogenesis, in which cell proliferation, cell
differentiation, and morphogenesis
must be spatially and temporally regulated in a precise and coordinated
manner. Cells communicate
with one another through the secretion and uptake of diverse types of
signaling molecules such as
hormones, growth factors, neuropeptides, and cytokines.
Hormones
Hormones are signaling molecules that coordinately regulate basic
physiological processes
from embryogenesis throughout adulthood. These processes include metabolism,
respiration,
reproduction, excretion, fetal tissue differentiation and organogenesis,
growth and development,
homeostasis, and the stress response. Hormonal secretions and the nervous
system are tightly
integrated and interdependent. Hormones are secreted by endocrine glands,
primarily the
hypothalamus and pituitary, the thyroid and parathyroid, the pancreas, the
adrenal glands, and the
ovaries and testes.
The secretion of hormones into the circulation is tightly controlled. Hormones
are often
secreted in diurnal, pulsatile, and cyclic patterns. Hormone secretion is
regulated by perturbations in
blood biochemistry, by other upstream-acting hormones, by neural impulses, and
by negative
feedback loops. Blood hormone concentrations are constantly monitored and
adjusted to maintain
optimal, steady-state levels. Once secreted, hormones act only on those target
cells that express
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specific receptors.
Most disorders of the endocrine system are caused by either hyposecretion or
hypersecretion
of hormones. Hyposecretion often occurs when a hormone's gland of origin is
damaged or otherwise
impaired. Hypersecretion often results from the proliferation of tumors
derived from hoimone-
secreting cells. Inappropriate hormone levels may also be caused by defects in
regulatory feedback
loops or in the processing of hormone precursors. Endocrine malfunction may
also occur when the
target cell fails to respond to the hormone.
Hormones can be classified biochemically as polypeptides, steroids,
eicosanoids, or amines.
Polypeptide hormones, which include diverse hormones such as insulin and
growth hormone, vary in
size and function and are often synthesized as inactive precursors that are
processed intracellularly
into mature, active forms. Amine hormones, which include epinephrine and
dopamine, are amino
acid derivatives that function in neuroendocrine signaling. Steroid hormones,
which include the
cholesterol-derived hormones estrogen and testosterone, function in sexual
development and
reproduction. Eicosanoid hormones, which include prostaglandins and
prostacyclins, are fatty acid
derivatives that function in a variety of processes. Most polypeptide hormones
and some amine
hormones are soluble in the circulation where they are highly susceptible to
proteolytic degradation
within seconds after their secretion. Steroid hormones and eicosanoid hormones
are insoluble and
must be transported in the circulation by carrier proteins. The following
discussion will focus
primarily on polypeptide hormones.
Hormones secreted by the hypothalamus and pituitary gland play a critical role
in endocrine
function by coordinately regulating hormonal secretions from other endocrine
glands in response to
neural signals. Hypothalamic hormones include thyrotropin-releasing hormone,
gonadotropin-
releasing hormone, somatostatin, growth-hormone releasing factor,
corticotropin-releasing hormone,
substance P, dopamine, and prolactin-releasing hormone. These hormones
directly regulate the
secretion of hormones from the anterior lobe of the pituitary. Hormones
secreted by the anterior
pituitary include adrenocorticotropic hormone (ACTH), melanocyte-stimulating
hormone,
somatotropic hormones such as growth hormone and prolactin, glycoprotein
hormones such as
thyroid-stimulating hormone, luteinizing hormone (LH), and follicle-
stimulating hormone (FSH), (3-
lipotropin, and (3-endorphins. These hornones regulate hormonal secretions
from the thyroid,
pancreas, and adrenal glands, and act directly on the reproductive organs to
stimulate ovulation and
spermatogenesis. The posterior pituitary synthesizes and secretes antidiuretic
hormone (ADH,
vasopressin) and oxytocin.
Disorders of the hypothalamus and pituitary often result from lesions such as
primary brain
tumors, adenomas, infarction associated with pregnancy, hypophysectomy,
aneurysms, vascular
malformations, thrombosis, infections, immunological disorders, and
complications due to head
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trauma. Such disorders have profound effects on the function of other
endocrine glands, Disorders
associated with hypopituitarism include hypogonadism, Sheehan syndrome,
diabetes insipidus,
I~allman's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease,
sarcoidosis, empty sella
syndrome, and dwarfism. Disorders associated with hyperpituitaxism include
acromegaly, giantism,
and syndrome of inappropriate ADH secretion (SIADH), often caused by benign
adenomas.
Hormones secreted by the thyroid and parathyroid primarily control metabolic
rates and the
regulation of serum calcium levels, respectively. Thyroid hormones include
calcitonin, somatostatin,
and thyroid hormone. The parathyroid secretes parathyroid hormone. Disorders
associated with
hypothyroidism include goiter, myxedema, acute thyroiditis associated with
bacterial infection,
subacute thyroiditis associated with viral infection, autoimmune thyroiditis
(Hashimoto's disease),
and cretinism. Disorders associated with hyperthyroidism include
thyrotoxicosis and its various
forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid
carcinoma, and
Plummer's disease. Disorders associated with hyperparathyroidism include Conn
disease (chronic
hypercalemia) leading to bone resorption and parathyroid hyperplasia.
Hormones secreted by the pancreas regulate blood glucose levels by modulating
the rates of
carbohydrate, fat, and protein metabolism. Pancreatic hormones include
insulin, glucagon, amylin, y-
aminobutyric acid, gastrin, somatostatin, and pancreatic polypeptide. The
principal disorder
associated with pancreatic dysfunction is diabetes mellitus caused by
insufficient insulin activity.
Diabetes mellitus is generally classified as either Type I (insulin-dependent,
juvenile diabetes) or
Type II (non-insulin-dependent, adult diabetes). The treatment of both forms
by insulin replacement
therapy is well known. Diabetes mellitus often leads to acute complications
such as hypoglycemia
(insulin shock), coma, diabetic ketoacidosis, lactic acidosis, and chronic
complications leading to
disorders of the eye, kidney, skin, bone, joint, cardiovascular system,
nervous system, and to
decreased resistance to infection.
The anatomy, physiology, and diseases related to hormonal function are
reviewed in
McCance, I~.L. and Huether, S.E. (1994) Pathophysiology: The Biological Basis
for Disease in
Adults and Children, Mosby-Year Book, Inc., St. Louis, MO; Greenspan, F.S. and
Baxter, J.D.
(1994) Basic and Clinical Endocrinolo~y, Appleton and Lange, East Norwalk, CT.
Growth Factors
Growth factors are secreted proteins that mediate intercellular communication.
Unlike
hormones, which travel great distances via the circulatory system, most growth
factors are primarily
local mediators that act on neighboring cells. Most growth factors contain a
hydrophobic N-terminal
signal peptide sequence which directs the growth factor into the secretory
pathway. Most growth
factors also undergo post-translational modifications within the secretory
pathway. These
modifications can include proteolysis, glycosylation, phosphorylation, and
intramolecular disulfide
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
bond formation. Once secreted, growth factors bind to specific receptors on
the surfaces of
neighboring target cells, and the bound receptors trigger intracellular signal
transduction pathways.
These signal transduction pathways elicit specific cellular responses in the
target cells. These
responses can include the modulation of gene expression and the stimulation or
inhibition of cell
division, cell differentiation, and cell motility.
Growth factors fall into at least two broad and overlapping classes. The
broadest class
includes the large polypeptide growth factors, which are wide-ranging in their
effects. These factors
include epidermal growth factor~(EGF), fibroblast growth factor (FGF),
transforming growth factor- (3
(TGF-(3), insulin-like growth factor (IGF), nerve growth factor (NGF), and
platelet-derived growth
factor (PDGF), each defining a family of numerous related factors. ~ The large
polypeptide growth
factors, with the exception of NGF, act as mitogens on diverse cell types to
stimulate wound healing,
bone synthesis and remodeling, extracellular matrix synthesis, and
proliferation of epithelial,
epidermal, and connective tissues. Members of the TGF-(3, EGF, and FGF
families also function as
inductive signals in the differentiation of embryonic tissue. NGF functions
specifically as a
neurotrophic factor, promoting neuronal growth and differentiation.
Another class of growth factors includes the hematopoietic growth factors,
which are narrow
in their target specificity. These factors stimulate the proliferation and
differentiation of blood cells
such as B-lymphocytes, T-lymphocytes, erythrocytes, platelets, eosinophils,
basophils, neutrophils,
macrophages, and their stem cell precursors. These factors include the colony-
stimulating factors
(G-CSF, M-CSF, GM-CSF, and CSF1-3), erythropoietin, and the cytokines. The
cytokines are
specialized hematopoietic factors secreted by cells of the immune system and
are discussed in detail
below.
Growth factors play critical roles in neoplastic transformation of cells in
vitro and in tumor
progression in vivo. Overexpression of the large polypeptide growth factors
promotes the
proliferation and transformation of cells in culture. Inappropriate expression
of these growth factors
by tumor cells in vivo may contribute to tumor vascularization and metastasis.
Inappropriate activity
of hematopoietic growth factors can result in anemias, leukemias, and
lymphomas. Moreover,
growth factors are both structurally and functionally related to oncoproteins,
the potentially cancer-
causing products of proto-oneogenes. Certain FGF and PDGF family members are
themselves
homologous to oncoproteins, whereas receptors for some members of the EGF,
NGF, and FGF
families are encoded by proto-oncogenes. Growth factors also affect the
transcriptional regulation of
both proto-oncogenes and oncosuppressor genes. (Pimentel, E. (1994) Handbook
of Growth Factors,
CRC Press, Ann Arbor, MI; McKay, I. and Leigh, L, eds. (1993) Growth Factors:
A Practical
Approach, Oxford University Press, New York, NY; Habenicht, A., ed. (1990)
Growth Factors,
Differentiation Factors, and Cytokines, Springer-Verlag, New York, NY.)
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WO 02/057454 PCT/US02/01339
In addition, some of the large polypeptide growth factors play crucial roles
in the induction of
the primordial germ layers in the developing embryo. This induction ultimately
results in the
formation of the embryonic mesoderm, ectoderm, and endoderm which in turn
provide the
framework for the entire adult body plan. Disruption of this inductive process
would be catastrophic
to embryonic development.
Small Peptide Factors - Neuropeptides and Vasomediators
Neuropeptides and vasomediators (NP/VM) comprise a family of small peptide
factors,
typically of 20 amino acids or less. These factors generally function in
neuronal excitation and
inhibition of vasoconstriction/vasodilation, muscle contraction, and hormonal
secretions from the
brain and other endocrine tissues. Included in this family are neuropeptides
and neuropeptide
hormones such as bombesin, neuropeptide Y, neurotensin, neuromedin N,
melanocortins, opioids,
galanin, somatostatin, tachykvlins, urotensin II and related peptides involved
in smooth muscle
stimulation, vasopressin, vasoactive intestinal peptide, and circulatory
system-borne signaling
molecules such as angiotensin, complement, calcitonin, endothelins, formyl-
methionyl peptides,
glucagon, cholecystokinin, gastrin, and many of the peptide hormones discussed
above. NP/VMs can
transduce signals directly, modulate the activity or release of other
neurotransmitters and hormones,
and act as catalytic enzymes in signaling cascades. The effects of NP/VMs
range from extremely
brief to long-lasting. (Reviewed in Martin, C.R. et al. (1985) Endocrine Ph,
si~y, Oxford
University Press, New York NY, pp. 57-62.)
Cytokines
Cytokines comprise a family of signaling molecules that modulate the immune
system and
the inflammatory response. Cytokines are usually secreted by leukocytes, or
white blood cells, in
response to injury or infection. Cytokines function as growth and
differentiation factors that act
primarily on cells of the immune system such as B- and T-lymphocytes,
monocytes, macrophages,
and granulocytes. Like other signaling molecules, cytokines bind to specific
plasma membrane
receptors and trigger intracellular signal transduction pathways which alter
gene expression patterns.
There is considerable potential for the use of cytokines in the treatment of
inflammation and immune
system disorders.
Cytokine structure and function have been extensively characterized in vitro.
Most cytokines
are small polypeptides of about 30 kilodaltons or less. Over 50 cytokines have
been identified from
human and rodent sources. Examples of cytokine subfamilies include the
interferons (IFN- a, -(3, and
-y), the interleukins (1L1-IL13), the tumor necrosis factors (TNF-a and -(3),
and the chemokines.
Many cytokines have been produced using recombinant DNA techniques, and the
activities of
individual cytokines have been determined in vitro. These activities include
regulation of leukocyte
proliferation, differentiation, and motility. '
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The activity of an individual cytokine in vitro may not reflect the full scope
of that cytokine's
activity in vivo. Cytokines are not expressed individually in vivo but are
instead expressed in
combination with a multitude of other cytokines when the organism is
challenged with a stimulus.
Together, these cytokines collectively modulate the immune response in a
manner appropriate for
that particular stimulus. Therefore, the physiological activity of a cytokine
is determined by the
stimulus itself and by complex interactive networks among co-expressed
cytokines which may
demonstrate both synergistic and antagonistic relationships.
Chemokines comprise a cytokine subfamily with over 30 members. (Reviewed in
Wells,
T.N.C. and Peitsch, M.C. (1997) J. Leukoc. Biol. 61:545-550.) Chemokines were
initially identified
as chemotactic proteins that recruit monocytes and macrophages to sites of
inflammation. Recent
evidence indicates that chemokines may also play key roles in hematopoiesis
and HIV-1 infection.
Chemokines axe small proteins which range from about 6-15 kilodaltons in
molecular weight.
Chemokines are further classified as C, CC, CXC, or CX3C based on the number
and position of
critical cysteine residues. The CC chemokines, for example, each contain a
conserved motif
consisting of two consecutive cysteines followed by two additional cysteines
which occur
downstream at 24- and 16-residue intervals, respectively (ExPASy PROSITE
database, documents
PS00472 and PDOC00434). The presence and spacing of these four cysteine
residues are highly
conserved, whereas the intervening residues diverge significantly. However, a
conserved tyrosine
located about 15 residues downstream of the cysteine doublet seems to be
important for chemotactic
activity. Most of the human genes encoding CC chemokines are clustered on
chromosome 17,
although there are a few examples of CC chemokine genes that map elsewhere.
Other chemokines
include lymphotactin (C chemokine); macrophage chemotactic and activating
factor (MCAF/MCP-1;
CC chemokine); platelet factor 4 and IL-8 (CXC chemokines); and fractalkine
and neurotractin
(CX3C chemokines). (Reviewed in Luster, A.D. (1998) N. Engl. J. Med. 338:436-
445.)
Chromogranins and secretogranins are acidic proteins present in the secretory
granules of
endocrine and neuro-endocrine cells (Huttner, W.B. et al. (1991) Trends
Biochem. Sci. 16:27-30)
(Simon, J.-P. et al. (1989) Biochem. J. 262:1-13). Granins may be precursors
of biologically-active
peptides, or they may be helper proteins in the packaging of peptide hormones
and neuropeptides -
their precise role is unclear.
The discovery of new receptors and membrane-associated proteins, and the
polynucleotides
encoding them, satisfies a need in the art by providing new compositions which
are useful in the
diagnosis, prevention, and treatment of cell proliferative,
autoimmune/inflammatory, neurological,
metabolic, developmental, and endocrine disorders, and in the assessment of
the effects of exogenous
compounds on the expression of nucleic acid and amino acid sequences of
receptors and membrane-
associated proteins.
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WO 02/057454 PCT/US02/01339
SUMMARY OF THE INVENTION
The invention features purified polypeptides, receptors and membrane-
associated proteins,
referred to collectively as "REMAP" and individually as "REMAP-1," "REMAP-2,"
"REMAP-3,"
"REMAP-4," "REMAP-5," "REMAP-6," "REMAP-7," "REMAP-8," "REMAP-9," "REMAP-10,"
"REMAP-11," "REMAP-12," "REMAP-13," "REMAP-14," and "REMAP-15." In one aspect,
the
invention provides an isolated polypeptide selected from the group consisting
of a) a polypeptide
comprising an amino acid sequence selected from the group consisting of SEQ ID
N0:1-15, b) a
polypeptide comprising a naturally occurring amino acid sequence at least 90%
identical to an amino
acid sequence selected from the group consisting of SEQ ID NO:1-15, c) a
biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-15, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-15. In one alternative, the
invention provides an
isolated polypeptide comprising the amino acid sequence of SEQ ID N0:1-15.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected
from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ m N0:1-15, b) a polypeptide comprising a naturally
occurring amino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
ID N0:1-15, c) a biologically active fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ ID N0:1-15, and d) an irninunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ m NO:1-15.
In one alternative, the polynucleotide encodes a polypeptide selected from the
group consisting of
SEQ ID NO:1-15. In another alternative, the polynucleotide is selected from
the group consisting of
SEQ ID N0:16-30.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ~ N0:1-15, b) a polypeptide comprising a naturally occurring amino acid
sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO:1-15, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-15, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ m NO:1-15. In one
alternative, the
invention provides a cell transformed with the recombinant polynucleotide. In
another alternative, the
invention provides a transgenic organism comprising the recombinant
polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
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WO 02/057454 PCT/US02/01339
of SEQ ID NO:1-15, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO:1-15, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ )D NO: l-15, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ >D N0:1-15. The method
comprises a)
culturing a cell under conditions suitable for expression of the polypeptide,
wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ m NO:1-15, b) a polypeptide
comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ )D NO: l-15, c) a biologically active
fragment of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ m NO:1-
15, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m N0:1-15.
The invention further provides an isolated polynucleotide selected from the
group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ m N0:16-30, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ m
N0:16-30, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
In one alternative, the
polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
selected from the group
consisting of a) a polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of SEQ )D N0:16-30, b) a polynucleotide comprising a naturally
occurring polynucleotide
sequence at least 90°Io identical to a polynucleotide sequence selected
from the group consisting of
SEQ >D N0:16-30, c) a polynucleotide complementary to the polynucleotide of
a), d) a
polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). The
method comprises a) hybridizing the sample with a probe comprising at least 20
contiguous
nucleotides comprising a sequence complementary to said target polynucleotide
in the sample, and
which probe specifically hybridizes to said target polynucleotide, under
conditions whereby a
hybridization complex is formed between said probe and said target
polynucleotide or fragments
thereof, and b) detecting the presence or absence of said hybridization
complex, and optionally, if
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
present, the amount thereof. In one alternative, the probe comprises at least
60 contiguous
nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ m N0:16-30, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ m
N0:16-30, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method
comprises a) amplifying said target polynucleotide or fragment thereof using
polymerase chain
reaction amplification, and b) detecting the presence or absence of said
amplified target
polynucleotide or fragment thereof, and, optionally, if present, the amount
thereof.
The invention further provides a composition comprising an effective amount of
a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ m NO:1-15, b) a polypeptide
comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ m N0:1-15, c) a biologically active fragment
of a polypeptide
having an anuno acid sequence selected from the group consisting of SEQ m NO:1-
15, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m NO:1-15, and a pharmaceutically acceptable excipient. In
one embodiment, the
composition comprises an amino acid sequence selected from the group
consisting of SEQ m NO:1-
15. The invention additionally provides a method of treating a disease or
condition associated with
decreased expression of functional REMAP, comprising administering to a
patient in need of such
treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ m NO:1-15, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ m NO:1-15, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ m NO:1-
15, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m N0:1-15. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
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WO 02/057454 PCT/US02/01339
treating a disease or condition associated with decreased expression of
functional REMAP,
comprising administering to a patient in need of such treatment the
composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1-15, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-15, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ m N0:1-15,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ m NO:1-15. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a composition comprising an antagonist compound identified
by the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
REMAP, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-15, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-15, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ m N0:1-
15, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-15. The method comprises a) combining the
polypeptide with at least
one test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ m NO:1-15, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ m N0:1-15, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ )D NO:
l-15, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m NO: l-15. The method comprises a) combining the
polypeptide with at least
one test compound under conditions permissive for the activity of the
polypeptide, b) assessing the
activity of the polypeptide in the presence of the test compound, and c)
comparing the activity of the
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WO 02/057454 PCT/US02/01339
polypeptide in the presence of the test compound with the activity of the
polypeptide in the absence
of the test compound, wherein a change in the activity of the polypeptide in
the presence of the test
compound is indicative of a compound that modulates the activity of the
polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of SEQ ID N0:16-30,
the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, b) detecting
altered expression of the target polynucleotide, and c) comparing the
expression of the target
polynucleotide in the presence of varying amounts of the compound and in the
absence of the
compound.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:16-30, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:16-30,
iii) a
polynucleotide having a sequence complementary to i), iv) a polynucleotide
complementary to the
polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization
occurs under conditions
whereby a specific hybridization complex is formed between said probe and a
target polynucleotide
in the biological sample, said target polynucleotide selected from the group
consisting of i) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:16-30, ii) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:16-30,
iii) a polynucleotide complementary to the polynucleotide of i), iv) a
polynucleotide complementary
to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
Alternatively, the target
polynucleotide comprises a fragment of a polynucleotide sequence selected from
the group consisting
of i)-v) above; c) quantifying the amount of hybridization complex; and d)
comparing the amount of
hybridization complex in the treated biological sample with the amount of
hybridization complex in
an untreated biological sample, wherein a difference in the amount of
hybridization complex in the
treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
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Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability scores for the
matches between each
polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection with the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
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DEFINITIONS
"REMAP" refers to the amino acid sequences of substantially purified REMAP
obtained
from any species, particularly a mammalian species, including bovine, ovine,
porcine, marine, equine,
and human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
REMAP. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of REMAP either by
directly interacting with
REMAP or by acting on components of the biological pathway in which REMAP
participates.
An "allelic variant" is an alternative form of the gene encoding REMAP.
Allelic variants
may result from at least one mutation in the nucleic acid sequence and may
result in altered mRNAs
or in polypeptides whose structure or function may or may not be altered. A
gene may have none,
one, or many allelic variants of its naturally occurring form. Common
mutational changes which give
rise to allelic variants are generally ascribed to natural deletions,
additions, or substitutions of
nucleotides. Each of these types of changes may occur alone, or in combination
with the others, one
or more times in a given sequence.
"Altered" nucleic acid sequences encoding REMAP include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as REMAP or
a polypeptide with at least one functional characteristic of REMAP. Included
within this definition
are polymorphisms which may or may not be readily detectable using a
particular oligonucleotide
probe of the polynucleotide encoding REMAP, and improper or unexpected
hybridization to allelic
variants, with a locus other than the normal chromosomal locus for the
polynucleotide sequence
encoding REMAP. 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 REMAP. Deliberate amino acid substitutions may be made on the basis
of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the
residues, as long as the biological or immunological activity of REMAP is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid
CA 02435260 2003-07-17
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sequence to the complete native amino acid sequence associated with the
recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerise chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of REMAP. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of REMAP either by
directly interacting with REMAP or by acting on components of the biological
pathway in which
REMAP participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind REMAP polypeptides can be prepared using intact
polypeptides or using
fragments containing small peptides of interest as the immunizing antigen. The
polypeptide or
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KI,H). The coupled peptide is
then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an in vitro evolutionary
process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large
combinatorial libraries.
Aptamer compositions rnay be double-stranded or single-stranded, and may
include
deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other
nucleotide-like molecules.
The nucleotide components of an aptamer may have modified sugar groups (e.g.,
the 2'-OH group of a
ribonucleotide may be replaced by 2'-F or 2'-NHZ), which may improve a desired
property, e.g.,
resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
e.g., a high molecular weight carrier to slow clearance of the aptamer from
the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by
photo-activation of a
31
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cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-
13.)
The term "intramer" refers to an aptamer which is expressed in vivo. For
example, a vaccinia
virus-based RNA expression system has been used to express specific RNA
aptamers at high levels in
the cytoplasm of leukocytes (Blind, M. et al. ( 1999) Proc. Natl Acad. Sci.
USA 96:3606-3610).
The term "spiegelmer" refers to an aptamer wluch includes L-DNA, L-RNA, or
other left-
handed nucleotide derivatives or nucleotide-like molecules. Aptamers
containing left-handed
nucleotides are resistant to degradation by naturally occurring enzymes, which
normally act on
substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic REMAP, or
of any oligopeptide
thereof, to induce a specific immune response in appropriate animals or cells
and to bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide
or amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding REMAP or fragments
of REMAP may
be employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
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"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELV1EW fragment assembly system
(GCG, Madison
Wn or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended and
assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conseryative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which
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retains at least one biological or immunological function of the natural
molecule. A derivative
polypeptide is one modified by glycosylation, pegylation, or any similar
process that retains at least
one biological or immunological function of the polypeptide from which it was
derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of REMAP or the polynucleotide encoding REMAP
which
is identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise
up to the entire length of the defined sequence, minus one nucleotide/amino
acid residue. For
example, a fragment may comprise from 5 to 1000 contiguous nucleotides or
amino acid residues. A
fragment used as a probe, primer, antigen, therapeutic molecule, or for other
purposes, may be at least
5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500
contiguous nucleotides or amino
acid residues in length. Fragments may be preferentially selected from certain
regions of a molecule.
For example, a polypeptide fragment may comprise a certain length of
contiguous amino acids
selected from the first 250 or 500 amino acids (or first 25% or 50%) of a
polypeptide as shown in a
certain defined sequence. Clearly these lengths are exemplary, and any length
that is supported by
the specification, including the Sequence Listing, tables, and figures, may be
encompassed by the
present embodiments.
A fragment of SEQ ID N0:16-30 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ m N0:16-30, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:16-30 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ m N0:16-30 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:16-30 and the region of SEQ ID N0:16-30 to which the fragment
corresponds are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ >D NO:1-15 is encoded by a fragment of SEQ >D N0:16-30. A
fragment
of SEQ ID NO:1-15 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:1-15. For example, a fragment of SEQ ID NO: l-15 is useful as an
immunogenic peptide
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for the development of antibodies that specifically recognize SEQ ID NO:1-15.
The precise length of
a fragment of SEQ ID NO: l-15 and the region of SEQ ID NO:1-15 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation
colon (e.g., methionine) followed by an open reading frame and a translation
termination colon. A
"full length" polynucleotide sequence encodes a "full length" polypeptide
sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between
two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLASTI. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
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WO 02/057454 PCT/US02/01339
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off.' SO
Expect: 10
Word Size: 1l
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length
supported by the sequences shown herein, in the tables, figures, or Sequence
Listing, may be used to
describe a length over whichpercentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the charge
and-hydrophobicity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
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WO 02/057454 PCT/US02/01339
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Operz Gap: 1l and Extension Gap: 1 penalties
Gap x drop-off.' S0
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1% (w/v) SDS, and about 100 ~,g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
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WO 02/057454 PCT/US02/01339
5°C to 20°C lower than the thermal melting point (Tin) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.
(1989) Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring
Harbor Press,
Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present
invention include wash conditions of 68°C in the presence of about 0.2
x SSC and about 0.1% SDS,
for 1 hour. Alternatively, temperatures of about 65°C, 60°C,
55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present
at about 0.1%.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents
include, for instance, sheared and denatured salmon sperm DNA at about 100-200
~,g/ml. Organic
solvent, such as formamide at a concentration of about 35-50% v/v, may also be
used under particular
circumstances, such as for RNA:DNA hybridizations. Useful variations on these
wash conditions
will be readily apparent to those of ordinary skill in the art. Hybridization,
particularly under high
stringency conditions, may be suggestive of evolutionary similarity between
the nucleotides. Such
similarity is strongly indicative of a similar role for the nucleotides and
their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"hnmune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, wluch may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of REMAP
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "imrnunogenic fragment" also includes any polypeptide or
oligopeptide fragment
of REMAP which is useful in any of the antibody production methods disclosed
herein or known in
the art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
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polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of REMAP. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of REMAP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin wluch may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an REMAP may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in
the art. These processes may occur synthetically or biochemically. Biochemical
modifications will
vary by cell type depending on the enzymatic milieu of REMAP.
"Probe" refers to nucleic acid sequences encoding REMAP, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerise chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
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or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual,
2°d ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
Institute/MTT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may also be obtained from their respective sources and modified to
meet the user's specific
needs.) The PrimeGen program (available to the public from the UK Human Genome
Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
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This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(LTTRs). Regulatory elements interact with host or viral proteins which
control transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing REMAP,
nucleic acids encoding REMAP, or fragments thereof may comprise a bodily
fluid; an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60°Io free,
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preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" or "expression profile" refers to the collective pattern
of gene
expression by a particular cell type or tissue under given conditions at a
given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Txansformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or
viral infection, electroporation, heat shock, lipofection, and particle
bombardment. The term
"transformed cells" includes stably transformed cells in which the inserted
DNA is capable of
replication either as an autonomously replicating plasmid or as part of the
host chromosome, as well
as transiently transformed cells which express the inserted DNA or RNA for
limited periods of time.
A "transgenic organism," as used herein, is any oxganism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook et al. (1989),
supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
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1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%; at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides will generally have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 91 %, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human receptors and membrane-
associated
proteins (REMAP), the polynucleotides encoding REMAP, and the use of these
compositions for the
diagnosis, treatment, or prevention of cell proliferative,
autoimmune/inflammatory, neurological,
metabolic, developmental, and endocrine disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Tncyte project identification number (Incyte Project m). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ m NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide m) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ >D NO:) and
an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide m)
as shown.
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Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBanle protein (genpept) database. Columns 1 and
2 show the
polypeptide sequence identification~number (Polypeptide SEQ D7 NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide 1D) for polypeptides of the
invention. Column 3
shows the GenBank identification number (GenBank ID NO:) of the nearest
GenBank homolog.
Column 4 shows the probability scores for the matches between each polypeptide
and its homolog(s).
Column 5 shows the annotation of the GenBank homolog(s) along with relevant
citations where
applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1
and 2 show the polypeptide sequence identification number (SEQ >D NO:) and the
corresponding
Incyte polypeptide sequence number (Incyte Polypeptide ID) for each
polypeptide of the invention.
Column 3 shows the number of amino acid residues in each polypeptide. Column 4
shows potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the
MOTIFS program of the GCG sequence analysis software package (Genetics
Computer Group,
Madison WI). Column 6 shows amino acid residues comprising signature
sequences, domains, and
motifs. Column 7 shows analytical methods for protein structure/function
analysis and in some cases,
searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are receptors and membrane-
associated proteins.
For example, SEQ >D NO:1 is 97% identical to rat retinoic acid receptor alpha
2 isoform (GenBank
m g3213188) as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.)
The BLAST probability score is 8.5e-245, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ >D N0:1 also contains a nuclear
hormone receptor
lignad-binding domain and a C4 type zinc finger as determined by searching for
statistically
significant matches in the hidden Markov model (HMM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN
analyses
provide further corroborative evidence that SEQ )D N0:1 is a retinoic acid
receptor. In an alternative
example, SEQ ID N0:2 is 31 % identical, from residue A3 to residue H575, to
human multiple
membrane spanning receptor TRC8 (GenBank m g3395787) as determined by BLAST
with a
probability score of 9.7e-61. (See Table 2.) Data from additional BLAST
analyses provide further
corroborative evidence that SEQ ID N0:2 is a multiple membrane spanning
receptor. In an
alternative example, SEQ )D N0:5 is 7G% identical, from residue V4 to residue
A479, to rat potential
ligand-binding protein (GenBank ID g57734) as determined by BLAST with a
probability score of
6.3e-187. (See Table 2.) Data from additional BLAST analyses provide further
corroborative
evidence that SEQ >D N0:5 is an olfactory ligand binding protein. In an
alternative example, SEQ >D
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NO:11 is 77% identical, from residue M1 to residue F310, to Canis familiaris
olfactory receptor
CfOLF2 (GenBank ID g1314663) as determined by BLAST with a probability score
of 9.7e-129.
(See Table 2.) SEQ ID N0:11 also contains a 7-transmembrane receptor
(rhodopsin family) active
site domain as determined by searching for statistically significant matches
in the HMM-based PFAM
database. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses
provide
further corroborative evidence that SEQ ll~ NO:11 is a G-protein coupled
receptor. In an alternative
example, SEQ ID N0:15 is 99% identical, from residue M5 to residue M328, and
is 89% identical,
from residue V313 to residue E410, to a human protein which is an ortholog of
the potential ligand-
binding protein RYA3 (GenBank ID g11877275) as determined by BLAST with a
probability score
of 3.3e-207. (See Table 2.) Data from BLIMPS and additional BLAST analyses
provide further
corroborative evidence that SEQ ID N0:15 is a ligand-binding protein. SEQ ID
NO:3-4, SEQ ID
N0:6-10, and SEQ ID N0:12-14 were analyzed and annotated in a similar manner.
The algorithms
and parameters for the analysis of SEQ ll~ NO:1-15 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Column 1 lists the polynucleotide
sequence
identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte
polynucleotide
consensus sequence number (Incyte ID) for each polynucleotide of the
invention, and the length of
each polynucleotide sequence in basepairs. Column 2 shows the nucleotide start
(5') and stop (3')
positions of the cDNA and/or genomic sequences used to assemble the full
length polynucleotide
sequences of the invention, and of fragments of the polynucleotide sequences
which are useful, for
example, in hybridization or amplification technologies that identify SEQ ID
N0:16-30 or that
distinguish between SEQ ID N0:16-30 and related polynucleotide sequences.
The polynucleotide fragments described in Column 2 of Table 4 may refer
specifically, for
example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from
pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2
may refer to GenBank
cDNAs or ESTs which contributed to the assembly of the full length
polynucleotide sequences. In
addition, the polynucleotide fragments described in column 2 may identify
sequences derived from
the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences
including the
designation "ENST"). Alternatively, the polynucleotide fragments described in
column 2 may be
derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those
sequences
including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence
Records (i.e., those
sequences including the designation "NP"). Alternatively, the polynucleotide
fragments described in
column 2 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together by
an "exon stitching" algorithm. For example, a polynucleotide sequence
identified as
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FL XXXXXX_NI IVz_YYYYY_N3 N4 represents a "stitched" sequence in which XXXXXX
is the
identification number of the cluster of sequences to which the algorithm was
applied, and YYYYY is
the number of the prediction generated by the algorithm, and NI,z,3...~ if
present, represent specific
exons that may have been manually edited during analysis (See Example V).
Alternatively, the
polynucleotide fragments in column 2 may refer to assemblages of exons brought
together by an
"exon-stretching" algorithm. For example, a polynucleotide sequence identified
as
FLXXXXXX_gAAAAA_gBBBBB_1 N is a "stretched" sequence, with XXXXXX being the
Incyte
project identification number, gAAAAA being the GenBank identification number
of the human
genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB
being the
GenBank identification number or NCBI RefSeq identification number of the
nearest GenBank
protein homolog, and N referring to specific exons (See Example V). In
instances where a RefSeq
sequence was used as a protein homolog for the "exon-stretching" algorithm, a
RefSeq identifier
(denoted by "NM," "NP," or "NT") may be used in place of the GenBank
identifier (i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis andJor examples of programs
GNN, GFG,Exon prediction from genomic sequences using,
for example,
ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UI~).
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences (see
Example V).
INCY Full length transcript and exon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the exons and
resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in Table
4 was obtained to confirm the final consensus polynucleotide sequence, but the
relevant Incyte cDNA
identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library is
the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences which
were used to assemble and confirm the above polynucleotide sequences. The
tissues and vectors
which were used to construct the cDNA libraries shown in Table 5 are described
in Table 6.
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The invention also encompasses REMAP variants. A preferred REMAP variant is
one which
has at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the REMAP amino acid sequence, and which contains at
least one functional or
structural characteristic of REMAP.
The invention also encompasses polynucleotides which encode REMAP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:16-30, which encodes REMAP. The
polynucleotide
sequences of SEQ )D N0:16-30, as presented in the Sequence Listing, embrace
the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced
with uracil, and the
sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
REMAP.
In particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at
least about 85%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding REMAP. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID
N0:16-30 which has at least about 70%, or alternatively at least about 85%, or
even at least about
95% polynucleotide sequence identity to a nucleic acid sequence selected from
the group consisting
of SEQ ID N0:16-30. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of REMAP.
In addition, or in the alternative, a polynucleotide variant of the invention
is a splice variant
of a polynucleotide sequence encoding REMAP. A splice variant may have
portions which have
significant sequence identity to the polynucleotide sequence encoding REMAP,
but will generally
have a greater or lesser number of polynucleotides due to additions or
deletions of blocks of sequence
arising from alternate splicing of exons during mRNA processing. A splice
variant may have less
than about 70%, or alternatively less than about 60%, or alternatively less
than about 50%
polynucleotide sequence identity to the polynucleotide sequence encoding REMAP
over its entire
length; however, portions of the splice variant will have at least about 70%,
or alternatively at least
about 85%, or alternatively at least about 95%, or alternatively 100%
polynucleotide sequence
identity to portions of the polynucleotide sequence encoding REMAP. For
example, a polynucleotide
comprising a sequence of SEQ ll~ N0:30 is a splice variant of a polynucleotide
comprising a
sequence of SEQ ID N0:20. Any one of the splice variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of REMAP.
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 REMAP, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally occurnng
gene, may be
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produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standaxd~triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring REMAP, and all such variations
are to be considered
as being specifically disclosed.
Although nucleotide sequences which encode REMAP and its variants are
generally capable
of hybridizing to the nucleotide sequence of the naturally occurring REMAP
under appropriately
selected conditions of stringency, it may be advantageous to produce
nucleotide sequences encoding
REMAP or its derivatives possessing a substantially different codon usage,
e.g., inclusion of non-
naturally occurring codons. Codons may be selected to increase the rate at
which expression of the
peptide occurs in a particular prokaryotic or eukaryotic host in accordance
with the frequency with
which particular codons are utilized by the host. Other reasons for
substantially altering the
nucleotide sequence encoding REMAP 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 REMAP
and
REMAP 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 REMAP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:16-30 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
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sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biolog~and Biotechnoloey, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding REMAP may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments
adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme
digestions and ligations may be used to insert an engineered double-stranded
sequence into a region
of unknown sequence before performing PCR. Other methods which may be used to
retrieve
unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991)
Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries
(Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need
to screen libraries
and is useful in finding intron/exon junctions. For all PCR-based methods,
primers may be designed
using commercially available software, such as OLIGO 4.06 primer analysis
software (National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the
template at temperatures of
about 68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
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
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emitted wavelengths. Outputllight intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode REMAP may be cloned in recombinant DNA molecules that direct
expression of
REMAP, 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 REMAP.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter REMAP-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of REMAP, such as its biological or
enzymatic activity or its ability
to bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding REMAP 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
CA 02435260 2003-07-17
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Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.)
Alternatively, REMAP itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solution-phase or
solid-phase techniques.
(See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Pro
erties, WH Freeman, New
York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.)
Automated synthesis
may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the
amino acid sequence of REMAP, or any part thereof, may be altered during
direct synthesis andlor
combined with sequences from other proteins, or any part thereof, to produce a
variant polypeptide or
a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active REMAP, the nucleotide sequences
encoding REMAP
or derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which
contains the necessary elements fox transcriptional and translational control
of the inserted coding
sequence in a suitable host. These elements include regulatory sequences, such
as enhancers,
constitutive and inducible promoters, and 5' and 3' untranslated regions in
the vector and in
polynucleotide sequences encoding REMAP. Such elements may vary in their
strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding REMAP. Such signals include the ATG initiation codon and
adjacent sequences,
e.g. the I~ozak sequence. In cases where sequences encoding REMAP and its
initiation codon and
upstream regulatory sequences are inserted into the appropriate expression
vector, no additional
transcriptional or translational control signals may be needed. However, in
cases where only coding
sequence, or a fragment thereof, is inserted, exogenous translational control
signals including an in-
frame ATG initiation codon should be provided by the vector. Exogenous
translational elements and
initiation codons may be of various origins, both natural and synthetic. The
efficiency of expression
may be enhanced by the inclusion of enhancers appropriate for the particular
host cell system used.
(See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding REMAP and appropriate transcriptional
and translational
control elements. These methods include in vitro recombinant DNA techniques,
synthetic techniques,
and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989)
Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-
17; Ausubel, F.M. et
al. (1995) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York
NY, ch. 9, 13, and
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16.)
A variety of expression vectorlhost systems may be utilized to contain and
express sequences
encoding REMAP. These include, but are not limited to, microorganisms such as
bacteria
transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast
transformed with yeast expression vectors; insect cell systems infected with
viral expression vectors
(e.g., baculovirus); plant cell systems transformed with viral expression
vectors (e.g., cauliflower
mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression
vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. (See, e.g., Sambrook, supra;
Ausubel, supra; Van Heeke,
G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et
al. (1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-
1945; Takamatsu,
N. (1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of Science and
Technolo~y (1992)
McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc.
Natl. Acad. Sci. USA
81:3655-3659; and Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.)
Expression vectors derived
from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from
various bacterial plasmids,
may be used for delivery of nucleotide sequences to the targeted organ,
tissue, or cell population.
(See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M.
et al. (1993) Proc.
Natl. Acad. Sci. USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature
317(6040):813-815;
McGregor, D.P. et al. (1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and
N. Somia (1997)
Nature 389:239-242.) The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding REMAP. For
example, routine cloning,
subcloning, and propagation of polynucleotide sequences encoding REMAP can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding REMAP into the
vector's multiple
cloning site disrupts the lacZ gene, allowing a colorimetric screening
procedure for identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of REMAP are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of REMAP may be used.
For example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of REMAP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
astoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
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integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, s_ upra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and
Scorer, C.A. et al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of REMAP. Transcription of
sequences
encoding REMAP 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.
6:307-31I). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Brogue, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of
Science and Technolo~y
(1992) McGraw Hill, New York NY, pp. 191-196.) .
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding REMAP
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 REMAP 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 rnay also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of REMAP in cell lines is preferred. For example, sequences encoding REMAP can
be transformed
into cell lines using expression vectors which may contain viral origins of
replication and/or
endogenous expression elements and a selectable marker gene on the same or on
a separate vector.
Following the introduction of the vector, cells may be allowed to grow for
about 1 to 2 days in
enriched media before being switched to selective media. The purpose of the
selectable marker is to
confer resistance to a selective agent, and its presence allows growth and
recovery of cells which
successfully express the introduced sequences. Resistant clones of stably
transformed cells may be
propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
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include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk- and apr' cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; faeo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl: Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding REMAP is inserted within a marker gene sequence, transformed
cells containing
sequences encoding REMAP can be identified by the absence of marker gene
function. Alternatively,
a marker gene can be placed in tandem with a sequence encoding REMAP under the
control of a
single promoter. Expression of the marker gene in response to induction or
selection usually
indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding REMAP
and that
express REMAP may be identified by a variety of procedures known to those of
skill in the art.
These procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations, PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution, or
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
Immunological methods for detecting and measuring the expression of REMAP
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 REMAP is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, Humana
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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 REMAP
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding REMAP, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding REMAP may be
cultured under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors
containing polynucleotides which encode REMAP may be designed to contain
signal sequences
which direct secretion of REMAP through a prokaryotic or eukaryotic cell
membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding REMAP 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 REMAP protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of REMAP
activity. Heterologous protein
and peptide moieties may also facilitate purification of fusion proteins using
commercially available
affinity matrices. Such moieties include, but are not limited to, glutathione
S-transferase (GST),
CA 02435260 2003-07-17
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maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-r~2yc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their
cognate fusion proteins on immobilized glutathione, maltose, phenylarsine
oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffinity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies
that specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the REMAP encoding sequence and the
heterologous
protein sequence, so that REMAP may be cleaved away from the heterologous
moiety following
purification. Methods for fusion protein expression and purification are
discussed in Ausubel (1995,
supra, ch. 10). A variety of commercially available kits may also be used to
facilitate expression and
purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled REMAP may
be achieved
in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
REMAP of the present invention.or fragments thereof may be used to screen for
compounds
that specifically bind to REMAP. At least one and up to a plurality of test
compounds may be
screened for specific binding to REMAP. Examples of test compounds include
antibodies,
oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
REMAP, e.g., a ligand or fragment thereof, a natural substrate, a structural
or functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which REMAP
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express REMAP,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosonhila, or
E. coli. Cells expressing REMAP or cell membrane fractions which contain REMAP
are then
contacted with a test compound and binding, stimulation, or inhibition of
activity of either REMAP
or the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
REMAP, either in
solution or affixed to a solid support, and detecting the binding of REMAP to
the compound.
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Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carried out using cell-free
preparations, chemical
libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to a
solid support.
REMAP of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of REMAP. Such compounds may include agonists,
antagonists, or partial
or inverse agonists. In one embodiment, an assay is performed under conditions
permissive for
REMAP activity, wherein REMAP is combined with at least one test compound, and
the activity of
REMAP in the presence of a test compound is compared with the activity of
REMAP in the absence
of the test compound. A change in the activity of REMAP. in the presence of
the test compound is
indicative of a compound that modulates the activity of REMAP. Alternatively,
a test compound is
combined with an in vitro or cell-free system comprising REMAP under
conditions suitable for
REMAP activity, and the assay is performed. In either of these assays, a test
compound which
modulates the activity of REMAP may do so indirectly and need not come in
direct contact with the
test compound. At least one and up to a plurality of test compounds may be
screened.
In another embodiment, polynucleotides encoding REMAP or their mammalian
homologs
may be "knocked out" in an animal model system using homologous recombination
in embryonic
stem (ES) cells. Such techniques are well known in the art and are useful for
the generation of animal
models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent
No. 5,767,337.) For
example, mouse ES cells, such as the mouse 1291SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding
region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding REMAP may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
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(1998) Science 282:1145-1147).
Polynucleotides encoding REMAP can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a
region of a polynucleotide encoding REMAP is injected into animal ES cells,
and the injected
sequence integrates into the animal cell genome. Transformed cells are
injected into blastulae, and
the blastulae are implanted as described above. Transgenic progeny or inbred
lines are studied and
treated with potential pharmaceutical agents to obtain information on
treatment of a human disease.
Alternatively, a mammal inbred to overexpress REMAP, e.g., by secreting REMAP
in its milk, may
also serve as a convenient source of that protein (Janne, J. et al. (1998)
Biotechnol. Annu. Rev. 4:55-
74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of REMAP and receptors and membxane-associated pxoteins. In
addition, examples
of tissues expressing REMAP can be found in Table 6. Therefore, REMAP appears
to play a role in
cell proliferative, autoimmune/inflammatory, neurological, metabolic,
developmental, and endocrine
disorders. In the treatment of disorders associated with increased REMAP
expression or activity, it is
desirable to decrease the expression or activity of REMAP. In the treatment of
disorders associated
with decreased REMAP expression or activity, it is desirable to increase the
expression or activity of
REMAP.
Therefore, in one embodiment, REMAP 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 REMAP. 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, 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
autoimmune/inflammatory
disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease,
adult respiratory
distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
polyendocrinopathy-
candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact
dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
episodic lymphopenia
with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic
gastritis,
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glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis,
Werner syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral,
bacterial, fungal, parasitic, protozoal, and helminthic infections, and
trauma; a neurological disorder
such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral
neoplasms, Alzheimer's disease,
Pick's disease, Huntington's disease, dementia, Parkinson's disease and other
extrapyramidal
disorders, amyotrophic lateral sclerosis and other motor neuron disorders,
progressive neural
muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis
and other demyelinating
diseases, bacterial and viral meningitis, brain abscess, subdural empyema,
epidural abscess,
suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral
central nervous system
disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-
Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous
system disorders, cranial
nerve disorders, spinal cord diseases, muscular dystrophy and other
neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and polymyositis,
inherited, metabolic,
endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental
disorders including
mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD),
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial
frontotemporal dementia; a metabolic disorder such as Addison's disease,
cerebrotendinous
xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic
fibrosis, fatty
hepatocirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, goiter,
glucagonoma, glycogen
storage diseases, hereditary fructose intolerance, hyperadrenalism,
hypoadrenalism,
hyperparathyroidism, hypoparathyroidism, hypercholesterolemia,
hyperthyroidism, hypoglycemia,
hypothyroidism, hyperlipidemia, hyperlipemia, lipid myopathies,
lipodystrophies, lysosomal storage
diseases, mannosidosis, neuraminidase deficiency, obesity, osteoporosis,
phenylketonuria,
pseudovitamin D-deficiency rickets, disorders of carbohydrate metabolism such
as congenital type II
dyserythropoietic anemia, diabetes, insulin-dependent diabetes mellitus, non-
insulin-dependent
diabetes mellitus, galactose epimerase deficiency, glycogen storage diseases,
lysosomal storage
diseases, fructosuria, pentosuria, and inherited abnormalities of pyruvate
metabolism, disorders of
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lipid metabolism such as fatty liver, cholestasis, primary biliary cirrhosis,
carnitine deficiency,
carnitine palmitoyltransferase deficiency, myoadenylate deaminase deficiency,
hypertriglyceridemia,
lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-
Pick's disease,
metachromatic leukodystrophy, adrenoleukodystrophy, GMZ gangliosidosis, and
cexoid
lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia,
lipodystrophy,
lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa,
lipoid adrenal
hyperplasia, minimal change disease, lipomas, atherosclerosis,
hypercholesterolemia,
hypercholesterolemia with hypertriglyceridemia, primary
hypoalphalipoproteinemia, hypothyroidism,
renal disease, liver disease, lecithin:cholesterol acyltransferase deficiency,
cerebrotendinous
xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease,
SandhofF's disease,
hyperlipidemia, hyperlipemia, and lipid myopathies, and disorders of copper
metabolism such as
Menke's disease, Wilson's disease, and Ehlers-Danlos syndrome type IX
diabetes; a developmental
disorder such as renal tubular acidosis, anemia, Cushing's syndrome,
achondroplastic dwarfism,
Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wilms'
tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-
Magenis syndrome,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas, hereditary
neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,
hypothyroidism,
hydrocephalus, a seizure disorder such as Syndenham's chorea and cerebral
palsy, spina bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, and
sensorineural hearing loss; and
an endocrine disorder such as a disorder of the hypothalamus and/or pituitary
resulting from lesions
such as a primary brain tumor, adenoma, infarction associated with pregnancy,
hypophysectomy,
aneurysm, vascular malformation, thrombosis, infection, immunological
disorder, and complication
due to head trauma, a disorder associated with hypopituitarism including
hypogonadism, Sheehan
syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian
disease, Letterex-Siwe
disease, sarcoidosis, empty sella syndxome, and dwarfism, a disorder
associated with hyperpituitarism
including acromegaly, giantism, and syndrome of inappropriate antidiuretic
hormone (ADH)
secretion (SIADH) often caused by benign adenoma, a disorder associated with
hypothyroidism
including goiter, myxedema, acute thyroiditis associated with bacterial
infection, subacute thyroiditis
associated with viral infection, autoimmune thyroiditis (Hashimoto's disease),
and cretinism, a
disorder associated with hyperthyroidism including thyrotoxicosis and its
various forms, Grave's
disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and
Plummer's disease, a
disorder associated with hyperparathyroidism including Conn disease (chronic
hypercalemia), a
pancreatic disorder such as Type I or Type II diabetes mellitus and associated
complications, a
disorder associated with the adrenals such as hyperplasia, carcinoma, or
adenoma of the adrenal
cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia,
Cushing's disease,
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Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors,
and Addison's
disease, a disorder associated with gonadal steroid hormones such as: in
women, abnormal prolactin
production, infertility, endometriosis, perturbation of the menstrual cycle,
polycystic ovarian disease,
hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea,
galactorrhea, hermaphroditism,
hirsutism and virilization, breast cancer, and, in post-menopausal women,
osteoporosis, and, in men,
Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, a
hypergonadal disorder
associated with Leydig cell tumors, androgen resistance associated with
absence of androgen
receptors, syndrome of 5 a-reductase, and gynecomastia.
In another embodiment, a vector capable of expressing REMAP 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 REMAP including; but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
REMAP 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 REMAP
including, but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of REMAP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of REMAP including, but not limited to, those listed above.
In a further embodiment, an antagonist of REMAP may be administered to a
subject to treat
or prevent a disorder associated with increased expression or activity of
REMAP. Examples of such
disorders include, but are not limited to, those cell proliferative,
autoimmune/inflammatory,
neurological, metabolic, developmental, and endocrine disorders described
above. In one aspect, an
antibody which specifically binds REMAP may be used directly as an antagonist
or indirectly as a
targeting or delivery mechanism for bringing a pharmaceutical agent to cells
or tissues which express
REMAP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding REMAP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of REMAP including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
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An antagonist of REMAP may be produced using methods which are generally known
in the
art. In particular, purified REMAP may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind REMAP.
Antibodies to REMAP may
also be generated using methods that are well known in the art. Such
antibodies may include, but are
not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies,
Fab fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with REMAP or with any fragment or
oligopeptide thereof
which has irmnunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, arid surface active substances such as
lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among
adjuvants used in
humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
REMAP have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches
of REMAP 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 REMAP 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.
hnmunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (I985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
REMAP-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.)
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Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for REMAP may also be
generated.
For example, such fragments include, but are not limited to, F(ab'~2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab~2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
REMAP and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering REMAP epitopes is generally used, but a
competitive binding assay
may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for REMAP.
Affinity is expressed as an
association constant, Ka, which is defined as the molar concentration of REMAP-
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 REMAP epitopes, represents the average affinity, or
avidity, of the antibodies
for REMAP. The Ka determined for a preparation of monoclonal antibodies, which
are monospecific
for a particular REMAP epitope, represents a true measure of affinity. High-
affinity antibody
preparations with Ka ranging from about 109 to 10'2 L/mole axe preferred for
use in immunoassays in
which the REMAP-antibody complex must withstand rigorous manipulations. Low-
affinity antibody
preparations with Ka ranging from about 106 to 10' L/mole are preferred for
use in
immunopurification and similar procedures which ultimately require
dissociation of REMAP,
preferably in active form, from the antibody (Catty, D. (1988) Antibodies,
Volume I: A Practical
Approach, IRL, Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A
Practical Guide to
Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
' 35 example, a polyclonal antibody preparation containing at least 1-2 mg
specific antibody/ml,
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preferably 5-10 mg specific antibody/ml, is generally employed in procedures
requiring precipitation
of REMAP-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 REMAP, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, modifications
of gene expression can be achieved by designing complementary sequences or
antisense molecules
(DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory
regions of the gene
encoding REMAP. Such technology is well known in the art, and antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding REMAP. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics,
Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, I~.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C, et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding REMAP may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCE)-X1 disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inhexited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
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cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites
(e.g., against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA 93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falci ap rum and
Trypanosoma cruzi). In the
case where a genetic deficiency in REMAP expression or regulation causes
disease, the expression of
REMAP from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
REMAP are treated by constructing mammalian expression vectors encoding REMAP
and
introducing these vectors by mechanical means into REMAP-deficient cells.
Mechanical transfer
technologies fox use with cells in vivo or ex vitro include (i) direct DNA
microinjection into
individual cells, (ii) ballistic gold particle delivery, (iii) liposome-
mediated transfection, (iv) receptor-
mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and
W.F. Anderson
(1993) Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510;
Boulay, J-L. and H.
Recipon (1998) Curr. Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of REMAP include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
REMAP
may be expressed using (i) a constitutively active promoter, (e.g., from
cytomegalovirus (CMV),
Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin
genes), (ii) an inducible
promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard
(1992) Proc. Natl.
Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769;
Rossi, F.M.V. and
H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in
the T-REX plasmid
(Invitrogen)); the ecdysone-inducible promoter (available in the plasmids
PVGRXR and P1ND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone
inducible promoter
(Rossi, F.M.V. and H.M. Blau, supra)), or (iii) a tissue-specific promoter or
the native promoter of the
endogenous gene encoding REMAP from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require nninimal effort to
optimize experimental
parameters. In the alternative, transfoi-~nation is performed using the
calcium phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-4.67), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to REMAP expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding REMAP under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on~published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A, et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1.998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
cells), and the return of transduced cells to a patient are procedures well
known to persons skilled in
the art of gene therapy and have been well documented (Ranga, U. et al. (1997)
J. Virol. 71:7020-
7029; Bauer, G, et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)
Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding REMAP to cells which have one or more genetic
abnormalities with respect
to the expression of REMAP. The construction and packaging of adenovirus-based
vectors are well
known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven to
be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for
gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999)
Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature
18:389:239-242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding REMAP to target cells which have one or more genetic
abnormalities with
respect to the expression of REMAP. The use of herpes simplex virus (HSV)-
based vectors may be
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especially valuable for introducing REMAP to cells of the central nervous
system, for which HSV has
a tropism. The construction and packaging of herpes-based vectors are well
lmown to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of
recombinant HSV d92 which
consists of a genome containing at least one exogenous gene to be transferred
to a cell under the
control of the appropriate promoter for purposes including human gene therapy.
Also taught by this
patent are the construction and use of recombinant HSV strains deleted for
ICP4, ICP27 and ICP22.
For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and
Xu, H. et al. (1994)
Dev. Biol. 163:152-161, hereby incorporated by reference. The manipulation of
cloned herpesvirus
sequences, the generation of recombinant virus following the transfection of
multiple plasmids
containing different segments of the large herpesvirus genomes, the growth and
propagation of
herpesvirus, and the infection of cells with herpesvirus are techniques well
lrnown to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding REMAP to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
REMAP into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
REMAP-coding RNAs and the synthesis of high levels of REMAP in vector
transduced cells. While
alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
persistent infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN)
indicates that the lytic replication of alphaviruses can be altered to suit
the needs of the gene therapy
application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
allow the introduction of REMAP into a variety of cell types. The specific
transduction of a subset of
cells in a population may require the sorting of cells prior to transduction.
The methods of
manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA
transfections, and performing alphavirus infections, are well known to those
with ordinary skill in the
art.
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Oligonucleotides derived from the transcription initiation site, e.g., between
about positions
-10 and +10 from the start site, may also be employed to inhibit gene
expression. Similarly,
inhibition can be achieved using triple helix base-pairing methodology. Triple
helix pairing is useful
because it causes inhibition of the ability of the double helix to open
sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using
triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et
al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and hnmunolo~ic Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-
177.) A complementary sequence or antisense molecule may also be designed to
block translation of
mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding REMAP.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding REMAP. Such DNA sequences may be incorporated into a wide
variety of
vectors with suitable RNA polymerase promoters such as T7 or SP6.
Alternatively, these cDNA
constructs that synthesize complementary RNA, constitutively or inducibly, can
be introduced into
cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine,
queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine,
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cytidine, guanine, thymine, and uridine which are not as easily recognized by
endogenous
endonucleases.
An additional embodiment of the invention encompasses a method fox screening
for a
compound which is effective in altering expression of a polynucleotide
encoding REMAP.
Compounds which may be effective in altering expression of a specific
polynucleotide may include,
but are not limited to, oligonucleotides, antisense oligonucleotides, triple
helix-forming
oligonucleotides, transcription factors and other polypeptide transcriptional
regulators, and non-
macromolecular chemical entities which are capable of interacting with
specific polynucleotide
sequences. Effective compounds may alter polynucleotide expression by acting
as either inhibitors or
promoters of polynucleotide expression. Thus, in the treatment of disorders
associated with increased
REMAP expression or activity, a compound which specifically inhibits
expression of the
polynucleotide encoding REMAP may be therapeutically useful, and in the
treatment of disorders
associated with decreased REMAP expression or activity, a compound which
specifically promotes
expression of the polynucleotide encoding REMAP may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding REMAP is exposed to at least one test compound thus
obtained. The
sample may comprise, for example, an intact or permeabilized cell, or an in
vitro cell-free or
reconstituted biochemical system. Alterations in the expression of a
polynucleotide encoding
REMAP are assayed by any method commonly known in the art. Typically, the
expression of a
specific nucleotide is detected by hybridization with a probe having a
nucleotide sequence
complementary to the sequence of the polynucleotide encoding REMAP. The amount
of
hybridization may be quantified, thus forming the basis for a comparison of
the expression of the
polynucleotide both with and without exposure to one or more test compounds.
Detection of a
change in the expression of a polynucleotide exposed to a test compound
indicates that the test
compound is effective in altering the expression of the polynucleotide. A
screen for a compound
effective in altering expression of a specific polynucleotide can be carried
out, for example, using a
Schizosaccharom ces pombe gene expression system (Atkins, D. et al. (1999)
U.S. Patent No.
5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human
cell line such as HeLa
cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A
particular
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embodiment of the present invention involves screening a combinatorial library
of oligonucleotides
(such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and
modified o~igonucleotides)
for antisense activity against a specific polynucleotide sequence (Bruice,
T.W. et al. (1997) U.S.
Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechno1.15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition
which generally comprises an active ingredient formulated with a
pharmaceutically acceptable
excipient. Excipients may include, fox example, sugars, starches, celluloses,
gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the
latest edition of
Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
compositions may
consist of REMAP, antibodies to REMAP, and mimetics, agonists, antagonists, or
inhibitors of
REMAP.
The compositions utilized in tlus invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, intra-
arterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of
fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g. larger peptides
and proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the
lung have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton,
J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage
of administration
without needle injection, and obviates the need for potentially toxic
penetration enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
puzpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
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Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising REMAP or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of
the macromolecule. Alternatively, REMAP or a fragment thereof may be joined to
a short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwaxze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs,
monkeys, or pigs. An animal model may also be used to determine the
appropriate concentration
range and route of administration. Such information can then be used to
determine useful doses and
routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
REMAP or fragments thereof, antibodies of REMAP, and agonists, antagonists or
inhibitors of
REMAP, which ameliorates the symptoms or condition. Therapeutic efficacy and
toxicity may be
determined by standard pharmaceutical procedures in cell cultures or with
experimental animals, such
as by calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the
dose lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDso
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting compositions may be administered every 3 to 4
days, every week,
or biweekly depending on the half-life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about O. l ,ug to 100,000 ,ug, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
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inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind REMAP may be used
for the
diagnosis of disorders characterized by expression of REMAP, or in assays to
monitor patients being
treated with REMAP or agonists, antagonists, or inhibitors of REMAP.
Antibodies useful for
diagnostic purposes may be prepared in the same manner as described above for
therapeutics.
Diagnostic assays for REMAP include methods which utilize the antibody and a
label to detect
REMAP in human body fluids or in extracts of cells or tissues. The antibodies
may be used with or
without modification, and may be labeled by covalent or non-covalent
attachment of a reporter
molecule. A wide variety of reporter molecules, several of which are described
above, are known in
the art and may be used.
A variety of protocols for measuring REMAP, including ELISAs, RIAs, and FAGS,
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of REMAP expression.
Normal or standard values for REMAP expression are established by combining
body fluids or cell
extracts taken from normal mammalian subjects, for example, human subjects,
with antibodies to
REMAP under conditions suitable for complex formation. The amount of standard
complex
formation may be quantitated by various methods, such as photometric means.
Quantities of REMAP
expressed in subject, control, and disease samples from biopsied tissues are
compared with the
standard values. Deviation between standard and subject values establishes the
parameters for
diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding REMAP may
be used
for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of REMAP
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of REMAP, and to monitor regulation of REMAP levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding REMAP or closely related
molecules may be used
to identify nucleic acid sequences which encode REMAP. The specificity of the
probe, whether it is
made from a highly specific region, e.g., the 5'regulatory region, or from a
less specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding REMAP, allelic
variants, or related
sequences.
35- Probes may also be used for the detection of related sequences, and may
have at least 50%
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sequence identity to any of the REMAP encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:16-30 or from
genomic sequences including promoters, enhancers, and introns of the REMAP
gene.
Means for producing specific hybridization probes for DNAs encoding REMAP
include the
cloning of polynucleotide sequences encoding REMAP or REMAP derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerises and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 355,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding REMAP may be used for the diagnosis of
disorders
associated with expression of REMAP. 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, 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
autoimmunelinflammatory disorder such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis,
anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis,
autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),
bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis,
dermatomyositis, diabetes
mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's 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 neurological disorder such as epilepsy, ischemic cerebrovascular
disease, stroke, cerebral
neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease,
dementia, Parkinson's disease
and other extrapyramidal disorders, amyotrophic lateral sclerosis and other
motor neuron disorders,
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progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias,
multiple sclerosis and
other demyelinating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema,
epidural abscess, suppurative intracranial thrombophlebitis, myelitis and
radiculitis, viral central
nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob
disease, and Gerstmann-
Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous
system disorders, cranial
nerve disorders, spinal cord diseases, muscular dystrophy and other
neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and polymyositis,
inherited, metabolic,
endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental
disorders including
mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD),
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial
frontotemporal dementia; a metabolic disorder such as Addison's disease,
cerebrotendinous
xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic
fibrosis, fatty
hepatocirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, goiter,
glucagonoma, glycogen
storage diseases, hereditary fructose intolerance, hyperadrenalism,
hypoadrenalism,
hyperparathyroidism, hypoparathyroidism, hypercholesterolemia,
hyperthyroidism, hypoglycemia,
hypothyroidism, hyperlipidemia, hyperlipemia, lipid myopathies,
lipodystrophies, lysosomal storage
diseases, mannosidosis, neuraminidase deficiency, obesity, osteoporosis,
phenylketonuria,
pseudovitamin D-deficiency rickets, disorders of carbohydrate metabolism such
as congenital type II
dyserythropoietic anemia, diabetes, insulin-dependent diabetes mellitus, non-
insulin-dependent
diabetes mellitus, galactose epimerase deficiency, glycogen storage diseases,
lysosomal storage
diseases, fructosuria, pentosuria, and inherited abnormalities of pyruvate
metabolism, disorders of
lipid metabolism such as fatty liver, cholestasis, primary biliary cirrhosis,
carnitine deficiency,
carnitine palmitoyltransferase deficiency, myoadenylate deaminase deficiency,
hypertriglyceridemia,
lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-
Pick's disease,
metachromatic leukodystrophy, adrenoleukodystrophy, GMz gangliosidosis, and
ceroid
lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia,
lipodystrophy,
lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa,
lipoid adrenal
hyperplasia, minimal change disease, lipomas, atherosclerosis,
hypercholesterolemia,
hypercholesterolemia with hypertriglyceridemia, primary
hypoalphalipoproteinemia, hypothyroidism,
renal disease, liver disease, lecithin:cholesterol acyltransferase deficiency,
cerebrotendinous
xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease,
Sandhoff's disease,
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hyperlipidemia, hyperlipemia, and lipid myopathies, and disorders of copper
metabolism such as
Menke's disease, Wilson's disease, and Ehlers-Danlos syndrome type IX
diabetes; a developmental
disorder such as renal tubular acidosis, anemia, Cushing's syndrome,
achondroplastic dwarfism,
Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wihns'
tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-
Magenis syndrome,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas, hereditary
neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,
hypothyroidism,
hydrocephalus, a seizure disorder such as Syndenham's chorea and cerebral
palsy, spina bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, and
sensorineural hearing loss; and
an endocrine disorder such as a disorder of the hypothalamus and/or pituitary
resulting from lesions
such as a primary brain tumor, adenoma, infarction associated with pregnancy,
hypophysectomy,
aneurysm, vascular malformation, thrombosis, infection, immunological
disorder, and complication
due to head trauma a disorder associated with hypopituitarism including
hypogonadism, Sheehan
syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian
disease, Letterer-Siwe
disease, sarcoidosis, empty sella syndrome, and dwarfism, a disorder
associated with hyperpituitarism
including acromegaly, giantism, and syndrome of inappropriate antidiuretic
hormone (ADH)
secretion (SIADH) often caused by benign adenoma, a disorder associated with
hypothyroidism
including goiter, myxedema, acute thyroiditis associated with bacterial
infection, subacute thyroiditis
associated with viral infection, autoimmune thyroiditis (Hashimoto's disease),
and cretinism, a
disorder associated with hyperthyroidism including thyrotoxicosis and its
various forms, Grave's
disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and
Plummer's disease, a
disorder associated with hyperparathyroidism including Conn disease (chronic
hypercalemia), a
pancreatic disorder such as Type I or Type II diabetes mellitus and associated
complications, a
disorder associated with the adrenals such as hyperplasia, carcinoma, or
adenoma of the adrenal
cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia,
Cushing's disease,
Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors,
and Addison's
disease, a disorder associated with gonadal steroid hormones such as: in
women, abnormal prolactin
production, infertility, endometriosis, perturbation of the menstrual cycle,
polycystic ovarian disease,
hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea,
galactorrhea, hermaphroditism,
hirsutism and virilization, breast cancer, and, in post-menopausal women,
osteoporosis, and, in men,
Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, a
hypergonadal disorder
associated with Leydig cell tumors, androgen resistance associated with
absence of androgen
receptors, syndrome of 5 a-reductase, and gynecomastia. The polynucleotide
sequences encoding
REMAP may be used in Southern or northern analysis, dot blot, or other
membrane-based
technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-
like assays; and in
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microarrays utilizing fluids or tissues from patients to detect altered REMAP
expression. Such
qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding REMAP may be useful
in assays
that detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding REMAP may be labeled by standard methods and added to a
fluid or tissue
sample from a patient under conditions suitable for the formation of
hybridization complexes. After a
suitable incubation period, the sample is washed and the signal is quantified
and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to
a control sample then the presence of altered levels of nucleotide sequences
encoding REMAP in the
sample indicates the presence of the associated disorder. Such assays may also
be used to evaluate
the efficacy of a particular therapeutic treatment regimen in animal studies,
in clinical trials, or to
monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
REMAP, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding REMAP, 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 sarilples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding
REMAP 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
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encoding REMAP, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
REMAP, and will be employed undex optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding REMAP may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic
disease in humans. Methods of SNP detection include, but are not limited to,
single-stranded
conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In
SSCP,
oligonucleotide primers derived from the polynucleotide sequences encoding
REMAP are used to
amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived,
for example,
from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause
differences in the secondary and tertiary structures of PCR products in single-
stranded form, and
these differences are detectable using gel electrophoresis in non-denaturing
gels. In fSCCP, the
oligonucleotide primers are fluorescently labeled, which allows detection of
the amplimers in high-
throughput equipment such as DNA sequencing machines. Additionally, sequence
database analysis
methods, termed in silico SNP (isSNP), are capable of identifying
polymorphisms by comparing the
sequence of individual overlapping DNA fragments which assemble into a common
consensus
sequence. These computer-based methods filter out sequence variations due to
laboratory preparation
of DNA and sequencing errors using statistical models and automated analyses
of DNA sequence
chromatograms. In the alternative, SNPs may be detected and characterized by
mass spectrometry
using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San
Diego CA).
SNPs may be used to study the genetic basis of human disease. For example, at
least 16
common SNPs have been associated with non-insulin-dependent diabetes mellitus.
SNPs are also
useful for examining differences in disease outcomes in monogenic disorders,
such as cystic fibrosis,
sickle cell anemia, or chronic granulomatous disease. For example, variants in
the mannose-binding
lectin, MBL2, have been shown to be correlated with deleterious pulmonary
outcomes in cystic
fibrosis. SNPs also have utility in pharmacogenomics, the identification of
genetic variants that
influence a patient's response to a drug, such as life-threatening toxicity.
For example, a variation in
N-acetyl transferase is associated with a high incidence of peripheral
neuropathy in response to the
anti-tuberculosis drug isoniazid, while a variation in the core promoter of
the ALOXS gene results in
diminished clinical response to treatment with an anti-asthma drug that
targets the 5-lipoxygenase
pathway. Analysis of the distribution of SNPs in different populations is
useful for investigating
genetic drift, mutation, recombination, and selection, as well as for tracing
the origins of populations
and their migrations. (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
I~wok, P.-Y. and Z. Gu
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(1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr. Opin.
Neurobiol. 11:637-641.).
Methods which may also be used to quantify the expression of REMAP include
radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Tmmunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives
rapid quantitatidn.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and
effective treatment regimen for that patient. For example, therapeutic agents
which are highly
effective and display the fewest side effects may be selected for a patient
based on his/her
pharmacogenomic ,profile.
In another embodiment, REMAP, fragments of REMAP, or antibodies specific for
REMAP
may be used as elements on a microarray. The microarray may be used to monitor
or measure
protein-protein interactions, drug-target interactions, and gene expression
profiles, as described
above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
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Transcript images may be generated using transcripts isolated from tissues,
cell lines,
biopsies, or other biological samples. The transcript image may thus reflect
gene expression in vivo,
as in the case of a tissue or biopsy sample, or in vitro, as in the case of a
cell line.
Transcript images which profile the expression of the polynucleotides of the
present
invention may also be used in conjunction with in vitro model systems and
preclinical evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share
those toxic properties. These fingerprints or signatures are most useful and
refined when they contain
expression information from a large number of genes and gene families.
Ideally, a genome-wide
measurement of expression provides the highest quality signature. Even genes
whose expression is
not altered by any tested compounds are important as well, as the levels of
expxession of these genes
are used to normalize the rest of the expression data. The normalization
procedure is useful for
comparison of expression data after treatment with different compounds. While
the assignment of
gene function to elements of a toxicant signature aids in interpretation of
toxicity mechanisms,
knowledge of gene function is not necessary for the statistical matching of
signatures which leads to
prediction of toxicity. (See, for example, Press Release 00-02 from the
National Institute of
Environmental Health Sciences, released February 29, 2000, available at
http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a
proteome can be subjected individually to further analysis. Proteome
expression patterns, or profiles,
are analyzed by quantifying the number of expressed proteins and their
relative abundance under
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given conditions and at a given time. A profile of a cell's proteome may thus
be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample are
separated by isoelectric focusing in the first dimension, and then according
to molecular weight by
sodium dodecyl sulfate slab gel electrophoresis in the second dimension
(Steiner and Anderson,
su~a). The proteins are visualized in the gel as discrete and uniquely
positioned spots, typically by
staining the gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical
density of each protein spot is generally proportional to the level of the
protein in the sample. The
optical densities of equivalently positioned protein spots from different
samples, for example, from
biological samples either treated or untreated with a test compound or
therapeutic agent, are
compared to identify any changes in protein spot density related to the
treatment. The proteins in the
spots are partially sequenced using, for example, standard methods employing
chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein in a spot
may be determined by
comparing its partial sequence, preferably of at least 5 contiguous amino acid
residues, to the
polypeptide sequences of the present invention. In some cases, further
sequence data may be
obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for REMAP
to quantify
the levels of REMAP expression. In one embodiment, the antibodies are used as
elements on a
microarray, and protein expression levels are quantified by exposing the
microarray to the sample and
detecting the levels of protein bound to each array element (Lueking, A. et
al. (1999) Anal. Biochem.
270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection
may be performed
by a variety of methods known in the art, for example, by reacting the
proteins in the sample with a
thiol- or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at
each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to
rapid degradation of mRNA, so proteomic profiling may be more reliable and
informative in such
cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
CA 02435260 2003-07-17
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each protein is compared to the amount of the corresponding protein in an
untreated biological
sample. A difference in the amount of protein between the two samples is
indicative of a toxic
response to the test compound in the treated sample. Individual proteins are
identified by sequencing
the amino acid residues of the individual proteins and comparing these partial
sequences to the
polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are
incubated with antibodies specific to the polypeptides of the present
invention. The amount of
protein recognized by the antibodies is quantified. The amount of protein in
the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount of
protein between the two samples is indicative of a toxic response to the test
compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acid. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acid. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of nnicroarrays are
well known and thoroughly described in DNA Microarrays: A Practical Approach,
M. Schena, ed.
(1999) Oxford University Press, London, hereby expressly incorporated by
reference.
In another embodiment of the invention, nucleic acid sequences encoding REMAP
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding ox noncoding sequences may be used, and in some instances,
noncoding sequences may
be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a multi-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial
chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial
chromosomes
(BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See,
e.g., Harrington, J.J.
et al. (1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134;
and Trask, B.J.
(1991) Trends Genet. 7:149-154.) Once mapped, the nucleic acid sequences of
the invention may be
used to develop genetic linkage maps, for example, which correlate the
inheritance of a disease state
with the inheritance of a particular chromosome region or restriction fragment
length polymorphism
(RFLP). (See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl.
Acid. Sci. USA 83:7353-
7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
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map data. (See, e.g.; Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding REMAP on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
IO valuable to investigators searching for disease genes using positional
cloning or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to l 1q22-23,
any sequences mapping to that area may represent associated or regulatory
genes for further
investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The
nucleotide sequence of
the instant invention may also be used to detect differences in the
chromosomal location due to
translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, REMAP, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between REMAP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with REMAP,
or fragments thereof,
and washed. Bound REMAP is then detected by methods well known in the art.
Purified REMAP
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 REMAP specifically compete with a test compound
for binding
REMAP. In this manner, antibodies can be used to detect the presence of any
peptide which shares
one or more antigenic determinants with REMAP.
In additional embodiments, the nucleotide sequences which encode REMAP may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely on
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properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are,
therefore, to be construed as merely illustrative, and not limitative of the
remainder of the disclosure
in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below,
including U.S. Ser. No. 60/262,838, U.S. Ser. No. 60/265,927, U.S. Ser. No.
60/271,196, U.S. Ser.
No. 60/274,549, and U.S. Ser. No. 60/334,179, are expressly incorporated by
reference herein.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database
(Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in
guanidinium
isothiocyanate, while others were homogenized and lysed in phenol or in a
suitable mixture of
denaturants, such as TRIZOL (Life Technologies), a monophasic solution of
phenol and guanidine
isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or
extracted with
chloroform. RNA was precipitated from the lysates with either isopropanol or
sodium acetate and
ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
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PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid
(Invitrogen),
PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE
(Incyte
Genomics), or pINCY (Incyte Genomics), or derivatives thereof. Recombinant
plasmids were
transformed into competent E. coli cells including XLl-Blue, XLl-BIueMRF, or
SOLR from
Stratagene or DHSa, DHlOB, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell Iysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasnnid purification systems or the
R.E.A.L. PREP 96
plasmid purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-
200 thermal
cycler (MJ Research) in conjunction with the HYDRA rnicrodispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the techniques
disclosed in Example VllI.
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The polynucleotide sequences derived from Incyte eDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo
Sapiens,
Rattus norve~icus, Mus musculus, Caenorhabditis ele~ans, Saccharomyces
cerevisiae,
Schizosaccharom cues pombe, and Candida albicans (Incyte Genomics, Palo Alto
CA); and hidden
Markov model (HMM)-based protein family databases such as PFAM. (HMM is a
probabilistic
approach which analyzes consensus primary structures of gene families. See,
for example, Eddy,
S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The queries were performed
using programs based
on BLAST, FASTA, BLIMPS, and HM1VIER. The Incyte cDNA sequences were assembled
to
produce full length polynucleotide sequences. Alternatively, GenBank cDNAs,
GenBank ESTs,
stitched sequences, stretched sequences, or Genscan-predicted coding sequences
(see Examples IV
and V) were used to extend Incyte cDNA assemblages to full length. Assembly
was performed using
programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened
for open
reading frames using programs based on GeneMark, BLAST, and FASTA. The full
length
polynucleotide sequences were translated to derive the corresponding full
length polypeptide
sequences. Alternatively, a polypeptide of the invention may begin at any of
the methionine residues
of the full length translated polypeptide. Full length polypeptide sequences
were subsequently
analyzed by querying against databases such as the GenBank protein databases
(genpept), SwissProt,
the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden
Markov
model (HMM)-based protein family databases such as PFAM. Full length
polynucleotide sequences
are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering,
South San
Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide
sequence
alignments are generated using default parameters specified by the CLUSTAL
algorithm as
incorporated into the MEGALIGN multisequence alignment program (DNASTAR),
which also
calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and
threshold parameters. The first column of Table 7 shows the tools, programs,
and algorithms used,
the second column provides brief descriptions thereof, the third column
presents appropriate
references, all of which are incorporated by reference herein in their
entirety, and the fourth column
presents, where applicable, the scores, probability values, and other
parameters used to evaluate the
strength of a match between two sequences (the higher the score or the lower
the probability value,
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the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide
and polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ
m N0:16-30. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization
and amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative receptors and membrane-associated proteins were initially identified
by running the
Genscan gene identification program against public genomic sequence databases
(e.g., gbpri and
gbhtg). Genscan is a general-purpose gene identification program which
analyzes genomic DNA
sequences from a variety of organisms (See Burge, C. and S. Karlin (1997) J.
Mol. Biol. 268:78-94,
and Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The
program concatenates
predicted exons to form an assembled cDNA sequence extending from a methionine
to a stop colon.
The output of Genscan is a FASTA database of polynucleotide and polypeptide
sequences. The
maximum range of sequence for Genscan to analyze at once was set to 30 kb. To
determine which of
these Genscan predicted cDNA sequences encode receptors and membrane-
associated proteins, the
encoded polypeptides were analyzed by querying against PFAM models for
receptors and membrane-
associated proteins. Potential receptors and membrane-associated proteins were
also identified by
homology to Incyte cDNA sequences that had been annotated as receptors and
membrane-associated
proteins. These selected Genscan-predicted sequences were then compared by
BLAST analysis to the
genpept and gbpri public databases. Where necessary, the Genscan-predicted
sequences were then
edited by comparison to the top BLAST hit from genpept to correct errors in
the sequence predicted
by Genscan, such as extra or omitted exons. BLAST analysis was also used to
find any Incyte cDNA
or public cDNA coverage of the Genscan-predicted sequences, thus providing
evidence for
transcription. When Incyte cDNA coverage was available, this information was
used to correct or
confirm the Genscan predicted sequence. Full length polynucleotide sequences
were obtained by
assembling Genscan-predicted coding sequences with Incyte cDNA sequences
and/or public cDNA
sequences using the assembly process described in Example III. Alternatively,
full length
polynucleotide sequences were derived entirely from edited or unedited Genscan-
predicted coding
sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
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based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an~interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along.their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for
homologous genomic sequences from the public human genome databases. Partial
DNA sequences
were therefore "stretched" or extended by the addition of homologous genomic
sequences. The
resultant stretched sequences were examined to determine whether it contained
a complete gene.
VI. Chromosomal Mapping of REMAP Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:16-30 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:16-30 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
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Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM
distances are based on genetic markers mapped by Genethon which provide
boundaries for radiation
hybrid markers whose sequences were included in each of the clusters. Human
genome maps and
other resources available to the public, such as the NCBI "GeneMap'99" World
Wide Web site
(http://www.ncbi.nlin.nih.gov/genemap/), can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel
(1995) supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the
computer search can be modified to determine whether any particular match is
categorized as exact or
similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
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BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding REMAP are analyzed with
respect to the
tissue sources from which they were derived. For example, some full length
sequences are
assembled, at least in part, with overlapping Incyte cDNA sequences (see
Example III). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following organ/tissue categories: cardiovascular
system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female;
genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal
system; nervous
system; pancreas; respiratory system; sense organs; skin; stomatognathic
system; unclassified/mixed;
or urinary tract. The number of libraries in each category is counted and
divided by the total number
of libraries across all categories. Similarly, each human tissue i's
classified into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
by the total number of libraries across all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding REMAP. cDNA sequences and cDNA
library/tissue
information are found in the LIF'ESEQ GOLD database (Incyte Genomics, Palo
Alto CA).
VIII. Extension of REMAP Encoding Polynucleotides
Full length polynucleotide sequences were also 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 was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)ZS04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
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(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, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
l: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68 ° C, 5 min; Step 7: storage at 4 ° C.
The concentration of DNA in each well was determined by dispensing 100 ~.1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~.1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ,u1 to 10 ~1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37°C in
384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise
(Amersham Pharmacia Biotech) and Pfu DNA polymerise (Stratagene) with the
following
parameters: Step 1: 94 ° C, 3 min; Step 2: 94 ° C, 15 sec; Step
3: 60 ° C, 1 min; Step 4: 72 ° C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5' regulatory sequences using the above procedure along
with oligonucleotides
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designed for such extension, and an appropriate genomic library.
IX. Identification of Single Nucleotide Polymorphisms in REMAP Encoding
Polynucleotides
Common DNA sequence variants known as single nucleotide polymorphisms (SNPs)
were
identified in SEQ )I? N0:16-30 using the LIFESEQ database (Incyte Genomics).
Sequences from the
same gene were clustered together and assembled as described in Example III,
allowing the
identification of all sequence variants in the gene. An algorithm consisting
of a series of filters was
used to distinguish SNPs from other sequence variants. Preliminary filters
removed the majority of
basecall errors by requiring a minimum Phred quality score of 15, and removed
sequence alignment
errors and errors resulting from improper trimming of vector sequences,
chimeras, and splice
variants. An automated procedure of advanced chromosome analysis analysed the
original
chromatogram files in the vicinity of the putative SNP. Clone error filters
used statistically generated
algorithms to identify errors introduced during laboratory processing, such as
those caused by reverse
transcriptase, polymerase, or somatic mutation. Clustering error filters used
statistically generated
algorithms to identify errors resulting from clustering of close homologs or
pseudogenes, or due to
contamination by non-human sequences. A final set of filters removed
duplicates and SNPs found in
immunoglobulins or T-cell receptors.
Certain SNPs were selected for further characterization by mass spectrometry
using the high
throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at
the SNP sites in
four different human populations. The Caucasian population comprised 92
individuals (46 male, 46
female), including 83 from Utah, four French, three Venezualan, and two Amish
individuals. The
African population comprised 194 individuals (97 male, 97 female), all African
Americans. The
Hispanic population comprised 324 individuals (162 male, 162 female), all
Mexican Hispanic. The
Asian population comprised 126 individuals (64 male, 62 female) with a
reported parental breakdown
of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
Allele
frequencies were first analyzed in the Caucasian population; in some cases
those SNPs which showed
no allelic variance in this population were not further tested in the other
three populations.
X. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:16-30 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~sCi of
[y 3zP] adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
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SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
XI. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena (1999),
supra). Suggested substrates include silicon, silica, glass slides, glass
chips, and silicon wafers.
Alternatively, a procedure analogous to'a dot or slot blot may also be used to
arrange and link
elements to the surface of a substrate using thermal, W, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinaxy skill in the art and may contain any appropriate number of
elements. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalom D. et al. (1996) Genome
Res. 6:639-645;
Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element
on the microarray may be assessed. In one embodiment, nucroarray preparation
and usage is
described in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
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poly(A)~ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~.1 oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/,ul RNase inhibitor, 500 ~M dATP, 500 ,uM
dGTP, 500 ,uM dTTP, 40
~,M dCTP, 40 ~,M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium
hydroxide and
incubated for 20 minutes at 85° C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 mI of glycogen (1 mg/ml), 60 m1 sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 ~,l 5X SSC/0.2% SDS.
Microarra~paration
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
~,g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water,
and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides
are cured in a
110°C oven.
Array.elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~,l of the array
element DNA, at an average
concentration of 100 ngl~,l, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are ITV-crosslinked using a STRA.TALINI~ER W-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in
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0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 ~1 of sample mixture consisting of 0.2 ,ug
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC; 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered
with an 1.8 cmz coverslip. The arrays are transferred to a waterproof chamber
having a cavity just
slightly larger than a microscope slide. The chamber is kept at 100% humidity
internally by the
addition of I40 ~.I of 5X SSC in a corner of the chamber. The chamber
containing the arrays is
incubated for about 6.5 hours at 60° C. The arrays are washed for 10
min at 45 ° C in a first wash
buffer (IX SSC, O.I% SDS), three times for IO minutes each at 45° C in
a second wash buffer (0.1X
SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser Iight is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x I.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are
differentially expressed, the calibration is done by labeling samples of the
calibrating cDNA with the
two fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
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(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each
spot is centered in each element of the grid. The fluorescence signal within
each element is then
integrated to obtain a numerical value corresponding to the average intensity
of the signal. The
software used for signal analysis is the GEMTOOLS gene expression analysis
program (Tncyte).
XII. Complementary Polynucleotides
Sequences complementary to the REMAP-encoding sequences, or any parts thereof,
are used
to detect, decrease, or inhibit expression of naturally occurring REMAP.
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 REMAP.
To inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5'
sequence and used to prevent promoter binding to the coding sequence. To
inhibit translation, a
complementary oligonucleotide is designed to prevent ribosomal binding to the
REMAP-encoding
transcript. .
XIII. Expression of REMAP
Expression and purification of REMAP is achieved using bacterial or virus-
based expression
systems. For expression of REMAP 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
try-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express REMAP upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of REMAP in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autog-raphica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding REMAP by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
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Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, REMAP is synthesized as a fusion protein with,
e.g., glutathione
S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His,
permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-
kilodalton enzyme from Schistosoma japonicum, 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
REMAP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity
purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues, enables
purification on metal-chelate
resins (QIAGEN). Methods for protein expression and purification are discussed
in Ausubel (1995,
supra, ch. 10 and 16). Purified REMAP obtained by these methods can be used
directly in the assays
1S shown in Examples XVII, XVIII, and XIX, where applicable.
XIV. Functional Assays
REMAP function is assessed by expxessing the sequences encoding REMAP at
physiologically elevated levels in mammalian cell culture systems. cDNA is
subcloned into a
mammalian expression vector containing a strong promoter that drives high
levels of cDNA
expression. Vectors of choice include PCMV SPORT (Life Technologies) and
PCR3.1 (Invitrogen,
Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ,ug of
recombinant vector
are transiently transfected into a human cell line, for example, an
endothelial or hematopoietic cell
line, using either liposome formulations or electroporation. 1-2 ,ug of an
additional plasmid
containing sequences encoding a marker protein are co-transfected. Expression
of a marker protein
2S provides a means to distinguish transfected cells from nontransfected cells
and is a reliable predictor
of cDNA expression from the recombinant vector. Marker proteins of choice
include, e.g., Green
Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow
cytometry (FCM),
an automated, laser optics-based technique, is used to identify transfected
cells expressing GFP or
CD64-GFP and to evaluate the apoptotic state of the cells and other cellular
properties. FCM detects
and quantifies the uptake of fluorescent molecules that diagnose events
preceding or coincident with
cell death. These events include changes in nuclear DNA content as measured by
staining of DNA
with propidium iodide; changes in cell size and granularity as measured by
forward light scatter and
90 degree side light scatter; down-regulation of DNA synthesis as measured by
decrease in
bromodeoxyuridine uptake; alterations in expression of cell surface and
intracellular proteins as
measured by reactivity with specific antibodies; and alterations in plasma
membrane composition as
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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 CytometrX, Oxford,
New York NY.
The influence of REMAP on gene expression can be assessed using highly
purified
populations of cells transfected with sequences encoding REMAP and either CDG4
or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind
to conserved regions
of human immunoglobulin G (IgG). Transfected cells are efficiently separated
from nontransfected
cells using magnetic beads coated with either human IgG or antibody against
CD64 (DYNAL, Lake
Success NY). mRNA can be purified from the cells using methods well known by
those of skill in
the art. Expression of mRNA encoding REMAP and other genes of interest can be
analyzed by
northern analysis or microarray techniques.
XV. Production of REMAP Specific Antibodies
REMAP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the REMAP amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
I~LH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide and anti-REMAP activity by, for example, binding the peptide or
REMAP to a substrate,
blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat
anti-rabbit IgG.
XVI. Purification of Naturally Occurring REMAP Using Specific Antibodies
Naturally occurring or recombinant REMAP is substantially purified by
immunoaffinity
chromatography using antibodies specific for REMAP. An immunoaffinity column
is constructed by
covalently coupling anti-REMAP 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 REMAP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of REMAP (e.g.,
high ionic strength
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CA 02435260 2003-07-17
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buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/REMAP 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 REMAP is collected.
XVII, Identification of Molecules Which Interact with REMAP
REMAP, or biologically active fragments thereof, are labeled with izsI Bolton-
Hunter reagent.
(See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a mufti-well plate are incubated with the
labeled REMAP, washed,
and any wells with labeled REMAP complex are assayed. Data obtained using
different
concentrations of REMAP are used to calculate values for the number, affinity,
and association of
REMAP with the candidate molecules.
Alternatively, molecules interacting with REMAP are analyzed using the yeast
two-hybrid
system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
REMAP may also be used in the PATHCALLING process (CuraGen Corp., New Haven
CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
XVIII. Demonstration of REMAP Activity
An assay for REMAP activity measures the expression of REMAP on the cell
surface. cDNA
encoding REMAP is transfected into an appropriate mammalian cell line. Cell
surface proteins are
labeled with biotin as described (de la Fuente, M.A. et al. (1997) Blood
90:2398-2405).
Immunoprecipitations are performed using REMAP-specific antibodies, and
immunoprecipitated
samples are analyzed using sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE)
and immunoblotting techniques. The ratio of labeled immunoprecipitant to
unlabeled
immunoprecipitant is proportional to the amount of REMAP expressed on the cell
surface.
In the alternative, an assay for REMAP activity is based on a prototypical
assay for
ligand/receptor-mediated modulation of cell proliferation. This assay measures
the rate of DNA
synthesis in Swiss mouse 3T3 cells. A plasmid containing polynucleotides
encoding REMAP is
added to quiescent 3T3 cultured cells using transfection methods well known in
the art. The
transiently transfected cells axe then incubated in the presence of
[3H]thymidine, a radioactive DNA
precursor molecule. Varying amounts of REMAP ligand are then added to the
cultured cells.
Incorporation of [3H]thymidine into acid-precipitable DNA is measured over an
appropriate time
interval using a radioisotope counter, and the amount incorporated is directly
proportional to the
amount of newly synthesized DNA. A linear dose-response curve over at least a
hundred-fold
REMAP ligand concentration range is indicative of receptor activity. One unit
of activity per
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WO 02/057454 PCT/US02/01339
milliliter is defined as the concentration of REMAP producing a 50% response
level, where 100%
represents maximal incorporation of [3H]thymidine into acid-precipitable DNA
(McKay, I. and T.
Leigh, eds. (1993) Growth Factors: A Practical Approach, Oxford University
Press, New York NY, p.
73.)
Tn a further alternative, the assay for REMAP activity is based upon the
ability of GPCR
family proteins to modulate G protein-activated second messenger signal
transduction pathways (e.g.,
CAMP; Gaudin, P. et al. (1998) J. Biol. Chem. 273:4990-4996). A plasmid
encoding full length
REMAP is transfected into a mammalian cell line (e.g., Chinese hamster ovary
(CHO) or human
embryonic kidney (HEK-293) cell lines) using methods well-known in the art.
Transfected cells are
grown in 12-well trays in culture medium for 48 hours, then the culture medium
is discarded, and the
attached cells are gently washed with PBS. The cells are then incubated in
culture medium with or
without ligand for 30 minutes, then the medium is removed and cells lysed by
treatment with 1 M
perchloric acid. The cAMP levels in the lysate are measured by
radioimmunoassay using methods
°well-known in the art. Changes in the levels of cAMP in the lysate
from cells exposed to ligand
compared to those without ligand are proportional to the amount of REMAP
present in the
transfected cells.
To measure changes in inositol phosphate levels, the cells are grown in 24-
well plates
containing 1x105 cells/well and incubated with inositol-free media and
[3H]myoinositol, 2 ~,Ci/well,
for 48 hr. The culture medium is removed, and the cells washed with buffer
containing 10 mM LiCI
followed by addition of ligand. The reaction is stopped by addition of
perchloric acid. Inositol
phosphates are extracted and separated on Dowex AGl-X8 (Bio-Rad) anion
exchange resin, and the
total labeled inositol phosphates counted by liquid scintillation. Changes in
the levels of labeled
inositol phosphate from cells exposed to ligand compared to those without
ligand are proportional to
the amount of REMAP present in the transfected cells.
In a further alternative, the ion conductance capacity of REMAP is
demonstrated using an
electrophysiological assay. REMAP is expressed by transforming a mammalian
cell line such as
COS7, HeLa or CHO with a eukaryotic expression vector encoding REMAP.
Eukaryotic expression
vectors are commercially available, and the techniques to introduce them into
cells are well known to
those skilled in the art. A small amount of a second plasmid, which expresses
any one of a number of
marker genes such as (3-galactosidase, is co-transformed into the cells in
order to allow rapid
identification of those cells which have taken up and expressed the foreign
DNA. The cells are
incubated for 48-72 hours after transformation under conditions appropriate
for the cell line to allow
expression and accumulation of REMAP and (3-galactosidase. Transformed cells
expressing (3-
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
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WO 02/057454 PCT/US02/01339
conductance due to various ions by electrophysiological techniques that are
well known in the art.
Untransformed cells, and/or cells transformed with either vector sequences
alone or (3-galactosidase
sequences alone, are used as controls and tested in parallel. The contribution
of REMAP to canon or
anion conductance cay be shown by incubating the cells using antibodies
specific for either REMAP.
The respective antibodies will bind to the extracellular side of REMAP,
thereby blocking the pore in
the ion channel, and the associated conductance.
In a further alternative, REMAP transport activity is assayed by measuring
uptake of labeled
substrates into Xenopus laevis oocytes. Oocytes at stages V and VI are
injected with REMAP mRNA
(10 ng per oocyte) and incubated for 3 days at 18 °C in OR2 medium
(82.5 mM NaCI, 2.5 mM KCI, 1
mM CaClz, 1 mM MgCIz, 1 mM Na.,HP04, 5 mM Hepes, 3.8 mM NaOH , 50 ~,g/ml
gentamycin, pH
7.8) to allow expression of REMAP protein. Oocytes are then transferred to
standard uptake medium
(I00 mM NaCI, 2 mM KCI, I mM CaCl2, I nnM MgCl2, 10 mM HepeslTris pH 7.5).
Uptake of
various substrates (e.g., amino acids, sugars, drugs, and neurotransmitters)
is initiated by adding a 3H
substrate to the oocytes. After incubating for 30 minutes, uptake is
terminated by washing the
oocytes three times in Na+-free medium, measuring the incorporated 3H, and
comparing with
controls. REMAP activity is proportional to the level of internalized 3H
substrate.
In a further alternative, REMAP protein kinase (PK) activity is measured by
phosphorylation
of a protein substrate using gamma-labeled [32P]-ATP and quantitation of the
incorporated
radioactivity using a gamma radioisotope counter. REMAP is incubated with the
protein substrate,
[32P]-ATP, and an appropriate kinase buffer. The 32P incorporated into the
product is separated from
free [3zP]-ATP by electrophoresis and the incorporated 32P is counted. The
amount of 32P recovered is
proportional to the PK activity of REMAP in the assay. A determination of the
specific amino acid
residue phosphorylated is made by phosphoamino acid analysis of the hydrolyzed
protein.
XIX. Identification of REMAP Ligands
REMAP is expressed in a eukaryotic cell line such as CHO (Chinese Hamster
Ovary) or HEK
(Human Embryonic Kidney) 293 which have a good history of GPCR expression and
which contain a
wide range of G-proteins allowing for functional coupling of the expressed
REMAP to downstream
effectors. The transformed cells are assayed for activation of the expressed
receptors in the presence
of candidate ligands. Activity is measured by changes in intracellular second
messengers, such as
cyclic AMP or Caz~. These may be measured directly using standard methods well
known in the art,
or by the use of reporter gene assays in which a luminescent protein (e.g.
firefly luciferase or green
fluorescent protein) is under the transcriptional control of a promoter
responsive to the stimulation of
protein kinase C by the activated receptor (Milligan, G. et al. (1996) Trends
Pharmacol. Sci. 17:235-
237). Assay technologies are available for both of these second messenger
systems to allow high
throughput readout in mufti-well plate format, such as the adenylyl cyclase
activation FlashPlate
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CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Assay (NEN Life Sciences Products), or fluorescent Ca2+ indicators such as
Fluo-4 AM (Molecular
Probes) in combination with the FLIPR fluorimetric plate reading system
(Molecular Devices). In
cases where the physiologically relevant second messenger pathway is not
known, REMAP may be
coexpressed with the G-proteins Gaisns which have been demonstrated to couple
to a wide range of G-
proteins (Offermanns, S. and M.I. Simon (1995) J. Biol. Chem. 270:15175-
15180), in order to funnel
the signal transduction of the REMAP through a pathway involving phospholipase
C and Caz+
mobilization. Alternatively, REMAP may be expressed in engineered yeast
systems which lack
endogenous GPCRs, thus providing the advantage of a null background for REMAP
activation
screening. These yeast systems substitute a human GPCR and Ga protein for the
corresponding
components of the endogenous yeast pheromone receptor pathway. Downstream
signaling pathways
are also modified so that the normal yeast response to the signal is converted
to positive growth on
selective media or to reporter gene expression (Broach, J.R. and J. Thorner
(1996) Nature 384
(suppl.):14-16). The receptors are screened against putative ligands including
known GPCR ligands
and other naturally occurring bioactive molecules. Biological extracts from
tissues, biological fluids
and cell supernatants are also screened.
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the
invention. Although the invention has been described in connection with
certain embodiments, it
should be understood that the invention as claimed should not be unduly
limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying
out the invention
which are obvious to those skilled in molecular biology or related fields are
intended to be within the
scope of the following claims.
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CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
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CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
<110> INCYTE GENOMICS, TNC.
LEE, Ernestine A.
WALIA, Narinder K.
BAUGHN, Mariah R.
AZIMZAI, Yalda
TANG, Y. Tom
YUE, Henry
THANGAVELU, Kavitha
XU, Yuming
ARVIZU, Chandra
WARREN, Bridget A.
YAO, Monique G.
AU-YOUNG, Janice
HAFALIA, April J.A.
ELLIOTT, Vicki S.
KALLICK, Deborah A.
GANDHI, Ameena R.
RICHARDSON, Thomas W.
KHAN, Farrah A.
LU, Yan
SWARNAKAR, Anita
RAMKUMAR, Jayala~ani
NGUYEN, Danniel B.
GRAUL, Richard
LU, Dyung Aina M.
<120> RECEPTORS AND MEMBRANE-ASSOCIATED PROTEINS
<130> PI-0346 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/262,838; 60/265,927; 60/271,196; 60/274,549; 60/334,179
<151> 2001-01-19; 2001-02-02; 2001-02-23; 2001-03-09; 2001-11-28
<160> 30
<170> PERL Program
<210> 1
<211> 457
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 71924779CD1
<400> 1
Met Tyr G1u Ser Val Glu Val G1y Gly Pro Thr Pro Asn Pro Phe
1 5 10 15
Leu Val Val Asp Phe Tyr Asn G1n Asn Arg Ala Cys Leu Leu Pro
20 25 30
Glu Lys Gly Leu Pro A1a Pro G1y Pro Tyr Ser Thr Pro Leu Arg
35 40 45
Thr Pro Leu Trp Asn Gly Ser Asn His Ser Ile Glu Thr Gln Ser
50 55 60
Ser Ser Ser Glu Glu Ile Val Pro Ser Pro Pro Ser Pro Pro Pro
65 70 75
Leu Pro Arg Ile Tyr Lys Pro Cys Phe Val Cys Gln Asp Lys Ser
80 85 90
1/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Ser Gly Tyr His Tyr Gly Val Ser Ala Cys Glu Gly Cys Lys Gly
95 100 105
Phe Phe Arg Arg Ser Ile Gln Lys Asn Met Val Tyr Thr Cys His
110 115 120
Arg Asp Lys Asn Cys Ile Ile Asn Lys Val Thr Arg Asn Arg Cys
125 130 135
Gln Tyr Cys Arg Leu Gln Lys Cys Phe Glu Val Gly Met Ser Lys
140 145 150
Glu Ser Va1 Arg Asn Asp Arg Asn Lys Lys Lys Lys Glu Val Pro
155 160 165
Lys Pro Glu Cys Ser Glu Ser Tyr Thr Leu Thr Pro Glu Val Gly
170 175 180
Glu Leu Ile Glu Lys Val Arg Lys Ala His Gln Glu Thr Phe Pro
185 190 195
Ala Leu Cys Gln Leu Gly Lys Tyr Thr Thr Asn Asn Ser Ser Glu
200 205 210
Gln Arg Val Ser Leu Asp Ile Asp Leu Trp Asp Lys Phe Ser Glu
215 220 225
Leu Ser Thr Lys Cys Ile Ile Lys Thr Val Glu Phe Ala Lys Gln
230 235 240
Leu Pro Gly Phe Thr Thr Leu Thr Ile Ala Asp Gln Ile Thr Leu
245 250 255
Leu Lys Ala Ala Cys Leu Asp Ile Leu Ile Leu Arg Ile Cys Thr
260 265 270
Arg Tyr Thr Pro Glu Gln Asp Thr Met Thr Phe Ser Asp Gly Leu
275 280 285
Thr Leu Asn Arg Thr Gln Met His Asn Ala Gly Phe Gly Pro Leu
290 295 300
Thr Asp Leu Val Phe Ala Phe Ala Asn Gln Leu Leu Pro Leu Glu
305 310 315
Met Asp Asp Ala Glu Thr Gly Leu Leu Ser Ala Ile Cys Leu Ile
320 325 330
Cys Gly Asp Arg Gln Asp Leu Glu Gln Pro Asp Arg Val Asp Met
335 340 345
Leu Gln Glu Pro Leu Leu Glu Ala Leu Lys Val Tyr Val Arg Lys
350 355 360
Arg Arg Pro Ser Arg Pro His Met Phe Pro Lys Met Leu Met Lys
365 370 375
Ile Thr Asp Leu Arg Ser Ile Ser Ala Lys Gly Ala Glu Arg Va1
380 385 390
Ile Thr Leu Lys Met Glu I1e Pro Gly Ser Met Pro Pro Leu Ile
395 400 405
Gln Glu Met Leu Glu Asn Ser Glu Gly Leu Asp Thr Leu Ser Gly
410 415 420
Gln Pro Gly Gly Gly Gly Arg Asp Gly Gly Gly Leu Ala Pro Pro
425 430 435
Pro Gly Ser Cys Ser Pro Ser Leu Ser Pro Ser Ser Asn Arg Ser
440 445 450
Ser Pro Ala Thr His Ser Pro
455
<210> 2
<211> 663
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2319430CD1
<400> 2
Met Ala A1a Lys Glu Lys Leu Glu Ala Val Leu Asn Val Ala Leu
1 5 10 15
2/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Arg Val Pro Ser Ile Met Leu Leu Asp Val Leu Tyr Arg Trp Asp
20 25 30
Val Ser Ser Phe Phe Gln Gln Ile Gln Arg Ser Ser Leu Ser Asn
35 40 45
Asn Pro Leu Phe Gln Tyr Lys Tyr Leu Ala Leu Asn Met His Tyr
50 ~ 55 60
Val Gly Tyr Ile Leu Ser Val Val Leu Leu Thr Leu Pro Arg Gln
65 70 75
His Leu Val Gln Leu Tyr Leu Tyr Phe Leu Thr Ala Leu Leu Leu
80 85 90
Tyr Ala Gly His Gln Ile Ser Arg Asp Tyr Val Arg Ser Glu Leu
95 100 105
Glu Phe Ala Tyr G1u Gly Pro Met Tyr Leu Glu Pro Leu Ser Met
110 115 120
Asn Arg Phe Thr Thr Ala Leu Ile Gly Gln Leu Val Val Cys Thr
125 130 135
Leu Cys Ser Cys Val Met Lys Thr Lys Gln Ile Trp Leu Phe Ser
140 145 150
Ala His Met Leu Pro Leu Leu Ala Arg Leu Cys Leu Val Pro Leu
155 160 165
Glu Thr Ile Val Ile Ile Asn Lys Phe Ala Met Ile Phe Thr Gly
270 275 180
Leu Glu Val Leu Tyr Phe Leu Gly Ser Asn Leu Leu Val Pro Tyr
185 290 195
Asn Leu Ala Lys Ser Ala Tyr Arg Glu Leu Val Gln Val Val Glu
200 205 210
Val Tyr Gly Leu Leu Ala Leu Gly Met Ser Leu Trp Asn Gln Leu
215 220 225
Va1 Val Pro Val Leu Phe Met Val Phe Trp Leu Val Leu Phe Ala
230 235 240
Leu Gln Ile Tyr Ser Tyr Phe Ser Thr Arg Asp Gln Pro Ala Ser
245 250 255
Arg Glu Arg Leu Leu Phe Leu Phe Leu Thr Ser Ile Ala Glu.Cys
260 265 270
Cys Ser Thr Pro Tyr Ser Leu Leu Gly Leu Val Phe Thr Val Ser
275 280 285
Phe Val A1a Leu G1y Val Leu Thr Leu Cys Lys Phe Tyr Leu Gln
290 295 300
Gly Tyr Arg Ala Phe Met Asn Asp Pro Ala Met Asn Arg Gly Met
305 310 315
Thr Glu Gly Val Thr Leu Leu Ile Leu Ala Val Gln Thr G1y Leu
320 325 330
Ile Glu Leu Gln Val Val His Arg Ala Phe Leu Leu Ser Ile Ile
335 340 345
Leu Phe Ile Val Val Ala Ser Ile Leu Gln Ser Met Leu Glu Ile
350 355 360
Ala Asp Pro Ile Val Leu Ala Leu Gly Ala Ser Arg Asp Lys Ser
365 370 375
Leu Trp Lys His Phe Arg Ala Val Ser Leu Cys Leu Phe Leu Leu
380 385 390
Val Phe Pro Ala Tyr Met Ala Tyr Met Ile Cys Gln Phe Phe His
395 400 405
Met Asp Phe Trp Leu Leu Ile Ile Ile Ser Ser Ser Ile Leu Thr
410 41.5 420
Ser Leu Gln Val Leu Gly Thr Leu Phe Ile Tyr Val Leu Phe Met
425 430 435
Val Glu Glu Phe Arg Lys Glu Pro Val Glu Asn Met Asp Asp Val
440 445 450
Ile Tyr Tyr Val Asn Gly Thr Tyr Arg Leu Leu Glu Phe Leu Val
455 460 465
Ala Leu Cys Val Val Ala Tyr Gly Val Ser G1u Thr Ile Phe Gly
470 475 480
3/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Glu Trp Thr Val Met Gly Ser Met Ile Ile Phe Ile His Ser Tyr
485 490 495
Tyr Asn Val Trp Leu Arg Ala Gln Leu Gly Trp Lys Ser Phe Leu
500 505 510
Leu Arg Arg Asp Ala Val Asn Lys Ile Lys Ser Leu Pro Ile Ala
515 520 525
Thr Lys Glu Gln Leu Glu Lys His Asn Asp I1e Cys Ala Ile Cys
530 535 540
Tyr Gln Asp Met Lys Ser Ala Val Ile Thr Pro Cys Ser His Phe
545 550 555
Phe His Ala Gly Cys Leu Lys Lys Trp Leu Tyr Val Gln Glu Thr
560 565 570
Cys Pro Leu Cys His Cys His Leu Lys Asn Ser Ser Gln Leu Pro
575 580 585
Gly Leu Gly Thr Glu Pro Val Leu Gln Pro His Ala Gly Ala Glu
590 595 . 600
Gln Asn Val Met Phe Gln Glu Gly Thr Glu Pro Pro Gly Gln Glu
605 610 615
His Thr Pro Gly Thr Arg Ile Gln Glu Gly Ser Arg Asp Asn Asn
620 625 630
Glu Tyr Ile Ala Arg Arg Pro Asp Asn Gln G1u Gly Ala Phe Asp
635 640 645
Pro Lys Glu Tyr Pro His Ser Ala Lys Asp Glu Ala His Pro Val
650 655 660
Glu Ser Ala
<210> 3
<211> 504
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7291877CD1
<400> 3
Met Lys Gly Ile Arg Lys Gly Glu Ser Arg Ala Lys Glu Ser Lys
1 5 10 15
Pro Trp Glu Pro Gly Lys Arg Arg Cys Ala Lys Cys Gly Arg Leu
20 25 30
Asp Phe Ile Leu Met Lys Lys Met Gly Ile Lys Ser Gly Phe Thr
35 40 45
Phe Trp Asn Leu Val Phe Leu Leu Thr Val Ser Cys Val Lys Gly
50 55 60
Phe Ile Tyr Thr Cys Gly Gly Thr Leu Lys Gly Leu Asn Gly Thr
65 70 75
Ile Glu Ser Pro Gly Phe Pro Tyr Gly Tyr Pro Asn Gly A1a Asn
80 85 90
Cys Thr Trp Val Ile Ile Ala Glu Glu Arg Asn Arg Ile Gln Ile
95 100 105
Val Phe Gln Ser Phe Ala Leu Glu Glu Glu Tyr Asp Tyr Leu Ser
110 115 120
Leu Tyr Asp Gly His Pro His Pro Thr Asn Phe Arg Thr Arg Leu
125 130 135
Thr Gly Phe His Leu Pro Pro Pro Val Thr Ser Thr Lys Ser Val
140 145 150
Phe Ser Leu Arg Leu Thr Ser Asp Phe Ala Val Ser Ala His Gly
155 160 165
Phe Lys Val Tyr Tyr Glu Glu Leu Gln Ser Ser Ser Cys Gl.y Asn
170 175 180
Pro Gly Val Pro Pro Lys Gly Val Leu Tyr Gly Thr Arg Phe Asp
185 190 195
4/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Val Gly Asp Lys Ile Arg Tyr Ser Cys Val Thr Gly Tyr Ile Leu
200 205 220
Asp Gly His Pro Gln Leu Thr Cys Ile Ala Asn Ser Val Asn Thr
215 220 225
Ala Ser Trp Asp Phe Pro Val Pro Ile Cys Arg Ala Glu Asp Ala
230 235 240
Cys Gly Gly Thr Met Arg Gly Ser Ser G1y Ile Ile Ser Ser Pro
245 250 255
Ser Phe Pro Asn Glu Tyr His Asn Asn Ala Asp Cys Thr Trp Thr
260 265 270
Ile Val Ala Glu Pro Gly Asp Thr Ile Ser Leu Ile Phe Thr Asp
275 280 285
Phe Gln Met G1u Glu Lys Tyr Asp Tyr Leu Glu Ile Glu Gly Ser
290 295 300
Glu Pro Pro Thr Ile Trp Leu Ser Gly Met Asn Ile Pro Pro Pro
305 310 315
Ile Ile Ser Asn Lys Asn Trp Leu Arg Leu His Phe Val Thr Asp
320 325 330
Ser Asn His Arg Tyr Arg Gly Phe Ser Ala Pro Tyr Gln Gly Ser
335 340 345
Ser Thr Leu Thr His Thr Thr Ser Thr Gly Glu Leu Glu Glu His
350 355 360
Asn Arg Thr Thr Thr Gly Ala Ile Ala Val Ala Ser Thr Pro Ala
365 370 375
Asp Va1 Thr Val Ser Ser Val Thr Ala Val Thr Ile His Arg Leu
380 385 390
Ser Glu Glu Gln Arg Val Gln Val Thr Ser Leu Arg Asn Ser Gly
395 400 405
Leu Asp Pro Asn Thr Ser Lys Asp Gly Leu Ser Pro His Pro Ala
410 415 420
Asp Thr Gln Ser Thr Arg Arg Arg Pro Arg His Ala G1u Gln Ile
425 430 435
Glu Arg Thr Lys Glu Leu Ala Val Val Thr His Arg Gly His Cys
440 445 450
Asn Arg VaI Glu Asp Ile Glu Lys Pro Ile Leu VaI Val Gln Asp
455 460 465
Arg Phe Cys Lys Met Asn Ser Asp Gln Ser Thr Lys Glu Val Thr
470 ' 475 480
Val Cys Met Gln Arg Val Ser Leu Leu Ser Tyr Phe Phe Asn Glu
485 490 495
Leu Val Asn Asn Arg Lys Pro Ile Ala
500
<210> 4
<211> 1114
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1218126CD1
<400> 4
Met Ala Pro Thr Leu Phe Gln Lys Leu Phe Ser Lys Arg Thr G1y
1 5 10 15
Leu Gly Ala Pro Gly Arg Asp Ala Arg Asp Pro Asp Cys Gly Phe
20 25 30
Ser '~rp Pro Leu Pro Glu Phe Asp Pro Ser Gln Ile Arg Leu Ile
35 40 45
Val Tyr Gln Asp Cys Glu Arg Arg Gly Arg Asn Val Leu Phe Asp
50 55 60
Ser Ser Val Lys Arg Arg Asn Glu Asp Ile Ser Va1 Ser Lys Leu
65 70 75
5/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Cys Ser Asp Ala Gln Val Lys Val Phe Gly Lys Cys Cys Gln Leu
80 85 90
Lys Pro Gly Gly Asp Ser Ser Ser Ser Leu Asp Ser Ser Val Thr
95 100 105
Ser Ser Ser Asp Ile Lys Asp Gln Cys Leu Lys Tyr Gln Gly Ser
210 115 120
Arg Cys Ser Ser Asp Ala Asn Met Leu Gly Glu Met Met Phe Gly
125 130 135
Ser Val Ala Met Ser Tyr Lys Gly Ser Thr Leu Lys Ile His Gln
140 145 150
Ile Arg Ser Pro Pro Gln Leu Met Leu Ser Lys Val Phe Thr Ala
155 160 165
Arg Thr Gly Ser Ser Ile Cys Gly Ser Leu Asn Thr Leu Gln Asp
170 175 180
Ser Leu Glu Phe Ile Asn Gln Asp Asn Asn Thr Leu Lys A1a Asp
185 190 195
Asn Asn Thr Val Tle Asn Gly Leu Leu Gly Asn Ile Ala Ser Leu
200 205 210
Ser Ser Leu Leu Tle Thr Pro Phe Pro Ser Pro Asn Ser Ser Leu
215 220 225
Thr Arg Ser Cys Ala Ser Ser Tyr Gln Arg Arg Trp Arg Arg Ser
230 235 240
Gln Thr Thr Ser Leu Glu Asn Gly Val Phe Pro Arg Trp Ser Ile
245 250 255
Glu Glu Ser Phe Asn Leu Ser Asp Glu Ser Cys Gly Pro Asn Pro
260 265 270
Gly Ile Val Arg Lys Lys Lys Ile Ala Ile Gly Val Ile Phe Ser
275 280 285
Leu Ser Lys Asp Glu Asp Glu Asn Asn Lys Phe Asn Glu Phe Phe
290 295 300
Phe Ser His Phe Pro Leu Phe Glu Ser Tyr Met Asn Lys Leu Lys
305 310 315
Ser Ala Ile G1u Gln Ala Met Lys Met Ser Arg Arg Ser A1a Asp
320 325 330
Ala Ser Gln Arg Ser Leu Ala Tyr Asn Arg Ile Val Asp Ala Leu
335 340 345
Asn G1u Phe Arg Thr Thr Ile Cys Asn Leu Tyr Thr Met Pro Arg
350 355 360
Ile G1y Glu Pro Val Trp Leu Thr Met Met Ser Gly Thr Pro Glu
365 370 375
Lys Asn His Leu Cys Tyr Arg Phe Met Lys Glu Phe Thr Phe Leu
380 385 390
Met Glu Asn Ala Ser Lys Asn Gln Phe Leu Pro Ala Leu Ile Thr
395 400 405
Ala Val Leu Thr Asn His Leu Ala Trp Val Pro Thr Val Met Pro
410 415 420
Asn Gly Gln Pro Pro Ile Lys Ile Phe Leu Glu Lys His Ser Ser
425 430 435
G1n Ser Val Asp Met Leu A1a Lys Thr His Pro Tyr Asn Pro Leu
440 445 450
Trp A1a Gln Leu Gly Asp Leu Tyr Gly Ala Ile Gly Ser Pro Val
455 460 465
Arg Leu Ala Arg Thr Val Val Va1 Gly Lys Arg Gln Asp Met Val
470 475 480
Gln Arg Leu Leu Tyr Phe Leu Thr Tyr Phe Ile Arg Cys Ser Glu
485 490 495
Leu Gln Glu Thr His Leu Leu Glu Asn Gly Glu Asp Glu Ala Ile
500 505 510
Val Met Pro Gly Thr Val Ile Thr Thr Thr Leu Glu Lys Gly Glu
515 520 525
Ile GIu Glu Ser Glu Tyr Val Leu Val Thr Met His Arg Asn Lys
530 535 540
6/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Ser Ser Leu Leu Phe Lys Glu Ser Glu Glu Ile Arg Thr Pro Asn
545 550 555
Cys Asn Cys Lys Tyr Cys Ser His Pro Leu Leu Gly Gln Asn Val
560 565 570
Glu Asn Ile Ser Gln Gln Glu Arg Glu Asp Ile Gln Asn Ser Ser
575 580 585
Lys Glu Leu Leu Gly Ile Ser Asp Glu Cys Arg Met Ile Ser Pro
590 595 600
Ser Asp Cys Gln Glu Glu Asn Ala Val Asp Val Lys Gln Tyr Arg
605 610 615
Asp Lys Leu Arg Thr Cys Phe Asp Ala Lys Leu Glu Thr Val Val
620 625 630
Cys Thr Gly Ser Val Pro Val Asp Lys Cys Ala Leu Ser Glu Ser
635 640 645
Gly Leu Glu Ser Thr Glu Glu Thr Trp Gln Ser Glu Lys Leu Leu
650 655 660
Asp Ser Asp Ser His Thr Gly Lys Ala Met Arg Ser Thr Gly Met
665 670 675
Val Val Glu Lys Lys Pro Pro Asp Lys Ile Val Pro Ala Ser Phe
680 685 690
Ser Cys Glu Ala Ala Gln Thr Lys Val Thr Phe Leu I1e Gly Asp
695 700 705
Ser Met Ser Pro Asp Ser Asp Thr Glu Leu Arg Ser Gln Ala Val
710 715 720
Val Asp Gln Ile Thr Arg His His Thr Lys Pro Leu Lys Glu Glu
725 730 735
Arg Gly Ala Ile Asp Gln His Gln Glu Thr Lys Gln Thr Thr Lys
740 745 750
Asp Gln Ser Gly Glu Ser Asp Thr Gln Asn Met Val Ser Glu Glu
755 760 765
Pro Cys Glu Leu Pro Cys Trp Asn His Ser Asp Pro Glu Ser Met
770 775 780
Ser Leu Phe Asp Glu Tyr Phe Asn Asp Asp Ser Ile Glu Thr Arg
785 790 795
Thr I1e Asp Asp Val Pro Phe Lys Thr Ser Thr Asp Ser Lys Asp
800 805 810
His Cys Cys Met Leu Glu Phe Ser Lys Ile Leu Cys Thr Lys Asn
815 820 825
Asn Lys Gln Asn Asn Glu Phe Cys Lys Cys Ile Glu Thr Val Pro
830 835 840
Gln Asp Ser Cys Lys Thr Cys Phe Pro Gln Gln Asp Gln Arg Asp
845 850 855
Thr Leu Ser Ile Leu Val Pro His Gly Asp Lys Glu Ser Ser Asp
860 865 870
Lys Lys Ile Ala Val Gly Thr Glu Trp Asp Ile Pro Arg Asn Glu
875 880 885
Ser Ser Asp Ser Ala Leu Gly Asp Ser Glu Ser Glu Asp Thr G1y
890 895 900
His Asp Met Thr Arg Gln Val Ser Ser Tyr Tyr Gly Gly Glu Gln
905 910 915
Glu Asp Trp Ala Glu Glu Asp Glu Ile Pro Phe Pro Gly Ser Lys
920 925 930
Leu Ile Glu Val Ser Ala Val Gln Pro Asn Ile Ala Asn Phe Gly
935 940 945
Arg Ser Leu Leu Gly Gly Tyr Cys Ser Ser Tyr Va1 Pro Asp Phe
950 955 960
Val Leu Gln Gly Ile Gly Ser Asp Glu Arg Phe Arg Gln Cys Leu
965 970 975
Met Ser Asp Leu Ser His Ala Val Gln His Pro Val Leu Asp Glu
980 985 990
Pro Ile Ala Glu Ala Val Cys Ile Ile Ala Asp Met Asp Lys Trp
995 1000 1005
7/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Thr Val Gln Val Ala Ser Ser Gln Arg Arg Val Thr Asp Asn Lys
1010 1015 1020
Leu Gly Lys Glu Val Leu Val Ser Ser Leu Val Ser Asn Leu Leu
1025 1030 1035
His Ser Thr Leu Gln Leu Tyr Lys His Asn Leu Ser Pro Asn Phe
1040 1045 1050
Cys Val Met His Leu Glu Asp Arg Leu Gln G1u Leu Tyr Phe Lys
1055 1060 2065
Ser Lys Met Leu Ser Glu Tyr Leu Arg Gly Gln Met Arg Val His
1070 1075 1080
Val Lys Glu Leu Gly Val Val Leu Gly Ile Glu Ser Ser Asp Leu
1085 1090 1095
Pro Leu Leu Ala Ala Val Ala Ser Thr His Ser Pro Tyr Val Ala
1100 1.105 1110
G1n Ile Leu Leu
<210> 5
<221> 479
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7479161CD1
<400> 5
Met Gln Pro Val Met Leu Ala Leu Trp Ser Leu Leu Leu Leu Trp
1 5 10 15
Gly Leu Ala Thr Pro Cys Gln Glu Leu Leu Glu Thr Val Gly Thr
20 25 30
Leu Ala Arg Ile Asp Lys Asp Glu Leu Gly Lys Ala Ile G1n Asn
35 40 45
Ser Leu Val Gly Glu Pro Ile Leu Gln Asn Val Leu Gly Ser Val
50 55 60
Thr AIa Val Asn Arg Gly Leu Leu G1y Ser Gly Gly Leu Leu Gly
65 70 75
Gly Gly Gly Leu Leu Gly His Gly Gly Val Phe Gly Val Val Glu
80 85 90
Glu Leu Ser Gly Leu Lys Tle Glu Glu Leu Thr Leu Pro Lys Val
95 100 105
Leu Leu Lys Leu Leu Pro Gly Phe Gly Val Gln Leu Ser Leu His
110 115 120
Thr Lys Val Gly Met His Cys Ser Gly Pro Leu Gly Gly Leu Leu
125 130 135
GIn Leu Ala Ala Glu Val Asn Val Thr Ser Arg Val AIa Leu Ala
140 145 150
Val Ser Ser Arg Gly Thr Pro Ile Leu Ile Leu Lys Arg Cys Ser
155 160 165
Thr Leu Leu Gly His Ile Ser Leu Phe Ser Gly Leu Leu Pro Thr
170 175 180
Pro Leu Phe Gly Val Val Glu Gln Met Leu Phe Lys Va1 Leu Pro
185 190 195
Gly Leu Leu Cys Pro Val Va1 Asp Ser Val Leu Gly Val Val Asn
200 205 210
Glu Leu Leu Gly Ala Val Leu Gly Leu Val Ser Leu Gly Ala Leu
215 220 225
Gly Ser Val Glu Phe Ser Leu Ala Thr Leu Pro Leu Ile Ser Asn
230 235 240
Gln Tyr Ile Glu Leu Asp Ile Asn Pro Ile Val Lys Ser Val Ala
245 250 255
G1y Asp Ile Ile Asp Phe Pro Lys Ser Arg Ala Pro Ala Lys Va1
260 265 270
8/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Pro Pro Lys Lys Asp His Thr Ser Gln Val Met Val Pro Leu Tyr
275 280 285
Leu Phe Asn Thr Thr Phe Gly Leu Leu Gln Thr Asn Gly Ala Leu
290 295 300
Asp Met Asp Ile Thr Pro Glu Leu Val Pro Ser Asp Val Pro Leu
305 310 315
Thr Thr Thr Asp Leu Ala Ala Leu Leu Pro Glu Ala Leu Gly Lys
320 325 330 .
Leu Pro Leu His Gln Gln Leu Leu Leu Phe Leu Arg Val Arg Glu
335 340 345
Ala Pro Thr Val Thr Leu His Asn Lys Lys Ala Leu Val Ser Leu
350 355 360
Pro Ala Asn Ile His Val Leu Phe Tyr Val Pro Lys Gly Thr Pro
365 370 375
Glu Ser Leu Phe Glu Leu Asn Ser Val Met Thr Val Arg Ala Gln
380 385 390
Leu Ala Pro Ser Ala Thr Lys Leu His I1e Ser Leu Ser Leu Glu
395 400 405
Arg Leu Ser Val Lys Val Ala Ser Ser Phe Thr His Ala Phe Asp
410 415 420
Gly Ser Arg Leu Glu Glu Trp Leu Ser His Val Val Gly Ala Val
425 430 435
Tyr Ala Pro Lys Leu Asn Val Ala Leu Asp Val Gly Ile Pro Leu
440 445 450
Pro Lys Val Leu Asn Ile Asn Phe Ser Asn Ser Val Leu Glu Ile
455 460 465
Va1 Glu Asn Ala Val Ala Ala Leu Tyr Val Leu Val Val Ala
470 475
<210> 6
<211> 1774
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7722591CD1
<400> 6
Met Ala Pro Val Ser Met Leu Ser Pro Ala Ser Ser Leu Ser His
1 5 10 15
Pro Ala Gly Ala Tyr Arg G1y Thr Ser Gln Gly Pro Ser Val Gly
20 25 30
Val Thr Ala Pro Cys Gly Val Gly Glu Gly Leu Gly Ala Ser Arg
35 40 45
Gly Pro A1a Leu Pro Val Trp Ala Tyr Ala Arg Cys Pro Asp Val
50 55 60
Asp Glu Cys Arg Leu Gly Leu Ala Arg Cys His Pro Arg Ala Thr
65 70 75
Cys Leu Asn Thr Pro Leu Ser Tyr Glu Cys His Cys Gln Arg Gly
80 85 90
Tyr Gln Gly Asp Gly Ile Ser His Cys Asn Arg Thr Cys Leu Glu
95 100 105
Asp Cys Gly His Gly Val Cys Ser Gly Pro Pro Asp Phe Thr Cys
110 115 120
Val Cys Asp Leu Gly Trp Thr Ser Asp Leu Pro Pro Pro Thr Pro
125 130 135
Ala Pro Gly Pro Pro Ala Pro Arg Cys Ser Arg Asp Cys Gly Cys
140 145 150
Ser Phe His Ser His Cys Arg Lys Arg Gly Pro Gly Phe Cys Asp
155 160 165
Glu Cys Gln Asp Trp Thr Trp Gly Glu His Cys Glu Arg Cys Arg
170 175 180
9/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Pro Gly Ser Phe Gly Asn Ala Thr Gly Ser Arg Gly Cys Arg Pro
185 190 195
Cys Gln Cys Asn Gly His G1y Asp Pro Arg Arg Gly His Cys Asp
200 205 210
Asn Leu Ser Gly Leu Cys Phe Cys G1n Asp His Thr Glu Gly Ala
215 220 225
His Cys Gln Leu Cys Ser Pro Gly Tyr Tyr Gly Asp Pro Arg Ala
230 235 240
Gly Gly Ser Cys Phe Arg Glu Cys Gly Gly Arg Ala Leu Leu Thr
245 250 255
Asn Val Ser Ser Val Ala Leu Gly Ser Arg Arg Val Gly Gly Leu
260 265 270
Leu Pro Pro Gly Gly Gly Ala Ala Arg Ala Gly Pro Gly Leu Ser
275 280 285
Tyr Cys Val Trp Val Val Ser Ala Thr Glu Glu Leu Gln Pro Cys
290 295 300
Ala Pro Gly Thr Leu Cys Pro Pro Leu Thr Leu Thr Phe Ser Pro
305 ~ 310 315
Asp Ser Ser Thr Pro Cys Thr Leu Ser Tyr Val Leu Ala Phe Asp
320 325 330
Gly Phe Pro Arg Phe Leu Asp Thr Gly Val Val Gln Ser Asp Arg
335 340 345
Ser Leu Ile Ala Ala Phe Cys Gly Gln Arg Arg Asp Arg Pro Leu
350 355 360
Thr Va1 Gln Ala Leu Ser Gly Leu Leu Val Leu His Trp Glu Ala
365 370 375
Asn Gly Ser Ser Ser Trp G1y Phe Asn Ala Ser Val Gly Ser Ala
380 385 390
Arg Cys Gly Ser Gly Gly Pro Gly Ser Cys Pro Val Pro Gln Glu
395 400 405
Cys Val Pro Gln Asp Gly Ala A1a Gly A1a Gly Leu Cys Arg Cys
410 415 420
Pro G1n G1y Trp Ala Gly Pro His Cys Arg Met Ala Leu Cys Pro
425 430 435
Glu Asn Cys Asn Ala His Thr Gly Ala Gly Thr Cys Asn Gln Ser
440 445 450
Leu Gly Val Cys Ile Cys Ala Glu Gly Phe Gly Gly Pro Asp Cys
455 460 465
Ala Thr Lys Leu Asp Gly Gly Gln Leu Val Trp Glu Thr Leu Met
470 475 480
Asp Ser Arg Leu Ser Ala Asp Thr Ala Ser Arg Phe Leu His Arg
485 490 495
Leu Gly His Thr Met Val Asp Gly Pro Asp Ala Thr Leu Trp Met
500 505 510
Phe Gly G1y Leu G1y Leu Pro Gln Gly Leu Leu Gly Asn Leu Tyr
515 520 525
Arg Tyr Ser Val Ser Glu Arg Arg Trp Thr Gln Met Leu Ala Gly
530 535 540
Ala Glu Asp Gly Gly Pro Gly Pro Ser Pro Arg Ser Phe His Ala
545 550 555
Ala Ala Tyr Val Pro Ala Gly Arg Gly Ala Met Tyr Leu Leu Gly
560 565 570
Gly Leu Thr Ala Gly Gly Val Thr Arg Asp Phe Trp Val Leu Asn
575 580 585
Leu Thr Thr Leu Gln Trp Arg Gln Glu Lys Ala Pro Gln Thr Val
590 595 600
Glu Leu Pro Ala Va1 Ala Gly His Thr Leu Thr Ala Arg Arg Gly
605 610 615
Leu Ser Leu Leu Leu Val Gly Gly Tyr Ser Pro Glu Asn Gly Phe
620 625 630
Asn Gln Gln Leu Leu Glu Tyr Gln Leu Ala Thr Gly Thr Trp Va1
635 640 645
10/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Ser Gly Ala Gln Ser Gly Thr Pro Pro Thr Gly Leu Tyr Gly His
650 655 660
Ser Ala Val Tyr His Glu Ala Thr Asp Ser Leu Tyr Val Phe Gly
665 670 675
Gly Phe Arg Phe His Val Glu Leu Ala A1a Pro Ser Pro Glu Leu
680 685 690
Tyr Ser Leu His Cys Pro Asp Arg Thr Trp Ser Leu Leu Ala Pro
695 700 705
Ser Gln Gly Ala Lys Pro Arg Pro Arg Leu Phe His Ala Ser Ala
710 715 720
Leu Leu Gly Asp Thr Met Val Val Leu Gly Gly Arg Ser Asp Pro
725 730 735
Asp Glu Phe Ser Ser Asp Val Leu Leu Tyr Gln Val Asn Cys Asn
740 745 750
Ala Trp Leu Leu Pro Asp Leu Thr Arg Ser Ala Ser Val Gly Pro
755 760 765
Pro Met Glu Glu Ser Val Ala His Ala Val Ala Ala Va1 Gly Ser
770 775 780
Arg Leu Tyr Ile Ser Gly Gly Phe Gly Gly Val Ala Leu Gly Arg
785 790 795
Leu Leu Ala Leu Thr Leu Pro Pro Asp Pro Cys Arg Leu Leu Ser
800 805 810
Ser Pro Glu A1a Cys Asn Gln Ser Gly Ala Cys Thr Trp Cys His
815 820 825
Gly Ala Cys Leu Ser Gly Asp Gln Ala His Arg Leu Gly Cys Gly
830 835 840
Gly Ser Pro Cys Ser Pro Met Pro Arg Ser Pro Glu Glu Cys Arg
845 850 855
Arg Leu Arg Thr Cys Ser Glu Cys Leu Ala Arg His Pro Arg Thr
860 865 870
Leu Gln Pro Gly Asp Gly Glu Ala Ser Thr Pro Arg Cys Lys Trp
875 880 885
Cys Thr Asn Cys Pro Glu Gly Ala Cys Ile Gly Arg Asn Gly Ser
890 895 900
Cys Thr Ser Glu Asn Asp Cys Arg Ile Asn Gln Arg Glu Va1 Phe
905 910 915
Trp Ala Gly Asn Cys Ser Glu A1a Ala Cys Gly Ala Ala Asp Cys
920 925 930
Glu Gln Cys Thr Arg Glu Gly Lys Cys Met Trp Thr Arg Gln Phe
935 940 945
Lys Arg Thr Gly Glu Thr Arg Arg Ile Leu Ser Val Gln Pro Thr
950 955 960
Tyr Asp Trp Thr Cys Phe Ser His Ser Leu Leu Asn Val Ser Pro
965 970 975
Met Pro Val Glu Ser Ser Pro Pro Leu Pro Cys Pro Thr Pro Cys
980 985 990
His Leu Leu Pro Asn Cys Thr Ser Cys Leu Asp Ser Lys Gly Ala
995 1000 1005
Asp Gly Gly Trp Gln His Cys Val Trp Ser Ser Ser Leu Gln Gln
1010 1015 1020
Cys Leu Ser Pro Ser Tyr Leu Pro Leu Arg Cys Met Ala Gly Gly
1025 1030 1035
Cys Gly Arg Leu Leu Arg Gly Pro Glu Ser Cys Ser Leu Gly Cys
1040 1045 1050
Ala Gln Ala Thr Gln Cys Ala Leu Cys Leu Arg Arg Pro His Cys
1055 1060 1065
Gly Trp Cys Ala Trp Gly Gly Gln Asp Gly Gly Gly Arg Cys Met
1070 1075 1080
Glu G1y Gly Leu Ser Gly Pro Arg Asp Gly Leu Thr Cys Gly Arg
1085 1090 1095
Pro Gly Ala Ser Trp Ala Phe Leu Ser Cys Pro Pro Glu Asp Glu
1100 1105 1110
11/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Cys Ala Asn Gly His His Asp Cys Asn Glu Thr Gln Asn Cys His
1115 1120 1125
Asp Gln Pro His Gly Tyr Glu Cys Ser Cys Lys Thr Gly Tyr Thr
1130 1135 1140
Met Asp Asn Met Thr Gly Leu Cys Arg Pro Val Cys Ala Gln Gly
1145 1150 1155
Cys Val Asn Gly Ser Cys Val Glu Pro Asp His Cys Arg Cys His
1160 1165 1170
Phe Gly Phe Va1 G1y Arg Asn Cys Ser Thr Glu Cys Arg Cys Asn
1175 1180 1185
Arg His Ser Glu Cys Ala Gly Val Gly Ala Arg Asp His Cys Leu
1190 1195 1200
Leu Cys Arg Asn His Thr Lys G1y Ser His Cys Glu Gln Cys Leu
1205 1210 1215
Pro Leu Phe Val Gly Ser Ala Val Gly Gly Gly Thr Cys Arg Pro
1220 1225 1230
Cys His Ala Phe Cys Arg Gly Asn Ser His Ile Cys Ile Ser Arg
1235 1240 1245
Lys Glu Leu G1n Met Ser Lys Gly Glu Pro Lys Lys Tyr Ser Leu
1250 1255 1260
Asp Pro Glu Glu Ile Glu Asn Trp Val Thr Glu Gly Pro Ser Glu
1265 1270 1275
Asp Glu Ala Val Cys Val Asn Cys Gln Asn Asn Ser Tyr Gly Glu
1280 1285 1290
Lys Cys Glu Ser Cys Leu Gln G1y Tyr Phe Leu Leu Asp Gly Lys
1295 1300 1305
Cys Thr Lys Cys Gln Cys Asn G1y His Ala Asp Thr Cys Asn Glu
1310 1315 1320
Gln Asp Gly Thr Gly Cys Pro Cys Gln Asn Asn Thr G1u Thr Gly
1325 1330 1335
Thr Cys Gln Gly Ser Ser Pro Ser Asp Arg Arg Asp Cys Tyr Lys
1340 1345 1350
Tyr Gln Cys Ala Lys Cys Arg G1u Ser Phe His Gly Ser Pro Leu
1355 1360 1365
Gly Gly Gln Gln Cys Tyr Arg Leu Ile Ser Val Glu Gln Glu Cys
1370 1375 1380
Cys Leu Asp Pro Thr Ser Gln Thr Asn Cys Phe His Glu Pro Lys
1385 1390 1395
Arg Arg Ala Leu Gly Pro Gly Arg Thr Val Leu Phe Gly Val G1n
1'400 1405 1410
Pro Lys Phe Thr Asn Val Asp Ile Arg Leu Thr Leu Asp Val Thr
1415 1420 1425
Phe Gly Ala Va1 Asp Leu Tyr Val Ser Thr Ser Tyr Asp Thr Phe
1430 1435 1440
Val Val Arg Val Ala Pro Asp Thr Gly Val His Thr Val His Ile
1445 1450 1455
G1n Pro Pro Pro Ala Pro Pro Pro Pro Pro Pro Pro Ala Asp Gly
1460 1465 1470
Gly Pro Arg Gly Ala Gly Asp Pro Gly Gly Ala Gly Ala Ser Ser
1475 1480 1485
Gly Pro Gly Ala Pro Ala Glu Pro Arg Val Arg Glu Val Trp Pro
1490 1495 1500
Arg Gly Leu Ile Thr Tyr Val Thr Val Thr Glu Pro Ser Ala Val
1505 1510 1515
Leu Val Va1 Arg Gly Val Arg Asp Arg Leu Val Ile Thr Tyr Pro
1520 1525 1530
His Glu His His Ala Leu Lys Ser Ser Arg Phe Tyr Leu Leu Leu
1535 1540 1545
Leu Gly Val Gly Asp Pro Ser G1y Pro Gly Ala Asn Gly Ser Ala
1550 1555 1560
Asp Ser Gln Gly Leu Leu Phe Phe Arg Gln Asp Gln Ala His Ile
1565 1570 1575
12/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Asp Leu Phe Val Phe Phe Ser Val Phe Phe Ser Cys Phe Phe Leu
1580 1585 1590
Phe Leu Ser Leu Cys Val Leu Leu Trp Lys Ala Lys Gln Ala Leu
1595 1600 1605
Asp Gln Arg Gln Glu Gln Arg Arg His Leu Gln Glu Met Thr Lys
1610 1615 1620
Met Ala Ser Arg Pro Phe Ala Lys Val Thr Val Cys Phe Pro Pro
1625 1630 1635
Asp Pro Thr Ala Pro Ala Ser Ala Trp Lys Pro Ala Gly Leu Pro
1640 1645 1650
Pro Pro Ala Phe Arg Arg Ser Glu Pro Phe Leu Ala Pro Leu Leu
1655 1660 1665
Leu Thr Gly Ala Gly Gly Pro Trp Gly Pro Met Gly Gly Gly Cys
1670 1675 1680
Cys Pro Pro Ala Ile Pro Ala Thr Thr Ala Gly Leu Arg Ala Gly
1685 1690 1695
Pro Ile Thr Leu Glu Pro Thr Glu Asp Gly Met Ala Gly Val Ala
1700 1705 1710
Thr Leu Leu Leu Gln Leu Pro Gly Gly Pro His Ala Pro Asn Gly
1715 1720 1725
Ala Cys Leu Gly Ser Ala Leu Val Thr Leu Arg His Arg Leu His
1730 1735 1740
Glu Tyr Cys Gly G1y Gly Gly Gly Ala Gly Gly Ser Gly His Gly
1745 1750 1755
Thr Gly Ala Gly Arg Lys Gly Leu Leu Ser Gln Asp Asn Leu Thr
1760 1765 1770
Ser Met Ser Leu
<210> 7
<211> 393
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2173285CD1
<400> 7
Met Phe Ser Val Glu Ser Leu Glu Arg Ala Glu Leu Cys Glu Ser
1 5 10 15
Leu Leu Thr Trp Ile Gln Thr Phe Asn Val Asp Ala Pro Cys Gln
20 25 30
Thr Val Glu Asp Leu Thr Asn Gly Val Val Met Ala Gln Val Leu
35 40 45
Gln Lys 21e Asp Pro Ala Tyr Phe Asp Glu Asn Trp Leu Asn Arg
50 55 60
Ile Lys Thr Glu Val Gly Asp Asn Trp Arg Leu Lys Ile Ser Asn
65 70 75
Leu Lys Lys Ile Leu Lys Gly Ile Leu Asp Tyr Asn His Glu Ile
80 85 90
Leu Gly Gln Gln Ile Asn Asp Phe Thr Leu Pro Asp Val Asn Leu
95 100 105
Ile Gly Glu His Ser Asp Ala Ala Glu Leu Gly Arg Met Leu Gln
110 115 120
Leu Ile Leu Gly Cys Ala Val Asn Cys Glu Gln Lys Gln Glu Tyr
125 130 135
Ile Gln Ala Ile Met Met Met Glu Glu Ser Val Gln His Val Val
140 145 150
Met Thr Ala Ile Gln Glu Leu Met Ser Lys Glu Ser Pro Val Ser
155 160 165
Ala Gly Asn Asp Ala Tyr Val Asp Leu Asp Arg Gln Leu Lys Lys
170 175 180
13/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Thr Thr Glu Glu Leu Asn Glu Ala Leu Ser Ala Lys Glu Glu T1e
185 190 195
Ala Gln Arg Cys His Glu Leu Asp Met Gln Val Ala Ala Leu Gln
200 205 210
Glu Glu Lys Ser Ser Leu Leu Ala Glu Asn Gln Val Leu Met Glu
215 220 225
Arg Leu Asn Gln Ser Asp Ser Ile Glu Asp Pro Asn Ser Pro Ala
230 235 240
Gly Arg Arg His Leu Gln Leu Gln Thr Gln Leu Glu Gln Leu Gln
245 250 255
Glu Glu Thr Phe Arg Leu Glu Ala Ala Lys Asp Asp Tyr Arg Ile
260 265 270
Arg Cys Glu Glu Leu Glu Lys Glu Ile Ser G1u Leu Arg Gln Gln
275 280 285
Asn Asp Glu Leu Thr Thr Leu Ala Asp Glu Ala Gln Ser Leu Lys
290 295 300
Asp Glu Ile Asp VaI Leu Arg His Ser Ser Asp Lys VaI Ser Lys
305 310 315
Leu Glu Gly Gln Val Glu Ser Tyr Lys Lys Lys Leu Glu Asp Leu
320 325 330
Gly Asp Leu Arg Arg Gln Val Lys Leu Leu Glu Glu Lys Asn Thr
335 340 345
Met Tyr Met Gln Asn Thr Val Ser Leu Glu Glu Glu Leu Arg Lys
350 355 360
Ala Asn Ala Ala Arg Ser Gln Leu Glu Thr Tyr Lys Arg Gln Val
365 370 375
Lys Glu Thr Gln His Leu Asp Asp Gly Phe Arg Gln Ala Leu Ser
380 385 390
Tyr Asp Met
<210> 8
<211> 311
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7487619CD1
<400> 8
Met Ser Asn Ala Ser Leu Val Thr Ala Phe Ile Leu Thr Gly Leu
l 5 10 15
Pro His Ala Pro Gly Leu Asp Ala Pro Leu Phe Gly Ile Phe Leu
20 25 30
Val Val Tyr Val Leu Thr Val Leu G1y Asn Leu Leu Ile Leu Leu
35 40 45
Val Ile Arg Va1 Asp Ser His Leu His Thr Pro Met Tyr Tyr Phe
50 55 60
Leu Thr Asn Leu Ser Phe Ile Asp Met Trp Phe Ser Thr Val Thr
65 70 75
Val Pro Lys Met Leu Met Thr Leu Val Ser Pro Ser Gly Arg Ala
80 85 90
Ile Ser Phe His Ser Cys Val Ala Gln Leu Tyr Phe Phe His Phe
95 100 105
Leu Gly Ser Thr Glu Cys Phe Leu Tyr Thr Val Met Ala Tyr Asp
110 215 120
Arg Tyr Leu Ala Ile Ser Tyr Pro Leu Arg Tyr Thr Ser Met Met
125 130 135
Thr Gly Arg Ser Cys Thr Leu Leu Ala Thr Ser Thr Trp Leu Ser
140 145 150
Gly Ser Leu His Ser Ala Val Gln Ala Ile Leu Thr Phe His Leu
155 160 ' 165
14/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Pro Tyr Cys Gly Pro Asn Trp I1e Gln His Tyr Leu Cys Asp Ala
170 175 . 180
Pro Pro Ile Leu Lys Leu Ala Cys Ala Asp Thr Ser Ala Ile Glu
185 190 195
Thr Val Ile Phe Val Thr Val Gly Ile Val Ala Ser Gly Cys Phe
200 205 210
Val Leu Ile Val Leu Ser Tyr Val Ser Ile Val Cys Ser Ile Leu
215 220 225
Arg Ile Arg Thr Ser Glu Gly Lys His Arg Ala Phe Gln Thr Cys
230 235 240
Ala Ser His Cys Ile Val Val Leu Cys Phe Phe Gly Pro Gly Leu
245 250 255
Phe Ile Tyr Leu Arg Pro Gly Ser Arg Lys Ala Val Asp Gly Val
260 265 270
Va1 Ala Va1 Phe Tyr Thr Val Leu Thr Pro Leu Leu Asn Pro Val
275 280 285
Val Tyr Thr Leu Arg Asn Lys Glu Val Lys Lys Ala Leu Leu Lys
290 295 300
Leu Lys Asp Lys Val Ala His Ser Gln Ser Lys
305 310
<210> 9
<211> 318
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7487607CD1
<400> 9
Met Glu Trp G1u Asn His Thr Ile Leu Val Glu Phe Phe Leu Lys
1 5 10 15
Gly Leu Ser Gly His Pro Arg Leu Glu Leu Leu Phe Phe Val Leu
20 25 30
21e Phe Ile Met Tyr Val Val Tle Leu Leu G1y Asn Gly Thr Leu
35 40 45
Ile Leu I1e Ser Ile Leu Asp Pro His Leu His Thr Pro Met Tyr
50 55 60
Phe Phe Leu Gly Asn Leu Ser Phe Leu Asp Ile Cys Tyr Thr Thr
65 70 75
Thr Ser Ile Pro Ser Thr Leu Val Ser Phe Leu Ser Glu Arg Lys
80 85 90
Thr Ile Ser Leu Ser Gly Cys Ala Va1 Gln Met Phe Leu Gly Leu
95 100 105
Ala Met Gly Thr Thr Glu Cys Va1 Leu Leu G1y Met Met Ala Tyr
110 115 120
Asp Arg Tyr Val Ala Ile Cys Asn Pro Leu Arg Tyr Pro Ile Ile
125 130 135
Met Ser Lys Asp Ala Tyr Val Pro Met Ala Ala Gly Ser Trp Ile
140 145 150
21e Gly Ala Val Asn Ser Ala Val Gln Ser Val Phe Val Val Gln
155 160 265
Leu Pro Phe Cys Arg Asn Asn Ile Ile Asn His Phe Thr Cys Glu
170 175 180
Ile Leu Ala Val Met Lys Leu Ala Cys Ala Asp Ile Ser Asp Asn
185 190 195
Glu Phe Ile Met Leu Val Ala Thr Thr Leu Phe Ile Leu Thr Pro
200 205 210
Leu Leu Leu Ile Ile Val Ser Tyr Thr Leu Ile Ile Val Ser Ile
215 220 225
Phe Lys Ile Ser Ser Ser Glu Gly Arg Ser Lys Ala Ser Ser Thr
230 235 240
15/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Cys Ser Ala His Leu Thr Val Val Ile Ile Phe Tyr Gly Thr Ile
245 250 255
Leu Phe Met Tyr Met Lys Pro Lys Ser Lys Glu Thr Leu Asn Ser
260 265 270
Asp Asp Leu Asp Ala Thr Asp Lys Tle Ile Ser Met Phe Tyr Gly
275 280 285
Val Met Thr Pro Met Met Asn Pro Leu Ile Tyr Ser Leu Arg Asn
290 295 300
Lys Asp Val Lys Glu Ala Val Lys His Leu Leu Asn Arg Arg Phe
305 310 315
Phe Ser Lys
<210> 10
<211> 311
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7487616CD1
<400> 10
Met Ser Asn Ala Ser Leu Val Thr Ala Phe Ile Leu Thr Gly Leu
1 5 10 15
Pro His Ala Pro Gly Leu Asp Ala Pro Leu Phe Gly Ile Phe Leu
20 25 30
Val Va1 Tyr Val Leu Thr Val Leu Gly Asn Leu Leu Ile Leu Leu
35 40 45
Val Ile Arg Val Asp Ser His Leu His Thr Pro Met Tyr Tyr Phe
50 55 60
Leu Thr Asn Leu Ser Phe Ile Asp Met Trp Phe Ser Thr Va1 Thr
65 70 75
Val Pro Lys Met Leu Met Thr Leu Val Ser Pro Ser Gly Arg Thr
80 85 90
Ile Ser Phe His Ser Cys Val Ala Gln Leu Tyr Phe Phe His Phe
95 100 105
Leu Gly Ser Thr Glu Cys Phe Leu Tyr Thr Val Met Ser Tyr Asp
110 115 120
Arg Tyr Leu Ala Ile Ser Tyr Pro Leu Arg Tyr Thr Asn Met Met
125 130 135
Thr Gly Arg Ser Cys Ala Leu Leu Ala Thr Gly Thr Trp Leu Ser
140 145 150
Gly Ser Leu His Ser Ala Val Gln Thr Ile Leu Thr Phe His Leu
155 160 165
Pro Tyr Cys GIy Pro Asn Gln Ile Gln His Tyr Phe Cys Asp Ala
170 175 180
Pro Pro Ile Leu Lys Leu Ala Cys Ala Asp Thr Ser Ala Asn Glu
185 190 195
Met Val Ile Phe Val Asn Ile Gly Leu Val Ala Ser Gly Cys Phe
200 205 210
Val Leu Ile Val Leu Ser Tyr Val Ser Ile Val Cys Ser Ile Leu
215 220 225
Arg Ile Arg Thr Ser Glu Gly Arg His Arg Ala Phe Gln Thr Cys
230 235 240
Ala Ser His Cys Ile Val Val Leu Cys Phe Phe Gly Pro Gly Leu
245 250 255
Phe Ile Tyr Leu Arg Pro Gly Ser Arg Asp A1a Leu His Gly Val
260 265 270
Val Ala Val Phe Tyr Thr Thr Leu Thr Pro Leu Phe Asn Pro Val
275 280 285
Val Tyr Thr Leu Arg Asn Lys Glu Val Lys Lys Ala Leu Leu Lys
290 295 300
16133
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Leu Lys Asn Gly Ser Val Phe Ala Gln Gly Glu
305 310
<210> 11
<211> 310
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7483204CD1
<400> 11
Met Asp Trp Glu Asn Cys Ser Ser Leu Thr Asp Phe Phe Leu Leu
l 5 10 15
Gly Ile Thr Asn Asn Pro Glu Met Lys Val Thr Leu Phe Ala Val
20 25 30
Phe Leu Ala Va1 Tyr Ile Ile Asn Phe Ser Ala Asn Leu Gly Met
35 40 45
Ile Val~Leu Ile Arg Met Asp Tyr Gln Leu His Thr Pro Met Tyr
50 55 60
Phe Phe Leu Ser His Leu Ser Phe Cys Asp Leu Cys Tyr Ser Thr
65 70 75
Ala Thr Gly Pro Lys Met Leu Val Asp Leu Leu Ala Lys Asn Lys
80 85 90
Ser Ile Pro Phe Tyr Gly Cys Ala Leu Gln Phe Leu Val Phe Cys
95 100 105
Ile Phe Ala Asp Ser Glu Cys Leu Leu Leu Ser Val Met Ala Phe
110 115 120
Asp Arg Tyr Lys Ala Ile Ile Asn Pro Leu Leu Tyr Thr Val Asn
125 130 135
Met Ser Ser Arg Val Cys Tyr Leu Leu Leu Thr Gly Val Tyr Leu
140 145 150
Val Gly I1e Ala Asp Ala Leu Ile His Met Thr Leu Ala Phe Arg
155 160 165
Leu Cys Phe Cys Gly Ser Asn Glu Ile Asn His Phe Phe Cys Asp
270 175 180
21e Pro Pro Leu Leu Leu Leu Ser Cys Ser Asp Thr Gln Val Asn
185 190 195
Glu Leu Val Leu Phe Thr Val Phe Gly Phe Ile Glu Leu Ser Thr
200 205 210
Ile Ser Gly Va1 Phe Ile Ser Tyr Cys Tyr Ile Ile Leu Ser Val
215 220 225
Leu Glu Ile His Ser Ala Glu Gly Arg Phe Lys Ala Leu Ser Thr
230 235 240
Cys Thr Ser His Leu Ser Ala Val Ala Ile Phe G1n Gly Thr Leu
245 250 255
Leu Phe Met Tyr Phe Arg Pro Ser Ser Ser Tyr Ser Leu Asp Gln
260 265 270
Asp Lys Met Thr Ser Leu Phe Tyr Thr Leu Val Val Pro Met Leu
275 280 285
Asn Pro Leu I1e Tyr Ser Leu Arg Asn Lys Asp Val Lys Glu Ala
290 295 300
Leu Lys Lys Leu Lys Asn Glu Ile Leu Phe
305 310
<210> 12
<211> 316
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
17/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
<223> Incyte ID No: 7472099CD1
<400> 12
Met Ser Ala Ser Ser 21e Thr Ser Thr His Pro Thr Ser Phe Leu
1 5 10 15
Leu Met Gly Ile Pro Gly Leu Glu His Leu His Ile Trp Ile Ser
20 25 30
Ile Pro Phe Ser Ala Tyr Thr Leu Ala Leu Leu Gly Asn Cys Thr
35 40 45
Leu Leu Leu Ile Ile Gln Ala Asp~Ala Ala Leu His Glu Pro Ile
50 55 60
Tyr Leu Phe Leu Ala Met Leu Ala Ala Ile Asp Leu Val Leu Ser
65 70 75
Ser Ser Ala Leu Pro Lys Met Leu Ala Ile Phe Trp Phe Arg Asp
80 85 90
Arg Glu Ile Asn Phe Phe Ala Cys Leu Val Gln Met Phe Phe Leu
95 100 105
His Ser Phe Ser Ile Met Glu Ser Ala Val Leu Leu A1a Met Ala
110 115 120
Phe Asp Arg Tyr Val Ala Ile Cys Lys Pro Leu His Tyr Thr Thr
125 130 135
Val Leu Thr Gly Ser Leu Ile Thr Lys Ile Gly Met Ala Ala Val
140 145 150
Ala Arg Ala Val Thr Leu Met Thr Pro Leu Pro Phe Leu Leu Arg
155 160 165
Cys Phe His Tyr Cys Arg Gly Pro Val Ile Ala Arg Cys Tyr Cys
170 175 180
Glu His Met Ala Val Val Arg Leu Ala Val Gly Thr Leu Gly Phe
185 190 195
Asn Asn Ile Tyr Gly I1e Ala Val Ala Met Phe Ile Gly Val Leu
200 205 210
Asp Leu Phe Phe Ile Ile Leu Ser Tyr Ile Phe Ile Leu Gln Ala
215 220 225
Val Leu Gln Leu Ser Ser Gln Glu Ala Arg Tyr Lys Ala Phe Gly
230 235 240
Thr Cys Val Ser His Ile Gly Ala Ile Leu Ala Phe Tyr Thr Pro
245 250 255
Ser Val Ile Ser Ser Val Met His Arg,Val Ala Arg Cys Ala Val
260 265 270
Pro His Val His Ile Leu Leu Ala Asn Phe Tyr Leu Leu Phe Pro
275 280 285
Pro Met Val Asn Pro Ile Ile Tyr Gly Val Lys Thr Lys Gln Ile
290 295 300
Arg Asp Ser Leu Gly Ser Ile Pro Glu Lys Gly Cys Val Asn Arg
305 310 315
Glu
<210> 13
<211> 318
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7485443CD1
<400> 13
Met Glu Trp Glu Asn His Thr Ile Leu Val Glu Phe Phe Leu Lys
1 5 10 15
Gly Leu Ser Gly His Pro Arg Leu Glu Leu Leu Phe Phe Val Leu
20 25 30
18/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
I1e Phe I1e Met Tyr Val Val Tle Leu Leu Gly Asn Gly Thr Leu
35 40 45
Ile Leu Ile Ser Ile Leu Asp Pro His Leu His Thr Pro Met Tyr
50 55 60
Phe Phe Leu Gly Asn Leu Ser Phe Leu Asp Ile Cys Tyr Thr Thr
65~ 70 75
Thr Ser Ile Pro Ser Thr Leu Val Ser Phe Leu Ser Glu Arg Lys
80 85 90
Thr Ile Ser Phe Ser Gly Cys Ala Val Gln Met Phe Leu Gly Leu
95 100 105
Ala Met Gly Thr Thr Glu Cys Val Leu Leu Gly Met Met Ala Phe
110 115 120
Asp Arg Tyr Val Ala Ile Cys Asn Pro Leu Arg Tyr Pro Ile Ile
125 130 135
Met Ser Lys Asn Ala Tyr Val Pro Met Ala Val Gly Ser Trp Phe
140 145 150
Ala Gly Ile Val Asn Ser Ala Val Gln Thr Thr Phe Val Val Gln
155 160 165
Leu Pro Phe Cys Arg Lys Asn Val Ile Asn His Phe Ser Cys Glu
170 175 180
Ile Leu Ala Val Met Lys Leu Ala Cys Ala Asp Ile Ser Gly Asn
185 190 195
Glu Phe Leu Met Leu Val Ala Thr Ile Leu Phe Thr Leu Met Pro
200 205 210
Leu Leu Leu Ile Val Ile Ser Tyr Ser Leu Ile Ile Ser Ser Ile
215 220 225
Leu Lys Ile His Ser Ser Glu Gly Arg Ser Lys Ala Phe Ser Thr
230 235 240
Cys Ser Ala His Leu Thr Val Val Ile Ile Phe Tyr Gly Thr Ile
245 250 255
Leu Phe Met Tyr Met Lys Pro Lys Ser Lys G1u Thr Leu Asn Ser
260 265 270
Asp Asp Leu Asp Ala Thr Asp Lys Ile Ile Ser Met Phe Tyr Gly
275 280 285
Val Met Thr Pro Met Met Asn Pro Leu I1e Tyr Ser Leu Arg Asn
290 295 300
Lys Asp Val Lys Glu Ala Va1 Lys His Leu Pro Asn Arg Arg Phe
3 05 310 315
Phe Ser Lys
<210> 14
<211> 321
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3090414CD1
<400> 14
Met Leu Thr Leu Asn Lys Thr Asp Leu I1e Pro Ala Ser Phe I1e
1 5 10 15
Leu Asn Gly Val Pro Gly Leu Glu Asp Thr Gln Leu Trp Ile Ser
20 25 30
Phe Pro Phe Cys Ser Met Tyr Val Val Ala Met Val Gly Asn Cys
35 40 45
Gly Leu Leu Tyr Leu Ile His Tyr Glu Asp Ala Leu His Lys Pro
50 55 60
Met Tyr Tyr Phe Leu Ala Met Leu Ser Phe Thr Asp Leu Val Met
65 70 75
Cys Ser Ser Thr Ile Pro Lys Ala Leu Cys Ile Phe Trp Phe His
80 85 90
19/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Leu Lys Asp Ile Gly Phe Asp Glu Cys Leu Val Gln Met Phe Phe
95 100 105
Ile His Thr Phe Thr Gly Met Glu Ser Gly Val Leu Met Leu Met
110 115 120
Ala Leu Asp Arg Tyr Val Ala Ile Cys Tyr Pro Leu Arg Tyr Ser
125 130 135
Thr Tle Leu Thr Asn Pro Val Tle Ala Lys Val Gly Thr Ala Thr
140 145 150
Phe Leu Arg Gly Val Leu Leu Ile Ile Pro Phe Thr Phe Leu Thr
155 160 165
Lys Arg Leu Pro Tyr Cys Arg G1y Asn Ile Leu Pro His Thr Tyr
170 175 180
Cys Asp His Met Ser Val Ala Lys Leu Ser Cys Gly Asn Val Lys
185 190 195
Val Asn Ala Ile Tyr Gly Leu Met Val Ala Leu Leu Ile Trp Gly
200 205 210
Phe Asp Ile Leu Cys Ile Thr Ile Ser Tyr Thr Met Ile Leu Arg
215 220 225
Ala Val Val Ser Leu Ser Ser Ala Asp Ala Arg Gln Lys Ala Phe
230 235 240
Asn Thr Cys Thr Ala His Ile Cys Ala Ile Val Phe Ser Tyr Thr
245 250 255
Pro Ala Phe Phe Ser Phe Phe Ser His Arg Phe Gly Glu His Ile
260 265 270
Ile Pro Pro Ser Cys His Ile Ile Val Ala Asn Ile Tyr Leu Leu
275 280 285
Leu Pro Pro Thr Met Asn Pro Ile Va1 Tyr Gly Val Lys Thr Lys
290 295 300
Gln Ile Arg Asp Cys Val Ile Arg Ile Leu Ser Gly Ser Lys Asp
305 310 315
Thr Lys Ser Tyr Ser Met
320
<210> 15
<211> 422
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No; 7503710CD1
<400> 15
Met Gln Pro Val Met Leu Ala Leu Trp Ser Leu Leu Leu Leu Trp
1 5 10 15
Gly Leu Ala Thr Pro Cys Gln Glu Leu Leu Glu Thr Val Gly Thr
20 25 30
Leu Ala Arg Ile Asp Lys Asp Glu Leu Gly Lys Ala Ile Gln Asn
35 40 45
Ser Leu Val Gly Glu Pro Ile Leu Gln Asn Val Leu Gly Ser Val
50 55 60
Thr Ala Val Asn Arg G1y Leu Leu Gly Ser Gly Gly Leu Leu Gly
65 70 75
Gly Gly Gly Leu Leu Gly His Gly Gly Val Phe Gly Val Val Glu
80 85 90
Glu Leu Ser Gly Leu Lys Ile Glu Glu Leu Thr Leu Pro Lys Val
95 100 105
Leu Leu Lys Leu Leu Pro Gly Phe Gly Val Gln Leu Ser Leu His
110 115 120
Thr Lys Val Gly Met His Cys Ser G1y Pro Leu Gly Gly Leu Leu
125 130 135
Gln Leu Ala Ala Glu Val Asn Val Thr Ser Arg Val Ala Leu Ala
140 145 150
20/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
Val Ser Ser Arg G1y Thr Pro Ile Leu Tle Leu Lys Arg Cys Ser
155 160 165
Thr Leu Leu Gly His Ile Ser Leu Phe Ser Gly Leu Leu Pro Thr
170 175 180
Pro Leu Phe Gly Val Val Glu Gln Met Leu Phe Lys Val Leu Pro
185 190 195
Gly Leu Leu Cys Pro Val Val Asp Ser Val Leu Gly Val Val Asn
200 205 210
Glu Leu Leu Gly Ala Val Leu Gly Leu Val Ser Leu Gly Ala Leu
215 220 225
Gly Ser Val Glu Phe Ser Leu Ala Thr Leu Pro Leu Ile Ser Asn
230 235 240
Gln Tyr Ile Glu Leu Asp Ile Asn Pro Ile Val Lys Ser Va1 Ala
245 250 255
Gly Asp Ile Ile Asp Phe Pro Lys Ser Arg Ala Pro Ala Lys Val
260 265 270
Pro Pro Lys Lys Asp His Thr Ser Gln Val Met Val Pro Leu Tyr
275 280 285
Leu Phe Asn Thr Thr Phe Gly Leu Leu Gln Thr Asn Gly Ala Leu
290 295 300
Asp Met Asp Ile Thr Pro Glu Leu Val Pro Ser Asp VaI Pro Leu
305 310 315
Thr Thr Thr Asp Leu Ala Ala Leu Leu Pro Glu Val Met Thr Val
320 325 330
Arg Ala Gln Leu Ala Pro Ser Ala Thr Lys Leu His Ile Ser Leu
335 340 345
Ser Leu Glu Arg Leu Ser Val Lys Val Ala Ser Ser Phe Thr His
350 355 360
A1a Phe Asp Gly Ser Arg Leu Glu Glu Trp Leu Ser His Val Val
365 370 375
Gly Ala Val Tyr Ala Pro Lys Leu Asn Val Ala Leu Asp Val Gly
380 385 390
Ile Pro Leu Pro Lys Val Leu Asn Ile Asn Phe Ser Asn Ser Val
395 400 405
Leu Glu I1e Val Glu Asn Ala Val Ala Ala Leu Tyr Val Leu Val
410 415 420
Val Ala
<210> 16
<211> 2192
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 71924779CB1
<400> 16
aggtggcgcg taccaccaga gaccgagcga gtcgccagct gcccctggcc tggcgggggc 60
ggaaccgcgc gggatcccca cccccacccg gaatcctcgc cacggagaat ccctggagaa 120
gccccggatc cccggctggg aggaggaagt gctcgttgac ccccagcccc gcgctgatcc 180
cgcccccggc ctgcggactt ggggagccgc tgtactctgc ctcggacgcc acgagactct 240
agacgggagt cccctcgagg tgaagccgct gagttcccgg gccccgccag gcttccctgg 300
gagagccgac ggaccccccc tcccagcaca cacaacttcc ctgcttttca ccgggactgg 360
cggagcggcc ggcggactta gacgcgggga cttcagggca gggggcgccc cctgcccggg 420
tcaccagtcg gggcgagggg acgtctcctc tcccccagct gctctgctcg gatggcgccg 480
ccggctgagt gacgggggcg gcgcgcagga cttcccagct cggacctctt gccttcgagg 540
ggaaagatgt acgagagtgt agaagtgggg ggtcccaccc ctaatccctt cctagtggtg 600
gatttttata accagaaccg ggcctgtttg ctcccagaga aggggctccc cgccccgggt 660
ccgtactcca ccccgctccg gactccgctt tggaatggct caaaccactc cattgagacc 720
cagagcagca gttctgaaga gatagtgccc agccctccct cgccaccccc tctaccccgc 780
atctacaagc cttgctttgt ctgtcaggac aagtcctcag gctaccacta tggggtcagc 840
21/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
gcctgtgagg gctgcaaggg cttcttccgc cgcagcatcc agaagaacat ggtgtacacg 900
tgtcaccggg acaagaactg catcatcaac aaggtgaccc ggaaccgctg ccagtactgc 960
cgactgcaga agtgctttga agtgggcatg tccaaggagt ctgtgagaaa cgaccgaaac 1020
aagaagaaga aggaggtgcc caagcccgag tgctctgaga gctacacgct gacgccggag 1080
gtgggggagc tcattgagaa ggtgcgcaaa gcgcaccagg aaaccttccc tgccctctgc 1140
cagctgggca aatacactac gaacaacagc tcagaacaac gtgtctctct ggacattgac 1200
ctctgggaca agttcagtga actctccacc aagtgcatca ttaagactgt ggagttcgcc 1260
aagcagctgc ccggcttcac caccctcacc atcgccgacc agatcaccct cctcaaggct 1320
gcctgcctgg acatcctgat cctgcggatc tgcacgcggt acacgcccga gcaggacacc 1380
atgaccttct cggacgggct gaccctgaac cggacccaga tgcacaacgc tggcttcggc 1440
cCCCtcaccg acctggtctt tgccttcgcc aaccagctgc tgcccctgga gatggatgat 1500
gcggagacgg ggctgctcag cgccatctgc ctcatctgcg gagaccgcca ggacctggag 1560
cagccggacc gggtggacat gctgcaggag ccgctgctgg aggcgctaaa ggtctacgtg 1620
cggaagcgga ggcccagccg cccccacatg ttccccaaga tgctaatgaa gattactgac 1680
ctgcgaagca tcagcgccaa gggggctgag cgggtgatca cgctgaagat ggagatcccg 1740
ggctccatgc cgcctctcat ccaggaaatg ttggagaact cagagggcct ggacactctg 1800
agcggacagc cggggggtgg ggggcgggac gggggtggcc tggccccccc gccaggcagc 1860
tgtagcccca gcctcagccc cagctccaac agaagcagcc cggccaccca ctccccgtga 1920
ccgcccacgc cacatggaca cagccctcgc cctccgcccc ggcttttctc tgctttctac 1980
cagacattgt gaccccgcac cagccctggc cccactgcct ccgggcagta ctggcgactt 2040
ccctggggac ggggagggag gagaagatct tggacagagg ctggcctcag ggatgcctgt 2100
cccagctggt gaataaagcg aggcgaagat gagccggccg gttcaaaggt cgggccgttc 2160
aaacctgccg ccatttaaga aggggcccaa as 2192
<210> 17
<211> 3614
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2319430CB1
<400> 17
cgttgagggc aacccagagg ccatgccccg ctcctcccct ctatcccgct ccggctgtta 60
agggctcggc cccgagcgcc tctgcccgcg gacgatggtg accgtacggg ccgggccact 120
gccgctgcct ccgcctcccc agagagccac atccgaggct cggcgcagaa gagccgccgc 180
tgtgaccgtg ccgtaccggc cccctgcctc cgcccgagga gaacgggagg gcgggcgaga 240
gagccgggga gttgcggagc ccgccccccg gcagcgccgc tcccccggga gggagtcccc 300
cagcctgagg tcttctccca gaaaaaaaaa aagaaaaaaa aaaacaacat ggctgcaaag 360
gagaaactgg aggcagtgtt aaatgtggcc ctgagggtgc caagcatcat gctgttggat 420
gtcctgtaca gatgggatgt cagctccttt ttccagcaga tccaaagaag tagccttagt 480
aataaccctc ttttccagta taagtatttg gctcttaata tgcattatgt aggttatatc 540
ttaagtgtgg tgctgctaac attgcccagg cagcatctgg ttcagcttta tctatatttt 600
ttgactgctc tgctcctcta tgctggacat caaatttcca gggactatgt tcggagtgaa 660
ctggagtttg cctatgaggg accaatgtat ttagaacctc tctctatgaa tcggtttacc 720
acagccttaa taggtcagtt ggtggtgtgt actttatgct cctgtgtcat gaaaacaaag 780
cagatttggc tgttttcagc tcacatgctt cctctgctag cacgactctg ccttgttcct 840
ttggagacaa ttgttatcat caataaattt gctatgattt ttactggatt ggaagttctc 900
tattttcttg ggtctaatct tttggtacct tataaccttg ctaaatctgc atacagagaa 960
ttggttcagg tagtggaggt atatggcctt ctcgccttgg gaatgtccct gtggaatcaa 1020
ctggtagtcc ctgttctttt catggttttc tggctcgtct tatttgctct tcagatttac 1080
tcctatttca gtactcgaga tcagcctgca tcacgtgaga ggcttctttt cctttttctg 1140
acaagtattg cggaatgctg cagcactcct tactctcttt tgggtttggt cttcacggtt 1200
tcttttgttg ccttgggtgt tctcacactc tgcaagtttt acttgcaggg ttatcgagct 1260
ttcatgaatg atcctgccat gaatcggggc atgacagaag gagtaacgct gttaatcctg 1320
gcagtgcaga ctgggctgat agaactgcag gttgttcatc gggcattctt gctcagtatt 1380
atccttttca ttgtcgtagc ttctatccta cagtctatgt tagaaattgc agatcctatt 1440
gttttggcac tgggagcatc tagagacaag agcttgtgga aacacttccg tgctgtaagc 1500
ctttgtttat ttttattggt attccctgct tatatggctt atatgatttg ccagtttttc 1560
cacatggatt tttggcttct tatcattatt tccagcagca ttcttacctc ~tcttcaggtt 1620
ctgggaacac tttttattta tgtcttattt atggttgagg aattcagaaa agagccagtg 1680
gaaaacatgg atgatgtcat ctactatgtg aatggcactt accgcctgct ggagtttctt 1740
22/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
gtggccctct gtgtggtggc ctatggcgtc tcagagacca tctttggaga atggacagtg 1800
atgggctcaa tgatcatctt cattcattcc tactataacg tgtggcttcg ggcccagctg 1860
gggtggaaga gctttcttct ccgcagggat gctgtgaata agattaaatc gttacccatt 1920
gctacgaaag agcagcttga gaaacacaat gatatttgtg ccatctgtta tcaggacatg 1980
aaatctgctg tgatcacgcc ttgcagtcat tttttccatg caggctgtct taagaaatgg 2040
ctgtatgtcc aggagacctg ccctctgtgc cactgccatc tgaaaaactc ctcccagett 2100
ccaggattag gaactgagcc agttctacag cctcatgctg gagctgagca aaacgtcatg 2160
tttcaggaag gtactgaacc cccaggccag gagcatactc cagggaccag gatacaggaa 2220
ggttccaggg acaataatga gtacattgcc agacgaccag ataaccagga aggggctttt 2280
gaccccaaag aatatcctca cagtgcgaaa gatgaagcac atcctgttga atcagcctag 2340
aggagaagca gcaggaatga tgctttgata ctctggagga gaagttaact caagatggaa 2400
ttcatgttct gatttgagga atgaaaatga gatgatcagg caggaaactg acattccaag 2460
gatctaatcc aggaagtact ctcagtgggg accacctgct ttcatcccct gacattgtgg 2520
gagaaatttt gcaatgtatg ctaatcaaaa tgtatttata tgttctctgc tgatgtttta 2580
tagaggtttg tgaagaaaat tcaacctcag caacttcaga aactgcccct gatacgtgtg 2640
agagagaaat aaaatcagat tttgagtgtt gaagggactg aggaagtgag gataaagagc 2700
atgaggacag catggaaaga aggaggcaga agtggaactg aactttcact ctccatggga 2760
cagatcaatc tcattatcaa gtctgaatag caaccagccc tctcctccac cccgtttctc 2820
ctcagttaat tggagctcag tcaggtgatt attgagtctt gtacagcact gaaatgaaat 2880
caaagatgaa gaagcattga ttgtattcaa agattgaagc acgctcatac tttgtatgtg 2940
ctttagggaa ggggtgggtg ggcacttggg ccttgcgggt gcattcatgt aatctgagac 3000
tcttgaactt tatgacggag tcttcaatat tttgatgtat atgaaacttt tgttaaatat 3060
gttgtatact tcgctggctg tgtgaagtaa actaaaactc tgatgaacac tttggagtct 3120
gctttagtga aggagaccaa agtgggaagg gctttagggc actgatagag gccctgggtg 3180
tacttttcaa tcctgtgtaa tgtttaattc ttgcaactga atcaaaacag tgttaaatta 3240
tggcaatatt tgcactttgg gaatgagtac ataactgtat gatcacactc tgcaaatgcc 3300
acttttaaag ctgttaatag actttgcacc ttttctttga caaggatgtg tcatatttaa 3360
atttttacat tcatcatggc tacaggtaga actggggagg ggggaatgta attttttatg 3420
ggaattttga tatgaaaaga aactagtcat ttatttatac aataggcttg gctcaaaaag 3480
tgtttttcag acctcggtat tcctaatgtg ggatgtgact ttattttatt tttagtagca 3540
aatttggatg tagactgaca gacatagctg aatgtcttaa taaatttaaa tttgaagata 3600
aaaaaaaaaa aaaa 3614
<210> 18
<211> 1585
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7291877C81
<400> 18
cgcgcaccaa caacagcaac aactccactg cgccgggctg aggagcagga attaggagct 60
cgcgaataat atgaaaggga tccgcaaagg ggaaagccga gcaaaggaat ccaaaccctg 120
ggagcctggc aagcgaagat gcgctaaatg tggccgccta gacttcatcc tgatgaagaa 180
aatggggatt aaaagtggat ttacgttttg gaacctcgtc tttttattga cggtgtcttg 240
tgtgaaagga tttatttata catgtggtgg aactttaaaa ggacttaatg gcactataga 300
aagccctggt tttccatatg gatatccaaa tggtgcaaac tgcacatggg taataatagc 360
agaagaacga aatagaatac aaattgtttt tcagtcattt gctctagaag aagaatacga 420
ctacttatca ttatatgatg gacatcctca tcctacaaac tttaggacaa ggttaacagg 480
attccatctg ccacctccag tgacaagtac caaatctgtg ttctcactac gtttgaccag 540
tgattttgca gttagtgctc atggatttaa ggtatattac gaagaattgc agagtagctc 600
ttgtggaaat cctggtgttc cacccaaagg tgtattatat ggcacaagat tcgacgtcgg 660
ggacaagatc cgctacagct gtgtaactgg atacatcctt gatggccacc ctcagctcac 720
ctgcatagcc aattcagtta atacagcttc gtgggatttt cctgttccta tctgtagagc 780
tgaagatgct tgtggaggaa caatgagagg atccagtggc atcatatcca gccctagttt 840
tcctaatgag taccataaca atgctgattg cacttggacc attgtagcag agcctgggga 900
cacaatttca ctcatattta ctgattttca aatggaagag aaatatgatt acttagaaat 960
agaaggttct gagccaccta ccatatggtt atctggaatg aatataccac caccaattat 1020
cagcaacaaa aactggctca gactgcattt tgttacagac agcaatcatc gataccgtgg 1080
atttagtgct ccctatcaag gttcttctac attgacccac actacctcca ctggtgagtt 1140
agaggagcat aacaggacta ccactggtgc tattgctgtt gctagcacac ctgcagatgt 1200
23/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
tactgtatcc agtgttacag ctgtcaccat ccatagactt tccgaggaac agcgagtgca 1260
agttacgagt ctcagaaatt caggtctgga ccccaacacg tccaaggacg ggctctctcc 1320
tcatccagca gatacacaaa gtaccaggag aagaccaaga catgctgaac agatagaaag 1380
aactaaagag cttgcagttg ttactcatag aggacattgc aatagagtcg aggacataga 1440
aaaacccatt ctagtggtac aagatagatt ttgtaaaatg aattctgatc aaagtactaa 1500
agaagttaca gtgtgtatgc agagagtgag tcttttaagt tactttttca atgagttggt 1560
aaacaaccga aaaccaattg cttaa 1585
<210> 19
<211> 5618
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1218126CB1
<400> 19
gatatgactg aggcgcccat gggggtggcg gggcggctgt aggagcaggg gcctagcaag 60
cgcccagcgg agcgacccct gcctggccgt ggctagcatg gcccctacgc tgttccagaa 120
gctcttcagc aagaggaccg ggctgggcgc gcccggccgc gacgcccggg acccagattg 180
cgggttcagt tggcctttac cagagtttga tccaagccag attcgactga ttgtatatca 240
agactgtgaa agacgaggga gaaatgtttt gtttgactcc agtgttaaga gaagaaatga 300
ggacatatca gtatcgaaac tctgcagtga tgctcaagtt aaagtctttg ggaaatgctg 360
ccaactgaaa cctggaggag acagttcttc ctctttagat agttctgtga cttcatcttc 420
tgatataaaa gaccagtgtc ttaagtacca gggttctcgg tgctcttctg atgccaatat 480
gcttggagag atgatgtttg gctcagtagc aatgagctac aaaggatcca ccttaaaaat 540
tcatcagatt cgttcccctc cacagctcat gctcagcaaa gtgtttactg ctcggactgg 600
cagcagtatt tgtgggagtc tcaatacgct acaagatagt cttgaattca tcaatcagga 660
caacaataca ttaaaggctg ataataacac agttattaat ggactgcttg gaaatatagc 720
atctctcagc agcttgctga tcactccatt tccttcccca aactcctcac ttacccgaag 780
ttgtgccagc agctaccagc gacgttggcg acgcagccaa acaacaagtt tggaaaatgg 840
ggtatttcct agatggtcta tagaagaaag ctttaatctc tcagatgaaa gctgtggccc 900
taacccagga attgtgcgga aaaagaagat tgcaattggg gtaatctttt cattgtccaa 960
agatgaagat gaaaataaca aatttaatga attctttttt tcacattttc ctctctttga 1020
aagctacatg aacaaattaa agagtgcaat agaacaggct atgaaaatga gccggagatc 1080
agctgatgcc agtcagagaa gtttggcata taatcgaata gttgatgccc taaatgaatt 1140
cagaacaaca atttgtaatc tttacacgat gccacgaatt ggagaacctg tctggcttac 1200
aatgatgtcg gggactccag aaaagaacca cctttgctat cgtttcatga aggagttcac 1260
ctttctaatg gaaaatgctt ccaaaaatca attcttgcca gctctcatta ctgcagttct 1320
gaccaatcat cttgcctggg ttccaacagt catgccaaat ggacaaccac ctataaaaat 1380
atttttagaa aagcattcct ctcagagtgt ggacatgttg gcaaagactc atccatataa 1440
cccactttgg gcacaactgg gagacttgta tggcgctatt ggctctcccg tacggttagc 1500
aaggactgtg gtagttggca aacgacaaga catggtccag aggctacttt attttcttac 1560
ttattttata agatgctctg aacttcaaga aa.cgcatctt ttagaaaatg gagaagatga 1620
agccatcgtt atgccaggca cagtaattac taccacttta gagaaaggtg aaatagaaga 1680
atcagagtat gtccttgtca caatgcatag aaacaaaagc agtttgctct ttaaagagtc 1740
agaagaaatt agaactccca attgtaactg taaatattgc agtcatccac tccttgggca 1800
aaatgtagag aacatttcac aacaagagag agaagatatt caaaacagct ctaaggagct 1860
gctaggaatt tcagatgagt gccggatgat ttctccttct gactgccaag aagaaaatgc 1920
tgttgatgtt aaacagtaca gagataaatt aagaacttgc tttgacgcca agttagagac 1980
agttgtttgc acaggatctg ttccagtaga caaatgtgca ttgtcagagt caggcttaga 2040
gtcaacagag gaaacatggc agagtgagaa gttgctggat tcagacagtc acacaggcaa 2100
agcaatgaga tccacaggaa tggttgtgga aaaaaaacct ccagataaga ttgtgcctgc 2160
ttcattttct tgtgaggctg cccagacaaa ggttactttc ctgattgggg attctatgtc 2220
acctgattca gatactgagc ttcgaagtca ggcagtggtg gatcagatta ccagacatca 2280
caccaaacca ttgaaggaag aaagaggggc tattgatcag catcaagaaa ctaaacaaac 2340
aaccaaggac caatctggag agtctgatac acagaacatg gtttctgaag agccctgtga 2400
acttccctgt tggaatcatt cagacccaga aagcatgagc ttattcgacg aatattttaa 2460
tgatgattca atcgaaacca ggactattga tgatgttcca tttaaaacaa gtacagatag 2520
taaagaccat tgctgtatgt tagagttttc aaaaatattg tgtacaaaaa ataacaagca 2580
gaacaatgaa ttttgtaaat gtatagaaac agttccccaa gattcatgta aaacctgctt 2640
tcctcagcag gaccaaagag atacactctc cattcttgtc ccccatgggg ataaagagag 2700
24/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
ttcagataaa aaaattgctg taggaactga atgggacatt ccaagaaatg aaagttcaga 2760
cagtgccctt ggggatagtg aaagtgaaga tacaggtcat gatatgacta gacaagttag 2820
cagttattat ggaggagagc aagaagattg ggcagaagag gatgagatac cttttcctgg 2880
gtcaaagtta atcgaagtga gtgctgttca gcccaacatt gccaacttcg ggaggtcctt 2940
gctgggtggc tactgctcat cttatgtgcc tgactttgtt cttcaaggaa ttgggagtga 3000
tgagaggttc cgtcagtgtc tgatgtcaga tttatctcat gctgtgcagc atccagtttt 3060
ggatgaacca atagcagaag ctgtctgtat tatagctgac atggataaat ggactgttca 3120
agtggccagt agccagagac gagtgacaga taataaattg ggaaaggaag tattggtttc 3180
cagtcttgtt tccaatctgc ttcattccac acttcagctt tataagcata acttgtctcc 3240
aaatttttgt gtaatgcatc ttgaagaccg gttgcaggag ctatacttca aaagtaaaat 3300
gctgtctgaa tacctgaggg ggcagatgcg tgttcatgtc aaggagctgg gagtggttct 3360
ggggattgaa tccagtgatc ttccacttct ggctgctgta gcaagcactc actctccata 3420
tgttgcacaa atactccttt aatataccta aaaattgtta gaaattggtg ggaaaatagg 3480
tagaaaccaa ggaagcagac acaacatgca tttatggaga ttctttttcc cttttagact 3540
tccatctgaa tgagtcagtc accagggtat tctgcatagc attgtatatt ctgtgtatgt 3600
cagatggctt tttctttttg actggacttt tgggtggtgg tagattttta aacaaatgaa 3660
attaaagcaa caataatttt gaagcatttg aaaaagccaa agtgtacggt agaaatttct 3720
acaaaatgaa tattatcaag agtttcatgt gatcactgca gtgttgtcac agctcataaa 3780
tagcaacagt gtttcatgat ttaatggctc agaaatagtt attcattagt ttttaatttt 3840
taatttctaa ggtacagaga tctataaaac cttgattatt tgttagtttt gcaattcaaa 3900
acagctaatg tctggttatt tctcaaagta agtattttaa acagcctgtt aattataaga 3960
aactcagaat aatgagtgta aatgtgttat gttatccacc caagtgtaca tatgtaccta 4020
ttttttttta aaaagcagaa atagaaatac aagactggta aacatgcctt taaaaatata 4080
tatattttca actagtattg tctataatgc tgaaatatta cttattggtg atttttctgt 4140
ttcacacact ctaaaatata agtaaagcca accttttttt taaggctgag attcccaaaa 4200
tgagaatact actttatacc atttgtttat aagtatgaac tgttcttata aatattaata 4260
tttacatatt cactaattta acataaatga aaattaggat taaaaattgc accaaagcat 4320
cggcaaaaac aatactatat tctttaaaag tgctcaggta gccaaggccc ttgcttttgg 4380
tatcaaccct catgaaccca taggagctga tatttgtttc actgcttaat aatcctcaat 4440
ttacactatt cataactctt aaaattattc tctttttttc taagagcccc tcccttccaa 4500
aagtgtattt ttttcaaaga ttttcacttc tcaattgttg cctttgtaca tactatagag 4560
tgttgcttgt aagaaaggct aatatggaac caaactcttg taagtaatgt aaatagaaag 4620
gtgggtggat aaagttttca atactttcta ctacctcagt ttacttgagt actacattat 4680
agtttattct ttgcttatct ggtctaagag acttttaatg ctagtagtaa agttggtttc 4740
tgctttcatt gactattttc atcataattt catcattgat taaaaaaaga aaaccacttg 4800
tttattcagt tattaaatat atttactata taacacatcc attcttgctg tttaaatttt 4860
caatagttaa tggaaagttg tctttgacct tgaatttaca gcattgggtc acattttgcc 4920
ttgctgtgta tgtattcaag agacttccaa ctagacaaag aaaaaattgt tgttttaatg 4980
gaatgtaaac ctgaaattgg,~gtgtctgca atctgtttgg cccatgacct tttacctagt 5040
cccagttatt acctgagtct cccatggatg acttgctgcc aaggagtgtt tgtggatata 5100
ttttctttgg cttaattttc ttattctgtg cattaacaaa attatccagt tgtctgattt 5160
tggaattcta tgagtcaatc tttttggcag aattcagaat attaaaaagt tcatacattt 5220
gcgggccatt gtaccttttt tttttttttt ttttttttga cggagtttca ctcttgttac 5280
ccaggctgga gttgcaatgg tgcgatctca gctcactgca acctccgcct cccagttcaa 5340
gtgattctcc gtgcctcagc ccccaagtta gctgggatta caatttgtgc gccaccacac 5400
ccagctaatt ttttattttt agtagagatg agatttcacc atgttttggt caggctggtc 5460
ttgaactcct gaactcaagt gatccaccgt gcctcgacct cccaaagtac tgggattaca 5520
ggcgtgagcc atgtgtgccc agccttgtac tttttttttt tttaatggta gctctgttta 5580
gcattggggc atatgtcggg gtgtctcttt aaccttaa 5618
<210> 20
<211> 1641
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7479161CB1
<400> 20
ccgacaggag gccgggagcc cccacctacc ccttgtggag ctgcaggagc aagggcatgc 60
agccagtcat gctggccctg tggtccctgc ttctgctctg gggcctggcg actccatgcc 120
aggagctgct agagacggtg ggcacgctcg ctcggattga caaggatgaa ctcggcaaag 180
25/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
ccatccagaa ctcactggtt ggggagccca ttctgcagaa tgtgctggga tcggtcacag 240
ctgtgaaccg gggcctcttg ggctcaggag ggctgcttgg aggaggcggc ttgctgggcc 300
acggaggggt ttttggcgtt gtcgaggagc tctctggtct gaagattgag gagctcacgc 360
tgccaaaggt gttgctgaag ctgctgccgg gatttggggt gcagctgagc ctgcacacca 420
aagtgggcat gcattgctct ggcccccttg gtggccttct gcagctggct gcggaggtga 480
acgtgacatc gcgggtggcg ctggccgtga gctcaagggg cacacccatc cttatcctca 540
agcgctgcag cacgctcctg ggccacatca gcctgttctc agggctgctg cccacaccac 600
tctttggggt cgtggaacag atgctcttca aggtgcttcc gggactgctg tgccccgtgg 660
tggacagtgt gctgggtgtg gtgaatgagc tcctgggggc tgtgctgggc ctggtgtccc 720
ttggggctct tgggtccgtg gaattctctc tggccacatt gcctctcatc tccaaccagt 780
acatagaact ggacatcaac cctatcgtga agagtgtagc tggtgatatc attgacttcc 840
ccaagtcccg tgccccagcc aaggtgcccc ccaagaagga ccacacatcc caggtgatgg 900
tgccactgta cctcttcaac accacgtttg gactcctgca gaccaacggc gccctcgaca 960
tggacatcac ccctgagctg gttcccagcg atgtcccact gacaactaca gacctggcag 1020
ctttgctccc tgaggccctg gggaagctgc ccctgcacca gcaactccta ctgttcctgc 1080
gggtgaggga agctcccacg gtcacactcc acaacaagaa ggccttggtc tccctcccag 1140
ccaacatcca tgtgctgttc tatgtcccta aggggacccc tgaatccctc tttgagctga 1200
actccgtcat gactgtgcgt gcccagctgg ctccctcggc taccaagctg cacatctccc 1260
tgtccctgga acggctcagt gtcaaggtgg cctcctcctt tacccatgcc tttgacggat 1320
cgcgtttaga agaatggctc agccatgtgg tcggggcagt gtatgcacca aagcttaacg 1380
tggccctgga tgttggaatt cccctgccta aggttcttaa tatcaatttt tccaattcag 1440
ttctggagat cgtagagaat gctgtggcag ctctctatgt ccttgtagta gcatagaaga 1500
tggtgttctt ctcagatcag tggactatgc catgttattt tgttcttgga ctaaggccct 1560
gtgaggtgca actggtccac tttcattttt ggtcagagat ggagaataag gaattatatg 2620
ttggtactag cactggaata g 1641
<210> 21
<211> 6056
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7722591CB1
<400> 21
gactgacctg tgaggactgc ctggccaact ctagccagtg cgcctggtgc cagtccaccc 60
acacctgctt cctgtttgct gcctacttgg cccggtaccc acacgggggc tgtcgaggct 120
gggacgacag gtatggtccc tggggcaggg ctaacagagg aagattcccc accggcaagg 180
ggctggggct ctgaccccca cccctgccat cctgcagtgt acactcggag ccacggtgcc 240
ggagctgcga tggcttcctg acctgccatg agtgtctgca gagccacgag tgtggctggt 300
gtggcaatga ggacaacccc acactgggac ggtgagcccg ggcaggtggg tgggcagggt 360
gcccggctgt gtccttcctc catgaccggt cattctaatg gcctctttgc ttctctgccc 420
tcttgcccat cccatggcca ccttcccttt tccgtactgt ttttctgttg tttattttac 480
CCtCCCgCtC ttttttCtCC atttttCCtC CttttCCggC tCtCtgCgat tcgttttctt 540
tctccttgtc tgtttctgtc tctccgctct ccctttcact gcatctctgt ctatgtctct 600
cttctgtctt ccaaaattgt ttttgtctgc gacttctcct ggtttctctg tctctctttc 660
caaatttgta tttgcatccc tctccttcca attggcctcc tctctccctg tcattgtttc 720
tatgtatggc tcctgtttct atgttgtCCC CCgCttCttC aCtCtCCCaC CCtgCaggtg 78O
cctacagggg gacttctcag ggcccctcgg tgggggtaac tgctccctgt ggtgtggggg 840
agggcctggg tgcttcccgt ggccctgccc tgcccgtctg ggcatacgcc cgctgtcctg 900
acgtggatga gtgtcgcctg ggcctggccc ggtgccaccc gcgggcgacc tgcctgaaca 960
cgcccctcag ctacgagtgt cactgccagc ggggctacca gggtgatggc atctcacact 1020
gcaaccgcac gtgcttggag gactgtggcc atggtgtgtg cagtggcccc ccggacttta 1080
cctgcgtgtg tgacctaggc tggacatcag acctgccccc tcccacacct gccccgggtc 1140
cgccagcccc ccgctgctcc cgggactgtg gctgcagctt ccacagccac tgccgcaagc 1200
ggggccctgg cttctgcgac gagtgccagg actggacatg gggggagcac tgcgaacgat 1260
gccggcccgg cagcttcggc aacgccacag gctctagggg ctgccggccc tgccagtgca 1320
acgggcacgg ggacccacgc cgtggccact gcgacaacct cagtgggctc tgcttctgcc 1380
aggaccacac cgagggtgcc cactgccagc tctgctcccc aggctattat ggggatccca 1440
gggccggtgg ttcctgcttt cgggagtgtg ggggtcgcgc cctcctcacc aacgtgtcct 1500
cagtggcact gggctcacgc cgggtcgggg ggctgctgcc tccaggtggc ggggctgcaa 1560
gagccgggcc tggcctgtcc tactgtgtgt gggttgtctc ggccactgag gagctacagc 1620
26/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
cctgtgctcc cgggaccctc tgtcccccac tcaccctcac cttctccccc gacagcagca 1680
CCCCCtgCaC gctgagctac gtcctggcgt ttgatggatt cccacgcttc ctggacactg 1740
gtgttgtcca gtcggaccgc agcctcatag ctgccttctg cggccagcga cgggacaggc 1800
ccctcactgt tcaggccctg tctgggctgc tcgtgctgca ctgggaggcc aatggctcct 1860
catcctgggg cttcaatgct tcggtgggct ctgcccgctg tgggtcaggg ggccccggga 1920
gctgtcccgt cccccaggaa tgcgtgcccc aggacggtgc tgcaggtgcg gggctctgcc 1980
gatgtcctca gggctgggct ggcccacact gccgcatggc tctgtgtcct gagaactgca 2040
atgcccacac tggggcagga acttgtaacc agagcctggg tgtgtgcatc tgtgccgagg 2100
gcttcggggg ccccgactgc gccaccaagc tggatggcgg gcagctggtc tgggagaccc 2160
tcatggacag ccgcctctca gccgacactg ccagccgctt cctgcaccgc ctgggccaca 2220
ccatggtgga tggacccgat gccaccttgt ggatgtttgg gggcctgggc ctgccccagg 2280
ggctgctggg aaacctgtac aggtactcag tgagtgagcg gcggtggaca cagatgctgg 2340
cgggagccga ggacgggggc ccaggcccat cgccccgctc cttccatgca gctgcatatg 2400
tgcccgctgg ccgtggtgcc atgtatctgc tggggggact taccgctgga ggcgtcaccc 2460
gtgatttctg ggtcctcaac ctcaccaccc tgcaatggcg gcaggagaag gccccccaga 2520
ccgtggagct gccagccgtt gctggtcaca cccttactgc ccgccgaggc ctgtctctgc 2580
tcctggtggg cggttactcc ccggaaaatg gcttcaacca gcagctgctg gagtaccagc 2640
tggcaaccgg cacctgggtg tcaggagccc agagtgggac accccccaca ggtctctatg 2700
gtcactctgc tgtctaccac gaggccaccg actccctcta cgtgtttggg gggttccgat 2760
tccatgtgga gctggcggcc CCatCCCCCg agctctactc cctgcactgt cctgaccgca 2820
cctggagtct gctggcccct tctcaggggg caaagccccg cccccggctt ttccacgcct 2880
cagccctgtt aggggacacc atggtggttc ttggggggcg ctcggaccct gacgagttca 2940
gcagcgacgt tctgctctac caggtcaact gcaatgcctg gcttctgccc gacctcaccc 3000
gctcggcctc tgtggggccc ccaatggagg agtctgtggc ccatgctgtg gcagcagtcg 3060
ggagccgcct gtatatctct gggggtttcg ggggagtggc cctgggccgc ctgctggcac 3120
tgaCCCtgCC CCCtgaCCCC tgccgcctgc tgtcctcacc tgaagcttgt aaccagtctg 3180
gggcctgcac ctggtgccat ggggcctgct tgtccgggga tcaggcccac aggctgggct 3240
gcgggggctc cccctgctcc ccaatgcctc gctccccgga ggaatgtcga cgtctccgga 3300
cctgcagtga gtgCCtggCC CgCCatCCtC ggaccctgca acctggagat ggagaggcgt 3360
ccaccccccg ctgtaagtgg tgtaccaact gccccgaagg tgcttgcatt ggacgcaatg 3420
ggtcctgcac ctctgagaat gactgtcgga tcaaccagcg agaggtcttc tgggcaggga 3480
actgctccga ggctgcgtgc ggggctgctg actgcgagca gtgcacgcgg gagggcaagt 3540
gcatgtggac gcggcagttc aagaggacag gggagacccg ccgcatcctc tccgtgcagc 3600
ccacctatga ctggacgtgc ttcagccact ctctgctgaa tgtgtccccc atgccggtgg 3660
aatcatcacc cccactgccc tgCCCCaCCC CttgtCaCCt cctacccaac tgtacctcct 3720
gcctggactc taagggagca gatgggggct ggcagcactg tgtttggagc agcagcctgc 3780
agcagtgtct gagcccttcc tacctgcccc tgcgatgtat ggccggaggc tgtgggcggc 3840
tgctccgggg acctgagagc tgctccctgg gctgtgctca ggcaactcag tgcgccttgt 3900
gcctgcggcg cccccattgc ggctggtgtg cctggggggg ccaggatggg ggtggccgct 3960
gcatggaggg tggactcagc ggcccccgtg atgggctgac atgtgggcgt ccgggggcct 4020
cctgggcctt cctgtcctgc ccccctgagg acgagtgtgc aaacgggcac cacgactgca 4080
acgagacgca gaattgccac gaccagcccc acggctatga gtgcagctgc aagaccggct 4140
ataccatgga caacatgaca gggctgtgcc gccctgtgtg cgcccagggc tgcgtgaacg 4200
gctcatgtgt ggagcccgac cactgccgct gccactttgg ctttgtgggc cgcaactgct 4260
ccacggaatg ccgctgcaac cgccacagtg aatgcgctgg tgttggggcg cgtgaccact 4320
gcttgctctg ccgcaaccac accaagggca gccactgtga gcagtgcctc ccgctgtttg 4380
tgggttcagc tgtcggaggc gggacctgcc ggccctgcca cgccttttgt cgtggaaata 4440
gccacatctg catctccagg aaggagttac aaatgtccaa gggagagcca aagaagtact 4500
cactggaccc agaggagatt gaaaactggg tgacagaggg tcctagtgaa gacgaggccg 4560
tgtgcgtgaa ctgccagaat aacagctatg gggagaaatg cgagagctgc ctgcagggct 4620
acttcctcct ggacgggaag tgcaccaaat gccagtgtaa tggccacgcg gacacatgta 4&80
acgagcagga tgggacgggc tgtccatgtc agaataacac agagacgggc acatgccagg 4740
gcagctcccc cagtgaccgt cgagactgct acaagtacca gtgcgccaag tgccgggaat 4800
catttcacgg gagtccgctg ggcggccagc agtgctaccg cctcatctcg gtggagcagg 4860
agtgctgcct ggaccccacg tcccagacca actgcttcca tgagcccaaa cgccgggcgc 4920
taggccccgg ccgcactgtc ctctttggcg tgcagcccaa attcaccaac gtggacatcc 4980
gcctgacgct ggacgtgacc ttcggggccg tggacctcta tgtctccacc tcctatgaca 5040
CCttCgtggt ccgtgtggcc cctgacactg gcgtccatac tgtacacatc cagccacccc 5100
cagccccacc acctccacca ccccctgcag atggtgggcc ccggggggct ggggatccag 5160
gaggagcagg ggccagcagt gggccgggcg ccccagcaga gccacgggta cgggaggtat 5220
ggccgcgggg cctgattacc tacgtgacgg tgacggagcc gtcggcagtg ctggtggtcc 5280
gcggcgtgcg ggaccggctg gtcatcacct acccacacga gcaccatgcc ctcaagtcga 5340
gccgcttcta cctgctgctg ctgggcgtgg gagacccaag~tgggcccggc gccaacggct 5400
27/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
cagccgactc gcagggcctg ctcttcttcc ggcaggacca ggcccacatt gacctgtttg 5460
tCttCttCtC CgtCttCttC tCCtgCttCt tCCtCttCCt Ct CdCtCtgt gtgctcctct 5520
ggaaggccaa gcaggctctg gaccagcggc aggagcagcg ccggcacttg caggagatga 5580
ccaagatggc cagccgcccc ttcgccaagg tcaccgtctg cttcccacct gaccctactg 5640
ccccggcctc cgcctggaag ccggctgggc tcccacctcc cgccttccgc cgctctgagc 5700
ccttcctggc acccctgctg ctgacagggg ccggtgggcc ctggggaccc atggg~gggg 5760
gctgctgccc accagccatc cccgccacca ctgctgggct gcgagctggg cccatcactc 5820
tcgagcccac agaagatggc atggctggcg tggccacact gctgctccag ctgcctggcg 5880
ggccccatgc acccaacggc gcctgcctgg ggtcagccct cgtcacactg cggcacaggc 5940
tgcacgagta ctgtgggggt ggtgggggtg ctgggggcag tgggcatggg actggtgcgg 6000
gccggaaggg actgttgagc caggacaacc tcaccagcat gtccctctga catgcc 6056
<210> 22
<211> 1699
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2173285CB1
<400> 22
ggggcgctgg gggtgacggt gcggagccgc tgccagcgct gggcgagagt cggcggccgg 60
atccgaggag caggcgggcc tgaggccgag tcagctgcgc gggcccccgg atcccccgac 120
agagcggcgg cggtgtctgg ccaggcggta ggcgctgcct ggccgcggcg gggaagatgt 180
tcagcgtaga gtcgctggag cgggcggagc tgtgcgagag cctcctcact tggatccaga 240
catttaatgt ggatgcacca tgccagaccg tggaagattt aacgaatggg gttgtgatgg 300
cccaggttct tcaaaagata gatcctgcat attttgatga aaattggcta aacagaatca 360
aaactgaagt aggagataat tggaggctaa agataagcaa tttaaagaaa attttaaaag 420
gaatcttgga ttataatcat gagattttag gacagcaaat taatgacttt acccttcctg 480
atgtgaacct tattggggag cattctgatg cagcagagct tggaaggatg cttcagctca 540
tcttaggctg tgctgtgaac tgtgaacaga agcaagagta catccaagcc attatgatga 600
tggaggaatc tgttcaacat gttgtcatga cagccattca agagctgatg agtaaagaat 660
CtCCtgtCtC tgctggaaat gatgcctatg ttgaccttga tcgtcagctg aagaaaacta 720
cagaggaact aaatgaagct ttgtcagcaa aggaagaaat tgctcaaaga tgccatgaac 780
tggatatgca ggttgcagca ttgcaggaag agaaaagtag tttgttggca gagaatcagg 840
tattaatgga aagactcaat caatctgatt ctatagaaga ccctaacagt ccagcaggaa 900
gaaggcattt gcagctccag actcaattag aacagctcca agaagaaaca ttcagactag 960
aagcagccaa agatgattat cgaatacgtt gtgaagagtt agaaaaggag atctctgaac 1020
ttcggcaaca gaatgatgaa ctgaccactt tggcagatga agctcagtct ctgaaagatg 1080
agatcgacgt gctgagacat tcttctgata aagtatctaa actagaaggt caagtagaat 1140
cttataaaaa gaagctagaa gaccttggtg atttaaggcg gcaggttaaa ctcttagaag 1200
agaagaatac catgtatatg cagaatactg tcagtctaga ggaagagtta agaaaggcca 2260
acgcagcgcg aagtcaactt gaaacctaca agagacaggt aaaagaaaca cagcatcttg 1320
atgatggttt caggcaagct ctcagttatg acatgtagct taccaaaatt actaatttgt 1380
tttcatggta ttctgttttt taccttttct ttattgtatt gattcattta ggagactgag 1440
tctcactctg tcacccagcc tggagtgcag tggcatgatc tcagctcact gcaacctcca 1500
cctcccaggt tcaagctatt CttCtgCCCC agCCtCCtga gtagctggaa ctacagacgc 1560
atgctgccac acctggctaa ttttttgtat tttggtaaag acagggtttc actgtgttgc 1620
ccaggctggt cttgtactcc tgagctcaag tgatccacca gcctcagcct tccaaagtgc 1680
taggattaca agcgtgagc 1699
<210> 23
<211> 1661
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7487619CB1
<400> 23
ctatttggtc cagccttatc gccccggact cgtaaccttc ggccatgccg tattataggc 60
28/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
tcccccatcc agtagtacag acattgcaat taactggcag agtcttttcc ctgattgaga 120
agtaaccctg tcatgtcatg cgaaggccag tgaaggagga ttttaggacc ataggtgacc 180
tctgtaaaat gagattgata tgtcctgcca aaaccagcaa aagacagaga cctctgccca 240
aaactgcaag aatgggatac tgccaacacc tgcaatagac ttggaagagg actgtggtcc 300
tcccatgaga tttcagcttc tggggacaca ttgattgaga cctgttgaga ctctcagcag 360
agggtccagc tgacctggcc cacagagagg gtgagataaa aattttacac gttttatggc 420
actaagtttg tgttattttt ttaatatatc aatggaaatc taatatagtg tttctctact 480
ttcttctgca tgtgtgtctc tgtgtgtgtg cacctgtgtg catgtgtgtg agagaggctg 540
aaataatttc atcatcatct ctgtgaggga agctttgtaa caagcgaagt gcaggataac 600
tccagaatta tctacctggt tgatgcagtt tccacataga gaatggattc tcatttctca 660
attaagtgct aaatgctggg tgctctttat atccccagag ggagagagac caagggtgag 720
aagaaatgtc caacgccagc ctcgtgacag cgttcatcct cacgggcctt ccccatgccc 780
cagggctgga cgcccccctc tttggaatct tcctggtggt ttacgtgctc actgtgctgg 840
ggaacctcct catcctgctg gtgatcaggg tggattctca cctccacacc cccatgtact 900
acttcctcac caacctgtcc ttcattgaca tgtggttctc cactgtcacg gtgcccaaaa 960
tgctgatgac ettggtgtcc ccaagcggca gggetatctc cttccacagc tgcgtggctc 2020
agctctattt tttccacttc ctggggagca ccgagtgttt cctctacaca gtcatggcct 1080
acgatcgcta cctggccatc agttacccgc tcaggtacac cagcatgatg actgggcgct 1140
cgtgtactct tctggccacc agcacttggc tcagtggctc tctgcactct gctgtccagg 1200
ccatattgac tttccatttg ccctactgtg gacccaactg gatccagcac tatttgtgtg 1260
atgcaccgcc catcctgaaa ctggcctgtg cagacacctc agccatagag actgtcattt 1320
ttgtgactgt tggaatagtg gcctcgggct gctttgtcct gatagtgctg tcctatgtgt 1380
CCatCgtCtg ttCCatCCtg cggatccgca cctcagaggg gaagcacaga gcctttcaga 1440
cctgtgcctc ccactgtatc gtggtccttt gcttctttgg ccctggtctt ttcatttacc 1500
tgaggccagg ctccaggaaa gctgtggatg gagttgtggc cgttttctac actgtgctga 1560
cgccccttct caaccctgtt gtgtacaccc tgaggaacaa ggaggtgaag aaagctctgt 1620
tgaagctgaa agacaaagta gcacattctc agagcaaata g 1661
<210> 24
<211> 2033
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7487607CB1
<400> 24
caatttccgg acctcggtgg tctgcccgcc tcgccctccc aaagtactgg gatttacagg 60
tcacctgccc tgctgggtag tatagaaaag gacatgattc aggtggggat gtcgaggata 120
caaagatgaa ggaagaagaa tggctgtctt tctttctatt cagaaaagat cccagaattc 180
tacatttatt cagcagaaac tatgccatgt atactatgtg agaggaagat cagcagaaca 240
gctgttcgag cagaggaagc tgacttctct gcaggaagaa ggcaatatgt attttctcat 300
acttagagat aatataccct gtgtcatcat ggcttccaaa cagtaatccc ttcttcagaa 360
attcatcagc ttcacagaaa attttgttga acagcctaaa aacagaagat caaggcaaaa 420
caatctgctg tgtattgcaa cctaagaagt gagctgacct tccatttaga ggttaaatag 480
agagtaaaat ggaatgggaa aaccacacca ttctggtgga attttttctg aagggacttt 540
ctggtcaccc aagacttgag ttactctttt ttgtgctcat cttcataatg tatgtggtca 600
tccttctggg gaatggtact ctcattttaa tcagcatctt ggaccctcac ettcacaccc 660
ctatgtactt ctttctgggg aacctctcct tcttggacat ctgctacacc accacctcta 720
ttCCCtCCaC gctagtgagc ttcctttcag aaagaaagac catttccctt tctggctgtg 780
cagtgcagat gttcctcggc ttggccatgg ggacaacaga gtgtgtgctt ctgggcatga 840
tggcctatga cegctatgtg gctatctgca accctctgag atatcccatc atcatgagta 900
aggatgccta tgtacccatg gcagctgggt cctggatcat aggagctgtc aattctgcag 960
tacaatcagt gtttgtggta caattgcctt tctgcaggaa taacatcatc aatcatttca 1020
cctgtgaaat tctggctgtc atgaaactgg cctgtgctga catctcagac aatgagttca 1080
tcatgcttgt ggccacaaca ttgttcatat tgacaccttt gttattaatc attgtctctt 1140
acacgttaat cattgtgagc atcttcaaaa ttagctcttc cgaggggaga agcaaagctt 1200
cctctacctg ttcagcccat ctgactgtgg tcataatatt ctatgggacc atcctcttca 1260
tgtacatgaa gcccaagtct aaagagacac ttaattcgga tgacttggat gctaccgaca 1320
aaattatatc catgttctat ggggtgatga ctcccatgat gaatccttta atctacagtc 1380
ttagaaacaa ggatgtgaaa gaggcagtaa aacacctact gaacagaagg ttctttagca 1440
agtgagtgca aaatgtactg gaatatgaac acacttgata ttgttgaaac ttcagaatta 1500
29/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
tgttagaatt ttgggtactt ttactatttt tatgcatttt catatattat gttaaaataa 1560
tgagatacag catttcaaaa ttattgcatg tccactctag agaatttgca agatacaggg 1620
cagtaggatg aagaagaaag aggggttacc tattactcta ctagtgggaa atggccccgt 1680
ttcaacattt tgaacagtaa ctttcatatt atgggttttt ttttctgcat tggaattggg 1740
tgtgatgtgc ctttttatgt tcactttttt ccataatgtt atttcatagg caacatttca 1800
tagaatcttt caaaataaat aaagccctct gttgtagaaa aagcaaaaca gaaaaacccc 1860
aacatagtgt actcacattt tccagggaca agcctgtgtt atagtttcac attaatctcc 1920
agatcctgtt aaagccacta aataaccagt ttctttttct gtatttaaat tttggtgtcg 1980
ggtgtccagc ctccaggttt ctcgggacca tcccaaaggg gcgggaataa atg 2033
<210> 25
<211> 1659
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7487616CB1
<400> 25
ctattcaccc tgagcaaata ctgacccatt tttcagcttc aacagagctt tctttacctc 60
cttgtttctc agggtgtaca caacagggtt gaaaagagga gtcagcgtgg tgtagaaaac 120
ggccacaacc ccatgcaagg cgtccctgga gcctggcctc aggtaaatga aaagaccagg 180
gccaaagaag caaaggacca cgatacagtg ggaggcacag gtctgaaagg ctctgtgcct 240
cccctctgag gtgcggatcc gcaggatgga acagacgatg gacacatagg acagcactat 300
caggacaaag cagcccgagg ccactagccc aatattcaca aagatgacca tctcgttggc 360
tgaggtgtct gcacaggcca gtttcaggat gggcggtgcg tcacagaagt agtgctggat 420
ctggttgggt ccacagtagg gcaaatggaa agtcaatatg gtctggacag cagagtgcag 480
agagccactg agccaagtgc cggtggccag gagggcacac gagcgcccag tcatcatgtt 540
ggtgtacctg agcgggtaac tgatggccag gtagcgatca taggacatga ctgtgtagag 600
gaaacactcg gtgctcccca ggaagtggaa aaaatagagc tgagccacgc agctgtggaa 660
ggagatagtc ctgccgcttg gggacaccaa ggtcatcagc attttgggca ccgtgacagt 720
ggagaaccac atgtcaatga aggacaggtt ggtgaggaag tagtacatgg gggtgtggag 780
gtgagaatcc accctgatca ccagcaggat gaggaggttc cccagcacag tgagcacgta 840
aaccaccagg aagattccaa agaggggggc gtccagccct ggggcatggg gaaggcccgt 900
gaggatgaac gctgtcacga ggctggcgtt ggacatttct tctcaccctt ggtctctctc 960
cctctgggga tataaagagc acccagcatt tagcacttaa ttgagaaatg agaatccatt 1020
ctctatgtgg aaactgcatc aaccaggtag ataattctgg agttatcctg cacttcgctt 1080
gttacaaagc ttccctcaca gagatgatga tgaaattatt tcagcctctc tcacacacat 1140
gcacacaggt gcacacacac agagacacac atgcagaaga aagtagagaa acactatatt 1200
agatttccat tgatatatta aaaaaataac acaaacttag tgccataaaa cgtgtaaaat 1260
ttttatctca ccctctctgt gggccaggtc agctggaccc tctgctgaga gtctcaacag 1320
gtctcaatca atgtgtcccc agaagctgaa atctcatggg aggaccacag tcctcttcca 1380
agtctattgc aggtgttggc agtatcccat tcttgcagtt ttgggcagag gtctctgttt 1440
tgctggtttt ggcaggacat atcaatctca ttttacagag gtcacctatg gtcctaaaat 1500
cctccttcac tggccttcgc atgacatgac agggttactt ctcaatcagg gaaaagactc 1560
tgccagttaa ttgcaatgtc tgtactactg gatgggggag cctataatac ggcatggccg 1620
aaggttacga gtccggggcg ataaggctgg accaaatag 1659
<210> 26
<211> 1175
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7483204CB1
<400> 26
cccttttagg gttttttttt tttaatacag taataatgcc tctttgaaag tgacactcac 60
ctgggatact ttttgagggt aaagaagata atttacataa accaatcttg ttctacttta 120
cagataattt tttttttgat atcttggaaa gtcaagttct agcctgtcat tctcgtaatg 180
atttctgtag cagtttgaaa caagaacaag gaagaatgga ctgggaaaat tgctcctcat 240
30/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
taactgattt ttttctcttg ggaattacca ataacccaga gatgaaagtg accctatttg 300
ctgtattctt ggctgtttat atcattaatt tctcagcaaa tcttggaatg atagttttaa 360
tcagaatgga ttaccaactt cacacaccaa tgtatttctt cctcagtcat ctgtctttct 420
gtgatctctg ctattctact gcaactgggc ccaagatgct ggtagatcta cttgccaaga 480
acaagtcaat acccttctat ggctgtgctc tgcaattctt ggtcttctgt atctttgcag 540
attctgagtg tctactgctg tcagtgatgg cctttgatcg gtacaaggcc atcatcaacc 600
ccctgctcta tacagtcaac atgtctagca gagtgtgcta tctactcttg actggggttt 660
atctggtggg aatagcagat gctttgatac atatgacact ggccttccgc ctatgcttct 720
gtgggtctaa tgagattaat catttcttct gtgatatccc tcctctctta ttactctctt 780
gctcagatac acaggtcaat gagttagtgt tattcaccgt ctttggtttt attgaactga 840
gtaccatttc aggagttttc atttcttatt gttatatcat cctatcagtc ttggagatac 900
actctgctga ggggaggttc aaagctctct ctacatgcac ttcccactta tctgcggttg 960
caattttcca gggaactctg ctctttatgt atttccggcc aagttcttcc tattctctag 1020
atcaagataa aatgacctca ttgttttaca cccttgtggt tcccatgttg aaccccctga 1080
tttatagcct gaggaacaag gatgtgaaag aggccctgaa aaaactgaaa aatgaaattt 1140
tattttaagg aaatagtaaa aatacatgtt tatac 1175
<210> 27
<211> 1737
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472099CB1
<400> 27
tcagagttga gctgggggag gcttcagagg ctctgcatgc ccaggaggct tctctgagac 60
tgggaaggga gtggaaactc agtagcccgt ggtcctggtt gcgcgctccc tcccatgtct 120
tcattatctg atttggtaaa atatctacaa gacgggggag tccgggctgc tttctttgac 180
actggtcatc actccacacc tagacgctat gtccatgtct ggaagatgct tttaaggaag 240
ttttgatgca gagaagagtg gccggtgctc ttCCtCgCgt gCCaCCatCt gCttaCttCC 300
agaagctaat tattttcatg tgactgatgt gactaatatt ttttaacgcc tgcaggttcg 360
ttatgtacca gcaaatgttc atggtactga agatatacca gttgaagaga aagaatgtct 420
ggttgtcata gtacctattt gctacgaggt caaatcttgt ccctggaaga aatgtacaaa 480
tattaatact taaaacagta ttttccatta agaagagaat tttattctga taaggtgaag 540
gagcctatga agatcaacaa ggagaatttc caagagtcat gtcagcctcc agtatcacct 600
caacacatcc aacttccttc ttgttgatgg ggattccagg cctggagcac ctgcacatct 660
ggatctccat ccccttctca gcatatacac tggccctgct tggaaactgc accctccttc 720
tcatcatcca ggctgatgca gccctccatg agcccatata cctctttetg gccatgttgg 780
cagccatcga cctggtcctt tcctcctcag cattgcccaa aatgcttgcc atattctggt 840
tcagggatcg ggagatcaac ttttttgcct gtctggtcca gatgttcttc cttcactcct 900
tctccatcat ggagtcagca gtgctgctgg ccatggcctt tgaccgctat gtggccatct 960
gcaagccact gcactacacc acggtcctga ctgggtccct catcaccaag attggcatgg 1020
ctgctgtggc ccgggctgtg acactaatga ctccactccc cttcctgctg agatgtttcc 1080
actactgccg aggcccagtg attgcccgct gctactgtga acacatggct gtggtcaggc 1140
tggctgtggg aacactaggc ttcaacaata tctatggcat tgctgtggcc atgtttattg 1200
gagtgttgga tctattcttt atcatcctat cttatatctt tatccttcag gcagttctac 1260
aactctcctc tcaggaggcc cgctacaaag catttgggac atgtgtctct cacataggtg 1320
ccatcttagc cttctacaca ccttcagtca tctcttcagt catgcaccgt gtggcccgct 2380
gtgctgtgcc acacgtccac attctcctcg ccaatttcta tCtgCtCttC ccacccatgg 1440
tcaatcccat catctacggc gttaagacca agcagatccg tgacagtctt gggagtattc 1500
ccgagaaagg atgtgtgaat agagagtgag gaataagtgg aaaaagagtg gggcacagtg 1560
aatgctgtag tgggccaggg ctgtgctgag agtagatggg tgctagactc cacgtttagt 1620
tcttttcttg tattatgaaa agaataaatg atgtcctgaa gctcagtgcc acagtctgtt 1680
aagaattgtg ggtctttgcc ctcggtacct ctggattgaa ctggtgactg tgcggtc 1737
<210> 28
<211> 972 .
<212> DNA
<213> Homo Sapiens
<220>
31/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
<221> misc_feature
<223> Incyte ID No: 7485443CB1
<400> 28
taaatagaga gtaaaatgga atgggaaaac cacaccattc tggtggaatt ttttctgaag 60
ggactttctg gtcacccaag acttgagtta ctcttttttg tgctcatctt cataatgtat 120
gtggtcatcc ttctggggaa tggtactctc attttaatca gcatcttgga ccctcacctt 180
cacaceccga tgtacttctt tctggggaac ctetccttct tggacatctg ctacaccacc 240
acctctattc cctccacact agtgagcttc ctttcagaaa gaaagaccat ttccttttct 300
ggctgtgcag tgcagatgtt ccttggcttg gccatgggga caacagagtg tgtgcttctg 360
ggcatgatgg cctttgaccg ctatgtggct atctgcaacc ctctgagata tcccatcatc 420
atgagcaaga atgcctatgt acccatggct gttgggtcct ggtttgcagg gattgtcaac 480
tctgcagtac aaactacatt tgtagtacaa ttgcctttct gcaggaagaa tgtcatcaat 540
catttctcat gtgaaattct agctgtcatg aagttggcct gtgctgacat ctcaggcaat 600
gagttcctca tgcttgtggc cacaatattg ttcacattga tgccactgct cttgatagtt 660
atctcttact cattaatcat ttccagcatc ctcaagattc actcctctga ggggagaagc 720
aaagctttct ctacctgctc agcccatctg actgtggtca taatattcta tgggaccatc 780
ctcttcatgt atatgaagcc caagtctaaa gagacactta attcagatga cttggatgct 840
accgacaaaa ttatatccat gttctatggg gtgatgactc ccatgatgaa tcctttaatc 900
tacagtctta gaaacaagga tgtgaaagag gcagtaaaac acctaccgaa cagaaggttc 960
tttagcaagt ga 972
<210> 29
<211> 1592
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3090414CB1
<400> 29
tagactacag gcccagagac tggaaacttt tccacgctag gtcccagctt gagctgtgtc 60
ataaccaagt tctctaagat tcaactataa aaacttacta aggtatggag aagaagaaaa 120
cattaatctg cagaaagccc agacaaattt tgagctattt cataacctac cagacttatc 180
atgctaacac tgaataaaac agacctaata ccagcttcat ttattctgaa tggagtccca 240
ggactggaag acacacaact ctggatttcc ttcccattct gctctatgta tgttgtggct 300
atggtaggga attgtggact cctctacctc attcactatg aggatgccct gcacaaaccc 360
atgtactact tcttggccat gctttccttt actgaccttg ttatgtgctc tagtacaatc 420
cctaaagccc tctgcatctt ctggtttcat ctcaaggaca ttggatttga tgaatgcctt 480
gtccagatgt tcttcatcca caccttcaca gggatggagt ctggggtgct tatgcttatg 540
gccctggatc gctatgtggc catctgctac cccttacgct attcaactat cctcaccaat 600
cctgtaattg caaaggttgg gactgccacc ttcctgagag gggtattact cattattccc 660
tttactttcc tcaccaagcg cctgccctac tgcagaggca atatacttcc ccatacctac 720
tgtgaccaca tgtatgtagc caaattgtcc tgtggtaatg tcaaggtcaa tgccatctat 780
ggtctgatgg ttgccctcct gatttggggc tttgacatac tgtgtatcac catctcctat 840
accatgattc tccgggcagt ggtcagcctc tcctcagcag atgctcggca gaaggccttt 900
aatacctgca ctgcccacat ttgtgccatt gttttctcct atactccagc tttcttctcc 960
ttcttttccc accgctttgg ggaacacata atcccccctt cttgccacat cattgtagcc 1020
aatatttatc tgctcctacc acccactatg aaccctattg tctatggggt gaaaaccaaa 1080
cagatacgag actgtgtcat aaggatcctt tcaggttcta aggataccaa atcctacagc 1140
atgtgaatga acacttgcca ggagtgagaa gagaaggaaa gaattacttc tatttgcctc 1200
ttatgcagga gttcataaaa tctttctgga agtactgtat tgatcacaaa atggagtttg 1260
ttgactggtg cattctcaat aagtaccttg ggaatctcaa catcattgga aggcccacca 1320
acatttctat aaatttttta ccttctcact catgtgaagg accagtctaa taattaaacc 1380
atattttatt cgacaaaaaa aaaaaaaaaa aaaaaacggg ggggggcccg caactatgac 1440
gcccgcaacc ccggaatata ctccggcacg ggaaacaaca gggcgtaatt ctcgcacaaa 1500
ttttggcccc taaatggggc ccccgcgtgg gcgtccacct tgtactccca tcttgtgggg 1560
cgcccacggc ggggaaacct ccggccagga tg 1592
<210> 30
<211> 1480
<212> DNA
32/33
CA 02435260 2003-07-17
WO 02/057454 PCT/US02/01339
<213> Homo Sapiens
~220>
<221> misc_feature
<223> Incyte ID No: 7503710CB1
<400> 30
ggtggaggaa ccgacaggag gccgggagcc cccacctacc ccttgtggag ctgcaggagc 60
aagggcatgc agccagtcat gctggccctg tggtccctgc ttctgctctg gggcctggcg 120
actccatgcc aggagctgct agagacggtg ggcacgctcg ctcggattga caaggatgaa 180
ctcggcaaag ccatccagaa ctcactggtt ggggagccca ttctgcagaa tgtgctggga 240
tcggtcacag ctgtgaaccg gggcctcttg ggctcaggag ggctgcttgg aggaggcggc 300
ttgctgggcc acggaggggt ttttggcgtt gtcgaggagc tctctggtct gaagattgag 360
gagctcacgc tgccaaaggt gttgctgaag ctgctgccgg gatttggggt gcagctgagc 420
ctgcacacca aagtgggcat gcattgctct ggcccccttg gtggccttct gcagctggct 480
gcggaggtga acgtgacatc gcgggtggcg ctggccgtga gctcaagggg cacacccatc 540
cttatcctca agcgctgcag cacgctcctg ggccacatca gcctgttctc agggctgctg 600
cccacaccac tctttggggt cgtggaacag atgctcttca aggtgcttcc gggactgctg 660
tgccccgtgg tggacagtgt gctgggtgtg gtgaatgagc tcctgggggc tgtgctgggc 720
ctggtgtccc ttggggctct tgggtccgtg gaattctctc tggccacatt gcctctcatc 780
tccaaccagt acatagaact ggacatcaac cctatcgtga agagtgtagc tggtgatatc 840
attgacttcc ccaagtcccg tgccccagcc aaggtgcccc ccaagaagga ccacacatcc 900
caggtgatgg tgccactgta cctcttcaac accacgtttg gactcctgca gaccaacggc 960
gccctcgaca tggacatcac ccctgagctg gttcccagcg atgtcccact gacaactaca 1020
gacctggcag ctttgctccc tgaggtcatg actgtgcgtg cccagctggc tccctcggct 1080
accaagctgc acatctccct gtccctggaa cggctcagtg tcaaggtggc ctcctccttt 1140
acccatgcct ttgacggatc gcgtttagaa gaatggctca gccatgtggt cggggcagtg 1200
tatgcaccaa agcttaacgt ggccctggat gttggaattc ccctgcctaa ggttcttaat 1260
atcaattttt ccaattcagt tctggagatc gtagagaatg ctgtggcagc tctctatgtc 1320
cttgtagtag catagaagat ggtgttcttc tcagatcagt ggactatgcc atgttatttt 1380
gttcttggac taaggccctg tgaggtgcaa ctggtccact ttcatttttg gtcagagatg 1440
gagaataagg aattatatgt tggtactagc actggaatag ' 1480
33/33
