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CA 02443770 2003-10-15
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PROTEINS AND NUCLEIC ACIDS ENCODING SAME
FIELD OF THE INVENTTON
The invention relates to polynucleotides and the polypeptides encoded by such
polynucleotides, as well as vectors, host cells, antibodies and recombinant
methods for
producing the polypeptides and polynucleotides, and methods for using the
same.
BACKGROUND OF THE INVENTION
The invention generally relates to nucleic acids and polypeptides encoded
therefrom.
More specifically, the invention relates to nucleic acids encoding
cytoplasmic, nuclear,
membrane bound, and secreted polypeptides, as well as vectors, host cells,
antibodies, and
recombinant methods for producing these nucleic acids and polypeptides.
Heart disease is the primary cause of death in most western societies. Death
from heart
disease is often induced by platelet-dependent ischemic syndromes which are
initiated by
atherosclerosis and arteriosclerosis and include, but are not limited to,
acute myocardial
infarction, chronic unstable angina, transient ischemic attacks and strokes,
peripheral vascular
disease, arterial thrombosis, preeclampsia, embolism, restenosis and/or
thrombosis following
angioplasty, carotid endarterectomy, anastornosis of vascular grafts, and
chronic
cardiovascular devices (e.g., in-dwelling catheters or shunts "extracorporeal
circulating
devices"). These syndromes represent a variety of stenotic and occlusive
vascular disorders
thought to be initiated by platelet activation either on vessel walls or
within the lumen by
blood-borne mediators but are manifested by platelet aggregates which form
thrombi that
restrict blood flow.
For example, Thrombospondin-1-like proteins associate with the extracellular
matrix
and inhibits angiogenesis in vivo. In vitro, Thrombospondin-like proteins
block capillary-like
tube formation and endothelial cell proliferation. The antiangiogenic activity
is mediated by a
region that contains 3 type 1 (properdin or thrombospondin) repeats.
In addition, Selectin-like proteins such as P-selectin, also called GMP-140,
CD62, or
selectin P, is a 140-kD adhesion molecule, expressed at the surface of
activated cells, that
mediates the interaction of activated endothelial cells or platelets with
leukocytes. In
endothelial cells, the protein is localized to the membranes of Weibel-Palade
bodies, the
intracellular storage granules for von Willebrand factor.
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Many disease states are characterized by uncontrolled cell proliferation.
These diseases
involve a variety of cell types and include disorders such as cancer,
psoriasis, pulmonary
fibrosis, glomeruloneplmitis, atherosclerosis and restenosis following
angioplasty. Vital
cellular functions such as cell proliferation and signal transduction are
regulated in part by the
balance between the activities of protein-tyrosine kinases (PTI~) and protein-
tyrosine
phosphatases (PTPase). Oncogenesis can result from an imbalance.
SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of nucleic acid sequences
encoding
novel polypeptides. The novel nucleic acids and polypeptides are referred to
herein as NOVX,
or NOV1, NOV2, NOV3, NOV4, NOVS, NOV6, NOV7, NOVB, NOV9, NOVlOa, NOVlOb,
NOV 11, NOV 12, NOV 13, NOV 14, NOV 1 S, and NOV 16 nucleic acids and
polypeptides.
These nucleic acids and polypeptides, as well as variants, derivatives,
homologs, analogs and
fragments thereof, will hereinafter be collectively designated as "NOVX"
nucleic acid or
polypeptide sequences.
1 S In one aspect, the invention provides an isolated NOVX nucleic acid
molecule
encoding a NOVX polypeptide that includes a nucleic acid sequence that has
identity to the
nucleic acids disclosed in SEQ m NOS:1, 3, S, 7, 9, 11, 13, 1S, 17, 19, 21,
23, 2S, 27, 29, 31,
and 33. Tn some embodiments, the NOVX nucleic acid molecule will hybridize
under
stringent conditions to a nucleic acid sequence complementary to a nucleic
acid molecule that
includes a protein-coding sequence of a NOVX nucleic acid sequence. The
invention also
includes an isolated nucleic acid that encodes a NOVX polypeptide, or a
fragment, homolog,
analog or derivative thereof. For example, the nucleic acid can encode a
polypeptide at least
80% identical to a polypeptide comprising the amino acid sequences of SEQ m
NOS:2, 4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34. The nucleic acid
can be, for example,
a genomic DNA fragment or a cDNA molecule that includes the nucleic acid
sequence of any
of SEQ lD NOS:1, 3, S, 7, 9, 11, 13, 1S, 17, 19, 21, 23, 2S, 27, 29, 31, and
33.
Also included in the invention is an oligonucleotide, e.g., an oligonucleotide
which
includes at least 6 contiguous nucleotides of a NOVX nucleic acid (e.g., SEQ m
NOS:1, 3, S,
7, 9, 11, 13, 1 S, 17~ 19, 21, 23, 2S, 27, 29, 31, and 33) or a complement of
said
oligonucleotide.
Also included in the invention are substantially purified NOVX polypeptides
(SEQ m
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34). In
certain embodiments,
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the NOVX polypeptides include an amino acid sequence that is substantially
identical to the
amino acid sequence of a human NOVX polypeptide.
The invention also features antibodies that immunoselectively bind to NOVX
polypeptides, or fragments, homologs, analogs or derivatives thereof.
In another aspect, the invention includes pharmaceutical compositions that
include
therapeutically- or prophylactically-effective amounts of a therapeutic and a
pharmaceutically-
acceptable carrier. The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX
polypeptide,
or an antibody specific for a NOVX polypeptide. In a further aspect, the
invention includes, in
one or more containers, a therapeutically- or prophylactically-effective
amount of this
pharmaceutical composition.
In a further aspect, the invention includes a method of producing a
polypeptide by
culturing a cell that includes a NOVX nucleic acid, under conditions allowing
for expression
of the NOVX polypeptide encoded by the DNA. If desired, the NOVX polypeptide
can then
be recovered.
In another aspect, the invention includes a method of detecting the presence
of a
NOVX polypeptide in a sample. In the method, a sample is contacted with a
compound that
selectively binds to the polypeptide mzder conditions allowing for formation
of a complex
between the polypeptide and the compound. The complex is detected, if present,
thereby
identifying the NOVX polypeptide within the sample.
The invention also includes methods to identify specif c cell or tissue types
based on
their expression of a NOVX.
Also included in the invention is a method of detecting the presence of a NOVX
nucleic acid molecule in a sample by contacting the sample with a NOVX nucleic
acid probe
or primer, and detecting whether the nucleic acid probe or primer bound to a
NOVX nucleic
acid molecule in the sample.
In a further aspect, the invention provides a method for modulating the
activity of a
NOVX polypeptide by contacting a cell sample that includes the NOVX
polypeptide with a
compound that binds to the NOVX polypeptide in an amount sufficient to
modulate the
activity of said polypeptide. The compound can be, e.g., a small molecule,
such as a nucleic
acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other
organic (carbon
containing) or inorganic molecule, as further described herein.
Also within the scope of the invention is the use of a therapeutic in the
manufacture of
a medicament for treating or preventing disorders or syndromes including,
e.g., those
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described for the individual NOVX nucleotides and polypeptides herein, and/or
other
pathologies and disorders of the like.
The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or a
NOVX-
specific antibody, or biologically-active derivatives or fragments thereof.
For example, the
compositions of the present invention will have efficacy for treatment of
patients suffering
from the diseases and disorders disclosed below and/or other pathologies and
disorders of the
like. The polypeptides can be used as irrnnunogens to produce antibodies
specific for the
invention, and as vaccines. They can also be used to screen for potential
agonist and
antagonist compounds. For example, a cDNA encoding NOVX rnay be useful in gene
therapy, and NOVX may be useful when administered to a subject in need
thereof. By way of
non-limiting example, the compositions of the present invention will have
efficacy for
treatment of patients suffering from the diseases and disorders disclosed
above and/or other
pathologies and disorders of the like.
The invention further includes a method for screening for a modulator of
disorders or
syndromes including, e.g., the diseases and disorders disclosed above and/or
other pathologies
and disorders of the like. The method includes contacting a test compound with
a NOVX
polypeptide and determining if the test compound binds to said NOVX
polypeptide. Binding
of the test compound to the NOVX polypeptide indicates the test compound is a
modulator of
activity, or of latency or predisposition to the aforementioned disorders or
syndromes.
Also within the scope of the invention is a method for screening for a
modulator of
activity, or of latency or predisposition to an disorders or syndromes
including, e.g., the
diseases and disorders disclosed above andlor other pathologies and disorders
of the like by
administering a test compound to a test animal at increased risk for the
aforementioned
disorders or syndromes. The test animal expresses a recombinant polypeptide
encoded by a
NOVX nucleic acid. Expression or activity of NOVX polypeptide is then measured
in the test
animal, as is expression or activity of the protein in a control animal which
recombinantly-
expresses NOVX polypeptide and is not at increased risk for the disorder or
syndrome. Next,
the expression of NOVX polypeptide in both the test animal and the control
animal is
compared. A change in the activity of NOVX polypeptide in the test animal
relative to the
control animal indicates the test compound is a modulator of latency of the
disorder or
syndrome.
In yet another aspect, the invention includes a method for determining the
presence of
or predisposition to a disease associated with altered levels of a NOVX
polypeptide, a NOVX
nucleic acid, or both, in a subject (e.g., a human subject). The method
includes measuring the
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amount of the NOVX polypeptide in a test sample from the subject and comparing
the amount
of the polypeptide in the test sample to the amount of the NOVX polypeptide
present in a
control sample. An alteration in the level of the NOVX polypeptide in the test
sample as
compared to the control sample indicates the presence of or predisposition to
a disease in the
subject. Preferably, the predisposition includes, e.g., the diseases and
disorders disclosed
above and/or other pathologies and disorders of the like. Also, the expression
levels of the new
polypeptides of the invention can be used in a method to screen for various
cancers as well as
to determine the stage of cancers.
Tn a further aspect, the invention includes a method of treating or preventing
a
pathological condition associated with a disorder in a mammal by administering
to the subject
a NOVX polypeptide, a NOVX nucleic acid, or a NOVX-specific antibody to a subj
ect (e.g., a
human subject), in an amount sufficient to alleviate or prevent the
pathological condition. In
preferred embodiments, the disorder, includes, e.g., the diseases and
disorders disclosed above
and/or other pathologies and disorders of the like.
W yet another aspect, the invention can be used in a method to identity the
cellular
receptors and downstream effectors of the invention by any one of a number of
techniques
commonly employed in the art. These include but are not limited to the two-
hybrid system,
affinity purification, co-precipitation with antibodies or other specific-
interacting molecules.
Unless otherwise defined, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although methods and materials similar or equivalent to those
described herein can
be used in the practice or testing of the present invention, suitable methods
and materials are
described below. All publications, patent applications, patents, and other
references
mentioned herein are incorporated by reference in their entirety. In the case
of conflict, the
present specification, including definitions, will control. In addition, the
materials, methods,
and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the
following detailed
description and claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel nucleotides and polypeptides encoded
thereby.
Included in the invention are the novel nucleic acid sequences and their
polypeptides. The
sequences are collectively referred to as "NOVX nucleic acids" or "NOVX
polynucleotides"
and the corresponding encoded polypeptides are referred to as "NOVX
polypeptides" or
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"NOVX proteins." Unless indicated otherwise, "NOVX" is meant to refer to any
of the novel
sequences disclosed herein. Table A provides a summary of the NOVX nucleic
acids and
their encoded polypeptides.
TABLE A. Sequences and Corresponding SEQ ID Numbers
NOVX Internal SEQ SEQ gomolo
ID ID gy
Identification NO (nt)NO (aa)
1 CG93221-01 1 2 Paladin
Plasma membrane ring finger
2 CG93210-01 3 4 protein
Thrombospondin-1 domain
3 CG93275-01 5 6 containing protein
Protocadherin alpha C2
short
4 CG93187-O1 7 8 form
COR CG95083-01 9 10 Nuclear protein
6 COR CG94989-Ol 11 12 Secretory protein
Transmission-blocking
target
7 COR CG94978-O1 13 14 antigen 5230 precursor
8 COR CG94713-02 l5 16 Nuclear protein
9 COR CG94702-01 17 18 Hemicentin precursor
10a COR CG94661-01 19 20 Selectin
lOb COR CG94661-02 21 22 Selectin
11 COR CG94325-01 23 24 Nuclear protein
12 COR CG94282-O1 25 26 Plasma membrane protein
13 COR CG94399-01 27 28 BHLH Factor MATH6
Putative protein-tyrosine
14 COR CG94366-01 29 30 phosphatase
CG95387-02 31 32 LRR protein
16 CG95419-02 33 34 RhoGEF
NOVX nucleic acids and their encoded polypeptides are useful in a variety of
applications and contexts. The various NOVX nucleic acids and polypeptides
according to the
invention are useful as novel members of the protein families according to the
presence of
domains and sequence relatedness to previously described proteins.
Additionally, NOVX
10 nucleic acids and polypeptides can also be used to identify proteins that
are members of the
family to which the NOVX polypeptides belong.
The NOVX genes and their corresponding encoded proteins are useful for
preventing,
treating or ameliorating medical conditions, e.g., by protein or gene therapy.
Pathological
conditions can be diagnosed by determinng the amount of the new protein in a
sample or by
15 determining the presence of mutations in the new genes. Specific uses are
described for each
of the sixteen genes, based on the tissues in which they are most highly
expressed. Uses
include developing products for the diagnosis or treatment of a variety of
diseases and
disorders.
The NOVX nucleic acids and polypeptides can also be used to screen for
molecules,
which inhibit or enhance NOVX activity or function. Specifically, the nucleic
acids and
polypeptides according to the invention may be used as targets for the
identification of small
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molecules that modulate or inhibit, e.g., cell growth, cell metabolism, cell
differentiation, cell
proliferation, andlor cell signaling.
In one embodiment of the present invention, NOVX 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 NOVX. Examples of such disorders include, but are
not limited to,
cancers such as 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; neurological disorders 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-Takob 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,
akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,
dystonias, paranoid
psychoses, postherpetic neuralgia, and Tourette's disorder; and disorders of
vesicular transport
such as cystic fibrosis, glucose-galactose malabsorption syndrome,
hypercholesterolemia,
diabetes mellitus, diabetes insipidus, hyper- and hypoglycemia, Grave's
disease, goiter,
Cushing's disease, Addison's disease, gastrointestinal disorders including
ulcerative colitis,
gastric and duodenal ulcers, other conditions associated with abnormal vesicle
trafficking
including acquired immunodeficiency syndrome (A)DS), allergic reactions,
autoimmune
hemolytic anemia, proliferative glomerulonephritis, inflammatory bowel
disease, multiple
sclerosis, myasthenia gravis, rheumatoid arthritis, osteoarthritis,
scleroderma, Chediak-Higashi
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syndrome, Sjogren's syndrome, systemic lupus erythiematosus, toxic shock
syndrome,
traumatic tissue damage, and viral, bacterial, fungal, helininthic, and
protozoal infections, as
well as additional indications listed for the individual NOVX clones.
The NOVX nucleic acids and proteins of the invention are useful in potential
S diagnostic and therapeutic applications and as a research tool. These
include serving as a
specific or selective nucleic acid or protein diag~iostic and/or prognostic
marker, wherein the
presence or amount of the nucleic acid or the protein are to be assessed.
These also include
potential therapeutic applications such as the following: (i) a protein
therapeutic, (ii) a small
molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug
targeting/cytotaxic
antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene
ablation), (v) an agent
promoting tissue regeneration i~a vitro and in vivo, and (vi) a biological
defense weapon.
Additional utilities for the NOVX nucleic acids and polypeptides according to
the invention
are disclosed herein.
NOV1
A NOV 1 polypeptide has been identified as a Paladin-like protein (also
referred to as
CG93221-O1). The disclosed novel NOV1 nucleic acid (SECT m NO:l) of 2600
nucleotides is
shown in Table 1A. The novel NOV1 nucleic acid sequences maps to the
chromosome 10.
An ORF begins with an ATG initiation codon at nucleotides 15-17 and ends with
a
TAG cadon at nucleotides 2583-2585. A putative untranslated region and/or
downstream
from the termination codon is underlined in Table IA, and the start and stop
codons are in
bold letters.
Table 1A. NOV1 Nucleotide Sequence (SEQ ID NO:1)
GCTGCTGGCAGACTATGGGTACAACGGCCAGCACAGCCCAGCAGACGGTCTCGGCAGGCACCCCATT
TGAGGGCCTACAGGGCAGTGGCACGATGGACAGTCGGCACTCCGTCAGCATCCACTCCTTCCAGAGC
ACTAGCTTGCATAACAGCAAGGCCAAGTCCATCATCCCCAACAAGGTGGCCCCTGTTGTGATCACGT
ACAACTGCAAGGAGGAGTTCCAGATCCATGATGAGCTGCTCAAGGCTCATTACACGTTGGGCCGGCT
CTCGGACAACACCCCTGAGCACTACCTGGTGCAAGGCCGCTACTTCCTGGTGCGGGATGTCACTGAG
AAGATGGATGTGCTGGGCACCGTGGGAAGCTGTGGGGCCCCCAACTTCCGGCAGGTGCAGGGTGGGC
TCACTGTGTTCGGCATGGGACAGCCCAGCCTCTTAGGGTTCAGGCGGGTCCTCCAGAAACTCCAGAA
GGACGGACATAGGGAGTGTGTCATCTTCTGTGTGCGGGAGGAACCTGTGCTTTTCCTGCGTGCAGAT
GAGGACTTTGTGTCCTACACACCTCGAGACAAGCAGAACCTTCATGAGAACCTCCAGGGCCTTGGAC
CCGGGGTCCGGGTGGAGAGCCTGGAGCTGGCCATCCGGAAAGAGATCCACGACTTTGCCCAGCTGAG
CGAGAACACATACCATGTGTACCATAACACCGAGGACCTGTGGGGGGAGCCCCATGCTGTGGCCATC
CATGGTGAGGACGACTTGCATGTGACGGAGGAGGTGTACAAGCGGCCCCTCTTCCTGCAGCCCACCT
ACAGGTACCACCGCCTGCCCCTGCCCGAGCAAGGGAG'T'CCCCTGGAGGCCCAGTTGGACGCCTT'T'GT
CAGTGTTCTCCGGGAGACCCCCAGCCTGCTGCAGCTCCGTGATGCCCACGGGCCTCCCCCAGCCCTC
GTCTTCAGCTGCCAGATGGGCGTGGGCAGGACCAACC'T'GGGCATGGTCCTGGGCACCCTCATCCTGC
TTCACCGCAGTGGGACCACCTCCCAGCCAGAGGCTGCCCCCACGCAGGCCAAGCCCCTGCCTATGGA
GCAGTTCCAGGTGATCCAGAGCTTTCTCCGCATGGTGCCCCAGGGAAGGAGGATGGTGGAAGAGGTG
GACAGAGCCATCACTGCCTGTGCCGAGTTGCATGACCTGAAAGAAGTGGTCTTGGAAAACCAGAAGA
AGTTAGAAGGTATCCGACCGGAGAGCCCAGCCCAGGGAAGCGGCAGCCGACACAGCGTCTGGCAGAG
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GGCGCTGTGGAGCCTGGAGCGATACTTCTACCTGATCCTGTTTAACTACTACCTTCATGAGCAGTAC
CCGCTGGCCTTTGCCCTCAGTTTCAGCCGCTGGCTGTGTGCCCACCCTGAGCTGTACCGCCTGCCCG
TGACGCTGAGCTCAGCAGGCCCTGTGGCTCCGAGGGACCTCATCGCCAGGGGCTCCCTACGGGAGGA
CGATCTGGTCTCCCCGGACGCGCTCAGCACTGTCAGAGAGATGGATGTGGCCAACTTCCGGCGGGTG
CCCCGCATGCCCATCTACGGCACGGCCCAGCCCAGCGCCAAGGCCCTGGGGAGCATCCTGGCCTACC
TGACGGACGCCAAGAGGAGGCTGCGGAAGGTTGTCTGGGTGAGCCTTCGGGAGGAGGCCGTGTTGGA
GTGTGACGGGCACACCTACAGCCTGCGGTGGCCTGGGCCCCCTGTGGCTCCTGACCAGCTGGAGACC
CTGGAGGCCCAGCTGAAGGCCCATCTAAGCGAGCCTCCCCCAGGCAAGGAGGGCCCCCTGACCTACA
GGTTCCAGACCTGCCTTACCATGCAGGAGGTCTTCAGCCAGCACCGCAGGGCCTGTCCTGGCCTCAC
CTACCACCGCATCCCCATGCCGGACTTCTGTGCCCCCCGAGAGGAGGACTTTGACCAGCTGCTGGAG
GCCCTGCGGGCCGCCCTCTCCAAGGACCCAGGCACTGGCTTCGTGTTCAGCTGCCTCAGCGGCCAGG
GCCGTACCACAACTGCGATGGTGGTGGCTGTCCTGGCCTTCTGGCACATCCAAGGCTTCCCCGAGGT
GGGTGAGGAGGAGCTCGTGAGTGTGCCTGATGCCAAGTTCACTAAGGGTGAATTTCAGGTAGTAATG
AAGGTGGTGCAGCTGCTACCCGATGGGCACCGTGTGAAGAAGGAGGTGGACGCAGCGCTGGACACTG
TCAGCGAGACCATGACGCCCATGCACTACCACCTGCGGGAGATCATCATCTGCACCTACCGCCAGGC
GAAGGCAGCGAAAGAGGCGCAGGAAATGCGGAGGCTGCAGCTGCGGAGCCTGCAGTACTTGGAGCGC
TATGTCTGCCTGATTCTCTTCAACGCGTACCTCCACCTGGAGAAGGCCGACTCCTGGCAGAGGCCCT
TCAGCACCTGGATGCAGGAGGTGGCATCGAAGGCTGGCATCTACGAGATCCTTAACGAGCTGGGCTT
CCCCGAGCTGGAGAGCGGGGAGGACCAGCCCTTCTCCAGGCTGCGCTACCGGTGGCAGGAGCAGAGC
TGCAGCCTCGAGCCCTCTGCCCCCGAGGACTTGCTGTAGGGGGCCTTACTCCCT
Variant sequences of NOV1 are included in Example 3, Table 18. A variant
sequence
can include a single nucleotide polymorphism (SNP). A SNP can, in some
instances, be
referred to as a "cSNP" to denote that the nucleotide sequence containing the
SNP originates
as a cDNA.
The NOV 1 protein (SEQ m N0:2) encoded by SEQ >D NO:1 is 856 amino acid
residues in length and is presented using the one-letter amino acid code in
Table 1B. Psort
analysis predicts the NOV 1 protein of the invention to be localized in the
cytoplasm with a
certainty of 0.4500.
Table 1B. Encoded NOV1 protein sequence (SEQ ID NO:2)
MGTTASTAQQTVSAGTPFEGLQGSGTMDSRHSVSIHSFQSTSLHNSKAKSIIPNKVAPWITYNC
KEEFQIHDELLKAHYTLGRLSDNTPEHYLVQGRYFLVRDVTEKMDVLGTVGSCGAPNFRQVQGGL
TVFGMGQPSLLGFRRVLQKLQKDGHRECVIFCVREEPVLFLRADEDFVSYTPRDKQNLHENLQGL
GPGVRVESLELAIRKEIHDFAQLSENTYHVYHNTEDLWGEPHAVAIHGEDDLHVTEEWKRPLFL
QPTYRYHRLPLPEQGSPLEAQLDAFVSVLRETPSLLQLRDAHGPPPALVFSCQMGVGRTNLGMVL
GTLILLHRSGTTSQPEAAPTQAKPLPMEQFQVIQSFLRMVPQGRRMVEEVDRAITACAELHDLKE
WLENQKKLEGIRPESPAQGSGSRHSWQRALWSLERYFYLILFNYYLHEQYPLAFALSFSRWLC
AHPELYRLPVTLSSAGPVAPRDLIARGSLREDDLVSPDALSTVREMDVANFRRVPRMPIYGTAQP
SAKALGSILAYLTDAKRRLRKWWVSLREEAVLECDGHTYSLRWPGPPVAPDQLETLEAQLKAHL
SEPPPGKEGPLTYRFQTCLTMQEVFSQHRRACPGLTYHRIPMPDFCAPREEDFDQLLEALRAALS
KDPGTGFVFSCLSGQGRTTTAMWAVLAFWHIQGFPEVGEEELVSVPDAKFTKGEFQWMKWQL
LPDGHRVKKEVDAALDTVSETMTPMHYHLREIIICTYRQAKAAKEAQEMRRLQLRSLQYLERYVC
LILFNAYLHLEKADSWQRPFSTWMQEVASKAGIYEILNELGFPELESGEDQPFSRLRYRWQEQSC
SLEPSAPEDLL
Tn all BLAST alignments described herein, the "E-value" or "Expect" value is a
numeric indication of the probability that the aligned sequences could have
achieved their
similarity to the BLAST query sequence by chance alone, within the database
that was
searched. The Expect value (E) is a parameter that describes the number of
hits one can
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"expect" to see just by chance when searching a database of a particular size.
It decreases
exponentially with the Score (S) that is assigned to a match between two
sequences.
Essentially, the E value describes the random background noise that exists for
matches
between sequences.
The Expect value is used to create a significance threshold for reporting
results. The
default value used for blasting is typically set to 0.0001, with the filter to
remove low
complexity sequence turned off. In BLAST 2.0, the Expect value is also used
instead of the P
value (probability) to report the significance of matches. For example, an E
value of one
assigned to a hit can be interpreted as meaning that in a database of the
current size one might
expect to see one match with a similar score simply by chance. An E value of
zero means that
one would not expect to see any matches with a similar score simply by chance.
See, e.g.,
http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/. Occasionally, a string of
X's or N's
will result from a BLAST search. This is a result of automatic filtering of
the query for low-
complexity sequence that is performed to prevent artifactual hits The filter
substitutes any
low-complexity sequence that it finds with the letter "N" in nucleotide
sequence (e.g.,
" ") or the letter "X" in protein sequences (e.g., "XXX"). Low-complexity
regions can result in high scores that reflect compositional bias rather than
significant position-
by-position alignment. Wootton and Federhen, Methods Enzymol 266:554-571,
(1996).
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 1 C.
Table 1C. Patp results for NOVl
Smallest
Sum
eadingigh Prob
equences Frame Score P(N)
producing
High-scoring
Segment
Pairs:
>patp:AAB41108Human ORFX ORF872 polypeptide +1 4187 0.0
>patp:AAB35276Murine dual specificity phosphatase+1 120 5.2e-06
DSP-11
>patp:AAB73211Murine phosphatase AA023073 +1 120 5.2e-06
m
>patp:AAB73231Human phosphatase BAA91172 h +1 115 1.8e-05
>patp:AAG67455Amino acid sequence of a human +1 115 1.8e-0
polypeptide
In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 2063 of 2508 bases (82%) identical
to a
gb:GENBANK-m:MMPAL~acc:X99384.1 mRNA from Mus musculus (Paladin gene). The
full amino acid sequence of the protein of the invention was found to have 695
of 859 amino
acid residues (80%) identical to, and 754 of 859 amino acid residues (87%)
similar to, the 859
amino acid residue ptnr:SPTREMBL-ACC:P70261 protein from Mus rnusculus
(PALADIN
GENE). NOVl also has homology to the proteins shown in the BLASTP data in
Table 1D.
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Table 1D. BLAST
results for
NOV1
Gene Index/ Protein/ OrganismLength Identity PositivesExpect
Identifier (aa) (%) (%)
gi~6331287~dbj~BAABKIAA1274 protein752 752/752 752/752 0.0
6588.1 (AB033100)[Homo Sapiens] (100%) (100%)
gi~14738662~ref~XPKIAA protein 748 747/748 747/748 0.0
_ (similar to (99%) (99%)
046314.1 mouse
(XM 046314) paladin) (Homo
sapi ens]
gi~7305365~ref~NPpaladin [Mus 859 673/841 730/841 0.0
0
_ musculus] (80%) (86%)
38781.1
(NM 013753)
gi~15228672~refINPputative protein1232 207/821 340/821 7e-45
_ [Arabidopsis (25%) (41%)
191760.1
(NM 116066) thaliana]
gi~12836455~dbj~BABdata source:SPTR,144 24/60 33/60 Ze-04
23663.1I(AK004912)source (40%) (55%)
key:Q9NX48,
evidence:ISS-.homo
log to CDNA
FLJ20442 FIB,
CLONE
KAT04828-putative
[Mus musculus]
A multiple sequence alignment is given in Table 1E, with the NOV 1 protein
being
shown on line 1 in Table 1E in a ClustalW analysis, and comparing the NOV1
protein with the
related protein sequences shown in Table 1D. This BLASTP data is displayed
graphically in
the ClustalW in Table 1E.
Table 1E. ClustalW Analysis of NOVl
1) > NOVl; SEQ ID NO:2
2) > gi~6331287/ KIAA1274 protein [Hofrao Sapiens]; SEQ ID N0:35
3) > gi~1473866/ KIAA protein (similar to paladin) [Homo Sapiens]; SEQ ID
N0:36
4) > gi~7305365/ paladin [Mus musculus]; SEQ ID N0:37
5) > gi[1522867/ putative protein [Arabidopsis thaliana]; SEQ ID N0:38
6) > gi~1283645/ data source: SPTR, source key: Q9NX48, evidence: IBS-homolog
to cDNA FLJ20442
FIB clone KAT04828 putative [Mus muscudus]; SEQ ID N0:39
10 20 30 40 50
NOV1 MGTTASTAQQTVSAGTPFEGLQGSGT--MDSRHSVS-IHSFQSTSLHNSK
gi~6331287 -_________________________________________________
gi~1473866 __________________________________________________
gi~7305365 MGTTASTAQQTVSAGTSLEGLQGGSSSSMDSQHSLGGVQSFRATSLHNSK
gi~1522867 __________________________________________________
gi~1283645 __________________________________________________
2S 60 70 80 90 100
NOV1 AKSIIPNKVAPVVITYNCKEEFQIHDELLKAHYTLGRLSDNTPEHYLVQG
gi16331287 __________________________________________________
gi11473866 __________________________________________________
3O gi~7305365 AKSIIPNKVAPWITYNCKEEFQIHDELLKAHYRMGRLSDATPEHYLVQG
gi11522867 ----------------------------MSIPKEPEQVMKMRDGSVLGKK
gi~1283645 __________________________________________________
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NOV1 RYFLVRDV
$ gi~6331287 -------V
gi~1473866 ________
gi~7305365 RYFLVRDI
gi11522867 TILKSDHF
i 1283645 __________________________________________________
g
160 170 180 190 200
.~. .~.
NOV1 ~ ~ ~ ---HR -iL - ~ Q QG
71D ~
gi16331287 ~ ~ ~ ---HR L D ~ ~ Q QG
gi~1473866 ~ ~ ~ ---HR L D ~ ~ Q QG
gi~7305365 ~ ~T~ ---L I ' E ~ ~~ES RD
giI1522867 RHIGAHKDGKQVKVLWTSLVYINGRPF VLRDVEKPFT GIN
EYT
gi~1283645 _______-__________________________________________
210 220 230 240 250
NOV1
gi~6331287
gi~1473866
2$ gi~7305365
gi,1522867
gi~1283645 _______________________________________________-__
Nov1
gi1 6331287
gi~ 1473866
gi~ 7305365
3$ gi~ 1522867
gi~ 1283645_______ ___________________________________________
310
320
330
340
350
NOV1 .Q'~.. P ~~1 ILL. ~-____
gi1 6331287 Q ~ P ~i ILL t-----
'
gi1 1473866 Q ~ P V ILL ~-----
~
giI 7305365 P ~ L L F ~-----
~S H
R
gi~ 1522867------- --IN TEII~R ' I F
TT T
SD~GFPRN
4$ gi~ ~.283645_______ _,___________________-_____________________
...
Nov1 --------
$0 gi~6331287 --------
gi11473866 --------
giI7305365 --------
gi~1522867 NSFGRIF ~N'
gi11283645 __________________________________________________
$$
NOV1
gi16331287
gi~1473866
gi~7305365
gi~1522867
gi~1283645 __________________________________________________
110 120 130 140 150
.~. .I.
i-
TE ~, G " '~ Q T ~~ L
TE ~ G " '~iQ T ~~ L
__ ~ G .. ~~-iQ T v~ S
TE ~I L " '~ R P ~~ L
PGCQNKR~M PQIE '~ Y~~ S- R H AI~TA I~~
360 370 380 390 400
... .~.
_____p ...TQ. . . ~ ~ ~ L~ i~~ R
_____p ...TQ. . . ~ ~ ~ L~Mi~~ R
_____p ...TQ. . ~ ~ ~ L~ i~~ R
-----L ~~SPL~ ' ~ ~ ~G IC ~~ K
KAGENITVNL~ SEEAIRRGEYAVVR~LI~I1LE EGKRQ
12
260 270 280 290 300
410 420 430 440 450
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NOV1
gi~6331287
gi~1473866
gi~7305365
gi~1522867
gi~1283645 ________________,_________________________________
510 520 530 540 550
... .I.
NOV1 ___________________ ~ W ~R ~Em pw -_
gi16331287 ~ ____________________ , . .E " p,. __
1$ gi11473866 ~ ____________________ ~ , ~R ~E " P~~ __
gi~7305365 ~L-____________________ ~G, ~ E " L,~ __
gi~1522867 ~MGALGYAAMKPSLIKIAESTDG 'HEMSW ~SGAVLGSQTVLKSDH
gi~1283645 __________________________________________________
560 570 580 590 600
NOV1 _________ , . .. .-r~ .~~ . . . T,' .
gi~6331287 _________ , .,.. .~.~.r~, ii,. T,.
gi11473866 _________ , . .. ~ .~. . . . T,.
2$ giI7305365 ----_---- , . .~. . . . S,.
gi11522867 SPGCQILNLPE~ EGAP 'E ~GF~ ; ~TIDGIR IERVGSSR-
gi~1283645 __________________________________________________
610 620 630 640 650
.~.
NOV1 R ' 'S ' ~ Y ~W' " --------- ~'~Q T
gi16331287 R ' S ' ~ y ~W~ .._________ ..,Q T
gi~1473866 R ~ S ' ~ Y 'W' ~~--------- ~~~Q T
giI7305365 'Q IF ' ~ WP' --------L~~E
3$ giI1522867 GG~P F HN~PV~ KPFVmEVE ~YKNMLEYTGIDR~R~GM
gi~1283645 ---NFS L -------- RLAG~LORL~---------AHYQF LDQG
660 670 680 690 700
NOV1 ~~. ~ 'P~GKE ~LT ~.~ ___,___________ , ,
gi16331287 ~~ ~ ~P'GKE ~LTY~ ~ _______________ v v
gi~1473866 ~~ ~ ~P~GKE ~LT ' ~ _______________
gi~7305365 ~~ ~ ~ TKS~TAP~ ~K---------------- T~
4S gi~1283645 VRHLVS~T~EAKRYD IMVIHE-KDGQIFDLWENVDADSVQ_PL--YKP
710 720 730 740 750
NOV1
50 gi~6331287
gi~1473866
gi~7305365
gi~1522867
gi11283645
760 770 780 790 800
.~....~... ,I.
NOV1 ~ ~ . . .F Q ___________F. .,. _
gi~6331287 ~ ~ ~ . .F Q ___________F. .,. _
gi~1473866 ~ ~ ~ ~ ~F Q -----------F~ ~
gi~7305365 ~ ~ ~ ~ ~C ----------C~ ~
gi~1522867 M R~GTVIiC~KLR YGRPIKVLYDVLT IVD SS GGEETG
gi~1283645 L F ~ G-_-__TMrr::rr~~CYLVK-________________~G_____
13
460 470 480 490 500
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810 820 830 840 850
NOV1 _____________________ ~iiTiii~ ~~ R ~-i1~1
gi16331287 _____________________ ~ ~ ., R
gi~1473866 _____________________ ~ ~ ~~ R
gi~7305365 -____________________ ~
gi~1522867 SNNAEARPRNSGRRTEEEQG GMDDILLLW'~iTTR FDN~TESREAL~~
gi~1283645 ____________________________DATAETRR~R~G__________
860 870 880 890 900
Nov1 ~ .T _____________________________ .. .. .
gi16331287 ~ ~T ___________________________ ,
v
gi~1473866 ~ ~T _____________________________
gi~7305365 ~ ~I _____________________________
gi11522867 VI~RC QNIREAVLQYRKVFNQQHVEPRVRSAALKRGAEYLERYF~L
gi~1283645 __________________________________________________
910 920 930 940 950
NOV1 C ----------
gi~6331287 C __________
gi~1473866 C __________
2$ giI7305365 S ----------
gi~1522867 AF LGSKAFDGFFVEi
gi~1283645 S~E E-----------
960 970 980 990 1000
.~. .~. .~.
NOVl ~ ~ " Q ~S ~ --- E ~ G ~
gi~6331287 ~ ~~~ Q ~S ~ --- E G D~
gi~1473866 ~ ~~ Q ~S ~ --- E G ~
giI7305365 ~ R STS ~'~ ~T ~ ---- Q ~ I E~
3S gi11522867 IPEE RAQHES~HGDAVMESI ERS SVLSKGS KMYF ~GQRTSSRL
gi~1283645 _________-________________________________________
1010 1020 1030 1040 1050
~n
NOV1 ~F~~ ~ ~.CSL.~S~,~PE~L -------------------------
gi~6331287 ~F ~ ~ ~ CSL ~S~,~PE~L -------------------------
gi~1473866 ~F ' ~ ~ CSL ~S~PE~L -________________________
gi~7305365 ~L ' ~ ~ RDP ~CDVG~F -------------------------
gi11522867 QINGAPHVYKVDRYPVYSMiTPTISGAKKMLAYLGTKI~KEEGGGSTERIV
4$ gi~1283645 __________________________________________________
The NOV1 Clustal W alignment shown in Table 1E was modified to begin at amino
residue 1050. The data in Table 1E includes all of the regions overlapping
with the NOV 1
protein sequences.
The presence of identifiable domains in the protein disclosed herein was
determined by
searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and
then
determining the Interpro number by crossing the domain match (or numbers)
using the
Interpro website (htip:www.ebi.ac.uk/interpro~. Table 1F lists the domain
description from
DOMAIN analysis results against NOV 1.
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Table 1F Domain
Analysis
of NOVl
Model Region of Score (bits) E value
Homology
#PD396342 19-118 431 4e-43
PALADIN GENE
#PD148197 119-399 1175 e-129
PALADIN GENE
#PD222597 119-231 119 6e-07
DOMAIN
OF UNKNOWN
#PD306865 354-454 132 2e-08
PALADIN GENE
#PD024454 356-445 84 0.007
PLASMID ORFS
#PD325716 400-604 800 6e-86
PALADIN GENE
#PD326847 458-594 97 2e-04
CG18442
#PD222597 505-602 113 3e-06
bOMAIN
OF UNKNOWN
#PD277963 595-648 97 2e-04
HYDROLASE
KAT04828 FIS
CDNA
#PD148197 605-678 340 1e-32
PALADIN GENE
#PD306865 680-856 765 7e-82
PALADIN GENE
#PD325716 751-820 86 0.004
PALADIN GENE
Consistent with other known members of the Paladin-like family of proteins,
NOV 1
has, for example, multiple Paladin gene signature sequences and homology to
other members
of the Paladin-like Protein Family. NOV 1 nucleic acids, and the encoded
polypeptides,
according to the invention are useful in a variety of applications and
contexts. For example,
NOV 1 nucleic acids and polypeptides can be used to identify proteins that are
members of the
Paladin like family of proteins. The NOV 1 nucleic acids and polypeptides can
also be used to
screen for molecules, which inhibit or enhance NOV 1 activity or function.
Specifically, the
nucleic acids and polypeptides according to the invention may be used as
targets for the
identification of small molecules that modulate or inhibit, e.g., cellular
activation, cellular
metabolism and signal transduction. These molecules can be used to treat,
e.g.,
cardiomyopathy, atherosclerosis, hypertension, congenital heart defects,
aortic stenosis, atnial
septal defect (ASD),atrioventricular (A-V) canal defect, ductus arteriosus,
pulmonary stenosis,
subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous
sclerosis,
scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital
adrenal hyperplasia,
diabetes, Von Hippel-Lindau (VHL) syndrome, pancreatitis, obesity,
hyperthyroidism and
hypothyroidism, hypercalceimia, ulcers, cirrhosis, transplantation,
inflammatory bowel
disease, diverticular disease, hemophilia, hypercoagulation, idiopathic
thrombocytopenic
purpura, autoimmmne disease, allergies, immunodeficiencies, transplantation,
graft vesus host,
hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume
disease,
allergies, immunodeficiencies, transplantation, graft versus host disease
(GVHD),
lymphedema, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia,
Parkinson's
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disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome,
multiple
sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders,
addiction, anxiety,
pain, neuroprotection, cancer, trauma, regeneration (in vitro and irz vivo),
viral/bacteriallparasitic infections, as well as other diseases, disorders and
conditions.
In addition, various NOV 1 nucleic acids and polypeptides according to the
invention
are useful, izzter alia, as novel members of the protein families according to
the presence of
domains and sequence relatedness to previously described proteins. For
example, the NOV1
nucleic acids and their encoded polypeptides include structural motifs that
are characteristic of
proteins belonging to the Paladin-Iike protein family.
Paladin proteins are a family of protein-tyrosine phosphatases. The protein
phosphatases can be divided into 2 large families: the serinelthreonine
phosphatases, which are
metalloproteins, and the protein-tyrosine phosphatases, which proceed via a
thiol-phosphate
enzyme intermediate. The protein-tyrosine phosphatase family includes the VHl-
like dual-
specificity phosphatases. These phosphatases dephosphorylate phosphotyrosine-
as well as
phosphoserine- and phosphothreonine-containing substrates. Members of the dual-
specificity
phosphatase protein family inactivate mitogen-activated protein (MAP) kinase
through
dephosphorylation of critical threonine and tyrosine residues. Members of the
MAP kinase
family play a pivotal role in cellular signal transduction. Using a
subtractive screen of mouse
gastrulation, Pearce et al. (1996) identified a novel mouse gene, paladin,
with similarity to the
dual specificity protein phosphatase family.
The NOV1 nucleic acids and polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and diagnostic
applications in the
mediation of cardiac and endocrine physiology. As such, the NOV 1 nucleic
acids and
polypeptides, antibodies and related compounds according to the invention may
be used to
treat muscle and nervous system disorders, e.g., cardiomyopathy,
atherosclerosis,
hypertension, congenital heart defects, aortic stenosis, atrial septal defect
(ASD),atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary
stenosis, subaortic
stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis,
scleroderma,
obesity, transplantation, adrenoleukodystrophy, congenital adrenal
hyperplasia, diabetes, Von
Hippel-Lindau (VHL) syndrome, pancreatitis, obesity, hyperthyroidism and
hypothyroidism,
hypercalceimia, ulcers, cirrhosis, transplantation, inflammatory bowel
disease, diverticular
disease, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura,
autoimmume
disease, allergies, immunodeficiencies, transplantation, graft vesus host,
hemophilia,
hypercoagulation, idiopathic thrombocytopenic purpura, autoimmume disease,
allergies,
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immunodeflciencies, transplantation, graft versus host disease (GVHD),
lymphedema,
Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's
disease,
Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple
sclerosis,
ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction,
anxiety, pain,
neuroprotection, cancer, trauma, regeneration (ira vitro and iya vivo),
viral/bacterial/parasitic
infections, as well as other diseases, disorders and conditions.
The NOV1 nucleic acids and polypeptides are useful for detecting specific cell
types.
For example, expression analysis has demonstrated that a NOV 1 nucleic acid is
expressed in
brown adipose, heart, aorta, Ve111, umbilical vein, adrenal gland/suprarenal
gland, pancreas,
thyroid, salivary glands, parotid salivary glands, stomach, liver, gall
bladder, small intestine,
colon, bone marrow, lymphoid tissue, spleen, lymph node, tonsils, thymus,
cartilage, muscle,
brain, thalamus, hypothalamus, pituitary gland, amygdala, substantia nigra,
hippocampus,
spinal chord, cervix, mammary gland/breast, ovary, placenta, uterus, vulva,
prostate, testis,
lung, lung pleura, kidney, retina, dermis.
Additional utilities for NOV 1 nucleic acids and polypeptides according to the
invention are disclosed herein.
NOV2
A NOV2 polypeptide has been identified as a Plasma Membrane Ring Finger-like
protein (also referred to as CG93210-01). The disclosed novel NOV2 nucleic
acid (SEQ m
N0:3) of 1205 nucleotides is shown in Table 2A. The novel NOV2 nucleic acid
sequences
maps to the chromosome 22.
An ORF begins with an ATG initiation codon at nucleotides 17-19 and ends with
a
ATT codon at nucleotides 1149-1151. A putative untranslated region and/or
downstream from
the termination codon is underlined in Table 2A, and the start and stop codons
are in bold
letters.
Table 2A. NOV2 Nucleotide Sequence (SEQ ID N0:3)
CTCGCCGGGTCCGGCCATGGGCCCCGCCGCTCGCCCCGCGCTGAGATCGCCGCCGCCGCCTCCGCCG
CCGCCTCCGTCTCCGCTGCTGCTGCTGCTGCCCCTGCTGCCGCTGTGGCTGGGCCTGGCGGGGCCCG
GGGCCGCGGCGGACGGCAGCGAGCCGGCGGCCGGGGCGGGGCGGGGCGGAGCCCGCGCCGTGCGGGT
GGACGTGAGACTGCCGCGCCAGGACGCTCTGGTCCTGGAGGGCGTCAGGATCGGCTCCGAAGCCGAC
CCGGCGCCCCTGCTGGGCGGTCGTCTGCTGCTGATGGACATCGTGGATGCCGAGCAGGAGGCACCAG
TGGAAGGCTGGATTGCAGTGGCATACGTGGGCAAGGAGCAGGCGGCCCAGTTCCACCAGGAGAATAA
GGGCAGTGGCCCGCAGGCCTATCCCAAGGCCCTGGTCCAGCAGATGCGGCGGGCCCTCTTCCTGGGT
GCCTCTGCCCTGCTTCTTCTCATCCTGAACCACAACGTGGTCCGAGAGCTGGACATATCCCAGCTTC
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TGCTCAGGCCAGTGATCGTCCTCCATTATTCCTCCAATGTCACCAAGCTGTTGGATGCATTGCTGCA
GAGGACCCAGGCCACGGCTGAGATCACCAGCGGAGAGTCCCTGTCTGCCAATATCGAGTGGAAGTTG
ACCTTGTGGACCACCTGTGGCCTCTCCAAGGATGGCTATGGAGGATGGCAGGACTTGGTCTGCCTTG
GAGGCAGTCGTGCCCAGGAGCAGAAACCCCTGCAGCAGCTGTGGAACGCCATCCTGCTGGTGGCCAT
GCTCCTGTGCACAGGCCTCGTGGTCCAGGCCCAGCGGCAGGCGTCGCGGCAGAGCCAGCGGGAGCTC
GGAGGCCAGGTGGACCTGTTTAAGCGCCGCGTGGTGCGGAGACTGGCATCCCTCAAGACACGGCGCT
GCCGGCTGAGCAGGGCAGCGCAGGGCCTCCCAGATCCGGGTGCTGAGACCTGTGCGGTGTGCCTGGA
CTACTTCTGCAACAAACAGTGGCTCCGGGTGCTGCCCTGTAAGCACGAGTTTCACCGAGACTGTGTG
GACCCCTGGCTGATGCTCCAGCAGACCTGCCCACTGTGCAAATTCAACGTCCTGGGTGAGCACCGCT
ACTCCGATGATTAGCTGCCCAGCTGGACTCTGCACATGGGGATGGACCCCTCCTGCCTGCACCCCG
The NOV2 protein (SEQ ID N0:4) encoded by SEQ ID NO:3 is 378 amino acid
residues in length and is presented using the one-letter amino acid code in
Table 2B. Psort
analysis predicts the NOV2 protein of the invention to be localized at the
plasma membrane
with a certainty of 0.6400.
Table 2B. Encoded NOV2 protein sequence (SEQ ID N0:4)
MGPAARPALRSPPPPPPPPPSPLLLLLPLLPLWLGLAGPGAAADGSEPAAGAGRGGARAVRVDVR
LPRQDALVLEGVRIGSEADPAPLLGGRLLLMDIVDAEQEAPVEGWIAVAYVGKEQAAQFHQENKG
SGPQAYPKALVQQMRRALFLGASALLLLILNHNVVRELDISQLLLRPVIVLHYSSNVTKLLDALL
QRTQATAEITSGESLSANIEWKLTLWTTCGLSKDGYGGWQDLVCLGGSRAQEQKPLQQLWNAILL
VAMLLCTGLVVQAQRQASRQSQRELGGQVDLFKRRWRRLASLKTRRCRLSRAAQGLPDPGAETC
AVCLDYFCNKQWLRVLPCKHEFHRDCVDPWLMLQQTCPLCKFNVLGEHRYSDD
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 2C.
Table 2C. Pat results for
NOV2
Smallest
Sum
eadingigh Prob
equences Frame ScoreP(N)
producing
High-scoring
Segment
Pairs:
>patp:AAB42695HumanORFX ORF2459 polypeptide +1 1715 3.0e-276
>patp:AAM79288Humanprotein SEQ TD NO 1950 +1 612 2.3e-59
>patp:AAM80272Humanprotein SEQ TD NO 3918 +1 534 4.2e-51
>patp:AAU28202Novelhuman secretory protein +1 201 5.1e-13
>patp:ABB50251Humantranscription factor TRFX-102+1 148 5.5e-13
In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 287 of 489 bases (58%) identical
to a
gb:GENBANK-1D:SSI132828~acc:AJ132828.1 mRNA from Spermatozopsis similis (mRNA
for p210 protein, partial). The full amino acid sequence of the protein of the
invention was
found to have 341 of 379 amino acid residues (89%) identical to, and 355 of
379 amino acid
residues (93%) similar to, the 379 amino acid residue ptnr:SPTREMBL-ACC:Q9DCW1
protein from Mus musculus (0610009J22RIK PROTEIN).
NOV2 also has homology to the proteins shown in the BLASTP data in Table 2D.
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Table 2D. BLAST
results for
NOV2
Gene Index/ Protein/ OrganismLength Identity PositivesExpect
Identifier (aa) (%) (%)
gi~12832380~dbj~BABdata source:SPTR,379 340/380 354/380 e-173
22082.1 (AK002414)source (89%) (92%)
key:Q9Y6U7,
evidence:ISS~homo
log to
WUGSC:H DJ130H16.
6
PROTEIN(FRAGMENT)
putative
[Mus musculus]
gi~5441942~gb~AAD43supported by 347 336/336 336/336 e-148
187.1~AC004997 mouse EST (100%) (100%)
(AC004997) AA538043
(NID:g2284036)
[Homo Sapiens]
gi~17485136~refiXPsimilar to data272 271/283 272/283 e-146
_ source:SPTR, (95%) (95%)
066294.1
(XM 066294) source key:Q9Y6U7
evidence:ISS-homo
log to
WUGSC:H_DJ130H16.
6 PROTEIN
(FRAGMENT)-putati
ve [Homo Sapiens]
giI17861674~gb~AAL3GH20973p 461 26/57 42/57 9e-13
9314.1 (AY069169)[Drosophila (45%) (73%)
melanogaster]
gi~18485962~ref~XPsimilar to 461 26/57 42/57 1e-12
- goliath (H. (45%) (73%)
080778.1
(XM 080778) Sapiens)
[Drosophila
melanogaster]
A multiple sequence alignment is given in Table 2E, with the NOV2 protein
being
shown on line 1 in Table 2E in a ClustalW analysis, and comparing the NOV2
protein with the
related protein sequences shown in Table 2D. This BLASTP data is displayed
graphically in
the ClustalW in Table 2E.
19
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Table 2E. ClustalW Analysis of NOV2
1) > NOV2; SEQ 1D N0:4
2) >gi~12832380~/ data source:SPTR, source key:Q9Y6U7, evidence:ISS~homolog to
WITGSC:H DJ130HI6.6 PROTEIN(FRAGMENT)~putative [Mus musculus]; SEQ ID N0:40
S 3) >gi~5441942[/ supported by mouse EST AA538043 (NID:g2284036) [Homo
sapierrs]; SEQ ID
N0:41
4) >gi~17485136~/ similar to data source:SPTR, source key:Q9Y6U7
evidence:ISS~homolog to
WUGSC:H DJ130H16.6 PROTEIN (FRAGMENT)~putative [Homo Sapiens]; SEQ ID N0:42
S) >gi~17861674~/ GH20973p [D>"osophila rnelanogaster°]; SEQ ID
N0:43
6) >gi~18485962~/ similar to goliath (H. Sapiens) [Drosophila melarrogaster];
SEQ ID N0:44
10 20 30 40 50 60
1 S NOV2 1 -- GP ~~ P RSP--P ~ 'PPP PPLL ~:f'LPI#LPLWL ~ GPG ~~ . EP ' 52
gi~128323801 1 -- -GS~P RSPSLP " PPS PPLL ~LP~iLPLWL GPG ~EPAT E 54
gi I 5441942 I 1 -- --GP~ARP RSP--P "PPP PPLL T.iLPLPLWLG~AGPGAAAS,EP '.. 52
gi~17485136~ 1 ______~_____________________.____'___________________________ 1
gi ~ 17861674 ~ 1 MYIRKTLL~i1CLVL FGG--L"LTF ~,'TTT AAh?tSIANQD ERYFRPG THSFS
58
2O gi ~ 18485962 I 1 MYIRKTT~LL~CLVLBFGG--
L~LTF~A'~'TTT~AAN~SIANQDBERYFRPG~'~HSF~ 58
70 80 90 100 110 120
p...
NOV2 53 GRGG~i =R----------------VL1't7RLP~.QDj~~I,LVL ------ ~ GS~EAT7PP 87
2S gi ~ 12832380 ~ 55 GRGGf,~~1,PL R=---------------V~T~KLP~.QD~~,LVL ------ ~
GPEDGPEP 89
gi~5441942~ 53 GRGG~RA~R _______________~RLP~2QD~LVL ________G'EM3PP 87
1
gi 17485136 Z
gi~178616741 59 EDRI~i1VD YNYAFLNWSYVEHGNMLC~EFA~Q~~,RY KVLNVTGRLH T,ATDFD 118
gi~18485962~ 59 EDRI~~YNYAFLNWSYVEHGNMLC~EFAQFQ~RYG~KVLNVTGRL,~HBT~TDF~.S,D
118
30 '
NOV2 88 143
gi~12832380~ 90 145
3S gi~5441942~ 88 143
gi~17485136~ 1 48
gi~I7861674~ 119 177
gi~184859621 119 177
40 190 200 210 220 230 240
NOV2 144 201
gi~128323801 146 203
gi~54419421 144 201
4S gi~174851361 49 106
gi~17861674~ 178 237
gi~18485962~ 178 237
250 260 270 280 290 300
S0
NOV2 202 ~ 1'I' ~ _____________ ______~ "___ 236
gi~12832380~ 204 ~ S ~ _____________ ______~ ~~-__ 238
gi~54419421 202 ~ T _____________ ______~ w-__ 236
gi~17485136~ 107 ~ ~ _______________ _______, w-__ 141
SS gi ~ 17861674 ~ 238 VS FIVL~III~L LFY~'~~~,'IQRFRYMQAKDQQSRNL StrT
~KAIMKIPTKT KE~S~EKD 297
gi ~ 18485962 I 238 VS SFTVL~IISL L~FYIQRFRYMQAKDQQSRNL S~tt'I' ~KAIMKIPTKT
IC~'S~EKD 297
310 320 330 340 350 360
60 NOV2 236 287
289
gi~Z2832380~ 238
gi~5441942~ 236 287
gi117485136~ 141 192
gi~178616741 298 357
6S gi~18485962~ 298 357
130 140 150 160 170 180
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370 380 390 400 410 420
~y y ~ .I~ ~~~ ~I. ~~~ .y y y . ~~. y w y
v~~w~~ aw ' ~w
NOV2 288 sn-- ' '~~Q '~' CDY .KQWLRVLP 342
7~1 ~vV
gi~12832380~ 290 ~E~-- ~ ~~HS~~E''T CDY KQWLRVLP 344
gi~54419421 288 ~ ~-- ~~Q ~~~ C~iDY ------- 334
gi~17485136~ 193 ~ ~-- ' '~~Q ~~~ ~ LRVLP K-------- 239
.YY
gi ~ 17861674 ~ 358 SE~SILE~IYC~PDPP~L,V 'DESAD '~RDF ~FPRVFVLDSGCWGAREMLFPCR
417
gi~18485962~ 358 ~- --~XQTPSP~HTiP~AIEEVPV~UViVPH~~Q~LQPLQiSN~iSS~APSHYFQSSR
412
1~ 430 440 450 460 470
NOV2 343 CK---HEFHDC'VD LLsQC~ CF GEHRYS~_?~~D------------- 378
gi~12832380! 345 CK---HEFHR!DC~D L~IL~Q'~C~ CAF GHYSD~-------------- 379
gi~54419421 334 ________ K __~ ~ ApG ___ ____-_________ 347
15 gy 17485136 ~ 239 -----HEFH~DC~DQP1LL~QC~CF G1i.~T:~RYSD~~--------------
272
gi~17861674! 418 IPERSQSSLSLRQARDW,~SLMN E~QQ RMRND MQQVIK-------- 461
gi~18485962~ 413 SP---SSSVQQLTYQPHPQQAASERGRRISAPATMPHAITASHQVTDV 461
20 The presence of identifiable domains in the protein disclosed herein was
determined by
searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and
then
determining the Iliterpro number by crossing the domain match (or numbers)
using the
Interpro website (http:www.ebi.ac.uk/interpro~. Table 2F lists the domain
description from
DOMAIN analysis results against NOV2.
Table 2F Domain sis of
Anal NOV2
Model Region of Score E value
(bits)
Homology
Ring Finger 325-365 49.3 4.0e-07
zf-C3HC4 325-365 34.7 2.2e-09
PHD 324-368 -10.4 1.1
Consistent with other known members of the Membrane Ring Finger-like family of
proteins, NOV2 has, for example, a Ring Finger signature sequence and homology
to other
members of the Plasma Membrane Ring Finger-Iike Protein Family. NOV 2 nucleic
acids,
and the encoded polypeptides, according to the invention are useful in a
variety of applications
and contexts. For example, NOV2 nucleic acids and polypeptides can be used to
identify
proteins that are members of the Plasma Membrane Ring Finger-like Protein
Family. The
NOV2 nucleic acids and polypeptides can also be used to screen for molecules,
which inhibit
or enhance NOV 1 activity or function. Specifically, the nucleic acids and
polypeptides
according to the invention may be used as targets for the identification of
small molecules that
modulate or inhibit, e.g., cellular activation, cellular metabolism and signal
transduction.
These molecules can be used to treat, e.g., anemia, ataxia-telangiectasia,
autoimmume disease,
immunodeficiencies, diabetes, autoimmune disease, renal artery stenosis,
interstitial nephritis,
glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus,
renal tubular
acidosis, IgA nephropathy, hypercalceimia, Lesch-Nyhan syndrome, cancer,
trauma,
i
0
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regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, as
well as other diseases,
disorders and conditions.
In addition, various NOV2 nucleic acids and polypeptides according to the
invention
are useful, inter alia, as novel members of the protein families according to
the presence of
domains and sequence relatedness to previously described proteins. For
example, the NOV2
nucleic acids and their encoded polypeptides include structural motifs that
are characteristic of
proteins belonging to the Plasma Membrane Ring Finger-like Protein Family .
The NOV2 nucleic acids and polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and diagnostic
applications in the
mediation of immune and renal physiology. As such, the NOV2 nucleic acids and
polypeptides, antibodies and related compounds according to the invention may
be used to
treat muscle and nervous system disorders, e.g., anemia, ataxia-
telangiectasia, autoimmume
disease, immunodeficiencies, diabetes, autoimmune disease, renal artery
stenosis, interstitial
nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus
erythematosus, renal
tubular acidosis, IgA nephropathy, hypercalceimia, Lesch-Nyhan syndrome,
cancer, trauma,
regeneration (in vitro and in vdv0), viral/bacterial/parasitic infections, as
well as other diseases,
disorders and conditions.
The NOV2 nucleic acids and polypeptides are useful for detecting specific cell
types.
For example, expression analysis has demonstrated that a NOV2 nucleic acid is
expressed in
peripheral blood, and a pool of various mammalian tissues. Expression
information was
derived from the tissue sources of the sequences that were included in the
derivation of the
sequence of CuraGen Acc. No. CG93210-Ol.The sequence is predicted to be
expressed in the
following tissues because of the expression pattern of (GENBANK-1D: gb:GENBANK-
ID:SSI132828~acc:AJ132828.1) a closely related Spe~matozopsis similis mRNA for
p210
protein, partial homolog in species Spe~n2atozopsis sirnilis :kidney.
Additional utilities for NOV2 nucleic acids and polypeptides according to the
invention are disclosed herein.
NOV3
A NOV3 polypeptide has been identified as a Thrombospondin type 1 (tsp_1)
domain
containing protein (also referred to as CG93275-OI). The disclosed novel NOV3
nucleic acid
(SEQ ID NO:S) of 799 nucleotides is shown in Table 3A. The novel NOV3 nucleic
acid
sequences maps to the chromosome 16.
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An ORF begins with an ATG initiation codon at nucleotides 51-53 and ends with
a
TGA codon at nucleotides 744-746. A putative untranslated region and/or
downstream from
the termination codon is underlined in Table 3A, and the start and stop codons
are in bold
letters.
Table 3A. NOV3 Nucleotide Sequence (SEQ ID NO:S)
GAATATATTTAGTGTGTTGTTTTTTTTTTTAATGTGGCTACTGAAACCTAATGGGAATGCAAATAGA
ACTTTTTTGTCTTCTCAAGTGTTCCAAGACCTGTGGACGAGGGGTGAGGAAGCGTGAACTCCTCTGC
AAGGGCTCTGCCGCAGAAACCCTCCCCGAGAGCCAGTGTACCAGTCTCCCCAGACCTGAGCTGCAGG
AGGGCTGTGTGCTTGGACGATGCCCCAAGAACAGCCGGCTACAGTGGGTCGCTTCTTCGTGGAGCGA
GTGTTCTGCAACCTGTGGTTTGGGTGTGAGGAAGAGGGAGATGAAGTGCAGCGAGAAGGGCTTCCAG
GGAAAGCTGATAACTTTCCCAGAGCGAAGATGCCGTAATATTAAGAAACCAAATCTGGACTTGGAAG
AGACCTGCAACCGACGGGCTTGCCCAGCCCATCCAGTGTACAACATGGTAGCTGGATGGTATTCATT
GCCGTGGCAGCAGTGCACAGTCACCTGTGGGGGAGGGGTCCAGACCCGGTCAGTCCACTGTGTTCAG
CAAGGCCGGCCTTCCTCAAGTTGTCTGCTCCATCAGAAACCTCCGGTGCTACGAGCCTGTAATACAA
ACTTCTGTCCAGCTCCTGAAAAGAGAGAGGATCCATCCTGCGTAGATTTCTTCAACTGGTGTCACCT
AGTTCCTCAGCATGGTGTCTGCAACCACAAGTTTTACGGAAAACAATGCTGCAAGTCATGCACAAGG
AAGATCTGATCTTGGTGTCCTCCCCAGCCTTAGGGCCAGGGGCTTACCTTTCAACCTCTAGA
The NOV3 protein (SEQ ID N0:6) encoded by SEQ m N0:5 is 231 amino acid
residues in length and is presented using the one-letter amino acid code in
Table 3B. Psort
analysis predicts the NOV3 protein of the invention to be localized in the
cytoplasm with a
certainty of 0.4500.
Table 3B. Encoded NOV3 protein sequence (SEQ ID N0:6)
MGMQIELFCLLKCSKTCGRGVRKRELLCKGSAAETLPESQCTSLPRPELQEGCVLGRCPKNSRLQ
WVASSWSECSATCGLGVRKREMKCSEKGFQGKLITFPERRCRNIKKPNLDLEETCNRRACPAHPV
YNMVAGWYSLPWQQCTVTCGGGVQTRSVHCVQQGRPSSSCLLHQKPPVLRACNTNFCPAPEKRED
PSCVDFFNWCHLVPQHGVCNHKFYGKQCCKSCTRKI
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 3C.
Table 3C. Patp results for
NOV3
Smallest
Sum
eadingigh Prob
equences Frame ScoreP(N)
producing
High-scoring
Segment
Pairs:
>patp:AAE09696Humangene 7 encoding protein +1 1248 9.2e-127
HE8CY61
>patp:AAE09699Humangene 10 encoding protein +1 1245 1.9e-126
HUVHR16
>patp:AAU72893Humanmetalloprotease partial +1 1204 4.2e-122
sequence #5
>patp:AAU72891Humanmetalloprotease partial +1 693 3.5e-70
sequence #$3
>patp:AAB21253Humanmetalloproteinase KIAA0605 +1 327 5.5e-28
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In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 392 of 396 bases (98%) identical
to an EST
AA057409 mRNA from human). The full amino acid sequence of the protein of the
invention
was found to have 74 of 216 amino acid residues (34%) identical to, and 107 of
216 amino
acid residues (49%) similar to, the 237 amino acid residue ptnr:SPTREMBL-
ACC:Q9HBS6
protein from Homo Sapiens (HYPOTHETICAL 25.7 I~DA PROTEIN.
NOV3 also has homology to the proteins shown in the BLASTP data in Table 3D.
Table 3D. BLAST
results for
NOV3
Gene Index/ Protein/ OrganismLength Tdentity PositivesExpect
Tdentifier (aa) (%) (%)
gi~18598706~ref~XPhypothetical 1123 181/183 183/183 e-200
_ protein XP_091253 (98%) (99%)
091253.7.~(XM_091253
[Homo Sapiens]
gi~19171150~emb~CACADAMTS18 protein1081 61/62 62/62 4e-27
83612.1~(AJ311903)[Homo Sapiens] (98%) (99%)
gi~7662202~ref~NP_0ItIAA0605 gene 951 79/216 99/216 9e-23
55509.11(NM 014694)product (36%) (45%)
[Homo Sapiens]
gi~18561227IrefIXPhypothetical 1365 51/112 74/112 4e-21
_ protein XP_094442 (45%) (65%)
094442.1~(XM_094442
[Homo Sapiens]
gi~17432918Isp~Q9H3HUMAN ADAMTS-10223 74/223 104/223 5e-20
24IAT10' precursor (A (33% (46%)
disintegrin
and
metalloproteinase
with
thrombospondin
motifs 10) (ADAM-
TS 10) (ADAM-
TS10)(Fragment)
A multiple sequence alignment is given in Table 3E, with the NOV3 protein
being
shown on Iine 1 in Table 3E in a ClustalW analysis, and comparing the NOV3
protein with the
related protein sequences shown in Table 3D. This BLASTP data is displayed
graphically in
the ClustalW in Table 3E.
Table 3E. ClustalW Analysis of NOV3
1) > NOV3; SEQ ID N0:6
2) >gi~18598706~/ hypothetical protein XP_091253 [Homo Sapiens]; SEQ >D N0:45
3) >gi~19171150~/ ADAMTS18 protein [Homo Sapiens]; SEQ ID N0:46
4) >gi~7662202~/ I~IAA0605 gene product [Homo Sapiens]; SEQ ID N0:47
5) >gi~ 18561227/ hypothetical protein XP_094442 [Homo Sapiens]; SEQ ID N0:48
6) >gi~17432918~/ AT10_HCTMAN ADAMTS-10 precursor (A disintegrin and
metalloproteinase with
thrombospondin motifs 10) (ADAM-TS 10) (ADAM-TS10) (Fragment); SEQ IT3 N0:49
1330 1340 1350 1360 1370 1380
NOV3 1 _______________________________________________~__MQIE~iF~LL 11
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gi1185987061 769 GGVRSAKVLSLEEWIKSETTL-ARKEQQQPSTGWMPGEQS<TQa~KA~-
AeG'IGQQSKIQ~VQ 826
gi1191711501 872 ______________________,__________________________
--PAYTWSIVQ 881
gi176622021 686 -------------------------------PQWEMSE ~E TA GERSVVT~DSE 715
gi1185612271 986 QGGGCTPSPPADGLVQNQLRLGRHTQAQCLDQSCSVG S ~R.T =~GAQS~t,P~Q TR
1044
S gi 117432918 ~ 807 ______________________________________ Ti ~,AQ
yn~~rGSQV,A'(?'E RN 828
1390 1400 1410 1420 1430 1440
.1....1....1....1....1....1....1....1.. .1. .1. .1.. .1
NOV3 12 ~_________________________________________~ Kip~~ E~',,j,~ G 30
1O gi 118598706 1 827 ~t"jIKPFQKEEAVLHSLCPVST~TQVI~A~NSHA~PPQ~SLGP~SQ Kf
'I~ELL~ G 886
gi1191711501 882 S-_______________________________________E ~ YINVKA~ LR 901
gi176622021 716 DEKLCDP---------NT ' GE~~ TGPP RQ TVSD GP GQ RTI'H'~Y 1T 766
gi1185612271 1045 ~1HYDSE-PVPASLCPQP SSR~ SQS PP SAGP 1'I_' ~ S 1103
gi1174329181 829 LDSSAVAPHYCS-AHSKL KRQTEP PPD G SL R6 'S S ,;, QR 887
1S
1450 1460 1470 1480 1490 1500
.1....1. .1....1.. .1. .1. .1....1. .1. ..1. .1. .1
NOi3 1 31 ----AA'E.''T~ Q L E~.Q~G=~ LG..~KNSRL~ ~-~8S .E L ~ 85
~ V n VW n V 1.
.1, V V/1 V
gi 18598706 887 ---AAT E Q ~L E~ G LG 'KNSRLiSS E L 941
20 gi1191711501 902 D----QNTQ"NS F SATCT.~ TEPKI AFS ~ ---A 'PGE T _K8 Q~S'
953
v v - ~t c
gi176622021 767 ;----DGR .~E Q QMT ~LAIHP- GD ---- ~QD E T ~ 817
gi ~ 17432918 1 8884 RVSAAEE~L ~~A P~~ ~~L~A- LGPT ~KPK P~vLD E 'I'PEP ~~ '
9412
2S 1510 1520 1530 1540 1550 1560
.1. ..1....1....1....1. .1. ..1....1. .1. ..1....1.. .1
NOV3 86 E~I~ S ~GFQ ~LI~FPER RNT' ~.LDL~E~'---- --AHPVYNMVA~ Y 138
gi1185987061 942 E~ S 'GFQ' LI,~~,FPER RNA ~VLDL E~----- --AHPVYNMVA I1Y 994
gi 119171150 1 954 ICS ~ ~KPFQ ~EEAVLHSL P STST~--TQU~?'A---- S -PQ------- S
995
30 gi176622021 818 L'~L LA- PQ~RSGPE GLA --P EST--- FE'P F---------- Y 858
gi1185612271 1163 FI~~ ~YVS ~YRELASK SHIP ~SLELBR~!~'PHLLLRI~P~GAAGLPHPGLREV P
1222
gi1174329181 942 VjUL KS---ADHRADLPPAH SP°~ -PATMR-____~'"L.~_______p__
982
1570 1580 1590 1600 1610 1620
3S ....1....1....1. .1....1. ~1..~...1. ..1....1....1. .1....1
NOV3 139 S--L~ QI,~ ------r;.~ G----- tot's .Q-----------QG.~~ S-- 170
v v
gi1185987061 995 S--L~"QQ _____ G_____ ~T.8~ Q___________QG ~~ S-- 1026
gi1191711501 996 L-- ~ S~,'7 ---- V ~R----- ~ 'E~:i~KGSA--------AETL~E Q--
1030
gi176622021 859 TS--' S---- ~ ------ ' ~ YQG------------TDIVRG-- 890
gi1185612271 1223 LTRVL~"P PLIHLFRP SGSPCTVP VSYQHNKPIPRRREHPPREHLTQ~'SP 1282
gi1174329181 983 AG--E~G: --- AQ ---- Q~yQ~'7SVR~TS-----------HTGQ~H--E 1015
1630 1640 1650 1660 1670 1680
1.. ~~~~~1~.~ 1 .1. .1 ..1....1....1.. 1 1 ..1
4S NOV3 171 LLHQ ~PVL , ---- TF ~~PEKR--------------DPSC~DFF ~'~'''~~'CH- 206
gi1185987061 1027 LLHQ'~PVL . --------TF ~ PEKR~GEMQAELDSKLSGFQTIS~IWFESEG
1077
gi1191711501 1031 TSLP ELQ~,~,. V --------LG ~KNSRLQ-------------- WVASSG~SE-
1062
gi176622021 891 DPLVVGR~,I --------LQP ~TEPPD-----------------SCQDQPGTN- 923
gi1185612271 1283 AKRKYGQKTDLDTLPIPLWAPLS~SPEPRG----PEICEQ----QGLD TECP LLL
1334
SO gi1174329181 1016 TEAL~PTTf,~Q~EAK--- -CD,~PT~GDGPE--------------
CKD~NKVACP- 1053
1690 1700 1710 1720
NOV3 206 - 1.~PQHGV~NHKFYGKQCC~I--.TRKI--I_---1----1- 231
SS gi1185987061 1078 NERL~PSFSLHLGGKNGIQYPKRLPBEQKENMALAAIKMLQSTF 1123
gi1191711501 1062 - 'V'WIRSH~RRLRPSWLTQ---------------------- 1081
gi176622021 923 - C ~IKVNL GHWYYSKACC~-- RPPHS------------- 951
gi1185612271 1335 AIG- IiPCQARDTESRPQGPVP~;--P GQDIEK------------ 1365
gi1174329181 1053 --- ,.LKFQF~SRAYFRQMCC'-- QGH--------------- 1077
The NOV3 Clustal W alignment shown in Table 3E was modified to begin at amino
residue 1321. The data in Table 3E includes all of the regions overlapping
with the NOV3
protein sequences.
2S
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The presence of identifiable domains in the protein disclosed herein was
determined by
searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and
then
determining the Interpro number by crossing the domain match (or numbers)
using the
Interpro website (hrip:www.ebi.ac.uk/interpro/). Table 3F lists the domain
description from
DOMAIN analysis results against NOV3.
Table 3F Domain sis of
Anal NOV3
Model Region of Score E value
(bits)
Homology
tsp 1 12-58 -6.8 4.1
tsp 1 66-125 14.6 0.015
tsp 1 141-187 19.3 0.0041
Consistent with other known members of the Thrombospondin type 1 (tsp_1)
family of
proteins, NOV3 has, for example, three tsp_1 domain signature sequences and
homology to
other members of the tsp_1 Domain-containing Protein Family. NOV 3 nucleic
acids, and the
encoded polypeptides, according to the invention are useful in a variety of
applications and
contexts. For example, NOV3 nucleic acids and polypeptides can be used to
identify proteins
that are members of the tsp_1 Domain-containing Protein Family. The NOV3
nucleic acids
and polypeptides can also be used to screen for molecules, which inhibit or
enhance NOV3
activity or function. Specifically, the nucleic acids and polypeptides
according to the
invention may be used as targets for the identification of small molecules
that modulate or
inhibit, e.g., cellular activation, cellular metabolism, and signal
transduction. These molecules
can be used to treat, e.g., Von Hippel-Lindau (VHL) syndrome, diabetes,
tuberous sclerosis,
fertility, hypogonadism, as well as other diseases, disorders and conditions.
In addition, various NOV3 nucleic acids and polypeptides according to the
invention
are useful, inter alia, as novel members of the protein families according to
the presence of
domains and sequence relatedness to previously described proteins. For
example, the NOV3
nucleic acids and their encoded polypeptides include structural motifs that
are characteristic of
proteins belonging to the tsp-1 Domain-containing Protein Family.
Thrombospondin type 1 domain (TSP1, IPR000884) is a repeat found in the
thrombospondin protein where it is repeated 3 times. Likewise, the tsp_1
domain is repeated
three times in the NOV3 polypeptide. Now a number of proteins involved in the
complement
pathway (properdin, C6, C7, CBA, C8B, C9) (Patthy,L., J. Mol. Biol. 202: 689-
696 (1988)) as
well as extracellular matrix protein like mindin, F-spondin (Okamoto, et al.,
Developnaeht 126:
3637-3648 (1999)), SCO-spondin and even the circumsporozoite surface protein 2
and TRAP
proteins of Plasmodium (Wengelnik, et al., EMBO J. 18: 5195-5204 (1999);
Rogers, et al.,
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CA 02443770 2003-10-15
WO 02/085922 PCT/US02/11634
Mol. Bioclzefn. Parasitol. 53: 45-51 (1992)) contain one or more instance of
this repeat. It has
been involved in cell-cell interraction, inhibition of angiogenesis (Krutzsch,
et al., Circulation
100: 1423-1431 (1999)), apoptosis [Krutzsch, et al., Cancer Res. 57: 1735-1742
(1997)).
The NOV3 nucleic acids and polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and diagnostic
applications in the
mediation of urogenital physiology. As such, the NOV3 nucleic acids and
polypeptides,
antibodies and related compounds according to the invention may be used to
treat reproductive
and metabolic disorders, e.g., Von Hippel-Lindau (VHL) syndrome, diabetes,
tuberous
sclerosis, fertility, hypogonadism, as well as other diseases, disorders and
conditions.
The NOV3 nucleic acids and polypeptides are useful for detecting specific cell
types.
For example, expression analysis has demonstrated that a NOV3 nucleic acid is
expressed in
eye and testis.
Additional utilities for NOV3 nucleic acids and polypeptides according to the
invention are disclosed herein.
NOV4
A NOV4 polypeptide has been identified as a Protocadherin Alpha C2 Short Form-
like
protein (also referred to as CG93187-O1). The disclosed novel NOV4 nucleic
acid (SEQ 1D
NO:7) of 600 nucleotides is shown in Table 4A. The novel NOV4 nucleic acid
sequences
maps to the chromosome 11.
An ORF begins with an ATG initiation codon at nucleotides 41-43 and ends with
a
TAG codon at nucleotides 2546-2548. A putative untranslated region and/or
downstream
from the termination codon is underlined in Table 4A, and the start and stop
codons are in
bold letters.
Table 4A. NOV4 Nucleotide Sequence (SEQ ID N0:7)
CACCATAAAAGCTCAGAAAATAGACTTTTCCTCTGCCTCTATGGAGGGGCAGGCCAGATCTGGGGAA
GGGATGGGACAGCCTGGCATGAAGAGCCCCAGGCCCCACCTCCTGCTACCATTGCTGCTGCTGCTGC
TGCTGCTGCTGTCTTCGCCTCGCCGTGCACGCGTGCGCCTCCCAGAGGACCAGCCGCCTGGGCCCGC
GGCTGGCACGCTCCTAGCCCGCGACCCGCATCTGGGCGAGGCTGCACGCGTGTCCTATCGGCTGGCA
TCTGGCGGGGACGGCCACTTCCGGCTGCACTCAAGCACTGGAGCGCTGTCCGTGGTGCGGCCGTTGG
ACCGCGAACAACGAGCTGAGCACGTACTGACAGTGGTGGCCTCAGACCGAGCTCCCCGCCCGCGCTC
GGCCACGCAGGTCCTGACCGTCAGTGTCGCTGACGTCAACGACGAGGCGCCTACTTTCCAGCAGCAG
GAGTACAGCGTCCTCTTGCGTGAGAACAACCCTCCTGGCACATCTCTGCTCACCCTGCGAGCAACCG
ACCCCGACGTGGGGGCCAACGGGCAAGTGACTTATGGAGGCGTCTCTAGCGAAAGCTTTTCTCTGGA
TCCTGACACTGGTGTTCTCACGACTCTTCGGGCCCTGGATCGAGAGGAACAGGAGGAGATCAACCTG
ACAGTGTATGCCCAGGACAGGGGCTCACCTCCTCAGTTAACGCATGTCACTGTTCGAGTGGCTGTGG
AGGATGAGAATGACCATGCACCAACCTTTGGGAGTGCCCATCTCTCTCTGGAGGTGCCTGAGGGCCA
GGACCCCCAGACCCTTACCATGCTTCGGGCCTCTGATCCAGATGTGGGAGCCAATGGGCAGTTGCAG
TACCGCATCCTAGATGGGGACCCATCAGGAGCCTTTGTCCTAGACCTTGCTTCTGGAGAGTTTGGCA
CCATGCGGCCACTAGACAGAGAAGTGGAGCCAGCTTTCCAGCTGAGGATAGAGGCCCGGGATGGAGG
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CCAGCCAGCTCTCAGTGCCACGCTGCTTTTGACAGTGACAGTGCTGGATGCCAATGACCATGCTCCA
GCCTTTCCTGTGCCTGCCTACTCGGTGGAGGTGCCGGAGGATGTGCCTGCAGGGACCCTGCTGCTGC
AGCTACAGGCTCATGACCCTGATGCTGGAGCTAATGGCCATGTGACCTACTACCTGGGCGCCGGTAC
AGCAGGAGCCTTCCTGCTGGAGCCCAGCTCTGGAGAACTGGTGTTGCTTGAACCTCTAGACTTTGAA
AGCCTGACACAGTACAATCTAACAGTGGCTGCAGCTGACCGTGGGCAGCCACCCCAAAGCTCAGTCG
TGCCAGTCACTGTCACTGTACTAGATGTCAATGACAACCCACCTGTCTTTACCCGAGCATCCTACCG
TGTGACAGTACCTGAGGACACACCTGTTGGAGCTGAGCTGCTGCATGTAGAGGCCTCTGACGCTGA.C
CCTGCCCTCATGGCCTCCTCAGGCGACCCATCAGGGCTCTTTGAGCTGGATGAGAGCTCAGGCACCT
TGCGACTGGCCCATGCCCTGGACTGTGAGACCCAGGCTCGACATCAGCTTGTAGTACAGGCTGCTGA
CCCTGCTGGTGCACACTTTGCTTTGGCACCAGTGACAATTGAGGTCCAGGATGTGAATGATCATGGC
CCAGCCTTCCCACTGAACTTACTCAGCACCAGCGTGGCCGAGAATCAGCCTCCAGGCACTCTCGTGA
CCACTCTGCATGCAATCGACGGGGATGCTGGGGCTTTTGGGAGGCTCCGTTACAGCCTGTTGGAGGC
TGGGCCAGGACCTGAGGGCCGTGAGGCATTTGCACTGAACAGCTCAACAGGGGAGTTGCGTGCGCGA
GTGCCCTTTGACTATGAGCACACAGAAAGCTTCCGGCTGCTGGTGGGTGCTGCTGATGCTGGGAATC
TCTCAGCCTCTGTCACTGTGTCGGTGCTAGTGACTGGAGAGGATGAGTATGACCCTGTATTTCTGGC
ACCAGCTTTCCACTTCCAAGTGCCCGAAGGTGCCCGGCGTGGCCACAGCTTGGGTCACGTGCAGGCC
ACAGATGAGGATGGGGGTGCCGATGGCCTGGTTCTGTATTCCCTTGCCACCTCTTCCCCCTATTTTG
GTATTAACCAGACTACAGGAGCCCTGTACCTGCGGGTGGACAGTCGGGCACCAGGCAGCGGAACAGC
CACCTCTGGGGGTGGGGGCCGGACCCGGCGGGAAGCACCACGGGAGCTGGGGCTCCACCTGGACTCT
TACCAGAGTCACTCCAAGTCCTGTCTCAGGCAGAATACTCAGATCTATTCCAAGCACCTTCCCTGGG
ATCTCAGGCGCATACTGAGAACCAGTGGGACAGGGTTGAGAGAGAGAGCCAACCGAGAATCTCAAAT
GAACCAAACTGAGAAAGATGCCCCTCAGTGGGGCTACAGACCGACACCCCACCATGGGGCAACAGAA
AAACCAAGACCCCCTCCCCAAAGGAATCAAACCAATCGGGAAAAGGAAGGAGGCGTTGGCCGTGCCT
AGGATAT
The NOV4 protein (SEQ 1D N0:8) encoded by SEQ ID N0:7 is 835 amino acid
residues in length and is presented using the one-letter amino acid code in
Table 4B. Psort
analysis predicts the NOV4 protein of the invention to be localized at the
plasma membrane
with a certainty of 0.7900.
Table 4B. Encoded NOV4 protein sequence (SEQ ID N0:8)
MEGQARSGEGMGQPGMKSPRPHLLLPLLLLLLLLLSSPRRARVRLPEDQPPGPAAGTLLARDPHL
GEAARVSYRLASGGDGHFRLHSSTGALSVVRPLDREQRAEHVLTWASDRAPRPRSATQVLTVSV
ADVNDEAPTFQQQEYSVLLRENNPPGTSLLTLRATDPDVGANGQVTYGGVSSESFSLDPDTGVLT
TLRALDREEQEEINLTWAQDRGSPPQLTHVTVRVAVEDENDHAPTFGSAHLSLEVPEGQDPQTL
TMLRASDPDVGANGQLQYRILDGDPSGAFVLDLASGEFGTMRPLDREVEPAFQLRIEARDGGQPA
LSATLLLTVTVLDANDHAPAFPVPAYSVEVPEDVPAGTLLLQLQAHDPDAGANGHVTYYLGAGTA
GAFLLEPSSGELVLLEPLDFESLTQYNLTVAAADRGQPPQSSWPVTVTVLDVNDNPPVFTRASY
RVTVPEDTPVGAELLHVEASDADPALMASSGDPSGLFELDESSGTLRLAHALDCETQARHQLWQ
AADPAGAHFALAPVTIEVQDVNDHGPAFPLNLLSTSVAENQPPGTLVTTLHAIDGDAGAFGRLRY
SLLEAGPGPEGREAFALNSSTGELRARVPFDYEHTESFRLLVGAADAGNLSASVTVSVLVTGEDE
YDPVFLAPAFHFQVPEGARRGHSLGHVQATDEDGGADGLVLYSLATSSPYFGINQTTGALYLRVD
SRAPGSGTATSGGGGRTRREAPRELGLHLDSYQSHSKSCLRQNTQIYSKHLPWDLRRILRTSGTG
LRERANRESQMNQTEKDAPQWGYRPTPHHGATEKPRPPPQRNQTNREKEGGVGRA
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 4C.
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Table 4C. Patp results for
NOV4
Smallest
Sum
ReadingHigh Prob
Sequences producing Frame Score P(N)
High-scoring
Segment Pairs:
>patp:AAU07054 Flamingo protein +1 968 1.8e-98
Human
>patp:AAU07053 Flamingo polypeptide +1 968 2.0e-98
Human
>patp:ABG21921 human diagnostic protein +l 642 3.1e-64
Novel #21912
>patp:ABG21921 human diagnostic protein +1 642 3.1e-64
Novel #21912
In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 273 of 415 bases (65%) identical
to a
gb:GENBANI~-ID:AF061573~acc:AF061573.2 mRNA from Homo sapie~rs (protocadherin
(PCDHB) mRNA, complete cds). The full amino acid sequence of the protein of
the invention
was found to have 273 of 415 amino acid residues (65%) identical to, and 273
of 415 amino
acid residues (65%) similar to, the 4076 amino acid residue gb:GENBANK-
m:AF061573~acc:AF061573.2 protein from Homo Sapiens (protocadherin (PCDHB)
mRNA,
complete cds).
NOV4 also has homology to the proteins shown in the BLASTP data in Table 4D.
Table 4D. BLAST
results for
NOV4
Gene Index/ Protein/ OrganismLength Identity PositivesExpect
Identifier (aa) (%) (%)
gi~17461472~ref~XPsimilar to 1415 459/682 503/682 0.0
_ protocadherin (67%) (73%)
052786.2I(XM 16
052786
[Homo Sapiens]
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gi~16933557iref~NPprotocadherin 443/676 490/676 0.0
16
_ precursor; (65%) (71%)
003728.1~(NM_003737
fibroblast
cadherin FIB1;
cadherin 19;
fibroblast
cadherin 1;
dachsous
homologue
[Homo Sapiens]
gi~6753408~ref~NPcadherin EGF 3034 48/693 358/693 1e-98
0 LAG
_ seven-pass G-type (35%) (50%)
34016.1~(NM_009886)
receptor
[Mus musculus]
gi~13325064~ref~NPcadherin EGF 2923 246/679 345/679 2e-98
LAG
_ seven-pass G-type (36%) (50%)
001399.1~(NM
001408
~
receptor 2;
EGF-like-domain,
multiple 2;
epidermal growth
factor-like
2;
multiple
epidermal growth
factor-like
domains 3;
cadherin, EGF
LAG
seven-pass G-type
receptor 2,
flamingo
(Drosophila)homol
og
gi~10727655Igb~AAF5Stan gene product3606 241/700 361/700 3e-98
8763.2 (AE003828)[Drosophila (34% (51%)
melanogaster]
A multiple sequence alignment is given in Table 4E, with the NOV4 protein
being
shown on line 1 in Table 4E in a ClustalW analysis, and comparing the NOV4
protein with the
related protein sequences shown in Table 4D. This BLASTP data is displayed
graphically in
the ClustalW in Table 4E.
Table 4E. ClustalW Analysis of NOV4
1) > NOV4; SEQ >D N0:8
2) >gi~ 17461472/ similar to protocadherin 16 (H, Sapiens) [Homo Sapiens]; SEQ
)D N0:50
3) >gi~16933557~/ protocadherin 16 precursor; fibroblast cadherin FIB1;
cadherin 19; fibroblast
cadherin 1; dachsous homologue [Homo sapieras] ; SEQ >D N0:51
4) >gi~6753408~/ cadherin EGF LAG seven-pass G-type receptor [Mus rnusculus] ;
SEQ >D N0:52
5) >gi~13325064~/ cadherin EGF LAG seven-pass G-type receptor 2; EGF-like-
domain, multiple 2;
epidermal growth factor-like 2; multiple epidermal growth factor-like domains
3; cadherin, EGF LAG
seven-pass G-type receptor 2, flamingo (Drosoplaila) homolog; ; SEQ )D N0:53
6) >gi~10727655~gb~AAF58763.2~(AE003828) Stan gene product [Dr~osophila
rnelanogaster] ; SEQ >D
N0:54
1210 1220 1230 1240 1250 1260
rrov4 1 3
gi~ 17461472~81
139
ga.~ 16933557~1153 1211
g3.~ 67534081955 1014
133250641863 921
gi~
gi~ 10727655~1055
1113
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1270 1280 1290 1300 1310 1320
NOV4 4 QARSGE-GMGQ-_______________pGMKS~___________________________ 19
gi1174614721 140 L~ASGAAGGG P ;Q PAR ~P 'I'L~uTTI:iQ~i ~'~ EI GT L T'T~T- PGS
L S PH 198
gi I 16933557 I 1212 L~,ASGAAGGG'L~t'Pt2.~Q PIJR ~P TL~TT,LiQ~ ~' ~ E GT L TAT-
PGS S PH 1270
gi~6753408~ 1015 EKDE--- ~'E~F E~NS~ S IRi ~'~ P Q I Q~~E P Q LL 1068
gi ~ 13325064 ~ 922 Ej~DE--- FDV~F~E~NS~LA~AR~Ti,~~~,T~ ~ ~ T Q Q~VE IP _ Q IF
975
gi ~ 10727655 ~ 1114 ASDK--- ~CTI~Y PNS SV GE'IHi ~ ~ ~ VUH SI~I DSN S TRP
1167
1330 1340 1350 1360 1370 1380
NOV4 19 __________________ .HL~ _P-________ ___LL~,tL~sL~:,.SS-__ _______ 37
gi~174614721 199 --~ LTAAP IRA-E~~H T~S~HDQGSP~RSSLQ LVQ PSARLAP~~D 253
15 gi~16933557~ 1271 --LTAAP IRA-E~~H TS~,~HDQGSP~RS~SLQ~L. PSARLAFSP~~D 1325
gi I 6753408 ~ 1069 ---~7 RALVE FE- 'RD Qi~i,TS-- ~LVRAT4V~i~~, QN-- ~7 ~ ~E P
1117
gi ~ 13325064 ~ 976 -- TALVD YE-D' ~E Q~TS-- ~LVRAT~'HU7~,~r~DRN-- 't~ ~ G
1024
gi I 10727655 ~ 1168 SERA LTMTE YEST KR~'E u.~R~AS---P ~LRNDAIiE~L'S~TDVN---
~3N ~ R 1221
1390 1400 1410 1420 1430 1440
NOV4 37 ____________________________________________________________ 37
gi ~ 17461472 ~ 254 k~RDPAAPVPWLT GL ~GSL S~~P~ GVG' T ~L~~ ;PEGTF ~ 313
gi~169335571 1326 RDPAAPVPWLT EGL ~GSL S, ~P~~ GVG T TL~ PEGTF Afi1385
gi~67534081 1118 FQILFNN----Y KSNSF'SGV,~ ~~P~~,H~ LSD~~a 'PFQ BLS-LLL ~P,A'~
1172
gi I 13325064 ~ 1025 NFEILFNN----YRSSSF'GGA'I R~t P~iH>~~7' ~ ISDS T SFE ~ BLS-
LVL G'i' 1079
gi~10727655~ 1222 ~.IFQVIFN--------NFRDHF~SGEiI P~FADVSD RIS ~AN-LL 51272
1450 1460 1470 1480 1490 1500
NOV4 37 ___________________________________________________-_____p__ 38
gi 17461472 314 373
gi~16933557~ 1386 1445
giI6753408~ 1173 1231
giI13325064~ 1080 1138
gi~10727655~ 1273
1331
1510 1520 1530 1540 1550 1560
NOV4 38 ____________________________________________________________ 38
gi117461472~ 374 RTRSPAQRCTLSARRTPTAP TATCATACCARSRPCRASPGRAHRGVSSARPGPRD---
430
gi~16933557~ 1446 PE-NPEPGAALYTF~SDADG'-GPNSDVRY.LLRQEPPVP R'~.iDARTGALSAPRG---
1500
gi~6753408~ 1232 EKFLSPLLSLFVEG ~~TVLSTTKDDIFVFNI~NDTDVSS-NI ~ TFSALLPGGTRG--R
1288
giI13325064~ 1139 ERFLSPLLGLFIQAVTLAT~PDHWVFNV~RDTDAPGGHI YSLSVGQPPGPGGGPP
1198
gi~10727655~ 1332 EAFLSPLLNFFLDGL~IIPC KEHIFVFSItDDTDVSS-RI j SFSARRPDVSHE--E
1388
1570 1580 1590 1600 1610 1620
NOV4 38 -_________________ _ __ _ ___ __ R~~a~R~=~~RLP~T'JQP~ 52
gi~17461472~ 431 HSRAAAAGGSHRPA~QRQPPSCFSA;~TSR~QR~TRLS,---P~RLP~QP~ 485
gi ~ 16933557 ~ 1501 LDRETTPALL'~L~1EATDRPANARAARVSARVFVTDEN~3NAPVASPS
~=RLP1QP ~ 1560
gi ~ 6753408 ~ 1289 FFPSEDLQEQ~Y' ~ 'TLLTTI.~~ LP",DD C EPCE'~CV~LRF~SS ~ 1344
gi~133250641 1199 FLPSEDLQERIar9 'SLLTAIAQ LP DN~Cxr EPCERCVS=LRFSS ~ 1254
' ~ ~ i..
gi~10727655~ 1389 FYTPQYLQER~3Y' ILARLTVEVLP~DN~C,'EPCL~'EECLT~LKFGASE 1444
55 ~ ,.
1630 7.640 1650 1660 1670 1680
. .,I. .~~ ~~~~ .~. ..y y ...I. y . .I.. .y
~V
NOV4 52 GP, P~LGE ~ YRLAS.~ ------~GHF ~S TGAL ' 'P~.iDv -- 101
gi~17461472~ 486 GP~ ~DP LGE YRLAS ------~GHF S TGAL~ ~PhD~ -- 535
gi~16933557~ 1561 GP~~LH ~DPDLGE _YRLAS ------~GHF S TGAL~,~ 'PI~D~ -- 1610
gi~6753408~ 1345 FIST F'PI PITGL~CRCPPGFT ----DYCTEID CY PC ' CRS' GGY 1400
gi~13325064~ 1255 FI~SSF'PI~PVGGL'CRCPPGFT ----DYCTEVD CY RPCGPHGyyCRS~ GGY
1310
gi~10727655~ 1445 FIND F'PI'Y~PVNTFAC~CPEGFT SKEHYLCdTEVD CY DPCQ~1GGTC'tt '
GGY 1504
65 1690 1700 1710 1720 1730 1740
NOV4 101 ____I....~.. .~. .'....~ .._.i....~ I"~ I,, I ~ I,~ ~'
--Q ~ L DB=--~pRP ATQ T'.t~~~~SV~TD' FQ~QQY'VL . ~ E 151
gi ~ 17461472 ~ 535 ---_---Q L --~PRP~~ATQ T~S~'~' V~17:: ~ .F~~QQ Y~sVL ~E
585
gi I 16933557 I 1610 -------Q L DH---GSPP32u~ATQ TVS~ 'TF~,bQf~~Ys.~'r, VL 'E
1660
70 gi I 6753408 ~ 1401 TCECFEDFT CQVNV G~-CAGVC~GGTC4~tN,~L~I,GG~VC
~PG~"YHPYCE~TST 1459
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gi ~ 133250641 1311 TCLCRDGYTG~CEVSAR~ -CTPGVCGGTCVNT.~L~GGFKCC~GDFPYCQ~STTT
1369
gi 1107276551 1505 TCVCPSTHTG~IStCE~G~GHrLyPCPETCEGGLSC~fiTYP- ~
QPPPYTA~I'CE~~yA 1558
1750 1760 1770 1780 1790 1800
NOV4 252 211
gi1174614721 586 645
gi1169335571 1661 1720
gi167534081 1460 1518
gi1133250641 1370 1428
gi1107276551 1559 1617
1810 1820 1830 1840 1850 1860
IS NOV4 212 271
gi1174614721 646 705
gi1169335571 1721 1780
gi16753408~ 1519 1578
gi1133250641 1429 1488
gi1107276551 1618 1677
1870 1880 1890 1900 1910 1920
1.. .~....1....~....1....1....1. .1. ..1. 1....~.. .1
NOV4 272 ~i7GQ ....~;_________-____________-R__-_~.~ ~GAFVLD~' G~ G-Tv P 303
gi1174614721 706 ~iGQ ~1'~' ----------------------R-- D~GAFVLD GG-Tv P 737
gi1169335571 1781 iINGQ ~'----------------------R----~ ~~GAFVLD ' GG-T ~P 1812
gi167534081 1579 JAVA ~._GSYVGNYSCAAQGTQSGSKKSLDLTGPLL ~ LPEDFPH~RQ GC ~ 1638
gi1133250641 1489 TGV 'GSVLGNYSCAAQGTQGGSKKSLDLTGPL DLPESFP'9'RMRQ GC ' 1548
gi1107276551 1678 NRT~LDKRCSLLTETCH-------RFLDLTGPLQT?'?'G~VL~'RIPAHFP~TRGCSD
1730
1930 1940 1950 1960 1970 1980
NOV4 304 360
gi1174614721 738 794
gi1169335571 1813 1869
gi167534081 1639 1698
gi1133250641 1549 1608
giI107276551 1731 1790
1990 2000 2010 2020 2030 2040
.1....1. .1. ..1....1....1 1....1. ..~. ~.~..1....1
NOV4 360 ---------- ~.1'L ~QLQ ~PDAGANG~1V~'GAGT~,~~I,, ~ PSS LVLLEPLD 409
e'E
gi 117461472 1 794 ------- '~L QLQAH~PDAGANGHV'~Y~GAGT~,~, ' ~ PAS ~LR'.AAALD
843
gi 116933557 1 1869 ------- -~. TL QLQ ~PDAGANG~3"~'Y GAGTi ~ PAS LItAAALD
1918
gi167534081 1699 CEQAMPHPQRFT ES''V~t(LWSDL~ITISVPW'x7~GLMFRTRKEDG ~,
TSSRLHLQI 1758
gi1133250641 1609 CAQEMANPQHFL ~SAWHGLSLPISQPW~h~L_MFRTRQiDG
S~AI'~RGRS'~.'ITLQL 1668
gi1107276551 1791 CQDNIPAPWRFGS SFNPLLRPIQLPWTT'~5~RTRQKE~ IQI~NSAAVCL 1850
2050 2060 2070 2080 2090 2100
. ..1. ~_ ..1... 1 ..1....1....~ .1. ..1....1.. ~ m..1
NOV4 410 F.SLTQ. L~~ ~--- RG-QP-PQSSWP~Tj~TVLDV~NP------- PV3.:TRA 453
gi1174614721 844 QCPS TFVSA ~--- G,~-~aAGPLSTTVS T TVT~D HA------- PT~PTS 888
gi1169335571 1919 ' QCPS TFSA ~--- G3~-AGPLSTTVS~T T~I2,D~---------PTPTS 1963
gi 1 67534081 1759 LNSYI1;EVYGPS~ ASMQLKRITDGGWHHiL
E~.,~SAKGKDIKYLAVMTLD~'~','GMD 1818
gi1133250641 1669 GHVMLSVEGTGLQASSLRLEPGRANDGDWHHAQ~GASGGPG----HAILSFD~~GQQ
1724
gi110727655~ 1851 '~QGVLY~IFDGEP-----MYLG~SFLSDGEWHRVE~RWS~QGIH------FS-
VD'YaGQR 1898
2110 2120 2130 2140 2150 2160
1 ...1.. 1 . ~ ..1.,...1....1....1. 1 ...1....1. ..1
CO NOV4 454 SYR~~J"T~~~~PEDT~- '~GAEL2aHVEASDADP--------ALN~AS~,.'rGD--------
'SGL 491
gi 1174614721 889 PLRT~.~RPRP ~SFSTPT~iAI~AT~R.~.A~;bRDAGAN-----ASILY LF~ -----
- 'PPG 935
gi1169335571 1964 PLRRI~PRP ~SFSTPT~TRDAGAN-----ASILY'_ --------'PPG 2010
gi167534081 1819 QST~QGNQL~GLKMRTVTGG~VT~KVSVRHG--FRGCMQG ' GE STNIATLNMNDA
1876
gi1133250641 1725 RAEGN~I~GPRLHGLHLSN~T GG~PGPAGGVARG--FRGCLQG ~wD
PEGVNSLD~SHG 1782
65 gi1107276551 1899 SGS~P1~SQKVQGLYVGKT7GSPDGSIGAVPEASPFEGCIQD~ Z~GAG----
QSVLSRPT 1954
2170 2180 2190 2200 2210 2220
1.. .1....1....1....1....1. ..1 ...1....1. 1 ...1....1
NOV4 492 FE~'pESS~TLRL~~,HAL------DCETQARHQ. Q ~PAG-AHFALAPVT~!EVQDVN-- 542
70 gi1174614721 936 TT~~SYT~EIRV~RSP------V~LGPRDR IV~T~L~RPARSATG~II'VGLQGEA--
987
32 ~~ _~
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gi1169335571 2011 TTU[.1~SYT EIRViRSP------VLGPRDRV~~IVT~L
RPARSATG~iII'S~GLQGEA-- 2062
gi~67534081 1877 LRVKD CDVEDPCASSPCPPHRPCRDTWDS'~SCIC~ YFGKKCVDACL~iNPCKHVAA
1936
gi1133250641 1783 ESIt~VEQ CSLPDPCDSNPCPANYCSNDWDSSCC~P YYGDNCTNVCD~iNPCEHQSV
1842
gi I 107276551 1955 IRE;E~VED CESRi~IQCP-DHCPNHSCQSSWD~STCEC~S
YVGTDCAPCT:StRPCASG-V 2012
.~.
2230 2240 2250 2260 2270 2280
.I.. .1....1....1....1. ..I....I....I....I....~..f..," I....p,
NOV4 542 -DH ~ F~LNLLSTSVAE---NQPP ~L~ITTLHAIDGDAG-AF RLRYLF.~AGPGPEG597
gi1174614721 987 -E~ ~RF SEATIRE---NAPP P~~ S~RAVH- G-T ~ITYS~LS --- 1037
1o gi1169335571 2062 -E' ~RF SEATIRE---NAPP P VS~RAVH- 'G-TV ~ITY~TLS V - ,
217.2
gi167534081 1937 C ~S~ T GYCECGPG-HYGQYCEN d7L~CPKGWW PVC ~CHC~51'S~ F~PDC
1995
giI107276551 2013 CRANTSL HGYDCECNSSSRHGDYCE~~QQ~CPGGWW ERVC ~CRCD~.tA, YHPDC
2072
IS 2290 2300 2310 2320 2330 2340
.1....I.. .1....I... I ...I I....I....I. ..I.. I . .I
NOV4 598 EAFALNSS GEL,: ~'.AR------VPFD'~'EHTESF LVGAADAGNLSAS~TVSVLVTGEIE651
ga. 11174614721 1038 G"~'FSIQPS GAITVRSA----EGLDF'EVSPRL'~VLQAESGGAFAFT
LiTLTLQDA13N3 1092
giI169335571 2113 G'.~'FSIQPS GAITVRSA----
EGLD.~.~EVSPRL'~iVLQAESGGAFAFT~L~TLTLQDA~N 2167
2O gi167534081 1996 TNGQCQCKENYYPPAQDACLPCDCPHGSHS~ CDMDTGQCACKP ~GRQCNRCP2055
gi~133250641 1902 °'GECHCKENHY PPGSPTCLLCDC3~PTGSLS sCDPEDGQCPCKP
~GRQCDRCi~P' 1962
gi110727655~ 2073
TGQCYCKQNHYPPNETACLSCDC~'SIGSFSGACNPLTGQCECREGuGRRCDSCS~P2132
2350 2360 2370 2380 2390 2400
25 ....~....I....I....I....I....I....I ...I. .I. ..1....1....I
NOV4 652 DPVF PAFHFQVPEGARRGH----SLGHaj'QATDEk~GGAD L~7LYATSSP-------- 699
gi1174614721 1093 ~,~~1,PRF RPHY LPES~PL G--- LL~?EADDL~QGS QISY~AASQP ----
1143
gi1169335571 2168 ~~,PRF RPHY LPES~PL G----~LLt~EADDL~7QGS 'Q,~SY~cAASQP ----
2218
gi~67534081 2056 ~iE=uTSLGCE I'~NGCP~ F GIWW QTFGQPAAVPCP 'S~ GN~,RHCSGE~ -
WLPP 2114
3~ gi1133250641 1962 iiIEuiTTNGCE YDSCP~ I GIWW RTFGLPAAAPCP SFGT,,'RHCDE -
WLPP 2020
gi1107276551 2133 iEuT~SGCE~~DACP~SFAGGWW~RTPiGGVAI!GCPPPARGKGQRSCDVQS SWNTP
2192
2410 2420 2430 2440 2450 2460
.I.. I . .I... 1....I. ..1....I ...1....I. ..~....I....I
3S NOV4 700 YFGINQi~TG - ---- ------ .YLRVDS~ P ---G GT ------------- 725
gi1174614721 1144 LFHVDP~~TGT~TTTAIL ~EIW ~? LMA'D~GP G~ATL ------------ 1190
gi116933557~ 2219 LFHVDP~TGTTTTAILREIW T LMAD~GP GATL ------------ 2265
gi167534081 2115 ELFNCTSGSF~DLKAL LN T GN L L RN=TQGNS------------- 2161
gi~133250641 2021 NLFNCTTTFSELKGFF~~LQ ~ SGF~Q'L~LL RN~~~,TQH~A-------'-----
2067
gi110727655~ 2193 DMYNCT~EPFITELRRQLSLEKL LGE~TSFVAIEQ RCEAVDRRGASKDQKISGN
2252
2470 2480 2490 2500 2510 2520
.'..I....I....I....I....I....I....I....I....I....I....I....I
NOV4 725 ______________________________________________gGGG-GRTRREAPR 738
45 gi1174614721 1190 ____________________________________________DTNDNRPTIPQPW
1206
gi1169335571 2265
____________________________________________~7jOTT7DNRPTIPQPW 2281
gi167534081 2161 ----------------------------------------TLFGNDC~RTAYQLLARILQH
2181
gi1133250641 2067 ----------------------------------------GYFGSDKVAYQLATRLLAH
2087
gi1107276551 2253 GRPNRRYKMESSFLLSNGGNVWSHELEMDYLSDELKFTHDRLYGADLVTEGLLQELINY
2312
2530 2540 2550 2560 2570 2580
.I....I....I....I....I....I....I....I....I....I.'.'I....I
NOV4 739 ~GLHLDSYQS-----------------------HS----------------------KS 753
gi1174614721 1207 RVSED LG~IAQVTG~~DS P YVLSPGP~ DPFSVGRY RVS TGPL~FEQ 1266
gi ~ 16933557 1 2282 RVSED LGI~IAQVTGI~(1'~'DS P YVLSPGP~DPFSVGRY RVS TGPLFEQ
2341
w
gi167534081 2182 SRQQGFD REANFHEI~~HT S LAPATE~3SWQIQRSE-- Q LRHFAYF 2239
gi1133250641 2088 STQRGF SA'QDVHFTL'~""iR. S LDTANKRHW_LIQQTE-- TAW LQHYAYA
2145
gi1107276551 2313 QSGL SH~C.~DKYFI~L~AASVLDRKYEEWRRATELIQ- PDD DAFNKYL 2371
2590 2600 2610 2620 2630 2640
.I.. L~. I ...I....I....p....l....l....l....l....~....1
NOV4 754 CLRQNT~IYKHL Ia~_iRRILRTSGTG~iRRA~3RESQ----------------------- 790
gi1174614721 1267 CDRY~LvLLA;E~D ~GRANLT~ Em n APAFSQSI~Y'VML~~~EHTP~GS--------
1318
ga.11693355i ~ 2342 CDRYL~LLA'~iD ~GRANLT E~ ~ APAFSQS~.aYVMLEHTP~GS--------
2393
gi~6753408 2240 SNVAN~T XL F~TANM~ WF~KL~FTGAQVPFED'~~QEEL~RE-----LES 2294
gi I 133250641 2146 S NMR.I-~TYLS FT. ;
~VTPNI~3~;SWRL~KGFAGAK'~.tPYEA~iRGEQ~PD-----LET 2200
gi ~ 10727655 I 2372 W~S~ID~~'1'TS
~F~ZTQPNMA~GL~~VTTE~LFGYEPE~LSEYHRSKYLICPNAFTTES 2431
2650 2660 2670 2680 2690 2700
....I....I....I....I....I.'..1....1....1....1....1....1....1
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NOV4 790 - -MNQ EIi;D PQW YR-________-_____-______pT~H TE ~RPPPQRN823
gi1174614721 1319 A~LSVS~p G~ ~~~~~,SYH------------------LAS~ FSVD~ GTLF~'~'I
1359
giI16933557~ 2394 A'~Z.tSVS~,D G~~SYH--------------'---LAS~ FSVD~ GTLFTI 2434
gi~6753408~ 2295 StT;SFPAD FPPEKKE~P~VRLTNR--------------
RTTPLTAQPEPRAERETSS~~2340
gi~13325064~ 2201 TjVLPE'~UF~;----ETPPRP-----------------------
A~ePGEAQEPEELAR~'t 2233
gi~10727655~ 2432
V;VV11PDTGFLQHARQRPISFPKYNNYILDRRKFDQHTKVLV~LEMLGIT~PESDEI~,t~ 2491
2710 2720 2730 2740 2750 2760
l~ NOV4 824 TN~'yIEKEGG Ri-_______________________________________________ 835
gi~17461472~ 1360 VGTVALGHD SGAVD ~~ KHETTG------------------------------------
1383
gi~I6933557~ 2435 VGTVALGHD SGAVD LEARDHGAPGRAAR.ATVH-------------------VQLQDQ
2475
giI6753408~ 2341 R ~HPDEPGQFiiIVAL ;;,IYRTLGQLLPEHYDP--------------------------
-- 2372
gi~13325064~ 2234 Q HPELSQ~eE~iAS IYRTLAGLLPHNYDP----------------------------
2265
1S gi~10727655~ 2492
SG'RGSSHDHRiIVAYAQYKDVGQLLPDLYDETITRRWGVDVELATPILSLQILVPSMER 2551
The NOV4 Clustal W alignment shown in Table 4E was modified to begin at amino
residue 1201 and end at amino acid residue 2760. The data in Table 1E includes
all of the
20 regions overlapping with the NOV4 protein sequences.
The presence of identifiable domains in the protein disclosed herein was
determined by
searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and
then
determining the Interpro number by crossing the domain match (or numbers)
using the
Interpro website (http:www.ebi.ac.uk/interpro~. Table 4F lists the domain
description from
25 DOMAIN analysis results against NOV4.
Table 4F Domain
Anal sis
of NOV4
Model Region of Score (bits) E value
Homology
cadherin 41-131 97.7 2.4e-25
T25P N 16-223 ~ -I17.2 1.3
cadherin 145-233 104.1 2.7e-27
cadherin 247-337 78.1 1.8e-19
cadherin 351-441 112.9 6e-30
cadherin 455-539 64.7 2e-15
cadherin 553-646 77.5 2.8e-19
cadherin 660-745 15.4 0.036
Consistent with other known members of the Protocadherin Alpha C2 Short Form
Protein-like family of proteins, NOV4 has, for example, seven Cadherin domain
signature
30 sequences and homology to other members of the Protocadherin Alpha C2 Short
Form
Protein-Like Protein Family. NOV4 nucleic acids, and the encoded polypeptides,
according to
the invention are useful in a variety of applications and contexts. For
example, NOV4 nucleic
acids and polypeptides can be used to identify proteins that are members of
the Protocadherin
Alpha C2 Short Form Protein-like Protein Family. The NOV4 nucleic acids and
polypeptides
35 can also be used to screen for molecules, which inhibit or enhance NOV4
activity or function.
Specifically, the nucleic acids and polypeptides according to the invention
may be used as
targets for the identification of small molecules that modulate or inhibit,
e.g., cellular
34
CA 02443770 2003-10-15
WO 02/085922 PCT/US02/11634
activation, cellular metabolism, and signal transduction. These molecules can
be used to treat,
e.g., Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous
sclerosis,
hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy,
epilepsy, Lesch-
Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies,
behavioral
disorders, addiction, anxiety, pain, neurodegeneration, systemic lupus
erythematosus,
autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS, fertility,
endometriosis, hypogonadism, hemophilia, hypercoagulation, idiopathic
thrombocytopenic
purpura, autoimrnune disease, allergies, immunodeficiencies, transplantation,
graft versus host
disease (GVHD), lymphaedema, as well as other diseases, disorders and
conditions.
In addition, various NOV4 nucleic acids and polypeptides according to the
invention
are useful, i~atef° alia, as novel members of the protein families
according to the presence of
domains and sequence relatedness to previously described proteins. For
example, the NOV4
nucleic acids and their encoded polypeptides include structural motifs that
are characteristic of
proteins belonging to the Protocadherin Alpha C2 Short Form Protein-like
Protein Family.
Cadherins (Takeichi, Arahu. Rev. Biochem. 59: 237-252 (1990); Takeichi Trends
Genet. 3: 213-217 (1987)), first discovered in mouse teratocarcinoma cells
(Liaw, EMBO.I. 9:
2701-2708 (1990)), are a family of animal glycoproteins responsible for
calcium-dependent
cell-cell adhesion. Cadherins preferentially interact with themselves in a
homophilic manner in
connecting cells; thus acting as both receptor and ligand. There are a number
of different
isoforms distributed in a tissue-specific manner in a wide variety of
organisms. Cells
containing different cadherins tend to segregate ira vitro, while those that
contain the same
cadherins tend to preferentially aggregate together. This observation is
linked to the finding
that cadherin expression causes morphological changes involving the positional
segregation of
cells into layers, suggesting they may play an important role in the sorting
of different cell
types during morphogenesis, histogenesis and regeneration. They may also be
involved in the
regulation of tight and gap junctions, and in the control of intercellular
spacing. Cadherins are
evolutionary related to the desmogleins which are component of intercellular
desmosome
junctions involved in the interaction of plaque proteins.
Structurally, cadherins comprise a number of domains: these include a signal
sequence;
a propeptide of around 130 residues; an extracellular domain of around 600
residues; a single
transmembrane domain; and a well-conserved C-terminal cytoplasmic domain of
about 150
residues. The extracellular domain can be subdivided into 5 parts, 4 of which
are repeats of
about 110 residues, and the fifth contains 4 conserved cysteines. The calcium-
binding region
of cadherins is thought to be located in the extracellular domain. This
indicates that the
CA 02443770 2003-10-15
WO 02/085922 PCT/US02/11634
sequence of the invention has properties similar to those of other proteins
known to contain
this/these domains) and similar to the properties of these domains.
Maniatis et al. has identified 52 novel human cadherin-like genes organized
into three
closely linked clusters (Wu and Maniatis, Cell 97(6):779-90 (1999).)
Comparison of the
genomic DNA sequences with those of representative cDNAs reveals a striking
genomic
organization similar to that of immunoglobulin and T cell receptor gene
clusters. The N-
terminal extracellular and transmembrane domains of each cadherin protein are
encoded by a
distinct and unusually large exon. These exons are organized in a tandem
array. By contrast,
the C-terminal cytoplasmic domain of each protein is identical and is encoded
by three small
exons located downstream from the cluster of N-terminal exons. This unusual
organization
has interesting implications regarding the molecular code required to
establish complex
networks of neuronal connections in the brain and the mechanisms of cell-
specific cadherin-
like gene expression.
The NOV4 nucleic acids and polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and diagnostic
applications in the
mediation of urogenital, nerve, and endocrine physiology. As such, the NOV4
nucleic acids
and polypeptides, antibodies and related compounds according to the invention
may be used to
treat reproductive and nervous system disorders, e.g., Von Hippel-Lindau (VHL)
syndrome,
Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's
disease,
Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple
sclerosis,
ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction,
anxiety, pain,
neurodegeneration, systemic lupus erythematosus, autoimmune disease, asthma,
emphysema,
scleroderma, allergy, ARDS, fertility, endometriosis, hypogonadism,
hemophilia,
hypercoagulation, idiopathic thrombocytopenic purpura, autoimmune disease,
allergies,
immunodeficiencies, transplantation, graft versus host disease (GVHD),
lymphaedema, as
well as other diseases, disorders and conditions.
The NOV4 nucleic acids and polypeptides are useful for detecting specific cell
types.
For example, expression analysis has demonstrated that a NOV4 nucleic acid is
expressed in
Heart, Aorta, Umbilical Vein, Thyroid, Colon, Peripheral Blood, Spleen, Lymph
node, Bone,
Cartilage, Brain, Left cerebellum, Right Cerebellum, Parietal Lobe, Temporal
Lobe, Cerebral
Medulla/Cerebral white matter, Hippocampus, Cervix, Mammary gland/Breast,
Ovary,
Placenta, Uterus, Testis, Lung, and Retina.
Additional utilities for NOV4 nucleic acids and polypeptides according to the
invention are disclosed herein.
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CA 02443770 2003-10-15
WO 02/085922 PCT/US02/11634
NOVS
A NOVS polypeptide has been identified as a Nuclear protein-like protein (also
referred to as CG95083-O1). The disclosed novel NOVS nucleic acid (SEQ ID
N0:9) of 2322
nucleotides is shot~cni in Table SA.
An ORF begins with an ATG initiation codon at nucleotides 70-72 and ends with
a
TAA codon at nucleotides 2320-2322. A putative untranslated region and/or
downstream
from the termination codon is underlined in Table SA, and the start and stop
codons are in
bold letters.
Table SA. NOVS Nucleotide Sequence (SEQ ID N0:9)
GTGGAAGGACAGTCCAGAGCCCTTGTCATCGCACAGGAACTGCTATCTTCAGAGAAAGCATACGTGG
A_GATGCTCCAGCACTTAAATCTGTTCCTGGCAGAGCAGGCTATCAGCAGGAGAGGCCAGGGCTCCAA
AGCCCCAGGGGAAATCTGCCAAGGAGGACTTGTGCTCAGTCCTATCAACCTGTGGGTAACAGACCTT
TTGGTGTTTCAGGATTTCCATGGAGCTGTCATGAGGGCCTTGGATGACATGGACCATGAAGGCAGAG
ACACATTGGCCCGGGAGGAGCTGAGGCAGGGCCTGAGTGAACTCCCAGCCATCCACGACCTTCATCA
AGGCATCCTGGAGGAGCTGGAGGAAAGGCTGTCAAATTGGGAGAGCCAGCAGAAGGTAGCTGACGTC
TTCCTTGCCCGGGAGCAGGGGTTTGATCACCACGCCACTCACATCCTGCAGTTCGACAGGTACCTAG
GTCTGCTCAGTGAGAATTGCCTCCACTCTCCCCGGCTGGCAGCTGCTGTCCGTGAATTTGAGCAGAG
TGTACAAGGAGGCAGCCAGACTGCGAAGCATCGGCTGCTGCGGGTGGTTCAACGCCTCTTCCAGTAC
CAAGTGCTCCTCACAGACTATTTAAACAACCTTTGTCCGGACTCCGCCGAGTACGACAACACACAGG
GTGCACTGAGCCTCATCTCCAAAGTCACAGACCGTGCCAACGACAGCATGGAGCAAGGGGAAAACCT
GCAGAAGCTGGTCCACATTGAGCACAGCGTCCGGGGCCAAGGGGATCTCCTCCAGCCAGGAAGGGAG
TTTCTGAAGGAAGGGACGCTGATGAAAGTAACAGGGAAAAACAGACGGCCCCGGCACCTATTTCTGA
TGAACGATGTGCTCCTGTACACCTATCCCCAGAAGGATGGGAAGTACCGGCTGAAGAACACATTGGC
TGTGGCCAACATGAAGGCTCTTTACCATGGGGAAGGGGAAGGAGGAAGCACCTTTCTCAGCATGGAG
GTTTGTTCCCTTTTGGAACCAAAGGCTCCACCGAGGAGCCTGTTAGAAAAAGGCATGGGAGACGTGG
TCACTGGCAGGTACTTGTCCAACATGACAGTGCACCTGGGGTTGCCCGGGCTGGGCCCTGAGCATGA
CGCTCTGCAGCCTTCCCAGCGGTGGGTCAGCCGCCCTGTGATGGAGAAAGTGCCCTACGCTCTAAAG
ATTGAGACTTCCGAGTCCTGCCTGATGCTGTCTGCGAGGCTGCAGGTCAGGAAGTCCAAGGTCAAGG
CACTGACTGATTCGGTGTCTGCAGCCCTGGGAGTTAGGGGAATATCATTATTCCAGTGTAAGAAGAA
ACAGACCCAAGGACAGCTAATGGACCAGTGGTCTGCTCGTAAACCTAGTCTGGCAGGTGATCTCTTC
TTTGCTGGTGGTTCTGGGCAGTGTGAGAGGTGCAGGCTCAAGGGGCATCTGAGTGAGAACCTCATCC
ATGCCGAGATGGAGGCCCATGCCCGCAGCTCCTGTGCAGAGAGGGACGAGTGGTATGGCTGTCTGAG
CAGAGCCCTCCCTGAGGACTACAAGGCCCAGGCGCTGGCTGCATTCCACCATAGCGTGGAGATACGA
GAGAGGCTGGGGGTTAGCCTTGGGGAGAGGCCCCCCACCCTGGTGCCTGTCACACACGTCATGATGT
GCATGAACTGCGGCTGCGACTTCTCCCTCACCCTGCGGCGTCATCACTGTCACGCCTGTGGCAAGCA
GATCGTGTGCCGGAACTGTTCGCGGAACAAGTACCCGCTGAAGTACCTGAAGGACAGGATGGCCAAG
GTCTGCGACGGCTGCTTCGGGGAGCTGAAGAAGCGGGGCAGGGCTGTCCCGGGCCTGATGAGAGTTA
CAGAGCGGCCTGTGAGCATGAGCTTCCCGCTGTCTTCACCCCGCTTCTCGGGCAGTGCCTTTTCATC
CGTCTTCCAGAGCATTAACCCCTCGACCTTCAAGAAGCAGAAGAAAGTCCCTTCAGCCCTGACAGAG
GTAGCTGCCTCTGGAGAGGGCTCTGCCATCAGTGGCTATCTCAGCCGGTGTAAGAGGGGCAAGCGGC
ACTGGAAGAAGCTCTGGTTTGTCATCAAAGGCAAAGTTCTCTACACCTACATGGCCAGTGAGGACAA
AGTGGCCTTGGAGAGTATGCCTCTGCTAGGCTTCACCATTGCTCCAGAAAAGGAAGAGGGCAGCAGT
GAAGTAGGACCTATTTTTCACCTTTACCACAAGAAAACCCTATTTTATAGCTTCAAAGCAGAAGATA
CCAATTCATGGATCGAGGCCATGGAAGATGCGAGTGTGTTATAG
Variant sequences of NOVS are included in Example 3, Table 19. A variant
sequence
can include a single nucleotide polymorphism (SNP). A SNP can, in some
instances, be
referred to as a "cSNP" to denote that the nucleotide sequence containing the
SNP originates
as a cDNA.
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The NOVS protein (SEQ ID NO:10) encoded by SEQ ID N0:9 is 750 amino acid
residues in length and is presented using the one-letter amino acid code in
Table SB. Psort
analysis predicts the NOVS protein of the invention to be localized in the
nucleus with a
certainty of 0.3000.
Table 5B. Encoded NOVS protein sequence (SEQ ID N0:10)
MLQHLNLFLAEQAISRRGQGSKAPGEICQGGLVLSPINLWVTDLLVFQDFHGAVMRALDDMDHEG
RDTLAREELRQGLSELPAIHDLHQGILEELEERLSNWESQQKVADVFLAREQGFDHHATHILQFD
RYLGLLSENCLHSPRLAAAVREFEQSVQGGSQTAKHRLLRWQRLFQYQVLLTDYLNNLCPDSAE
YDNTQGALSLISKVTDRANDSMEQGENLQKLVHIEHSVRGQGDLLQPGREFLKEGTLMKVTGKNR
RPRHLFLMNDVLLYTYPQKDGKYRLKNTLAVANMKALYHGEGEGGSTFLSMEVCSLLEPKAPPRS
LLEKGMGDWTGRYLSNMTVHLGLPGLGPEHDALQPSQRWVSRPVMEKVPYALKIETSESCLMLS
ARLQVRKSKVKALTDSVSAALGVRGISLFQCKKKQTQGQLMDQWSARKPSLAGDLFFAGGSGQCE
RCRLKGHLSENLIHAEMEAHARSSCAERDEWYGCLSRALPEDYKAQALAAFHHSVEIRERLGVSL
GERPPTLVPVTHVMMCMNCGCDFSLTLRRHHCHACGKQIVCRNCSRNKYPLKYLKDRMAKVCDGC
FGELKKRGRAVPGLMRVTERPVSMSFPLSSPRFSGSAFSSVFQSINPSTFKKQKKVPSALTEVAA
SGEGSAISGYLSRCKRGKRHWKKLWFVIKGKVLYTYMASEDKVALESMPLLGFTIAPEKEEGSSE
VGPIFHLYHKKTLFYSFKAEDTNSWIEAMEDASVL
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table SC.
Table SC. Patp results for NOVS
Smallest
Sum
eadingigh Prob
equences FrameScore P(N)
producing
High-scoring
Segment
Pairs:
>patp:AAB93568Human protein sequence SEQ ID +1 577 1.7e-95
N0:12972
>patp:AAY51248Rat actin-binding protein frabin+1 312 1.9e-41
>patp:AAU21630Novel human neoplastic disease +l 256 1.6e-38
polypeptide
>patp:AAU27818Human full-length polypeptide +1 300 2.6e-29
#143
>patp:ABG00573Novel human diagnostic protein +1 261 1.8e-26
#564
In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 443 of 754 bases (58%) identical
to a
gb:GENBANI~-ID:AB037783~acc:AB037783.1 mRNA from H~~zo Sapiens (mRNA for
KIAA1362 protein, partial cds). The full amino acid sequence of the protein of
the invention
was found to have 114 of 263 amino acid residues (43%) identical to, and 173
of 263 amino
acid residues (65%) similar to, the 699 amino acid residue ptnr:SPTREMBL-
ACC:Q9P2I5
protein from Ho»ao Sapiens (KIAA1362 PROTElI~.
NOVS also has homology to the proteins shown in the BLASTP data in Table SD.
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CA 02443770 2003-10-15
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Table 5D. BLAST
results for
NOVS
Gene Index/ Protein/ OrganismLength Identity PositivesExpect
Identifier (aa) (%) (%)
gi~8922921~ref~NPhypothetical 432 135/284 169/284 5e-57
0
_ protein FLJ11183 (47%) (58%)
60821.1~(NM_018351)
[Homo Sapiens]
gi~16716345~ref~NPethanol decreased431 131/284 171/284 2e-55
_ 4 [Mus musculus] (46%) (60%)
444302.1~(NM
053072
gi~7243105~dbj~BAA9KIAA1362 protein699 111/251 166/251 2e-54
2600.1~(AB037783)[Homo Sapiens] (44%) (65%)
gi~13648298~refIXPhypothetical 204 115/222 141/222 1e-49
- protein FLJ11183 (51%) (62%),
012133.21
(XM 012133) [Homo Sapiens]
gi(15426438~gb~AAH1Similar to 376 103/221 129/221 4e-40
3319.1~AAH13319 hypothetical (46%) (57%)
(BC013319) protein FLJ11183
[Homo Sapiens]
A multiple sequence alignment is given in Table 5E, with the NOVS protein
being
shown on line 1 in Table SE in a ClustalW analysis, and comparing the NOVS
protein with the
related protein sequences shown in Table SD. This BLASTP data is displayed
graphically in
the ClustalW in Table SE.
Table 5E. ClustalW Analysis of NOVS
1) > NOVS; SEQ ID NO:10
2) >giJ8922921J/ hypothetical protein FLJ11183 [Homo Sapiens]; SEQ >D NO:55
3) >giJ16716345J/ ethanol decreased 4 [Mus musculus]; SEQ ID NO:56
4) >giJ7243105J/ KIAA1362 protein [Homo Sapiens]; SEQ ID N0:57
5) >giJ13648298J/ hypothetical protein FLJ11183 [Homo Sapiens]; SEQ ID N0:58
6) >giJ 15426438J/ Similar to hypothetical protein FLJ11183 [Homo Sapiens];
SEQ m N0:59
10 20 30 40 50 60
..
NOVS 1 ____________________________________________________________ 1
gi~8922921~ 1 ____________________________________________________________ 1
gi~16716345~ 1 ____________________________________________________________ 1
giI72431051 1 GIESDWQGLLVGEEKRSKPIKAYSTENYSLESQKKRKKSRGQTSAANGLRAESLDDQMLS 60
gi~7.3648298~ 1 ____________________________________________________________ 1
gi~15426438~ 1 ____________________________________________________________ 1
70 80 90 100 110 120
..
NOV5 1 ---------------------------------MLQHLNLFLAEQAISRRGQGS------ 21
gi~89229211 1 ____________________________________________________________ 1
gi~16716345~ 1 ________________________________________,______________,____ 1
3O gi~7243105~ 61 RESSSQAPYKSVTSLCAPEYENIRHYEEIPEYENLPFIMATRKTQELEWQNSSSMEDADA
120
gi~13648298~ 1 ____________________________________________________________ 1
gi~15426438~ 1 ____________________________________________________________ 1
130 140 150 160 170 180
....~....J...
NOV5 21 -----KAPGETCQGGLVLSP---------------------------------------- 36
gi~8922921J 1 ____________________________________________________________ 1
g1(16716345( l __________________________________________________________
gi~72431051 121 NVYEVEEPYEAPDGQLQLGPRHQHSSSGASQEEQNDLGLGDLPSDEEEIINSSDEDDVSS
180
gi~13648298~ 1 ____________________________________________________________ 1
gi~15426438~ 1 ____________________________________________________________ 1
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CA 02443770 2003-10-15
WO 02/085922 PCT/US02/11634
190
200
210
220
230
240
J
J
J
J
NOV5 36 .... 64
....
....J.,..J....J....
....J....J....J....J....J....
-----------------------INLWVTDLLVFQDFHGAVMRAL-----DDMDHE----
gi J8922921J1 -___________________________________________________________
1
gi J16716345J1 -___________________________________________________________
1
gi J7243105J181ESSKGEPDPLEDKQDEDNGMKSKVHHIAKEIMSSEKVFVDVLKLLHIDFRDAVAHASRQL
240
giJ13648298J1 ____________________________________________________________
1
giJ15426438J1 ____________________________________________________________
1
250
260
270
280
290
300
J
J
J
....
NOV5 65 ....J 124
....J....J....J....
....J....J....J....J....J....
GRDTLAREELRQGLSELPAIHDLHQGILEELEERLSNWESQQKVADVFLAREQGFDHHAT
giJ8922921J1 ____________________________________________________________
1
giJ16716345J1 ____________________________________________________________
1
IS giJ7243105J241GKPVIEDRILNQILYYLPQLYELNRDLLKELEERMLHWTEQQRIADIFVKKGPYLKMYST
300
gi~13648298J1 ____________________________________________________________
1
giJ15426438J1 ____________________________________________________________
1
310
320
330
340
350
360
2.0 ....J....J....~....~....~....~.
.J.
.~.
~
.
...J
.
..J
.~
L
~
.
.
NOVS 125HILQFDRYLGLLSENCLHSPRLAAAVREFEQ "~GG,QTA
. r 184
.
.
..
.
._
.
~V
r
~;tFr
Q~T
giJ892292111 _____________________________ . . r'.r
r 30
.
giJ16716345J1 _____________________________ . v
r 30
~ v
giJ7243105J301YIKEFDKNIALLDEQCKKNPGFAAVVREFEi C ~
r 360
r
25 giJ13648298J1 -___________________________________________________________
1
giJ15426438J1 -_____________________________~
30
.
.
r.
.r
.
r
370
380
390
400
410
420
30 NOVS l85 244
giJ8922921J31 90
giJ16716345J31 90
giJ7243105J361 420
giJ13648298J1 ____________________________________________________________
1
35 gi 1542643831 ~I~ilr 90
J J -,
r
r
r
r
'
r
v
r
v
v
r
r
430
440
450
460
470
480
.J. . .J....J. ...J. .~ . .J. . ..J,.. ~J...'....v..W ....J....J
NOV5 245E . T G~LR' E' . r ~~.... I E T YHGEGEG 304
Y ~ A~ i
'HL. QD
40 giJ8922921J91 I ~.. r ~ ~,t,~ 141
~~_________
giJ16716345J91 T ~ r ~ ~ -------- 141
giJ7243105J421 I t' r ~ -------- 471
giJ13648298J1 ____________________________________________________________
1
giJ15426438J91 ~I 141
r
r
r
--------
45
490
500
510
520
530
540
J
....J....J....J
NOV5 305....J....J....J....J....J....J....J....J....
364
GSTFLSMEVCSLLEPKAPPRSLLEKGMGDVVTGRYLSNMTVHLGLPGLGPEHDALQPSQR
giJ8922921Jl41____________________________________________________________
141
50 giJ16716345J141___________________________________-____,___________________
141
giJ7243105J471________________________________________-__________________
- 471
giI13648298Jl ____________________________________________________________
1
giJ15426438J141_____________________________________________
141
____________
S5 550
560
570
580
590
600
J
..J.. .J....~....J ....J
NOV5 365W.S~ . ~S SCL~ ....J....J....J....J....J....
424
RLQVRKSKVKALTDSVSAALGVRGISLFQCKKK
KVPY
giJ8922921J141- ~ ._______________________,_________
167
~
giJ16716345J141- ~ ._________________________________
167
~
60 giJ7243105J471- r ._________________________________
497
r
giJ13648298J1 ____________________________________________________________
1
giJ15426438J141- 167
~
~
._________________________________
610
620
630
640
650
660
65 ....J....J....J....J....J....J....J....J....J....J..
.~.
.J
..
NOV5 425QTQGQLMDQWSARKPSLAGDLFFAGGSGQCERCRLKGHLSENLIHAEME
C r 484
giI8922921J167____________________________________________________
~T r 174
giJ16716345J167____________________________________________________
r 174
giJ7243105J497____________________________________________________
T r 504
70 giI13648298J1 ____________________________________________________________
1
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gi1154264381
167__,__________________________________________________~T~
174
670 690 700
680 710
720
.1. ._...~....~'i, ..~.E.P~ ~. 540
S NOV5 485..~....~....~ VF~IR'~RLG- L_
'G
. 1.
YGCL ..
' -
~
T.~P
DY
~ALAAFHH
gi1 89229211175 ~ ~ i ~ .. .~ 234
..
gi1 167163451175~ S ~ -~ ~ 233
gi1 7243105)505~ ' ~ ~ S ' ~ 564
'
gi1 1364829811 _________________________ _____,______
____.___________ 6
-
154264381175' ~ S 234
gi1 ~ "
~
730 780
740
750
760
770
..
NOV5 541 VP 600
89229211235 --
gi1 286
gi1 167163451234 -- ~ 285
gi1 72431051565 --
616
gi1 1364829817 --
58
gi1 154264381235 --
286
790 840
800
810
820
830
..,.~. .~....~. ..~. .~. .~...
NOV5 601 RVTERPSMSFPLS ~RFS ~F FQ PS 660
gi189229211 287 ~ - ~ ~ 341
gi1167163451 286 ~L ~ - ~ 340
gi172431051 617 ~
671
gi1136482981 59 t - ~ 113
gi1154264381 287 ~ - ~
341
850 860 870 880 890 900
' ..1....1.,....1.,.. ,1....1
I 'I Y KVY
NOV5 661 ~ S ~ C ERG ~ ~H '1KL ~ ~ ~ :APE E . SEVGP~ 720
gi ~ 89229211 342 ~ ? ~ v . ,.I ~ 3'.. . Q ~ IQ ESK--~. 399
gi1167163451 341 ~ ~ L ~ ~~ ~ ~ Q~~TL ESK--~ 398
gi172431051 672 v v CCK-_-V~_____________________'__ 699
gi 1 13648298 ~ 114 ~ ~ ~ v ~ Q~FIQV~ytl.7~ESK--'~ l71
gi1154264381 342 ~ ~~ R,E~------- -- ------------ 376
910 920 930
NOV5 721 ~ ~ I~KT. ~ 15..:,~:T.. 1 .S~ ~..~. ... 1w~.-
; ~A. 750
gi189229211 400 Q L ~ ~E~ .SAQ ~~F~G'.I'~ 432
gi 1167163451 399 Q~L~N~~ r, STQ D~F~G~ 431
gi172431051 699 -_ __________ ___,____ '_ __ 699
gi 1136482981 172 Q~L~mlM~ESAQIt~EiFGT~ 204
gi~15426438~ 376 _________________________________ 376
The presence of identifiable domains in the protein disclosed herein was
determined by
searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and
then
determining the Interpro number by crossing the domain match (or numbers)
using the
Interpro website (http:www.ebi.ac.uk/interpro~. Table SF lists the domain
description from
DOMAIN ailalysis results against NOVS.
Table SF Domain
Analysis
of NOVS
Model Region of Score (bits) E value
Homology
RhoGEF 33-215 -1.9 1.2e-05
FYVE Ring 525-591 55.6 6.4e-14
Finger
Plekstrin 657-748 49.3 8.0e-7
(PH)
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Consistent with other known members of the Nuclear Protein-like family of
proteins,
NOVS has, for example, an RhoGEF signature sequence and a FYVE Zinc Finger
signature
sequence, aw well as homology to other members of the Nuclear Protein-like
Protein Family.
NOVS nucleic acids, and the encoded polypeptides, according to the invention
are useful in a
variety of applications and contexts. For example, NOVS nucleic acids and
polypeptides can
be used to identify proteins that are members of the Nuclear Protein-like
Protein Family. The
NOVS nucleic acids and polypeptides can also be used to screen for molecules,
which inhibit
or enhance NOVS activity or function. Specifically, the nucleic acids and
polypeptides
according to the invention may be used as targets for the identification of
small molecules that
modulate or inhibit, e.g., cellular activation, cellular replication, and
signal transduction.
These molecules can be used to treat, e.g., Cardiovascular diseases,
Cardiomyopathy,
Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis,
Atrial septal defect
(ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary
stenosis, Subaortic
stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis,
Scleroderma,
Obesity, Transplantation, Diabetes,Von Hippel-Lindau (VHL) syndrome ,
Pancreatitis,
Obesity, Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke,
Tuberous
sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral
palsy, Epilepsy,
Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia,
Leukodystrophies,
Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection as well as
other diseases,
disorders and conditions.
In addition, various NOVS nucleic acids and polypeptides according to the
invention
are useful, ihteY alia, as novel members of the protein families according to
the presence of
domains and sequence relatedness to previously described proteins. For
example, the NOVS
nucleic acids and their encoded polypeptides include structural motifs that
are characteristic of
proteins belonging to the Nuclear Protein-like Protein Family.
The NOVS nucleic acids and polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and diagnostic
applications in the
mediation of cardiac and nerve physiology. As such, the NOVS nucleic acids and
polypeptides, antibodies and related compounds according to the invention may
be used to
treat cardiovascular and nervous system disorders, e.g., Cardiovascular
diseases,
Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects,
Aortic stenosis,
Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus
arteriosus , Pulmonary
stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases,
Tuberous
sclerosis, Scleroderma, Obesity, Transplantation, Diabetes,Von Hippel-Lindau
(VHL)
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syndrome , Pancreatitis, Obesity, Von Hippel-Lindau (VHL) syndrome ,
Alzheimer's disease,
Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's
disease,
Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-
telangiectasia,
Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain,
Neuroprotection as well as
other diseases, disorders and conditions.
The NOVS nucleic acids and polypeptides are useful for detecting specific cell
types.
For example, expression analysis has demonstrated that a NOVS nucleic acid is
expressed in
Brown adipose, Vein, Umbilical Vein, Adrenal Gland/Suprarenal gland, Gall
Bladder, Small
Intestine, Colon, Lymphoid tissue, Spleen, Lymph node, Thymus, Brain, Temporal
Lobe,
Basal Ganglia/Cerebral nuclei, Substantia Nigra, Spinal Chord, Cervix, Ovary,
Uterus, Testis,
Lung, Lung Pleura, Larynx, Urinary Bladder, Kidney.
Additional utilities for NOVS nucleic acids and polypeptides according to the
invention are disclosed herein.
NOV6
A NOV6 polypeptide has been identified as a Secretory Protein-like protein
(also
referred to as CG949~9-O1). The disclosed novel NOV6 nucleic acid (SEQ m NO:l
1) of 2372
nucleotides is shown in Table 6A. The novel NOV6 nucleic acid sequences maps
to the
chromosome 17.
An ORF begins with an ATG initiation codon at nucleotides 99-101 and ends with
a
TAA codon at nucleotides 1710-1712. A putative untranslated region and/or
downstream
from the termination codon is underlined in Table 6A, and the start and stop
codons are in
bold letters.
Table 6A. NOV6 Nucleotide Sequence (SEQ ID NO:11)
CCGGCAAGGATGACGCCTCCGGAGGCCCTGGCCTCACTCCCACCTGGGCGCTAGGAGCCATCCCGGG
GCTCCAGCCAGGAGCCCTGCTGCCCAGGGGCATGGCCAAACCTTTCTTCCGACTCCAGAAGTTTCTC
CGCCGAACACAGTTCCTGCTGTTCTTCCTCACGGCTGCCTACCTGATGACCGGCAGCCTGCTGCTGC
TGCAGCGGGTCCGCGTGGCTCTCCCACAGGGCCCCCGGGCACCCGGCCCCCTGCAGACCTTGCCAGT
GGCCGCCGTGGCGCTGGGCGTGGGCTTGCTGGACAGCAGAGCCCTGCACGACCCTCGAGTCAGCCCA
GAGCTGCTGCTGGGTGTGGACATGCTGCAGAGCCCCCTGACCCGGCCCCGGCCCGGCCCCCGCTGGC
TCCGGAGCCGCAACTCGGAGCTGCGTCAGTTGCGTCGCCGCTGGTTCCACCACTTCATGAGTNGACT
CCCAGGGACCGCCCGCCCTGGGCCCCGAGGCTGCCAGGCCCGCCATCCACAGCCGAGGTCCTATGTC
TACGCCGGCTTGGAGGCCGGGGCGGAGTGTTACTGCGGGAACCGGCTGCCAGCGGTGAGCGTGGGGC
TGGAAGAGTGTAACCATGAGTGCAAAGGCGAGAAGGGCTCTGTGTGCGGGGCTGTGGACCGGCTCTC
CGTGTACCGTGTGGACGAGCTGCAGCCGGGCTCCAGGAAGCGGCGGACCGCCACCTACCGCGGATGC
TTCCGACTGCCAGAGAACATCACACATGCCTTCCCCAGCTCCCTGATACAGGCCAATGTGACCGTGG
GGACTTGCTCGGGCTTTTGTTCCCAGAAAGAGTTCCCCTTGGCCATTCTCAGGGGCTGGGAATGCTA
CTGTGCTTACCCTACCCCCCGGTTCAACCTGCGGGATGCCATGGACAGCTCAGTATGTGGCCAGGAC
CCTGAGGCACAGAGGCTGGCAGAATACTGTGAGGTCTACCAGACACCTGTGCAAGACACTCGTTGTA
CAGACAGGAGGTTCCTGCCTAACAAATCCAAAGTGTTTGTGGCTTTGTCAAGCTTCCCAGGAGCCGG
GAACACGTGGGCACGGCACCTCATTGAGCATGCCACTGGCTTCTATACAGGGAGCTACTACTTTGAT
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GGAACCCTCTACAACAAAGGGTTCAAGGGCGAAAAGGACCACTGGCGGAGCCGACGCACCATCTGTG
TCAAAACCCACGAGAGTGGCAGGAGGGAGATTGAGATGTTTGATTCAGCCATCCTGCTAATCCGGAA
CCCATACAGGTCCCTGGTGGCAGAATTCAACAGAAAATGTGCCGGGCACCTGGGATATGCAGCTGAC
CGCAACTGGAAGAGCAAAGAGTGGCCGGACTTTGTCAACAGCTACGCCTCGTGGTGGTCCTCGCACG
TCCTGGACTGGCTCAAGTACGGGAAGCGGCTGCTGGTGGTGCACTACGAGGAGCTGCGGCGCAGCCT
GGTGCCCACGTTACGGGAGATGGTGGCCTTCCTCAACGTGTCTGTGAGCGAGGAGCGGCTGCTCTGC
GTGGAGAACAACAAGGAGGGCAGCTTCCGGCGGCGCGGCCGGCGCTCCCACGACCCTGAGCCCTTCA
CCCCGGAGATGAAAGACTTGATCAATGGCTACATCCGGACGGTGGACCAAGCCCTGCGTGACCACAA
CTGGACGGGGCTGCCCAGGGAGTATGTGCCCAGATGATAGGCCTGGCCCACGCCGCCGCCCCCGCTG
AGTGACGCAATCGCACCACGGGGCTGCGCTCCCCACTCTGATGCTCAGGCCCGTGGCCTCACTGGGA
CGAACGGTGGGTGGGGGGCTCACCCTGGTGCTGCCTCCCGCACAAGGAGACCTGGACACAACAGACA
CACATCACAAGGCGAACACAAATGGACACACATACCTGGCCACGAACCCACACCTCCTCAGACACTC
AGACACCACTCCAGGCTCATAGCCCCGTCTTGATGCAGAGAAGCCACCCACGTGGGGTGTGCCAGGC
ACCCCCAGCTACAAATGCAGCCACGCACAGACGTAACACACAGGTGCCAGGCCGTGTGCTCCTGGAG
GCTGGCTGGCTGTCTCTCTCACACAGATACACGTGCGCTCCCTGGGATCCGGGAGGCCCTGGGCTTC
CTGTGTGTAGCCCTGGCATAGACTTGCTCGTCAGGGTGTTTGACTCTGGGATGCTGGGCCGGGCAGA
CATTTATGCTCTGAGCAGCAAGGACCATTGGGATGGAGGTGGGCACAAAGACTGCTGCTTCCAGGGT
GTGCGGCCCTGGCCGTGTGTCTGACATCCCATAAATGTGTGTGTGGTGTGACTACGGGCACCACAAA
CTCCGC
The NOV6 protein (SEQ m NO: I2) encoded by SEQ m NO:I 1 is 537 amino acid
residues in length and is presented using the one-letter amino acid code in
Table 6B. Psort
analysis predicts the NOV6 protein of the invention to be localized outside
the cell with a
certainty of 0.6997.
Table 6B. Encoded NOV6 protein sequence (SEQ ID N0:12)
MAKPFFRLQKFLRRTQFLLFFLTAAYLMTGSLLLLQRVRVALPQGPRAPGPLQTLPVAAVALGVG
LLDSRALHDPRVSPELLLGVDMLQSPLTRPRPGPRWLRSRNSELRQLRRRWFHHFMSXLPGTARP
GPRGCQARHPQPRSYVYAGLEAGAECYCGNRLPAVSVGLEECNHECKGEKGSVCGAVDRLSVYRV
DELQPGSRKRRTATYRGCFRLPENITHAFPSSLIQANVTVGTCSGFCSQKEFPLAILRGWECYCA
YPTPRFNLRDAMDSSVCGQDPEAQRLAEYCEVYQTPVQDTRCTDRRFLPNKSKVFVALSSFPGAG
NTWARHLIEHATGFYTGSYYFDGTLYNKGFKGEKDHWRSRRTICVKTHESGRREIEMFDSAILLI
RNPYRSLVAEFNRKCAGHLGYAADRNWKSKEWPDFVNSYASWWSSHVLDWLKYGKRLLVVHYEEL
RRSLVPTLREMVAFLNVSVSEERLLCVENNKEGSFRRRGRRSHDPEPFTPEMKDLINGYIRTVDQ
ALRDHNWTGLPREYVPR
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 6C.
Table 6C. Patp results for NOV6
Smallest
Sum
eading igh Prob
Sequences Score P(N)
producing
High-scoring
Segment
Pairs: Frame
>patp:ABB15485Human nervous system related polypeptide+1 92 0.0036
>patp:AAU50001Propionibacterium acnes immunogenic+1 82 0.042
protein
>patp:AAU50001Propionibacterium acnes immunogenic+1 82 0.042
protein
>patp:AAU18674Renal and cardiovascular-associated+1 79 0.085
protein
>patp:AAB95341Human protein sequence SEQ ID +1 99 0.17
N0:17621
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In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 2188 of 2189 bases (99%) identical
to a
gb:GENBANK-ID:AK000243~acc:AK000243.1 mRNA from Horrao sapiehs (cDNA
FLJ20236 fis, clone COLF5810, highly similar to ABOl 1095 Homo Sapiens mRNA
for
KIAA.0523 protein). The full amino acid sequence of the protein of the
invention was found to
have 395 of 395 amino acid residues (100%) identical to, and 395 of 395 amino
acid residues
(100%) similar to, the 468 amino acid residue ptnr:SPTREMBL-ACC:060276 protein
from
Homo Sapiens (KIAA0523 PROTEIN)(Fig. 3B).
NOV6 also has homology to the proteins shown in the BLASTP data in Table 6D.
Table 6D. BLAST
results for
NOV6
Gene Index/ Protein/ OrganismLength Identity PositivesExpect
Identifier (aa) (%) (%)
gi~14602977~gb~AAHOSimilar to 575 523/575 524/575 0.0
9975.1IAAH09975 KIAA0789 gene (90%) (90%)
(BC009975) product
[Homo Sapiens]
gi~3043570~dbj~BAA2KIAA0523 protein468 417/468 417/468 0.0
5449.1 (AB011095)[Homo Sapiens] (89%) (89%)
gi~18489296~ref~XPCG9164 317 76/206 15/206 3e-28
_ [Drosophila (36%) (54%)
082751.1I(XM
082751
melanogaster]
gi~16944644~emb~CADhypothetical 2117 43/131 62/131 5e-08
11404.11(AL513445)protein (32%) (46%)
[Neurospora
crassa]
gi~11359357~pir~~T4beta-1,3 1032 40/128 55/128 2e-05
3257 exoglucanase (31%) (42%)
(EC
3.2.1.-)
precursor -
fungus
(Trichoderma
harzianum)
A multiple sequence alignment is given in Table 6E, with the NOV6 protein
being
shown on line 1 in Table 6E in a ClustalW analysis, and comparing the NOV6
protein with the
related protein sequences shown in Table 6D. This BLASTP data is displayed
graphically in
the ClustalW in Table 6E.
Table 6E. ClustalW Analysis of NOV6
1) > NOV6; SEQ ID NO:12
2) >gi~14602977~/ Similar to KIAA0789 gene product [Homo sapiens]; SEQ ID
N0:60
3) >gi~3043570~/ KIAA0523 protein [Homo Sapiens]; SEQ ID N0:61
4) >gi~18489296~/ CG9164 [Drosoplaila melanogaster]; SEQ 117 N0:62
5) >gi~ 16944644/ hypothetical protein [Neurospora cf assa]; SEQ ID N0:63
6) >gi~ 11359357/ beta-1,3 exoglucanase (EC 3.2.1.-) precursor - fungus
[Trichodertna harzianum];
SEQ 1D N0:64
850 860 870 880 890 900
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p....
NOV6 1 ------------------------MAKPFFR2QKFItRRTQFLLFFLTAAYt'C.yMT~-=--SL 32
gi1146029771 1 ------------------------KPFFRIiQKF~RRTQFLLFFLTAAY~.,MT - --SL
32
gi130435701 1 ________________________-___________________________________ 1
gi 18489296 1 ____________________________________________________________ 1
gi ~ 16944644 I 841
GTSPGLTTTEVTIRFTNKGGSDLNLI~DKSKPP~'IGSV~iGAQNPSSDLFEGMV2'KP KSESAT 900
gi1113593571 235 TFNGGLIG--------------AA~GNQQYT~RNL~TFNN--CAQPLSAASxaGSB----
FT 274
910 920 930 940 950 960
....~....~....~....~.. .~....~.. .,1....1....1....1..~.1....1
NOV6 33 LLLQRVRV~PQGPRAPGPLQT~.iiPVAAVAL~G~LDSR-ALHDPRVSPE~LLGVDMLQSP 91
gi~14602977~ 33 LLLQRVRV PQGPRAPGPLQTPVAAVAL G~LDSR-ALHDPRVSPE LLGVDMLQSP 91
gi130435701 1 ____________________________________________________________ 1
gi1184892961 1 ____________________________________________________________ 1
IS gi1169446441 901 LFFTPGAA~ADPIVYSGAWTLTFG~Fi7NFIGTLRATKVGPT~PDGSARFKYL 960
gi~11359357~ 275 RAISINNC.','!~lGIDMTAAESITLkr~. SSISGTP G~KTSFRRNQSPATSNS
IVENLSLNNV 334
970 980 990 1000 1010 1020
NOV6 92 LTRPRPGPRWLRSRNS~LR---------------------------------------X7112
giI14602977~ 92 LTRPRPGPRWLRSRNSLR---------------------------------------',#~'
112
gi130435701 1 ________________LR-______________________________________~5
gi1184892961 1 ___________________________________________________________- 1
gi116944644~ 961 GCYRDSSANRLETTQAFPSDNDNGKCQQYAITNKAAFAGTQYTYECWVGRSIPPASLiF
1020
gi1113593571 335 PVAIQSSSGSTILAGGTTT----------------------------------------
354
1030 1040 1050 1060 1070 1080
NOV6 113 RRRWF ~'i~X------------------------------L~GT .~G--PR------- 133
3o gi~146029771 113 RRRWF~~'MT7SQGP--===____ _____= PA'I~ ~E IHRG--=T~I 146
gi 1 30435701 6 RRRWFE., FMSDSQGP-- PAIi ~E ~IHRG-- Trr~~I 39
gi1184892961 1 __ _____________________________________ _________ 1
gi~16944644~ 1021 DDYLCTj~IzCPGDKSQFCGGVGSYMMMWYDTTGYFPENGT'~FRP~ASIC~VVGDW~
1080
gi~11359357~ 355 AAWGQG~QYj~"PNGPTTFQG---------------- SItT SRPSLLGSN T 392
35 _.
1090 1100 1110 1120 1130 1140
.~....~....~....~....~....~....~. _ l~ ~I.
NOV6 134 ~QA--_______R_______________HpQp__ ~~y . ' ~ PA~~ 167
gi1146029771 147 FSDDGHERTLKGAVFYDLRKMTV-SHCQDAC~~'1,E Y~~' ~ PA205
gi~3043570~ 40 FSDDGHERTLKGAVFYDLRKMTV-SHCQDACiIE YV~ ' PA 98
gi~184892961 1 ________________________________~L~G~IR~'g S~TIII I -____'G__
21
gi1169446441 1081 ~RTDNSASPATRALNDRIVGQSSTNTIESCAQ~iCAG~S'F r .. PG~T 1140
gi~11359357~ 393 RSKPQYETLPVSSFRSVRSAGATGN-AVTDDTA~L~ATAC QI FDAG~ R'~~?S 451
45 1150 1160 1170 1180 1190 1200
.1. .1. .1. .1. .1.
NOV6 168 .E - E ~ 'E'EKGS~, VF.W . Z7DELQ~ _____________________ 202
gi~14602977~ 206 -- !E KGSi E L VDELQ~ _______________________ 240
gi~3043570! 99 ~ -- _E EKGS~Z~_ I VDELQ~ -____________________ 333
O gy 184892961 21 _ __________ __ __ __ V~~LSMNNI~
gi~16944644~ 1141 VA~KT~YV~ 1~PTEGSG~'yI~TCQKGTVI PSTGVSSSSGTASGTASATAS 1200
gi111359357i 452 T~S------IPP~ -AK~EYP~2IMSSGSFFND----------------------Q 479
1210 1220 1230 1240 1250 1260
. .1...~1. ~~~ .1...y y .~ . ~~. ~ ~~.. y
, c - v _ r v-
NOV6 203 R j' "I'~~TY~ C~RL~. . ----- !i! ' ~ ~5~~~~ ~ - '~!':2'C~GT S. -Q ~E
246
gi ~ 146029771 241 R ~TiiITY~ C RL~ _=---- TH~ ~ 5~~~ - --'i~T~TGT ;, -Q ~E
284
gi ~ 304357Q ~ 134 R WiTY~ C RL E ---- ~'H' ~ S~Ir~~ -- --,~T"~"sIGT ,~ -Q ~E
177
gi1184892961 34 HP4'PRIE~__ ________ -~ . __-_____ _ ___._ _p_ 53
gi 1 169446441 1201 STSSiIA~TPGN~Q'S'~GQYSSLGCY',~.",Di RSL~GKNTQSNVMSIiDD~T
!GY~yIY 1260
gi~11359357~ 480 SNP~PVVQ TPGQTG-------- QVE~SDM~TVSTQG- -----TQAGAVLIEWNL 520
1270 1280 1290 1300 1310 1320
.1. .1....1....1.. .1.~...~...1.~~ .1. ..1....1....1....1....1
NOV6 247 .PLAIL~~ E YCA ~T'P~tFNL~'~ n SS~ Gy P________________________ 282
gi114602977~ 285 PLAIL E YCAY~'T~P~R,FN~~W ySa GyP3~"'s -______-______________
320
gi~30435701 178 PLAIL E YCAY~'~P~FN~rw W9~G~',~,?rP~'." ______________________
213
gi1184892961 53 ____gp________ ~~gTI~ CR~LKYI ~- ________________________ 75
gi116944644~ 1261 ~GTEYSAECF~GNDLLNGAAP1~T~GRC~G QQQICGGSNGLSMYQLNPNGTSSSVT
1320
gi1113593571 521 ATSGTPSe~vIWDVHTRIGGFKGSNLQ'~'~1QCPVTAS~----------------------
---- 554
4~ _:
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1330 1340 1350 1360 1370 1380
..J.. .~....~.,...~ ..L ...~....~....~....~....~
NOV6 282 _______ ~QR(~..[~~ E~Q P~'ST;QD RC'DR~~2.F ~ K-___________________
311
gi~14602977~ 320 ------- Q ~ E EVY'Q PVQD RC DR~F ~ K-------------------- 349
gi~30435701 213 ------- fi~Q E EV~Q P~QD RC~DR~2.~' ~ K--------------------
242
gi 18489296 75 ___________ZPIKSD~'Y ;'PSDVSAAL~S ~ _____________________ 102
gi~16944644) 1321 ASGSATQSATA~GTASGTASSSS ' T TS~AVPT ~ VSVKCPDNNNGTYLSLNGKTF
1380
gi I 11359357 ~ 554 ------- TTt'V.NTA~IGAYMS ,:, I~ASASNL~'~'NIEI~-------------
-------- 582
1390 1400 1410 1420 1430 1440
NOV6 311 _____________________________ ~~ . ~ _______________ 325
w
gi~14602977~ 349 ______________________________. ~r _______________ 363
w
15 gi~30435701 242 ______________________________r~' S _______________ 256
gi~184892961 102 __-___________________________ pLT. ~ ________________ 114
gi~16944644~ 1381 LLECFTDHEGGDLALAYVDSYALCAEKCSTTDI,rC~ F~ GTGIQAPCYMKKSVGRGF
1440
gi~113593571 582 ______________________________~T~DHDIDDS--__________N___ 597
1450 1460 7.470 1480 1490 1500
....
NOV6 325 --------------------- F~ K 359
v
gi~14602977~ 363 _____________________ y F~ K IC 397
v
gi~30435701 256 --------------------- F~ K K 290
gi118489296~ 114 _____________________ I ~Y LKT P 4 148
gi~16944644~ 1441 SAAATMTSAASAMGSNSWGPS TGS TGSATDSTT 1500
gi~11359357~ 598 ------------------FWF ~..'AVEHH QYQ~ANT,638
1510 1520 1530 1540 1550 1560
NOV6 360 419
gi~146029771 398 457
gi~3043570~ 291 350
gi1184892961 148 206
gi~16944644~ 1501 1560
gi~11359357~ 639 697
1570 1580 1590 1600 1610 1620
4o NOV6 420 ----I----I----I 458
gi~14602977~ 458 --------------- 496
gi~3043570~ 351 _,_____________ 389
gi~18489296~ 207 _______________ 249
g3~16944644~ 1561 IAPSENTTPSASVAP 1619
gi~11359357~ 697 --------------- 729
1630 1640 1650 1660 1670 1680
NOV6 458 --------L PT. E1~3't~~ 5~- ERW ~ I$G---------------- 494
giI14602977f 496 -_______L PT ~ES ER,~,~ 'j ' ~G-_______________ 532
gi ~ 3043570 ~ 389 --------L PT ~E~'1'C7~ ~ g~,~,, yG---------------- 425
gi~18489296~ 249 _-______TERE S ~D QFPQLt: IMy" I ~ ~_________________ 284
gi~16944644~ 1620 SNSWPSDS~APSAS~I~iPSA~~ yS~ASV~PSTSIAHS SESVAPAESIAPSASVSS
1679
gi~11359357~ 729 --------N~FD~EGTTN~yNLGTVG,i~VI~IT~ LATS------S--------- 766
1690 1700 1710 1720 1730 1740
NOV6 494 --_________________________-_________-______________________ 494
gi~14602977~ 532 _-_________________________-________________________________
532
gi~3043570~ 425 ____________________________________________________________
425
gi118489296~ 284 ____________________________________________________________
284
gi~16944644~ 1680 GSNTGVAPTNSASVTPTNSASVATTISVSVAPTASDAPTTSITLSVAPGSSSSTTAPAW
1739
gi~11359357~ 766 ____________________________________________________________
766
1750 1760 1770 1780 1790 1800
NOV6 494 _______________________, ~~~~ PE.: L~~GYIv Q RDHNW G 529
gi~146029771 532 -_____________-_________ ~~E~F PE"IDL~~TGYI~ Q~ RDHNW G 567
gi~3043570~ 425 _________________________ . ~.E. PE ~CDI~Ii~GYI~ ' Q; RDHNW G
460
gi~18489296~ 284 -------------------------;_~LLSF~1~ ES ~AE~QNR ~I YGL GRQEP--
317
47 _ _
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gi~169446441 1740 STTLSAPTVTSVPPAAGTSTTTAAAVT~TTTTTTATSTT TPVLFTTSTT~T 1799
gi~11359357~ 766 -------------------------SNVFADVIALFLASGSGGV~PPP'S~STTKAQT
801
1810 1820 1830 1840 1850 1860
NOV6 530 LPREY~7'PR-___________,____-__________________________________ 537
giI14602977~ 568 LPRE'IV'PR-___________,____-
__________________________________ 575
gi13043570~ 461 LPREYPR-___________,___________________________-___________
468
gi118489296~ 317 ________________________________________________-___________
317
1O gi~16944644~ 1800
TAAAVATTAPTQATTTIATAATTSTTNIASASPTIPAVVNWDYQGCASDSNTAAPTARAL 1859
gi~11359357~ 802 TFSTI';TSSPPKQTG-------------------------WNFLGCYSDNVNGRTLANQV
836
The NOV6 Clustal W alignment shown in Table 6E was modified to begin at amino
residue 841 and end at amino acid residue 1860. The data in Table 6E includes
all of the
regions overlapping with the NOV6 protein sequences.
The presence of identifiable domains in the protein disclosed herein was
determined by
searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and
then
determining the Interpro number by crossing the domain match (or numbers)
using the
Interpro website (http:www.ebi.ac.uk/interpro~. Table 6F lists the domain
description from
DOMAIN analysis results against NOV6.
Table 6F Domain
Analysis of
NOV6
Model Region of Score (bits) E value
Homology
Disintegrin 151-159 5.8 0.73
WSC domain 120-186 36.8 5e-07
Peptidase family346-354 -0.2 8.4
M1
Sulfotransferase288-528 -143.3 0.14
proteins
Consistent with other known members of the Secretory Protein-like family of
proteins,
NOV6 has, for example, has homology to other members of the Secretory Protein-
like Protein
Family. NOV6 nucleic acids, and the encoded polypeptides, according to the
invention are
useful in a variety of applications and contexts. For example, NOV6 nucleic
acids and
polypeptides can be used to identify proteins that are members of the
Secretory Protein-like
Protein Family. The NOV6 nucleic acids and polypeptides can also be used to
screen for
molecules, which inhibit or enhance NOV6 activity or function. Specifically,
the nucleic acids
and polypeptides according to the invention may be used as targets for the
identification of
small molecules that modulate or inhibit, e.g., cellular activation and signal
transduction.
These molecules can be used to treat, e.g., Cardiovascular diseases,
Cardiomyopathy,
Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis,
Atrial septal defect
(ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus as well as other
diseases,
disorders and conditions.
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In addition, various NOV6 nucleic acids and polypeptides according to the
invention
are useful, intes~ alia, as novel members of the protein families according to
the presence of
domains and sequence relatedness to previously described proteins. For
example, the NOV6
nucleic acids and their encoded polypeptides include structural motifs that
are characteristic of
proteins belonging to the Secretory Protein-like Protein Family.
The NOV6 nucleic acids and polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and diagnostic
applications in the
mediation of cardiac physiology. As such, the NOV6 nucleic acids and
polypeptides,
antibodies and related compounds according to the invention may be used to
treat cardiac and
vascular system disorders, e.g., Cardiovascular diseases, Cardiomyopathy,
Atherosclerosis,
Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect
(ASD),
Atrioventricular (A-V) canal defect, Ductus arteriosus as well as other
diseases, disorders and
conditions
The NOV6 nucleic acids and polypeptides are useful for detecting specific cell
types.
For example, expression analysis has demonstrated that a NOV6 nucleic acid is
expressed in
Aorta.
Additional utilities for NOV6 nucleic acids and polypeptides according to the
invention are disclosed herein.
NOV7
A NOV7 polypeptide has been identified as a Transmission Blocking Target
Antigen
5230 Precursor-like protein (also referred to as CG9497~-O1). The disclosed
novel NOV7
nucleic acid (SEQ )D N0:13) of 1629 nucleotides is shown in Table 7A. The
novel NOV7
nucleic acid sequences maps to the chromosome 1.
An ORF begins with an ATG initiation codon at nucleotides 1-3 and ends with a
TGA
codon at nucleotides 1627-1629. A putative untranslated region and/or
downstream from the
termination codon is underlined in Table 7A, and the start and stop codons are
in bold letters.
Table 7A. NOV7 Nucleotide Sequence (SEQ ID N0:13)
ATGGCGGTGCCCGGCGAGGCGGAGGAGGAGGCGACAGTTTACCTGGTAGTGAGCGGTATCCCCTCCG
TGTTGCGCTCGGCCCATTTACGGAGCTATTTTAGCCAGTTCCGAGAAGAGCGCGGCGGTGGCTTCCT
CTGTTTCCACTACCGGCATCGGCCTGAGCGGGCCCCTCCGCAGGCCGCTCCTAACTCTGCCCTAATT
CCTACCGACCCAGCCGCTGAGGGCCAGCTTCTCTCTCAGACTTCGGCCACCGATGTCCGGCCTCTCT
CCACTCGAGACTCTACTCCAATCCAGACCCGCACCTGCTGCTGCGTCATCTCGGTAAGGGGGTTGGC
TCAAGCTCAGAGGCTTATTCGCATGTACTCGGGCCGCCGGTGGCTGGATTCTCACGGGACTTGGCTA
CCGGGTCGCTGTCTCATCCGCAGACTTCGGCTACCTACGGAGGCATCAGGTCTGGGCTCCTTTCCCT
TCAAGACCCGGAAGGAACTGCAGAGTTGGAAGGCAGAGAATGAAGCCTTCACCCTGGCTGACCTGAA
GCAACTGCCGGAGCTGAACCCACCAGTGCTGATGCCCAGAGGGAATGTGGGGACTCCCCTGCGGGTC
TTTTTGGAGTTGATCCGGGCCTGCCGCCTACCCCCTCGGATCATCACCCAGCTGCAGCTCCAGTTCC
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CCAAGACAGGTTCCTCCCGGCGCTACGGCAATGTGCCTTTTGAGTATGAGGACTCAGAGACTGTGGA
GCAGGAAGAGCTTGTGTATACAGCAGAGGGTGAAGAAATACCCCAAGGAACCTACCTGGCAGATATA
CCAGCCAGCCCCTGTGGAGAGCCTGAGGAAGAAGTGGGGAAGGAAGAGGAAGAAGAGTCTCACTCAG
ATGAGCTGTTCGGGTGTGCTGTGGTCATCCTCCCTGCGCACCTACAGCCGCAGACCGCCGGTGGGGG
GCGGGGGATGCCGGGCTGCCGCATCAGCGCCTGCGGCCCGGGGGCCCAGGAGGGGACGGCAGAGCAG
AGGTCGCCGCCGCCGCCCTGGGATCCCATGCCGTCCTCTCAGCCCCCGCCCCCAACTCCGACCTTGA
CTCCTACCCCGACCCCGGGTCAGTCCCCGCCGCTGCCGGACGCAGCTGGGGCTTCAGCAGGCGCGGC
CGAGGACCAGGAGCTGCAGCGCTGGCGCCAGGGCGCTAGCGGGATCGCGGGGCTCGCCGGCCCCGGA
GGGGGCTCTGGCGCGGCTGCGGGGGCGGGGGGCCGCGCGCTGGAGCTGGCCGAAGCACGGCGGCGGC
TGCTGGAGGTGGAGGGCCGCCGGCGCCTGGTGTCGGAGCTGGAGAGCCGCGTGCTGCAGCTGCACCG
CGTTTTCTTGGCGGCCGAGCTGCGCCTGGCGCACCGCGCGGAGAGCCTGAGCCGCCTGAGCGGCGGC
GTGGCGCAGGCCGAGCTCTACCTGGCGGCTCACGGGTCGCGCCTCAAGAAGGGCCCGCGCCGCGGCC
GCCGCGGCCGACCCCCCGCGCTGCTGGCCTCGGCGCTGGGCCTGGGCGGCTGCGTGCCCTGGGGTGC
CGGGCGACTGCGGCGCGGCCACGGCCCCGAGCCCGACTCGCCCTTCCGCCGCAGCCCGCCCCGCGGC
CCCGCCTCCCCGCAGCGCTGA
The NOV7 protein (SEQ m N0:14) encoded by SEQ m N0:13 is 542 amino acid
residues in length and is presented using the one-letter amino acid code in
Table 7B. Psort
analysis predicts the NOV7 protein of the invention to be localized in the
cytoplasm with a
certainty of 0.4500.
Table 7S. Encoded NOV7 protein sequence (SEQ ID N0:14)
MAVPGEAEEEATVYLVVSGIPSVLRSAHLRSYFSQFREERGGGFLCFHYRHRPERAPPQAAPNSA
LIPTDPAAEGQLLSQTSATDVRPLSTRDSTPIQTRTCCCVISVRGLAQAQRLIRMYSGRRWLDSH
GTWLPGRCLIRRLRLPTEASGLGSFPFKTRKELQSWKAENEAFTLADLKQLPELNPPVLMPRGNV
GTPLRVFLELIRACRLPPRIITQLQLQFPKTGSSRRYGNVPFEYEDSETVEQEELVYTAEGEEIP
QGTYLADIPASPCGEPEEEVGKEEEEESHSDELFGCAVVILPAHLQPQTAGGGRGMPGCRISACG
PGAQEGTAEQRSPPPPWDPMPSSQPPPPTPTLTPTPTPGQSPPLPDAAGASAGAAEDQELQRWRQ
GASGIAGLAGPGGGSGAAAGAGGRALELAEARRRLLEVEGRRRLVSELESRVLQLHRVFLAAELR
LAHRAESLSRLSGGVAQAELYLAAHGSRLKKGPRRGRRGRPPALLASALGLGGCVPWGAGRLRRG
HGPEPDSPFRRSPPRGPASPQR
A search against the Patp database, a proprietary database that'contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 7C.
Table 7C. Patp results for
NOV7
Smallest
Sum
eadingigh Prob
equences FrameScore P(N)
producing
High-scoring
Segment
Pairs:
>patp:AAU33166Novelhuman secreted protein #3657+1 1533 5.8e-157
>patp:AAE04880Humanprotease protein-7 (PRTS-7) +1 1533 5.8e-l57
>patp:AAB94023Humanprotein sequence SEQ ID N0:14157+1 1519 1.8e-155
>patp:AAU33124Novelhuman secreted protein #3615+1 390 7.7e-36
>patp:AAG02700Humansecreted protein, SEQ ID +1 268 3.5e-22
NO: 6781
In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 874 of 876 bases (99%) identical
to a
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gb:GENBANK-ID:AK022517~acc:AK022517.1 mRNA from Homo Sapiens (cDNA
FLJ12455 fis, clone NT2RM1000563, weakly similar to TRANSMISSION-BLOCKING
TARGET ANTIGEN 5230 PRECURSOR). The full amino acid sequence of the protein of
the
invention was found to have 290 of 292 amino acid residues (99%) identical to,
and 290 of
292 amino acid residues (99%) similar to, the 525 amino acid residue pW
r:SPTREMBL-
ACC:Q9H9Z3 protein from Homo Sapiens (CDNA FLJ12455 FIS, CLONE NT2RM1000563,
WEAKLY SIMILAR TO TRANSMISSION- BLOCKING TARGET ANTIGEN 5230
PRECURSOR).
NOV7 also has homology to the proteins shown in the BLASTP data in Table 7D~
Table 7D. BLAST
results for
NOV7
Gene Index/ Protein/ OrganismLength Identity PositivesExpect
Identifier (aa) (~) (%)
gi~18545154~ref~XPhypothetical 525 274/274 274/274 e-147
_ protein FLJ12455 (100%) (100%)
084046.1~(XM_084046
[Homo Sapiens]
gi~11545793IrefINPhypothetical 525 272/274 272/274 e-145
_ protein FLJ12455 (99%) (99%)
071361.1I(NM
022078
[Homo Sapiens]
gi~18545156~ref~XPsimilar to 107 83/84 84/84 6e-37
_ hypothetical (98%) (99%)
086159.1~(XM
086159
_ protein FLJ12455
[Homo Sapiens]
giI18545158~ref~XPhypothetical 141 135/137 136/137 2e-33
_ protein XP_097448 (98%) (98%)
097448.1~(XM_097448
[Homo Sapiens]
gi~~7.7562286IrefINPK07B1.7b.p 487 76/237 119/237 3e-30
_ [Caenorhabditis (32%) (50%)
505420.1~(NM_073019
e1 egans]
A multiple sequence alignment is given in Table 7E, with the NOV7 protein
being
shown on line 1 in Table 7E in a ClustalW analysis, and comparing the NOV7
protein with the
select related protein sequences shown in Table 7D. This BLASTP data is
displayed
graphically in the ClustalW in Table 7E.
Table 7E. ClustalW Analysis of NOV7
1) > NOV7; SEQ m N0:14
2) > gig 18545154/ hypothetical protein FLJ12455 [Horrao Sapiens); SEQ m N0:65
3) > gi~11545793~/ hypothetical protein FLJ12455 [Homo Sapiens]; SEQ ID N0:66
NOV7 l 60
gi1185451541 1 60
gi~11545793~ 1 60
70 80 90 100 110 120
y . ~~~.~.I~ .~~ ~I~ ~I. y
NOV7 61 ~~~ ~ ,~~~ ,. ~ ~ . .~ ~ . ~~w ~, 120
51
10 20 30 40 50 60
<IMG>
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TRANSMISSION-
FIS
#PD229850 55-90 179 4e-14
BLOCKING
NT2RM1000563
TRANSMISSION-
FIS
#PD138963 91-258 872 2e-94
BLOCKING
NT2RM1000563
TRANSMISSION-
FIS
Consistent with other known members of the Transmission Blocking Target
Antigen
5230 Precursor-like family of proteins, NOV7 has, for example, three Blocking
NT2RM1000563 Transmission-FIS Antigen Weakly Precursor Peptidase A2 signature
sequences and homology to other members of the Transmission Blocking Target
Antigen
5230 Precursor-like Protein Family. NOV7 nucleic acids, and the encoded
polypeptides,
according to the invention are useful in a variety of applications and
contexts. For example,
NOV7 nucleic acids and polypeptides can be used to identify proteins that are
members of the
Transmission Blocking Target Antigen 5230 Precursor-like Protein Family. The
NOV7
I O nucleic acids and polypeptides can also be used to screen for molecules,
which inhibit or
enhance NOV7 activity or function. Specifically, the nucleic acids and
polypeptides
according to the invention may be used as targets fox the identification of
small molecules that
modulate or inhibit, e.g., cellular activation and signal transduction. These
molecules can be
used to treat, e.g., Cardiovascular diseases, Cardiomyopathy, Atherosclerosis,
Hypertension,
Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD),
Atrioventricular (A-V)
canal defect, Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis,
Ventricular septal
defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity,
Transplantation,
Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Von Hippel-
Lindau
(VHL) syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis,
hypercalceimia,
Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-
Nyhan syndrome,
Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral
disorders, Addiction,
Anxiety, Pain, Neuroprotection
In addition, various NOV7 nucleic acids and polypeptides according to the
invention
are useful, irzte~ alia, as novel members of the protein families according to
the presence of
domains and sequence relatedness to previously described proteins. For
example, the NOV7
nucleic acids and their encoded polypeptides include structural motifs that
are characteristic of
proteins belonging to the Transmission Blocking Target Antigen 5230 Precursor-
like Protein
Family.
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The NOV7 nucleic acids and polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and diagnostic
applications in the
mediation of cardiac and nerve physiology. As such, the NOV7 nucleic acids and
polypeptides, antibodies and related compounds according to the invention may
be used to
treat cardiovascular and nervous system disorders, e.g., Cardiovascular
diseases,
Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects,
Aortic stenosis,
Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus
arteriosus , Pulmonary
stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases,
Tuberous
sclerosis, Scleroderma, Obesity, Transplantation, Diabetes,Von Hippel-Lindau
(VHL)
syndrome, Pancreatitis, Obesity, Von Hippel-Lindau (VHL) syndrome, Alzheimer's
disease,
Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's
disease,
Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-
telangiectasia,
Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain,
Neuroprotection
The NOV7 nucleic acids and polypeptides are useful for detecting specific cell
types.
For example, expression analysis has demonstrated that a NOV7 nucleic acid is
expressed in
Adipose, Heart, Aorta, Coronary Artery, Umbilical Vein, Pancreas, Liver, Gall
Bladder,
Colon, Bone Marrow, Thymus, Bone, Cartilage, Synovium/Synovial membrane,
Skeletal
Muscle, Brain, Left cerebellum, Right Cerebellum, Thalamus, Hypothalamus,
Pituitary Gland,
Frontal Lobe, Parietal Lobe, Cerebral Medulla/Cerebral white matter, Basal
Ganglia/Cerebral
nuclei, Substantia Nigra, Hippocampus, Cervix, Mammary gland/Breast, Uterus,
Oviduct/LTterine Tube/Fallopian tube, Prostate, Testis, Lung, Bronchus,
Larynx, Kidney,
Retina, Skin, Epidermis.
Additional utilities for NOV7 nucleic acids and polypeptides according to the
invention are disclosed herein.
NOV8
A NOV8 polypeptide has been identified as a Nuclear Protein-like protein (also
referred to as CG94713-O1). The disclosed novel NOV8 nucleic acid (SEQ ZD
NO:15) of 3807
nucleotides is shown in Table 8A. The novel NOV8 nucleic acid sequences maps
to the
chromosome 1.
An ORF begins with an ATG initiation codon at nucleotides 16-18 and ends with
a
TGA codon at nucleotides 3793-3795. A putative untranslated region and/or
downstream
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from the termination codon is underlined in Table 8A, and the start and stop
codons are in
bold letters.
Table 8A. NOV8 Nucleotide Sequence (SEQ ID NO:15)
ATGATAAAATAGAAGATGAATTGCAAACCTTCTTTACCAGTGATAAAGATGGAAATTACACATGCAT
ACAACCCGAAATCACCACCTACACAAAACTCTTCAGCCAGCAGTGTGAACTGGAATTCTGCCAACCC
AGATGACATGGTGGTTGATTATGAAACTGACCCTGCTGTAGTTACTGGTGAAAATATTTCTTTAAGC
CTTCAGGGTGTTGAAGTATTTGGTCATGAAAAGTCTTCTAGTGATTTCATTAGTAAGCAGGTGTTAG
ATATGCATAAAGATTCTATTTGTCAGTGTCCTGCACTTGTAGGTACTGAGAAGCCCAAATATCTGCA
ACACAGTTGTCATTCCCTAGAAGCAGTTGAGGGCCAGAGTGTTGAGCCATCTTTGCCTTTTGTGTGG
AAGCCTAATGACAATTTGAACTGTGCAGGCTACTGTGATGCCTTGGAGCTGAACCAAACATTTGACA
TGACAGTGGATAAAGTTAACTGCACCTTTATATCACATCATGCCATCGGAAAGAGTCAGTCCTTCCA
TACTGCTGGAAGCCTGCCACCAACTGGTAGGAGAAGTGGAAGTACATCTTCTTTATCCTATTCCACT
TGGACATCTTCCCATTCTGATAAGACGCATGCAAGAGAAACTACTTATGATAGAGAAAGCTTTGAAA
ACCCTCAAGTCACACCATCAGAAGCCCAAGACATGACTTACACAGCATTTTCTGATGTGGTGATGCA
AAGTGAGGTTTTTGTTTCAGATATTGGAAATCAGTGTGCATGTTCTTCAGGAAAGGTCACCAGTGAG
TACACAGATGGATCACAACAAAGACTAGTTGGAGAAAAAGAGACACAAGCACTAACACCAGTTTCTG
ATGGCATGGAAGTCCCCAATGATTCTGCATTACAAGAGTTCTTTTGTTTATCCCATGATGAATCCAA
TAGCGAACCACATTCACAGAGCTCATACAGGCACAAGGAAATGGGCCAAAATCTGAGAGAGACAGTG
TCCTATTGTCTTATTGATGATGAATGCCCTTTAATGGTGCCAGCTTTTGATAAGAGCGAAGCTCAAG
TGCTGAACCCAGAGCATAAAGTCACTGAGACTGAAGACACACAAATGGTCTCCAAAGGAAAGGATTT
GGGAACCCAAAATCATACCTCAGAATTGATTCTAAGTAGCCCGCCAGGACAAAAGGTGGGCTCGTCA
TTTGGACTGACTTGGGATGCAAATGATATGGTCATTAGCACAGACAAAACGATGTGCATGTCAACAC
CAGTCCTAGAACCCACAAAAGTAACCTTTTCTGTTTCACCGATTGAAGCGACGGAGAAATGTAAGAA
AGTGGAGAAGGGTAATCGAGGGCTTAAAAACATACCAGACTCGAAGGAGGCACCTGTGAACCTGTGT
AAACCCAGTTTAGGAAAATCAACAATCAAAACGAATACCCCAATAGGCTGCAAAGTTAGAAAAACTG
AAATTATAAGTTACCCAAGACCAAACTTCAAGAATGTCAAAGCAAAAGTTATGTCTAGAGCAGTGTT
GCAGCCCAAAGATGCTGCTTTATCAAAGGTCACGCCCAGACCTCAGCAGACCAGTGCCTCATCACCC
TCATCAGTGAATTCAAGACAACAAACAGTCTTGAGCAGAACACCGAGATCTGACTTGAATGCAGACA
AAAAAGCAGAAATTCTAATTAACAAGACACATAAGCAGCAGTTTAATAAACTCATTACTAGCCAGGC
TGTGCATGTTACAACTCATTCTAAAAATGCTTCACACAGGGTTCCAAGAACAACATCTGCCGTGAAA
TCGAATCAGGAAGATGTTGACAAAGCCAGTTCTTCTAACTCAGCATGCGAGACCGGGTCCGTTTCTG
CGTTGTTTCAGAAGATCAAAGGCATACTCCCTGTTAAAATGGAAAGTGCAGAATGTTTGGAAATGAC
CTATGTTCCCAACATTGATAGGATTAGCCCTGAAAAGAAGGGTGAAAAAGAAAATGGGACATCTATG
GAAAAACAAGAGCTGAAACAAGAGATTATGAATGAGACTTTTGAATATGGTTCTCTGTTTTTGGGCT
CTGCTTCAAAAACAACGACCACCTCAGGTAGGAATATATCCAAGCCTGACTCCTGCGGTTTGAGGCA
AATAGCTGCTCCAAAAGCCAAAGTGGGGCCCCCTGTTTCCTGTTTGAGGCGGAACAGTGACAATAGA
AATCCCAGTGCTGATCGAGCCGTATCTCCTCAGAGGATCAGGCGTGTGTCCAGTTCTGGAAAGCCTA
CATCCTTGAAAACTGCACAGTCGTCATGGGTGAATTTGCCTAGACCACTTCCTAAATCCAAAGCATC
TTTGAAAAGTCCTGCGCTGCGGAGGACAGGAAGCACCCCCTCAATAGCCAGCACCCACAGTGAGCTG
AGCACTTACAGCAACAATTCTGGTAATGCCGCTGTCATCAAATATGAGGAGAAACCTCCAAAACCAG
CATTTCAGAATGGTTCCTCAGGATCCTTTTATTTGAAGCCTTTGGTATCCAGGGCTCATGTTCACTT
GATGAAAACTCCTCCAAAAGGTCCTTCGAGAAAAAATTTATTTACAGCTCTTAATGCAGTTGAAAAG
AGCAGGCAAAAGAATCCTCGAAGCTTATGTATCCAGCCACAGACAGCTCCCGATGCGCTGCCCCCTG
AGAAAACACTTGAATTGACGCAATATAAAACAAAATGTGAAAACCAAAGTGGATTTATCCTGCAGCT
CAAGCAGCTTCTTGCCTGTGGTAATACCAAGTTTGAGGCATTGACAGTTGTGATTCAGCACCTGCTG
TCTGAGCGGGAGGAAGCACTGAAACAACACAAAACCCTATCTCAAGAACTTGTTAACCTCCGGGGAG
AGCTAGTCACTGCTTCAACCACCTGTGAGAAATTAGAAAAAGCCAGGAATGAGTTACAAACAGTGTA
TGAAGCATTCGTCCAGCAGCACCAGGCTGAAAAAACAGAACGAGAGAATCGGCTTAAAGAGTTTTAC
ACCAGGGAGTATGAAAAGCTTCGGGACACTTACATTGAAGAAGCAGAGAAGTACAAAATGCAATTGC
AAGAGCAGTTTGACAACTTAAATGCTGCGCATGAA.ACCTCTAAGTTGGAAATTGAAGCTAGCCACTC
AGAGAAACTTGAATTGCTAAAGAAGGCCTATGAAGCCTCCCTTTCAGAAATTAAGAAAGGCCATGAA
ATAGAAAAGAAATCGCTTGAAGATTTACTTTCTGAGAAGCAGGAATCGCTAGAGAAGCAAATCAATG
ATCTGAAGAGTGAAAATGATGCTTTAAATGAAAAATTGAAATCAGAAGAACAAAAAAGAAGAGCAAG
AGAAAAAGCAAATTTGAAAAATCCTCAGATCATGTATCTAGAACAGGAGTTAGAAAGCCTGAAAGCT
GTGTTAGAGATCAAGAATGAGAAACTGCATCAACAGGACATCAAGTTAATGAAAATGGAGAAACTGG
TGGACAACAACACAGCATTGGTTGACAAATTGAAGCGTTTCCAGCAGGAGAATGAAGAATTGAAAGC
TCGGATGGACAAGCACATGGCAATCTCAAGGCAGCTTTCCACGGAGCAGGCTGTTCTGCAAGAGTCG
CTGGAGAAGGAGTCGAAAGTCAACAAGCGACTCTCTATGGAAAACGAGGAGCTTCTGTGGAAACTGC
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ACAATGGGGACCTGTGTAGCCCCAAGAGATCCCCCACATCCTCCGCCATCCCTTTGCAGTCACCAAG
GAATTCGGGCTCCTTCCCTAGCCCCAGCATTTCACCCAGATGACACCTCCCCAAA
Variant sequences of NOVB are included in Example 3, Table 20. A variant
sequence
can include a single nucleotide polymorphism (SNP). A SNP can, in some
instances, be
referred to as a "cSNP" to denote that the nucleotide sequence containing the
SNP originates
as a cDNA.
The NOV8 protein (SEQ m N0:16) encoded by SEQ m NO:15 is 1259 amino acid
residues in length and is presented using the one-letter amino acid code in
Table 8B. Psort
analysis predicts the NOV8 protein of the invention to be localized in the
nucleus with a
certainty of 0.7600.
Table 8B. Encoded NOV8 protein sequence (SEQ ID N0:16)
MNCKPSLPVIKMETTHAYNPKSPPTQNSSASSVNWNSANPDDMVVDYETDPAWTGENISLSLQG
VEVFGHEKSSSDFTSKQVLDMHKDSICQCPALVGTEKPKYLQHSCHSLEAVEGQSVEPSLPFWK
PNDNLNCAGYCDALELNQTFDMTVDKVNCTFISHHAIGKSQSFHTAGSLPPTGRRSGSTSSLSYS
TWTSSHSDKTHARETTYDRESFENPQVTPSEAQDMTYTAFSDWMQSEVFVSDIGNQCACSSGKV
TSEYTDGSQQRLVGEKETQALTPVSDGMEVPNDSALQEFFCLSHDESNSEPHSQSSYRHKEMGQN
LRETVSYCLIDDECPLMVPAFDKSEAQVLNPEHKVTETEDTQMVSKGKDLGTQNHTSELILSSPP
GQKVGSSFGLTWDANDMVISTDKTMCMSTPVLEPTKVTFSVSPIEATEKCKKVEKGNRGLKNIPD
SKEAPVNLCKPSLGKSTIKTNTPIGCKVRKTEIISYPRPNFKNVKAKVMSRAVLQPKDAALSKVT
PRPQQTSASSPSSVNSRQQTVLSRTPRSDLNADKKAEILINKTHKQQFNKLITSQAVHVTTHSKN
ASHRVPRTTSAVKSNQEDVDKASSSNSACETGSVSALFQKIKGILPVKMESAECLEMTYVPNIDR
ISPEKKGEKENGTSMEKQELKQEIMNETFEYGSLFLGSASKTTTTSGRNISKPDSCGLRQIAAPK
AKVGPPVSCLRRNSDNRNPSADRAVSPQRIRRVSSSGKPTSLKTAQSSWVNLPRPLPKSKASLKS
PALRRTGSTPSIASTHSELSTYSNNSGNAAVIKYEEKPPKPAFQNGSSGSFYLKPLVSRAHVHLM
KTPPKGPSRKNLFTALNAVEKSRQKNPRSLCIQPQTAPDALPPEKTLELTQYKTKCENQSGFILQ
LKQLLACGNTKFEALTWIQHLLSEREEALKQHKTLSQELVNLRGELVTASTTCEKLEKARNELQ
TVYEAFVQQHQAEKTERENRLKEFYTREYEKLRDTYIEEAEKYKMQLQEQFDNLNAAHETSKLEI
EASHSEKLELLKKAYEASLSEIKKGHEIEKKSLEDLLSEKQESLEKQINDLKSENDALNEKLKSE
EQKRRAREKANLKNPQIMYLEQELESLKAVLEIKNEKLHQQDIKLMKMEKLVDNNTALVDKLKRF
QQENEELKARMDKHMAISRQLSTEQAVLQESLEKESKVNKRLSMENEELLWKLHNGDLCSPKRSP
TSSAIPLQSPRNSGSFPSPSISPR
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 8C.
Table 8C. results for
Patp NOV8
Smallest
Sum
eading igh Prob
equences Pairs: Frame ScoreP(N)
producing
High-scoring
Segment
>patp:AAG63542Aminoacidsequence human ATIP +1 6389 0.0
of a isoform
>patp:AAG63529Aminoacidsequence human ATIP +1 6233 0.0
of a isoform
>patp:AAG63541Aminoacidsequence human ATIP +1 3928 0.0
of a isoform
>patp:AAG63537Aminoacidsequence ATIP isoform +l 3279 0.0
of a
>patp:AAG63530Aminoacidsequence human ATIP +1 2954 1.5e-307
of a isoform
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In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 3751 of 3751 bases (100%)
identical to a
gb:GENBANI~-m:AB033114~acc:AB033114.1 mRNA from Homo Sapiens (mRNA for
KIAA1288 protein, partial cds). The full amino acid sequence of the protein of
the invention
was found to have 1245 of 1245 amino acid residues (100%) identical to, and
1245 of 1245
amino acid residues (100%) similar to, the 1245 amino acid residue
ptnr:SPTREMBL-
ACC:Q9IJLD2 protein from Horno Sapiens (I~IAA1288 PROTEIN).
NOV8 also has homology to the proteins shown in the BLASTP data in Table 8D.
Table 8D. BLAST
results for
NOV8
Gene Index/ Protein/ OrganismLength Identity PositivesExpect
Identifier (aa) (%) (%)
gi163314071dbjIBAABKIAA1288 protein1245 1245/12451245/12450.0
6602.11(AB033114)[Homo Sapiens] (100%) (100%)
gi1178656321refINPAT2 receptor- 436 404/436 409/436 0.0
065800.11(NM interacting (92%) (93%)
020749
protein 1
[Homo Sapiens]
gi1204367221dbjIBABunnamed protein240 239/240 239/240 e-107
14894.11 (AK024357)product (99%) (99%)
[Homo Sapiens]
gi138822691dbjIBAA3KIAA0774 protein1163 135/366 224/366 9e-49
4494.11(AB018317)[Homo Sapiens] (36%) (60%)
gi1174756301refIXPKIAA0774 protein901 135/366 224/366 3e-48
' [Homo Sapiens] (36%) (60%)
029364.31
(XM 029364)
A multiple sequence alignment is given in Table 8E, with the NOV8 protein
being
shown on line 1 in Table 8E in a ClustalW analysis, and comparing the NOVB
protein with the
related protein sequences shown in Table 8D. This BLASTP data is displayed
graphically in
the ClustalW in Table 8E.
Table 8E. ClustalW Analysis of NOVB
1) > NOVB; SEQ m N0:16
2) > gi~6331407~/ KIAA1288 protein [Homo Sapiens]; SEQ ID N0:67
3) > gi~17865632~/ AT2 receptor-interacting protein 1 [Homo Sapiens]; SEQ m
NO:68
4) > gi~10436722~/ unnamed protein product [Homo Sapiens]; SEQ m N0:69
5) > gi~3$82269~/ I~IAA0774 protein [Homo Sapiens]; SEQ ID N0:70
6) > gig 17475630/ KIAA0774 protein [Homo Sapiens]; SEQ m N0:71
ZO 20 30 40 50 60
....1....1....1....1....1....1....1....1....1....1....1....1
NOV8 1 -MNCKPSLPVIKMEITHAYNPKSPPTQNSSASSVNWNSANPDDMWDYETDPAWTG--- 56
gi163314071 1 ---------------THAYNPKSPPTQNSSASSVNWNSANPDDMWDYETDPAWTG--- 42
gi1178656321 1 ____________________________________________________________ 1
gi1104367221 1 ____________________________________________________________ 1
gi138822691 1 RGQIPGGGEGPQKTLPDHAVPAAFPATDSTSEGKSVRHPKPSTSESKQSTPSETQTVGAH 60
gi1174756301 1 ____________________________________________________________ 1
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70
80
90
100
110
120
....I....I....I....I....I....I....I....I....I....I....I....I
NOV8 56 ------ENISLSLQGVEVFGHEKSSSDFISKQVLDMHKDSICQCPALVGTEKPKYLQHSC
110
S gi I63314071 42 ---~---
ENISLSLQGVEVFGHEKSSSDFISKQVLDMHKDSICQCPALVGTEKPKYLQHSC
96
gi ~17865632~ 1 _____________________________-
______________________________ 1
gi ~10436722~ 1
____________________________________________________________
1
gi 13882269~ 61 VLQVCSEHTSHSAHPEPALNLTLASKEIPSKLEAQLGQGKGEAKLDLKYVPPRRVEQEGK
120
gi ~17475630~ 1
____________________________________________________________
1
130
140
150
160
170
180
NOV8 111HSLEAVEGQSVEPSLPFVWKPNDN----LNCAGYCDALELNQTFDMTVDKVNCTFISHHA
166
gi I63314071 97 HSLEAVEGQSVEPSLPFVWKPNDN----LNCAGYCDALELNQTFDMTVDKVNCTFISHHA
152
15gi 1178656321 1
____________________________________________________________
1
gi ~104367221 1
____________________________________________________________
1
gi ~3882269~ 121AAQEGYLGCHKEENLSALEGRDPCGEAHPEATDALGHLLNSDLHHLGVGRGNCEEKRGVN
180
gi I174756301 1
____________________________________________________________
1
190
200
210
220
230
240
NOV8 167IGKSQSFHT---AGSLPPTGRRSGSTSSLSYSTWTSSHSDKTHARETTYDRESFENPQVT
223
gi I6331407~ 153IGKSQSFHT---AGSLPPTGRRSGSTSSLSYSTWTSSHSDKTHARETTYDRESFENPQVT
209
gi ~17865632~ 1
____________________________________________________________
1
gi I104367221 1
____________________________________________________________
1
gi ~3882269~ 181PGEQDSLHTTPKQGSASLGGADNQPTGKISPCAGEKLGERTSSSFSPGDSHVAFIPNNLT
240
gi ~174756301 1
____________________________________________________________
1
250
260
270
280
290
300
..
NOV8 224PSEAQDMTYTAFSDVVMQSEVFV'SD'~~ QCACSGKVTSYT~GSQ
Q P 283
~ B ~ LVGEETAL
B
B
giI 6331407~ 210PSEAQDMTYTAFSDVVMQSEVFSD~G QCACSGKVTSYTGSQ
LVGEETAL P 269
Q
gi~ 17865632~ 1 _________________________.__________________________._______
1
gi~ 10436722~ 1 ___________________
1
__
____
____________________
____
3Sgi1 38822691 241DSKPLDVIEEERRLGSGNKDSVVL~?F PSVGE PL
SEAR~SKVT S 300
KTEVP PQS
gi 174756301 1 ~ ~ ~ ~ S 38
~ ------------------- ~ PL~pQS ~
VLV'F,,~~,, PSVGE SEARSK~'VT
~KTEVP
310
320
330
340
350
360
g 284~SDGMEU'PN~7~AL Q~FFCLS~
ESNE~HSQSSj~'RHKEGQNL'RE~SYCLID ~ECPL~ 343
i 270SIaGMEU'PN~:?~AL Q ESN HSQSS'Y'RHKE ~ECPL
329
8 FFCLS E GQNLE
I SYCLID
i
6331407
' __._______.______
gi~ 17865632~ 1 ___________________________________________
1
i 10436722 1 ____________________________________________________________
1
B
gi 3882269 301~~.''NRN~~.~iLENADK I~STSAR SVL~TI~--APLPETTiVNMTYP~T
PSSSFQ~S===~ 355
I I ~ ~ s
i 1747
630
I 5 39 NRN I SVLI --APLPETTNMTY PSSSFQ~ 93
g 1 LEN~K STS P T S
~
370
380
390
400
410
420
NOV8 344PAF~KSEAQ~ EHKVTETE QMVSKLGTQNHTSEILS PPGQKVGSSFGLTWD
403
.. C'~
S0gi~ 63314071 330PAF~KSEAQ~~ EHKVTETE ~QMVSKLGTQNHTSEILS
~PPGQKVGSSFGLTWD 389
gi~ 17865632~ 1
_____________________'_______________________________________
1
giI 10436722~ 1 ____________________________________________________________
1
gi1 3882269~ 356FG PTDSARLL SPKVPD KDTPSSQEGMENYQV
415
~ TCPSGIPKP'T;FTH
GSPL
P
g1~ 17475630~ 94 FGGSPL~P~ PTDSARLL ~SPKVPD~TCPSGIPKP~FTH
~KDTPSSQEGMENYQV 153
SS
430
440
450
460
470
480
NOVS 404ANDMVITDITjNjC 463
6331407 390ST
I LEPT
i TFSSPIEATEKCKKVEKGN
KNIDKE
L
T
~
A
~
EP~~
~
~
~
~
~
g NDMVITD T S DK 449
I !ICITFSSPIEATEKCKKVEKGNR L
VLEPT
KNI
gi~ 17865632~ 1 ____________________________________________________._______
1
gi~ 10436722~ 1 __________________________________________________________
gi 3882269 416EKTEER4LI~!:ET ~PI'IP KHVRPIITYRRNPQALGQVDASLVP
475
~ I ~ ~ PY
B P~''i'CT
~PH
~
~
~
~
gi 17475630 154EKTEERET P~'IPIKHVRP~PH 213
~ ~ I<ITY~C!RRNPQALGQVDASLVPV
PYA
P~CTl
65 490
500
510
520
530
540
~
.~....~...
.~..
.~.
_
giI 6331407~ 450C'~P~LKSTIK'hNTPIGCKVRKTIISYPRPNFKNV
PICD Si P 509
SRAVL~
gi~ 178656321 1 _____ 1
_________________~_____________
_____
__'_
_
_
70gi~ 104367221 1
____________________________________________________________
1
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gi~174756301 214 EK~GDLKP~ANLYEKFKPDLQKPRVFSSGLMVSGI~PPGHPFS~ S~KF~Q~DH~ 273
550
560
570
580
590
600
.
NOV8 524 Qi~TSAS SSVI~SRQQTVLS AD~CKAEIL'INTCTHKQQFN
583
gi 510 Q~TSAS ~SSVSRQQTVLS T ~ 569
~ VHV'~TH,,H.,
6331407 'TPR3S~'S'iD
~ ~TPRD~TAD~KAEIL2NT~THKQFNF~T~VHV~~TH
gi~1786563211 ___________'.'________,_________._____________________________
1
gi ~10436722~1 _ 1
_____
_____________________
_____
__
_________
__
_
IQ gi ~3882269i536 GEEFC PYAYEVPPTF
591
E --S
LKP~LGLGA~tRLPSA'ECSRT
~
RSAS
MS
~
YB
I
~
~
~
gi~17475630~274 G QLGLGA 329
EFC RLPSATtSRT
A
~
-RSAS
PYA
YEVPPTF
--S
LKP
,
610
620
630
640
650
660
I NOV8 584 KIt~ASHRrV~~PRT
642
S gi~6331407~ 570 . 628
V
QE
~
ShCETGS
.
FQK-IKG~I'LP
E'AEC
EM
ICASHRPRT~AVQEy~
~S~CETGSV~A
FQK-IKGLPV'~EAECEM
gi~1786563211 _=__________________________________________________________
1
gi~10436722~1 _.___________________________________-______________________
1
~ ~ ~
~
~
gi 17475630330 I LYSPS 389
~ I PPGPTTA y
KSNLPI
G
RPPGYSR
P
FGFRS
670
680
690
700
710
720
~S gi~63314071 629 Y~P~T~DRISPEKKG~ITSME~QELKQ
BIMNETFEYGS~ FLGS T~TTSGRNK 688
gi~1786563211 _______________.________.___________________________________
1
gi~10436722~1 ____________________________________________________________
1
gi 3882269 652 S~S~Cl'SSTQSGDSAP~ Q~RPATSTFG~EQ--====P~
KASLP~D~PKGAGR'V.A!P 705
I ~ ' ~
' '
gi 17475630390 ,~S Q RPAT~ t~. 443
~ I SF , KASLP
~SSTQSGDSA~P ,STFG tD
EQ-- PKGAGR
P P
30
730
740
750
760
770
780
NOV8 703 ~ D~CGLRQI~P~K~GP~CLRRNSDNRNPSADRAV~
~QRI~~SSG ~ 74
6331407 689 D ~
i GLR L
A
I '
'
LRR
DN
S
A
V
#~
P Q 8
g QI KT
( ~ ~
I GP QRI
NS
RNP
ADR
C
P~
C
V
35 gi~17865632~ 1 -------MLLSPFS--------------------
26
LTTHI~LTAKGLLRLR----
giI10436722~ 1
____________._________________________.______________.______
1
gi 706 S ------------ S 748
~ -SVTiIPR~;.SLT.iP ~ TSiI ~
3882269 ~ ~ GTR
~ ~ T~PKDD
KPAV
~
~
~~
gi 444 S--SVT 'I-------------- S 486
~ PR~SLPA TS
17475630 QKD~QD
~ KPAV
GT
40 790
800
Sl0
820
830
840
NOV8 763 SWVNLP ~PI~P~KAS ~
~R~t~,TGSTPSIASTHSELSTYSNNSGNAAVIKYEEKPPKPA 822
gi~63314071 749 SWVNLP~ P~.iP~KAS '
RtRTGSTPSIASTHSELSTYSNNSGNAAVIKYEEKPPKPA 808
gi~178656321 26 ____Lp____________SGFg,~'-
_______ST___________________________
35
gi~10436722~ 1
______________________=~_____________________________________
1
gi~3882269~ 748 ___ H GYP_-___________________________________
766
p
~
TT
gi~174756301 486 ____p~_TTI~ I-I~GYP_-
___________________________________ 504
850
860
870
880
890
900
50
NOV8 823 FQNGSSGSFYLKPLVSRAHVHLMKTPPKGPSRKNLFTALNAVEKS
'QKN P'LCTQ'Q TA882
giI6331407~ 809 FQNGSSGSFYLKPLVSRAHVHLMKTPPKGPSRKNLFTALNAVEKS
'QKN P' TA868
LCIQ'~
gi~178656321 35 ____________________________________wFHTVEKS
'QKN P' TA59
LCIQ'
giI10436722~ 1 _______________________________________-
_______________-____ 1
gi~3882269~ 766 __,________________________________________
T ~PD781
Q -GFP
I
gi~17475630~ 504 ___________________
~TF~~_GFP~I ~PD519
______________________Q
910
920
930
940
950
960
60 NOV8 883 942
gi~6331407~ 869 928
gi~17865632~ 60
119
gi~104367221 1 _______ 1
____
_-__
_
_______________________
_____
gi~38822691 782 Q~1RE Q R E QyRQ~GVAGE KRAICT ~FF S
840
B ~ L ~ ~ ~ ~~ ~~
~ ~ ~
~
gi 520 Q Q V R E Q KRAICGA~AT FF S 578
( RE L T'RQ7~ {R~
17475630 GVA~GE LV I~
I
970 980 990 1000 1010 1020
70 gi~63314071 929 HST Q~ .' 3.L~VTATT~. E ~~ .'TV . F~~____~H~, KT. .'~ 984
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gi I 17865632120 H~yT~Q~~GQI;VTATT~ETV~AFV~----~W
I KT~R~ICE 175
gi~10436722~1 _______ _-__ ___ _______ ________________________
__-__ 1
gi ~ 17475630579 E~E~I~~'~D~VAFHAIQ~EE~~RR~~DE~ L E' QL
~ I~RLGGV~Q~ LQ 638
~
1030 1040 1050 1060 1070 1080
NOV8 999 F~';T E T~ II~~AEK v ~ ... , 1058
' v v
giI63314071985 F'~T YE 1~TIEAEKY~ ~ ~ '' 1044
~
gi I 17865632176 F~'~T YE~i~T.I~~I~AEKY~ ~ 235
~ ~ ~
gi~10436722~1 __________=_'~_______v ~ .. . . 39
v ~
gi ~ 3882269901 Q E QE G t~LLSI~;C~H~~ QDD~DTiK
~ ~ S .7AL TVA'~T~ 960
~ ~ ,f"D T
gi ~ 17475630639 QLLSIC~ ~ r , QDD~iI7HK
f Qy~ , 698
"',E~Q~E~G vV,Ep T~S AL r a
. ~VAT~
1090 1100 1110 1120 1130 1140
NOV8 1059 1116
gi~6331407~1045 1202
gi~17865632~236 293
gi~10436722~40 97
gi~3882269,961 1020
gi~17475630~699 758
1150 1160 1170 1180 1190 1200
NOV8 1117 1176
gi~6331407~1103 1162
gi1178656321294 353
giI10436722~98 157
gi~3882269~1021 1080
gi~174756301759 818
1210 1220 1230 1240 1250 1260
NOV8 1177 1236
gi~633140711163 1222
gi~17865632~354 413
gi~10436722~158 217
gi13882269~1081 1140
gi~17475630~819 878
1270 1280
NOV8 1237 1259
gi~ 633140711223 1245
gi~ 17865632~414 43b
gi~ 104367221218 240
gi~ 3882269~1141 1163
gi~ 17475630~879 901
50
The presence of identifiable domains in the protein disclosed herein was
determined by
searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and
then
determining the Interpro number by crossing the domain match (or numbers)
using the
55 Interpro website (http:www.ebi.ac.uk/interpro~. Table 8F lists the domain
description from
DOMAIN analysis results against NOVB.
Table 8F Domain
Anal sis
of NOV8
Model Region of Score (bits) E value
Homology
RNA polymerase1008-1094 -12.8 5.7
omega subunit
Intermediate 967-1193 48.9 2.0e-06
filament
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Consistent with other known members of the Nuclear Protein-like family of
proteins,
NOV8 has, for example, an RNA polymerase omega subunit signature sequence and
homology to other members of the Nuclear Protein-like Protein Family. NOV8
nucleic acids,
and the encoded polypeptides, according to the invention are useful in a
variety of applications
and contexts. For example, NOV8 nucleic acids and polypeptides can be used to
identify
proteins that are members of the Nuclear Protein-like Protein Family. The NOVB
nucleic
acids and polypeptides can also be used to screen for molecules, which inhibit
or enhance
NOVB activity or function. Specifically, the nucleic acids and polypeptides
according to the
invention may be used as targets for the identification of small molecules
that modulate or
inhibit, e.g., cellular activation, cellular replication, and signal
transduction. These molecules
can be used to treat, e.g., Cardiovascular diseases, Cardiomyopathy,
Atherosclerosis,
Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect
(ASD),
Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary stenosis,
Subaortic
stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis,
Scleroderma,
Obesity, Transplantation, Diabetes,Von Hippel-Lindau (VHL) syndrome ,
Pancreatitis,
Obesity, Hyperparathyroidism, Hypoparathyroidism as well as other diseases,
disorders and
conditions.
In addition, various NOV8 nucleic acids and polypeptides according to the
invention
are useful, ihtef° alia, as novel members of the protein families
according to the presence of
domains and sequence relatedness to previously described proteins. For
example, the NOV8
nucleic acids and their encoded polypeptides include structural motifs that
are characteristic of
proteins belonging to the Nuclear Protein-like Protein Family.
The NOV8 nucleic acids and polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and diagnostic
applications in the
mediation of cardiac or endocrine physiology. As such, the NOVB nucleic acids
and
polypeptides, antibodies and related compounds according to the invention may
be used to
treat infection, cardiovascular system, immune system, and nervous system
disorders, e.g.,
Cardiovascular diseases, Cardiomyopathy, Atherosclerosis, Hypertension,
Congenital heart
defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V)
canal defect,
Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal
defect (VSD),
valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation,
Diabetes,Von
Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity, Hyperparathyroidism,
Hypoparathyroidism as well as other diseases, disorders and conditions.
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The NOV8 nucleic acids and polypeptides are useful for detecting specific cell
types.
For example, expression analysis has demonstrated that a NOV8 nucleic acid is
expressed in
Heart, Aorta, Coronary Artery, Vein, Umbilical Vein, Adrenal GlandlSuprarenal
gland,
Pancreas, Islets of Langerhans, Parathyroid Gland, Thyroid, Pineal Gland,
Tongue, Salivary
Glands, Stomach, Liver, Small Intestine, Colon, Ascending Colon, Lymphoid
tissue, Spleen,
Brain, Thalamus, Hypothalamus, Temporal Lobe, Amygdala, Cerebral
Medulla/Cerebral
white matter, Basal GangliaJCerebral nuclei, Substantia Nigra, Hippocampus,
Spinal Chord,
Cervix, Mammary gland/Breast, Ovary, Placenta, Uterus, Prostate, Testis, Lung,
Nasoepithelium, Larynx, Urinary Bladder, Kidney, Kidney Cortex, Retina, Skin,
Foreskin,
Epidermis, Dermis.
Additional utilities for NOV8 nucleic acids and polypeptides according to the
invention are disclosed herein.
NOV9
A NOV9 polypeptide has been identified as a Hemicentin precursor-like protein
(also
referred to as CG94702-OI). The disclosed novel NOV9 nucleic acid (SEQ ID
N0:17) of
11796 nucleotides is shown in Table 9A. The novel NOV9 nucleic acid sequences
maps to
the chromosome 9.
An ORF begins with an ATG initiation codon at nucleotides 1-3 and ends with a
TAA
codon at nucleotides 11794-11796. A putative untranslated region and/or
downstream from
the termination codon is underlined in Table 9A, and the start and stop codons
are in bold
letters.
Table 9A. NOV9 Nucleotide Sequence (SEQ ID N0:17)
ATGTCTGCCTTATTTGCAGCTGTGTACCAGATGCTAAA.ACCACGCCTGGTCCATAACAGCCCACATC
CGGTGACCTATCAAATTGAGGCAAGTTTAAAGCCAGAGCAGCCTGGTGTCACGCTGGTGTCCATCCC
AGTCTTCCTGGCACCTTCCTGGCACAAAGCCTCAGAGCTGATCCCGACCCAGTCCTTCCGAGCACAG
GGGGCAGGGAAGCAGCTCCTCGGCTCTCCTTGCCCCCAAGTGCCCCCCAGCATCCGGGAGGACGGGC
GCAAGGCCAACGTGTCGGGTATGGCCGGGCAGTCCCTGACGCTGGAGTGTGACGCGAACGGCTTTCC
AGTCCCTGAGATCGTGTGGCTGAAGGACGCGCAGCTGATTCCTAAGGTGGGCGGCCACCGCCTCCTG
GACGAGGGCCAGTCCCTCCACTTCCCCAGGATCCAGGAGGGTGATTCTGGGCTCTACTCCTGCCGGG
CAGAGAACCAGGCTGGCACCGCCCAGAGGGACTTCCATCTCCTTGTGCTCACCCCTCCTTCCGTGCT
TGGAGCCGGGGCCGCTCAGGAGGTGCTAGGATTGGCCGGTGCAGACGTGGAGCTGCAGTGTTGGACC
TCAGGGGTCCCCACGCCCCAGGTGGAGTGGACCAAGGACAGGCAGCCTGTCCTTCCGGGAGGCCCTC
ACCTGCAGGTCCAGGAGGATGGCCAGGTTCTCAGGATCACCGGCAGTCACGTGGGGGATGAGGGACG
ATACCAGTGCGTGGCCTTCAGCCCAGCTGGTCAGCAGGCCAGGGACTTCCAGCTCCGAGTTCATGCG
CCCCCCACTATCTGGGGCTCCAACGAGACAGGCGAGGTGGCCGTCATGGAGGACCACCTAGTGCAGC
TCCTGTGTGAGGCTCGAGGAGTGCCCACCCCAAACATCACCTGGTTCAAGGACGGGGCCCTGCTCCC
CACCAGCACCAAGGTGGTCTACACTAGGGGCGGTCGGCAGTTGCAGCTGGGGAGGGCCCAGAGCTCC
GATGCCGGCGTCTACACCTGCAAGGCCAGCAATGCTGTGGGGGCCGCAGAGAAGGCCACCAGGCTGG
ATGTTTATGTCCCACCTACCATCGAGGGCGCCGGTGGAAGACCATACGTGGTGAAGGCTGTGGCTGG
GAGGCCTGTGGCGCTGGAGTGCGTGGCCAGAGGCCACCCGTCCCCCACCCTCTCCTGGCACCACGAG
GGGCTGCCCGTGGCAGAGAGCAACGAGTCGCGGCTGGAGACAGACGGGAGTGTGCTGAGGCTGGAGA
62
CA 02443770 2003-10-15
WO 02/085922 PCT/US02/11634
GCCCGGGGGAGGCATCCAGTGGCCTGTACAGCTGTGTGGCCAGCAGTCCTGCCGGGGAAGCCGTCCT
GCAGTACTCCGTGGAGGTTCAGGTGCCCCCACAGCTCCTGGTGGCTGAAGGCTTGGGACAGGTGACC
ACCATCGTGGGACAGCCCCTGGAACTTCCCTGCCAGGCCTCAGGCTCCCCAGTACCCACTATCCAGT
GGCTGCAGAATGGCCGCCCAGCCGAGGAGCTGGCTGGGGTGCAGGTGGCCTCGCAGGGGACCACACT
GCACATTGACCATGTGGAGCTGGACCACTCAGGCCTCTTCGCCTGCCAGGCCACCAATGAGGCGGGC
ACTGCCGGGGCCGAGGTGGAGGTGTCTGTGCATGAGTTCCCATCGGTCAGTATCATTGGGGGTGAGA
ACATCACAGCTCCTTTCCTGCAGCCTGTGACCCTCCAGTGCATAGGGGATGGGGTGCCCACCCCAAG
CCTCCGTTGGTGGAAGGATGGTGTAGCCCTGGCAGCCTTTGGGGGGAACCTACAGATTGAGAAGGTG
GACCTGAGGGACGAGGGCATCTACACTTGTGCTGCTACCAACCTGGCTGGGGAGAGCAAGAGGGAAG
TGGCGCTGAAAGTTTTGGTGCCCCCCAACATCGAGCCAGGCCCAGTCAACAAGGCAGTGCTGGAAAA
TGCCTCAGTGACCTTGGAGTGTCTGGCTTCGGGCGTGCCCCCTCCTGATGTCTCCTGGTTCAAGGGC
CACCAACCTGTCTCTTCATGGATGGGAGTGACAGTATCAGTGGATGGGAGAGTTCTCCGCATTGAGC
AAGCCCAGCTTTCTGATGCTGGGAGCTACCGCTGTGTGGCATCCAATGTGGCAGGTAGCACAGAGCT
GCGGTATGGCCTACGGGTCAATGTGCCCCCTCGAATCACACTGCCACCCAGCCTGCCAGGCCCTGTG
TTGGTCAACACCCCTGTCCGGCTGACCTGCAATGCCACCGGTGCCCCCAGCCCCACACTGATGTGGC
TGAAGGATGGAAACCCTGTGTCCCCTGCAGGGACCCCTGGCCTGCAGGTCTTCCCTGGGGGCCGGGT
CCTCACCTTGGCTAGTGCCCGGGCCTCCGACTCTGGGAGGTACTCCTGCGTGGCTGTGAGCGCGGTG
GGCGAGGACCGCCAGGATGTTGTCCTGCAAGTCCACATGCCCCCGAGTATCCTTGGAGAAGAGCTGA
ATGTGTCCGTTGTGGCCAATGAGTCAGTGGCCCTGGAGTGCCAGAGCCACGCCATGCCCCCTCCTGT
GCTGAGCTGGTGGAAGGACGGGCGGCCCCTGGAACCACGGCCTGGAGTCCACCTCTCCGCAGACAAA
GCCTTGCTGCAGGTGGACAGAGCCGATGTGTGGGATGCGGGCCATTACACCTGTGAGGCACTGAACC
AGGCCGGCCACTCAGAGAAACACTACAATCTGAACGTCTGGGGTCAACCCCTCCCCGGGGAGGGGGC
TGGCCTCCAGCACGTGTCGGCTGTGGGGAGGCTGTTGTACCTGGGACAGGCCCAGCTGGCTCAGGAA
GGAACATACACCTGTGAATGCAGCAACGTGGTGGGGAACAGCAGCCAGGACCTGCAGCTGGAGGTGC
ACGTTCCCCCTCAGATTGCCGGTCCCCGGGAGCCTCCCACACAAGTCTCTGTGGTCCAGGATGGAGT
GGCCACTCTGGAGTGCAACGCCACAGGGAAACCCCCTCCGACAGTGACATGGGAGCGGGACGGCCAG
CCCGTGGGGGCTGAACTGGGCCTGCAGCTGCAGAACCAGGGTCAGAGCCTGCATGTGGAGCGGGCCC
AGGCTGCCCACACTGGACGCTACAGCTGTGTGGCCGAGAACCTGGCTGGGAGGGCAGAGAGGAAGTT
TGAGCTCTCCGTACTGGTGCCCCCAGAGCTCATTGGAGACTTGGACCCGCTGACCAACATCACTGCT
GCCTTGCACAGCCCCTTAACTCTGCTCTGTGAAGCCATGGGGATCCCACCTCCAGCCATCCGCTGGT
TCCGAGGGGAGGAGCCTGTCAGCCCCGGGGAGGACACCTACCTGCTGGCAGGTGGCTGGATGCTGAA
GATGACTCAGACACAGGAGCAAGACAGTGGCCTCTACTCATGCCTGGCAAGCAACGAGGCTGGGGAG
GCACGGAGGAACTTCAGTGTGGAGGTGCTGGTTCCTCCCAGTATTGAGAACGAGGACTTGGAGGAGG
TGATCAAGGTCCTTGATGGACAGACTGCCCATCTTATGTGCAACGTCACAGGCCACCCACAGCCCAA
GCTCACATGGTTCAAAGATGGCCGGCCTCTGGCTAGGGGAGATGCTCACCACATCTCCCCAGACGGA
GTCCTCCTGCAGGTCCTCCAGGCAAACCTGTCCAGTGCTGGCCACTACTCCTGCATTGCAGCCAACG
CTGTTGGGGAGAAGACCAAACACTTCCAGCTCAGTGTCCTGTTGGCTCCCACCATCCTGGGAGGGGC
CGAGGACAGTGCAGATGAGGAGGTGACCGTGACTGTCAACAACCCCATCTCTCTGATCTGCGAGGCC
CTGGCCTTCCCTTCCCCCAACATCACCTGGATGAAGGACGGGGCCCCGTTTGAGGCCTCCAGGAACA
TCCAGCTGCTCCCAGGTACCCACGGGCTGCAGATCCTGAATGCCCAGAAGGAAGATGCTGGCCAGTA
CACCTGCGTGGTCACCAATGAGCTCGGGGAGGCCGTGAAAAACTACCATGTGGAAGTGCTCATCCCC
CCTTCCATCTCCAAAGACGACCCCTTGGCGGAGGTCGGCGTGAAGGAGGTGAAGACCAAGGTCAACA
GCACCTTGACCTTGGAGTGTGAGAGCTGGGCTGTGCCCCCGCCCACCATCCGCTGGTACAAGGATGG
ACAGCCCGTGACCCCCAGCTCGCGGCTGCAGGTCCTGGGTGAAGGGCGACTGCTCCAGATCCAGCCC
ACACAGGTCTCAGACTCGGGGCGGTACCTGTGTGTGGCCACCAATGTGGCTGGCGAGGACGACCAGG
ACTTCAACGTGCTCATCCAGGTGCCCCCCATGTTCCAGAAGGTGGGTGATTTCAGTGCAGCCTTCGA
GATCCTGTCCCGGGAGGAGGAGGCCCGGGGCGGAGTCACGGAATACAGGGAGATCGTGGAGAACAAC
CCAGCCTACCTGTACTGCGACACCAACGCGATCCCACCCCCGGACCTCACCTGGTACAGAGAGGATC
AGCCCCTCTCGGCCGGGGATGAGGTGTCTGTGCTGCAAGGAGGCCGGGTCCTGCAGATCCCCCTGGT
GCGGGCAGAGAACGCCGGGAGGTACTCGTGCAAGGCCTCCAACGAGGTGGGCGAGGACTGGCTGCAC
TACGAGCTGCTGGTGCTGACCCCACCTGTGATCCTGGGTGACACAGAGGAGCTGGTGGAAGAGGTGA
CAGTCAATGCCAGCAGCACCGTCAGCCTGCAGTGCCCGGCCCTGGGAAACCCCGTGCCCACCATCTC
ATGGCTCCAGAATGGGCTGCCTTTCTCCCCGAGCCCACGGCTGCAGGTCCTGGAGGACGGGCAAGTC
TTGCAGGTTTCCACGGCAGAGGTGGCCGACGCCGCCAGCTACATGTGTGTGGCCGAGAACCAGGCGG
GCTCCGCTGAGAAGCTCTTCACCCTCAGGGTTCAAGGCCTGGACTTGGAGCAGGTCACTGCCATCCT
CAACAGCAGCGTCTCCCTCCCTTGCGACGTCCACGCTCACCCAAACCCCGAGGTCACGTGGTACAAG
GACAGCCAGGCCCTCTCCCTGGGTGAAGAGGTCTTCCTCCTGCCTGGCACCCACACGCTGCAGCTGG
GGAGAGCACGGCTGTCGGACTCCGGGATGTACACATGCGAAGCCCTCAATGCTGCCGGCCGAGACCA
GAAGCTGGTGCAGCTCAGTGTTCTGGTTCCCCCGGCCTTCAGGCAGGCTCCCAGAGGTCCCCAGGAT
GCGGTCCTGGTGAGGGTCGGGGACAAAGCTGTCCTGAGCTGCGAGACAGATGCGCTCCCTGAGCCAA
CTGTGACCTGGTACAAGGATGGGCAGCCCCTGGTCCTGGCACAGCGGACCCAGGCTCTGCGGGGTGG
GCAGAGGCTGGAGATCCAGGAAGCCCAGGTATCGGATAAAGGTTTATACAGCTGTAAAGTCAGCAAC
63
CA 02443770 2003-10-15
WO 02/085922 PCT/US02/11634
GTGGCTGGGGAGGCCGTGCGGACCTTCACCCTCACCGTCCAGGTGCCCCCAACATTTGAGAACCCCA
AGACAGAGACAGTGAGCCAGGTGGCTGGGAGCCCCCTGGTCCTGACCTGTGATGTGTCCGGGGTCCC
TGCACCCACGGTCACTTGGCTGAAGGACAGGATGCCTGTGGAGAGCAGCGCGGTGCACGGTGTGGTC
TCCCGGGGGGGCCGCCTCCAGCTGAGCCGCCTGCAACCGGCCCAGGCGGGCACCTACACGTGCGTGG
CTGAGAACACCCAGGCTGAGGCCCGCAAGGACTTCGTGGTAGCAGTGCTGGTGGCCCCCCGGATCCG
GAGCTCGGGCGTGGCGCGGGAGCACCATGTCTTGGAAGGGCAGGAGGTGCGGCTGGACTGTGAGGCC
GATGGGCAGCCGCCGCCGGACGTGGCCTGGCTGAAGGACGGCAGCCCGCTGGGCCAGGACATGGGCC
CCCACCTCCGGTTCTACCTGGACGGCGGCTCCCTGGTGCTAAAAGGCCTGAGGGCCTCGGACGCGGG
TGCCTACACCTGCGTGGCCCACAACCCAGCCGGGGAGGACGCCAGGCTGCACACGGTGAATGTGCTG
GTTCCTCCCACCATCAAGCAGGGAGCAGACGGCTCGGGGACCCTGGTGAGCAGGCCTGGGGAGCTGG
TGACCATGGTGTGCCCTGTGCGGGGCTCCCCGCCCATCCACGTGAGCTGGCTCAAGGACGGCCTGCC
CCTCCCGCTCTCCCAGCGCACCCTCCTCCACGGCTCTGGCCACACCCTCAGGATTTCCAAGGTGCAA
TTGGCAGACGCTGGCATCTTCACCTGTGTGGCCGCAAGCCCAGCTGGCGTGGCGGACAGGAACTTCA
CCTTGCAGGTGCAGGTGCCCCCTGTCCTGGAGCCGGTGGAGTTCCAGAATGACGTGGTGGTGGTTCG
TGGCTCCCTGGTGGAACTCCCGTGCGAGGCCCGGGGCGTTCCCCTGCCTCTCGTGTCGTGGATGAAG
GATGGGGAACCCTTGTTGTCCCAGAGCCTCGAGCAGGGGCCCAGCCTGCAGCTGGAGGCAGTGGGAG
CTGGTGACTCGGGGACCTACTCCTGTGTGGCCGTGAGCGAGGCGGGGGAAGCCAGGAGGCATTTCCA
GCTGACCGTCATGGAGCCCCCTCACATTGAGGACTCAGGCCAGCCTACAGAGCTGTCGCTGACCCCC
GGCGCCCCCATGGAGCTCCTCTGTGATGCCCAGGGCACCCCCCAGCCCAACATCACCTGGCATAAGG
ACGGGCAGGCCCTGACCAGGCTGGAGAACAACAGCAGAGCCACACGGGTGCTCCGGGTGGAGAATGT
GCAGGTTAGGGATGCTGGGCTGTACACTTGTCTGGCTGAAAGCCCTGCAGGTGCAATTGAGAAGAGC
TTCCGGGTCAGGGTTCAAGCCCCTCCAAACATTGTTGGGCCCCGAGGCCCCCGCTTTGTGGTCGGCC
TGGCCCCAGGGCAGCTGGTCCTGGAGTGTTCGGTGGAGGCAGAGCCAGCGCCCAAGATCACGTGGCA
CCGAGACGGCATTGTGCTGCAGGAGGACGCCCACACACAATTCCCGGAGCGGGGCAGGTTCCTCCAG
CTGCAGGCCCTGAGCACGGCTGACAGCGGCGACTACAGCTGCACAGCCCGCAACGCCGCAGGCAGCA
CTAGTGTCGCCTTCCGCGTGGAGATCCACACGGTGCCCACCATCCGGTCAGGACCACCTGCAGTGAA
CGTCTCAGTGAACCAGACAGCCCTGCTGCCTTGCCAGGCCGACGGCGTGCCCGCACCCCTCGTGAGC
TGGCGGAAGGACAGGGTCCCCCTGGATCCCAGGAGCCCCAGGGCAACCCCCATCCATTCTAGGTTTG
AAATTCTGCCTGAGGGTTCCCTGAGAATCCAGCCAGTCCTTGCCCAGGACGCCGGCCACTACCTCTG
CCTGGCATCCAACTCTGCTGGCTCCGATCGTCAAGGCCGTGACCTACGGGTCTTGGAGCCTCCAGCC
ATCGCCCCCAGCCCCTCCAACCTGACCCTGACCGCCCACACCCCAGCCTTGCTGCCCTGCGAGGCCA
GCGGCTCCCCTAAGCCCCTGGTGGTCTGGTGGAAGGACGGACAGAAGCTGGACTTCCGCCTGCAGCA
GGGCGCCTACCGGCTCCTGCCCTCCAACGCCCTGCTCCTCACGGCCCCCGGCCCCCAGGACTCAGCC
CAGTTTGAATGCGTGGTGAGCAATGAGGTGGGCGAGGCCCACAGGCTCTACCAGGTGACCGTCCATG
TGCCTCCCACCATTGCCGATGACCAGACAGACTTCACCGTGACCATGATGGCACCTGTGGTCCTCAC
ATGTCACAGCACGGGTATACCAGCTCCGACCGTGTCCTGGAGCAAGGCAGGCGCCCAGCTAGGAGCT
CGGGGGAGTGGCTATCGTGTCTCACCATCGGGCGCCCTGGAGATCGGGCAGGCCCTCCCCATCCACG
CAGGCCGCTACACCTGCTCAGCCCGCAACTCTGCCGGCGTAGCCCACAAGCACGTCTTCCTCACTGT
GCAAGCCTCCCCGGTGGTGAAGCCGCTGCCCAGCGTGGTTCGGGCAGTGGCAGAGGAGGAGGTGCTG
CTGCCCTGCGAGGCCTCAGGCATCCCCCGGCCGACCATCACCTGGCAGAAGGAAGGGCTCAACGTCG
CTACTGGAGTGAGTACCCAGGTCCTACCAGGCGGACAGCTGCGGATTGCCCATGCCAGCCCAGAGGA
TGCTGGAAACTATCTCTGCATCGCTAAGAACAGTGCGGGCAGTGCCATGGGGAAGACGCGGCTGGTG
GTGCAAGTCCCACCAGTGATCGAGAATGGCCTCCCAGACCTGTCCACCACCGAAGGCTCCCACGCCT
TCTTGCCTTGCAAGGCGAGGGGCAGTCCTGAGCCCAACATCACCTGGGACAAAGATGGCCAGCCTGT
GTCGGGCGCCGAGGGGAAGTTCACCATCCAGCCTTCTGGGGAGTTGCTGGTGAAGAACTTGGAGGGC
CAGGACGCAGGCACCTATACCTGTACCGCTGAGAACGCCGTGGGCCGGGCCCGCCGCCGCGTGCACC
TCACCATCCTGGTACTGCCTGTGTTCACCACCCTGCCTGGGGACCGCAGCCTGCGCCTTGGGGACAG
GCTGTGGCTTCGCTGTGCAGCCCGGGGCAGCCCCACCCCTCGCATTGGCTGGACTGTCAACGACCGG
CCAGTCACAGAAGGGGTGTCTGAGCAGGATGGAGGCAGCACGCTGCAGCGGGCCGCTGTCTCCAGAG
AAGACAGCGGGACCTATGTCTGCTGGGCGGAGAACAGAGTGGGCCGCACGCAGGCGGTCAGCTTCGT
CCACGTGAAGGAGGCTCCTGTCCTACAAGGGGAGGCTTTCTCCTACCTGGTGGAACCTGTAGGAGGC
AGCATTCAGCTAGACTGTGTGGTGCGTGGAGACCCAGTGCCGGACATCCACTGGATCAAAGATGGCC
TTCCACTGCGGGGCAGCCACCTCCGGCACCAGCTGCAGAATGGCTCGCTGACCATCCGCAGGACTGA
GGCAAGGCGGGGCCTGGCACCTTGGAGGGACGATGCGGGACGGTACCAGTGCCTGGCAGAGAATGAG
ATGGGCGTGGCGAAGAAAGTGGTGATCCTCGTCCTGCAGACCAGGATGGTGCCAGCAGAGCCCCACT
TGAAGCGCCAACTCCCACCGATCCCCAGCAATAATGAGGCACCCTCCCTGTTCCCGGGTGTCCATGG
AGGCCACGTGGGGAACCCGGACTTCCACTCTCATCTAGCAGAAGTTCTCGCCGTTCAGTTGCTGGCT
GGGTCCCTGCTCTTCTCAGCCAGGGCCATGCCGCAGGCCAGCACAGCAGCCATTTCCCTTTTGGCTC
CTACCAGTTTTGCCCCTTTTCCTGATGATATTTCTCAGGGCATACTTTCATCCTCTACTGCACATCA
AGGCAGCCCCCAGGGGTGGCAAAAGCTGCTGTTTTTCACAGCCATCCCTAATAAAACCACTGTGATG
GTCACGGTGGAGCCCCAGGACATGACAGTGAGATCTGGGGATGACGTGGCCCTGCGGTGCCAGGCCA
CTGGAGAGCCCACACCCACCATTGAATGGCTACAGGCGGGTCAACCCTTGCGGGCCAGCCGGCGGCT
64
CA 02443770 2003-10-15
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CCGGACCCTGCCCGATGGGAGCCTGTGGCTGGAGAACGTGGAGACTGGGGATGCAGGCACCTACGAC
TGCGTCGCTCACAACCTCCTGGGCTCTGCCACAGCCCGGGCGTTCCTGGTCTGTGCCAGCCACGCCA
TCGTGGGCTCCCGGCATTTCAGAGACCCACAGGTCTTCTGTGAGTTTGTGGTCCCGCCTCCTCATTT
TACAGGGGAGCCCCAGGGGAGCTGGGGCAGCATGACTGGGGTGATAAATGGCCGGAAATTTGGCGTG
GCCACACTCAACACCAGCGTGATGCAGGAGGCACACTCCGGGGTCAGCAGCATCCACAGCAGCATCC
GCCATGTCCCAGCAAACGTGGGGCCTCTGATGCGGGTGCTCGTGGTCACCATCGCCCCCATCTACTG
GGCCCTGGCCAGAGAGAGTGGGGAAGCCCTGAATGGCCACTCTCTGACTGGGGGCAGGTTCCGGCAG
GAGTCACACGTGGAGTTTGCTACAGGGGAGCTGCTCACGATGACCCAGGTGGCCCGGGGTCTGGATC
CCGATGGCCTCCTGCTCCTCGACGTGGTGGTCAATGGCGTTGTCCCCGAGAGCCTGGCTGACGCAGA
TCTTCAAGTGCAGGACTTTGAGGAGCACTACGTGCAAACAGGGCCTGGCCAGCTGTTCGTGGGCTCC
ACACAGCGCTTCTTCCAGGGCGGCCTCCCCTCGTTCCTACGCTGCAACCACAGCATCCAGTACAACG
CGGCCCGGGGCCCCCAGCCCCAGCTGGTGCAGCACCTGCGGGCCTCAGCTATCAGCTCGGCCTTTGA
TCCAGAGGCCGAGGCCCTGCGCTTCCAGCTCGCTACAGCCCTGCAGGCGGAGGAGAACGAGGTCGGC
TGCCCCGAGGGCTTTGAGCTGGACTCCCAGGGAGCGTTTTGTGTGGACAGGGACGAGTGCTCAGGAG
GCCCTAGCCCCTGCTCCCATGCCTGCCTTAATGCACCCGGCCGCTTCTCCTGCACCTGCCCCACTGG
CTTCGCCCTGGCCTGGGATGACAGGAACTGCAGAGATGTGGACGAGTGTGCGTGGGATGCTCACCTC
TGCCGAGAGGGACAGCGCTGTGTGAACCTGCTCGGGTCCTACCGCTGCCTCCCCGACTGTGGGCCTG
GCTTCCGGGTGGCTGATGGGGCCGGCTGTGAAGATGTGGACGAATGCCTGGAGGGGTTGGACGACTG
TCACTACAACCAGCTCTGCGAGAACACCCCAGGCGGTCACCGCTGCAGCTGCCCCAGGGGTTACCGG
ATGCAGGGCCCCAGCCTGCCCTGCCTAGATGTCAATGAGTGCCTGCAGCTGCCCAAGGCCTGCGCCT
ACCAGTGCCACAACCTCCAGGGCAGCTACCGCTGCCTGTGCCCCCCAGGCCAGACCCTCCTTCGCGA
CGGCAAGGCCTGCACCTCACTGGAGCGGAATGGACAAAATGTGACCACCGTCAGCCACCGAGGCCCT
CTATTGCCCTGGCTGCGGCCCTGGGCCTCGATCCCCGGTACCTCCTACCACGCCTGGGTCTCTCTCC
GTCCGGGTCCCATGGCCCTGAGCAGTGTGGGCCGGGCCTGGTGCCCTCCTGGTTTCATCAGGCAGAA
CGGAGTCTGCACAGACCTTGACGAGTGCCGCGTGAGGAACCTGTGTCAGCACGCCTGCCGCAACACT
GAGGGCAGCTACCAGTGCCTGTGCCCCGCCGGCTACCGTCTGCTCCCCAGCGGGAAGAACTGCCAGG
ACATCAACGAGTGCGAGGAGGAGAGCATCGAGTGTGGACCCGGCCAGATGTGCTTCAACACCCGTGG
CAGCTACCAGTGTGTGGACACACCCTGTCCTGCCACCTACCGGCAGGGCCCCAGCCCTGGGACGTGC
TTCCGGCGCTGCTCGCAGGACTGCGGCACGGGCGGCCCCTCTACGCTGCAGTACCGGCTGCTGCCGC
TGCCCCTGGGCGTGCGCGCCCACCACGACGTGGCCCGCCTCACCGCCTTCTCCGAGGTCGGCGTCCC
CGCCAACCGCACCGAGCTCAGCATGCTGGAGCCCGACCCCCGCAGCCCCTTCGCGCTGCGTCCGCTG
CGCGCGGGCCTTGGCGCGGTCTACACCCGTCGCGCGCTCACCCGCGCCGGCCTCTACCGGCTCACCG
TGCGTGCTGCGGCACCGCGCCACCAAAGCGTCTTCGTCTTGCTCATCGCCGTGTCCCCCTACCCCTA
CTAA
Variant sequences of NOV9 are included in Example 3, Table 21. A variant
sequence
can include a single nucleotide polymorphism (SNP). A SNP can, in some
instances, be
referred to as a "cSNP" to denote that the nucleotide sequence containing the
SNP originates
as a cDNA.
The NOV9 protein (SEQ m N0:18) encoded by SEQ 1D N0:17 is 3931 amino acid
residues in length and is presented using the one-letter amino acid code in
Table 9B. Psort
analysis predicts the NOV9 protein of the invention to be localized at the
plasma membrane
IO with a certainty of 0.7300.
Table 9B. Encoded NOV9 protein sequence (SEQ ID N0:18)
MSALFAAVYQMLKPRLVHNSPHPVTYQIEASLKPEQPGVTLVSIPVFLAPSWHKASELIPTQSFR
AQGAGKQLLGSPCPQVPPSIREDGRKANVSGMAGQSLTLECDANGFPVPETVWLKDAQLIPKVGG
HRLLDEGQSLHFPRIQEGDSGLYSCRAENQAGTAQRDFHLLVLTPPSVLGAGAAQEVLGLAGADV
ELQCWTSGVPTPQVEWTKDRQPVLPGGPHLQVQEDGQVLRITGSHVGDEGRYQCVAFSPAGQQAR
DFQLRVHAPPTIWGSNETGEVAVMEDHLVQLLCEARGVPTPNITWFKDGALLPTSTKVVYTRGGR
QLQLGRAQSSDAGVYTCKASNAVGAAEKATRLDVYVPPTIEGAGGRPYWKAVAGRPVALECVAR
GHPSPTLSWHHEGLPVAESNESRLETDGSVLRLESPGEASSGLYSCVASSPAGEAVLQYSVEVQV
CA 02443770 2003-10-15
WO 02/085922 PCT/US02/11634
PPQLLVAEGLGQVTTIVGQPLELPCQASGSPVPTIQWLQNGRPAEELAGVQVASQGTTLHIDHVE
LDHSGLFACQATNEAGTAGAEVEVSVHEFPSVSIIGGENITAPFLQPVTLQCIGDGVPTPSLRWW
KDGVALAAFGGNLQIEKVDLRDEGIYTCAATNLAGESKREVALKVLVPPNIEPGPVNKAVLENAS
VTLECLASGVPPPDVSWFKGHQPVSSWMGVTVSVDGRVLRIEQAQLSDAGSYRCVASNVAGSTEL
RYGLRVNVPPRITLPPSLPGPVLVNTPVRLTCNATGAPSPTLMWLKDGNPVSPAGTPGLQVFPGG
RVLTLASARASDSGRYSCVAVSAVGEDRQDWLQVHMPPSILGEELNVSWANESVALECQSHAM
PPPVLSWWKDGRPLEPRPGVHLSADKALLQVDRADWDAGHYTCEALNQAGHSEKHYNLNVWGQP
LPGEGAGLQHVSAVGRLLYLGQAQLAQEGTYTCECSNWGNSSQDLQLEVHVPPQIAGPREPPTQ
VSWQDGVATLECNATGKPPPTVTWERDGQPVGAELGLQLQNQGQSLHVERAQAAHTGRYSCVAE
NLAGRAERKFELSVLVPPELIGDLDPLTNITAALHSPLTLLCEAMGIPPPAIRWFRGEEPVSPGE
DTYLLAGGWMLKMTQTQEQDSGLYSCLASNEAGEARRNFSVEVLVPPSIENEDLEEVIKVLDGQT
AHLMCNVTGHPQPKLTWFKDGRPLARGDAHHISPDGVLLQVLQANLSSAGHYSCIAANAVGEKTK
HFQLSVLLAPTILGGAEDSADEEVTVTVNNPISLICEALAFPSPNITWMKDGAPFEASRNIQLLP
GTHGLQILNAQKEDAGQYTCWTNELGEAVKNYHVEVLIPPSISKDDPLAEVGVKEVKTKVNSTL
TLECESWAVPPPTIRWYKDGQPVTPSSRLQVLGEGRLLQIQPTQVSDSGRYLCVATNVAGEDDQD
FNVLIQVPPMFQKVGDFSAAFEILSREEEARGGVTEYREIVENNPAYLYCDTNAIPPPDLTWYRE
DQPLSAGDEVSVLQGGRVLQIPLVRAENAGRYSCKASNEVGEDWLHYELLVLTPPVILGDTEELV
EEVTVNASSTVSLQCPALGNPVPTISWLQNGLPFSPSPRLQVLEDGQVLQVSTAEVADAASYMCV
AENQAGSAEKLFTLRVQGLDLEQVTAILNSSVSLPCDVHAHPNPEVTWYKDSQALSLGEEVFLLP
GTHTLQLGRARLSDSGMYTCEALNAAGRDQKLVQLSVLVPPAFRQAPRGPQDAVLVRVGDKAVLS
CETDALPEPTVTWYKDGQPLVLAQRTQALRGGQRLEIQEAQVSDKGLYSCKVSNVAGEAVRTFTL
TVQVPPTFENPKTETVSQVAGSPLVLTCDVSGVPAPTVTWLKDRMPVESSAVHGWSRGGRLQLS
RLQPAQAGTYTCVAENTQAEARKDFWAVLVAPRIRSSGVAREHHVLEGQEVRLDCEADGQPPPD
VAWLKDGSPLGQDMGPHLRFYLDGGSLVLKGLRASDAGAYTCVAHNPAGEDARLHTVNVLVPPTI
KQGADGSGTLVSRPGELVTMVCPVRGSPPIHVSWLKDGLPLPLSQRTLLHGSGHTLRISKVQLAD
AGIFTCVAASPAGVADRNFTLQVQVPPVLEPVEFQNDWWRGSLVELPCEARGVPLPLVSWMKD
GEPLLSQSLEQGPSLQLEAVGAGDSGTYSCVAVSEAGEARRHFQLTVMEPPHIEDSGQPTELSLT
PGAPMELLCDAQGTPQPNITWHKDGQALTRLENNSRATRVLRVENVQVRDAGLYTCLAESPAGAI
EKSFRVRVQAPPNIVGPRGPRFWGLAPGQLVLECSVEAEPAPKITWHRDGIVLQEDAHTQFPER
GRFLQLQALSTADSGDYSCTARNAAGSTSVAFRVEIHTVPTIRSGPPAVNVSVNQTALLPCQADG
VPAPLVSWRKDRVPLDPRSPRATPIHSRFEILPEGSLRIQPVLAQDAGHYLCLASNSAGSDRQGR
DLRVLEPPAIAPSPSNLTLTAHTPALLPCEASGSPKPLWWWKDGQKLDFRLQQGAYRLLPSNAL
LLTAPGPQDSAQFECWSNEVGEAHRLYQVTVHVPPTIADDQTDFTVTMMAPWLTCHSTGIPAP
TVSWSKAGAQLGARGSGYRVSPSGALEIGQALPIHAGRYTCSARNSAGVAHKHVFLTVQASPWK
PLPSWRAVAEEEVLLPCEASGIPRPTITWQKEGLNVATGVSTQVLPGGQLRIAHASPEDAGNYL
CIAKNSAGSAMGKTRLWQVPPVIENGLPDLSTTEGSHAFLPCKARGSPEPNITWDKDGQPVSGA
EGKFTIQPSGELLVKNLEGQDAGTYTCTAENAVGRARRRVHLTILVLPVFTTLPGDRSLRLGDRL
WLRCAARGSPTPRIGWTVNDRPVTEGVSEQDGGSTLQRAAVSREDSGTYVCWAENRVGRTQAVSF
VHVKEAPVLQGEAFSYLVEPVGGSIQLDCWRGDPVPDIHWIKDGLPLRGSHLRHQLQNGSLTIR
RTEARRGLAPWRDDAGRYQCLAENEMGVAKKWILVLQTRMVPAEPHLKRQLPPIPSNNEAPSLF
PGVHGGHVGNPDFHSHLAEVLAVQLLAGSLLFSARAMPQASTAAISLLAPTSFAPFPDDISQGIL
SSSTAHQGSPQGWQKLLFFTAIPNKTTVMVTVEPQDMTVRSGDDVALRCQATGEPTPTIEWLQAG
QPLRASRRLRTLPDGSLWLENVETGDAGTYDCVAHNLLGSATARAFLVCASHAIVGSRHFRDPQV
FCEFWPPPHFTGEPQGSWGSMTGVINGRKFGVATLNTSVMQEAHSGVSSIHSSIRHVPANVGPL
MRVLWTIAPIYWALARESGEALNGHSLTGGRFRQESHVEFATGELLTMTQVARGLDPDGLLLLD
WVNGWPESLADADLQVQDFEEHYVQTGPGQLFVGSTQRFFQGGLPSFLRCNHSIQYNAARGPQ
PQLVQHLRASAISSAFDPEAEALRFQLATALQAEENEVGCPEGFELDSQGAFCVDRDECSGGPSP
CSHACLNAPGRFSCTCPTGFALAWDDRNCRDVDECAWDAHLCREGQRCVNLLGSYRCLPDCGPGF
RVADGAGCEDVDECLEGLDDCHYNQLCENTPGGHRCSCPRGYRMQGPSLPCLDVNECLQLPKACA
YQCHNLQGSYRCLCPPGQTLLRDGKACTSLERNGQNVTTVSHRGPLLPWLRPWASIPGTSYHAWV
SLRPGPMALSSVGRAWCPPGFIRQNGVCTDLDECRVRNLCQHACRNTEGSYQCLCPAGYRLLPSG
KNCQDTNECEEESIECGPGQMCFNTRGSYQCVDTPCPATYRQGPSPGTCFRRCSQDCGTGGPSTL
QYRLLPLPLGVRAHHDVARLTAFSEVGVPANRTELSMLEPDPRSPFALRPLRAGLGAWTRRALT
RAGLYRLTVRAAAPRHQSVFVLLIAVSPYPY
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 9C.
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Table 9C. Patp results for NOV9
Smallest
Sum
eadingigh Prob
equences FrameScore P(N)
producing
High-scoring
Segment
Pairs:
>patp:AAY53667Sequence gi/3328186 +1 1529 6.5e-244
>patp:AAY87206Human secreted protein sequence +1 2235 6.4e-230
ID N0:245
>patp:AAE06183Human gene 57 encoded secreted +1 2235 6.4e-230
protein
>patp:AAY87120Human secreted protein sequence +1 2235 6.4e-230
SEQ ID:159
>patp:AAE06097Human gene 57 secreted protein +1 2235 6.4e-230
HRACD80
In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 625 of 1067 bases (58%) identical
to a
gb:GENBANK-ID:HSLTGFBP4~acc:Y13622.1 mRNA from Homo sapieszs (mRNA for latent
transforming growth factor-beta binding protein-4). The full amino acid
sequence of the
protein of the invention was found to have 502 of 1665 amino acid residues
(30%) identical to,
and 767 of 1665 amino acid residues (46%) similar to, the 5198 amino acid
residue
ptnr:SPTREMBL-ACC:076518 protein from Cae~orhabditis elegaf~.s (HEMICENTIN
PRECURSOR).
NOV9 also has homology to the proteins shown in the BLASTP data in Table 9D.
Table 9D. BLAST
results for
NOV9
Gene Index/ Protein/ OrganismLength Identity PositivesExpect
Identifier (aa) (%) (%)
gi~14575679~gb~AAK6hemicentin 5636 1230/30171785/30170.0
8690.1~AF156100 [Homo Sapiens] (40%) (58%)
1
(AF156100)
gi118547943~reflXP_hemicentrin 3645 979/2379 1413/23790.0
053531.3~(XM [Homo Sapiens] (4l%) (59%)
053531
gi~17568539~refINP_Ig superfamily 5175 857/3077 1348/30770.0
509636.1~(NM_077235repeats (I-type) (27%) (42%)
[Caenorhabditis
e1 egans]
gi~17568541IrefINP_IG 5198 857/3077 1348/30770.0
509635.1I(NM (immunoglobulin) (27%) (42%)
077234
superfamily
(47
domains)
[Caenorhabditis
e1 egans]
gi~13872813~emb~CACfibulin-6 2673 552/1399 796/1399 0.0
37630.1~(AJ306906)[Homo Sapiens] (39%) (56%)
A multiple sequence alignment is given ix~ Table 9E, with the NOV9 protein
being
shown on line 1 in Table 9E in a ClustalW analysis, and comparing the NOV9
protein with the
related protein sequences shown in Table 9D. This BLASTP data is displayed
graphically in
the ClustalW in Table 9E.
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Table 9E. ClustalW Analysis of NOV9
1) > NOV9; SEQ ID N0:18
2) > gig 14575679~J hemicentin [Homo Sapiens]; SEQ 1D N0:72
3) > gi~18547943~/ hemicentrin [Homo Sapiens]; SEQ ID N0:73
4) > gig 17568539/ Ig superfamily repeats (I-type) [Caeraorlaabditfs
elegaras]; SEQ ID N0:74
5) > gig 17568541 ~/ IG (inununoglobulin) superfamily (47 domains)
[Caenorlaabditis elegans]; SEQ ID
N0:75
6) > gi~13872813~/ fibulin-6 [Homo Sapiens]; SEQ ID N0:76
4090 4130 4140
4100
4110
4120
..J....J....J....J....J.. ,.J....J. .
NOV9 2602 _________________________ _________ ____
2621
giJ14575679J3929 _________________________ _________ ____
3948
giJ18547943J1938 _________________________ _________ ____
1957
giJ17568539J3248 TRADEGKYSCIASNEAGTAVADFLI DVFTKPTFE THET
3307
giJ17568541J3248 TRADEGKYSCIASNEAGTAVADFLI DVFTKPTFE THET
3307
giJ13872813J966 _________________________ _________ ____
985
4150 4190 4200
4160
4170
4180
....J....J....J....J....J....J....J....J.. .J.,
J....J . .J
NOV9 2621---_______________________________________p
. GQ~,PI . 2639
..~
giJ14575679J3948-___,_____________________________________S
.~ TEL 3966
giJ18547943J1957-_________________________________________S
~ TvL '
1975
giJ17568539J3308TFNIVEGESAKIECKIDGHPKPTISWLKGGRPFNMDNIILSPRGDT
RFD L
3367
giJ17568541J3308TFNIVEGESAKIECKIDGHPKPTISWLKGGRPFNMDNIILSPRGDT
RFD L
3367
giJ13872813J985 __________________________________________S
TvL 1003
~.I
4210 4250 4260
4220
4230
4240
NOV9 2640 2698
giJ14575679J 3967 4025
giJ18547943J 1976
2034
giJ17568539J 3368 3427
giJ17568541J 3368 3427
giJ13872813J 1004 1062
4270 4280 4290 4300 4310 4320
NOV9 2699 2756
giJ14575679J 4026 4085
giJ18547943J 2035 2094
giJ17568539J 3428 3486
giJ17568541J 3428 3486
giJ13872813J 1063 1122
4330 4340 4350 4360 4370 4380
....~....J....J....J,...J....J....J....J...,J....J....J....J
NOV9 2756 2759
giJ14575679J 4086 4143
ga.J18547943~ 2095 2152
giJ17568539J 3487 3546
giJ17568541J 3487 3546
giJ13872813J 1123 1180
4390 4400 4410 4420 4430 4440
.J....J,...J....J:..,J....J....J....J. .J.. .J....J.. .J
NOV9 2759 __________________ _______ ___ DLTT~~ ~1F ~ ~ S~E~ 2783
H' I v VI r t ~ Vm
gi J 14575679 J 4144 T ~ ~ S STST Ti~H ~ RS'I!--E Y , v Q~iI ~ 'D ~T ~ v 4201
gi J 18547943 J 2153 T ' S STST T~~Ii ' RS,~--E ~Y_' QiI ~ iD ~T~ 2210
gi J 17568539 J 3547 I R~iS~E ~ T DIDLI ILL ' DIt'~'NTI PLAIVARTIY E PIS ~Q
~D'S~ 3606
giJ17568541J 3547 ' I RfiS EI~rT DIDLI ~L~~~ DKNTI PLAIVARTIY E PIS ~Q~ 3606
gi J 13872813 ~ 1181 H T ~ ~ S STST TtlIi ~ ~ RS!~--E YiT'VN~Qi~',T ~ iD ~T ~
1238
4450 4460 4470 4480 4490 4500
.J....J....J . . .. .. J....J....J...
NOV9 2784 T~D~GQP~IS,GAE~F'~IQ~S~-QLVK~LEGQ~T~TyE ~ ' ~RARRR~fi~I,L~ 2842
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gi~14575679~ 4202 4260
gi1185479431 2211 2269
gi1175685391 3607 3666
gi1175685411 3607
gi1138728131 1239 3666
1297
4510 4520 4530 4540 4550 4560
NOV9 2843 ~...T.:. I..RL1. .1. .~.~S~~____T ~I.~1 'RP~'TEGVSE-I--Q~I 2892
gi1145756791 4261 'T E ' r - ~Q S T I~-----~~KL F I PAHFDS---- 4310
gi1185479431 2270 ~T E ' r - ~Q~ S I'T I~---- 'KL, F~~I~PAHFDS--- 2319
gi ~ 17568539 ~ 3667 ~E DIHGTQP 'KEE ;T~T,T T PIKLAEDIADQ~; . D~t7~
KDRAI7GDLTDNVDIS~J17 3726
gi ~ 27568541 ~ 3667 ~E IHGTQPi~~.KRE TAT T PIKLAEDIADQ'~~'ID;tTS
K'~,~RA~iDGDLTDNVDIS~77D 3726
gi~13872813~ 1298 ~T E ' r 4~ DQfl S It~~lTneI~-____~,. gIpp,HFDS-___~ 1347
a
4570 4580 4590 4600 4610 4620
NOV9 2893 2951
gi1145756791 4311 4369
gi1185479431 2320 2378
gi1175685391 3727 3786
gi1175685411 3727 3786
gi1138728131 1348 1406
25 4630 4640 4650 4660 4670 4680
NOV9 2952 3010
gi1145756791 4370 4420
gi1185479431 2379 2429
30 gi~17568539~ 3787 3838
gi1175685411 3787 3838
gi1138728131 1407 1457
4690 4700 4710 4720 4730 4740
NOV9 3011 3062
gi1145756791 4421 4480
gi1185479431 2430 2489
gi1175685391 3839 3898
gi1175685411 3839 3898
gi1138728131 1458 1517
4750 4760 4770 4780 4790 4800
.'....,....1,...1....1....1....1....1. .1....1,.. .1....1
NOV9 3063 t~GNPr-------------------------------FH- LA'S~QL_' ------ 3083
gi1145756791 4481 SWDrR --S Y A~ ~ErTSF'E ~ L ~P~I Q GF----- 4533
1 7 v r'
gi ~ 18547943 ~ 2490 SWDrR ' Y 'yV--S Y ~A ErTS~.~'E V.~',' L V V P ~Q ~l GF---
-- 2542
gi1175685391 3899 FDSPDGAR,'~ LKG PHL ~Tr GI?Y'T Q~L 'SEAS S~~L PPEINR17GI
3958
gi 113872813 ~ 1518 SWD~RGLKG-A~ PHL[',J ;E~TS~E!E Q~LI L SEAS ~
P==~~''~~~''Q~~GFNRDGI 1570
4810 4820 4830 4840 4850 4860
.1. ..1. ..1....1....1....1. .1....1. ..1. I . .~.. .1
NOV9 3083 -- LLFj" .PQAST SLL~PTSF F~~IS~GILS~STAHQGSP ------ - 3132
gi I 14575679 ~ 4533 -- WSiiIW~ ~ C~'T GK ~IxCNQ L'L~ GG PCQ DIsEMR'~CQNPCP -
D'S 4589
gi ~ 18547943 I 2542 - ~WS~i~ 'CSV'T GK QR~CNQ L~ GC~PCQ D~;EM~CQNPCP -D S
2598
giI175685391 3959 DMSPLP~iQ L'~T.Q LAQG~PVPQ1~RWTLNGTALTHSTP ITASDGTFII SLSD
4018
g7. 175685411 3959 DMSP ~LP~iQ~~v"'L~'IiQ LAQGT~PVPQR.WTLNGTALTHSTP
IT~,ASDixTFI"~3I 'L, SLSD 4018
gi 113872813 ~ 1570 ---~5~'~" C~S"''~T GKGQ~KRR~iCNQ~LGG~T~'PCQ
~iEM'k'i.2~CQNIfPCP -D~S 1626
4870 4880 4890 4900 4910 4920
~. _..~....~....~....~....~....~
NOV9 3132 - --~QLLCLLFFTAIP~~ --------------- ----- T: TUE'---Q----- 3155
gi 114575679 ~ 4590 WSE~fL E ~CTRSCGRGi Q RTRTCNNPSVQH GPCEGNA'VE~~ ' ~C~ GA---
4646
gi118547943~ 2599 WSE~IL CTRSCGRG~QBRTRTCNNPSVQ GPCEGNAiSTE~ ,11 ~C~ GA---
2655
gi1175685391 4019 KGV~TC~I,~, AGSDNLMYNVDWQAPVISN T'KQVIEGE E L~EGY' PQVSWL
4078
gi1175685411 4019 KGVY','~CAGSDNLMYNVDWQAPVISN T~QVIEGE~ E L~EGY~ PQVSWL 4078
gi 113872813 1 1627 WSETL~ CTRSCGRG~f~RTRTCNNPSVQH~eG ,PCEGNA3 ,ELI 3 !R~C~ GA-
-- 1683
4930 4940 4950 4960 4970 4980
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NOV9 3155 ----------------D-------M'VRS DVAL~CATGE~-------------T~TI 3179
gi~14575679~ 4646 ------------ W~'v'AWQPWGTCESC KGTQT' r~LC~ ------------P~ F
4678
gi~18547943~ 2655 ------------ WAW~PWGTCESC KGTQT' _LC ~-------------P~ F 2687
gi~175685391 4079 RNGNRVETGVQGVRYVi!xDG~~MLTIIEARSLDSGIYLCSAT
EAGSAQQAYTLEVLVS~KI 4138
gi~17568541~ 4079 RNGNRVETGVQGVRYV"~'DGMLTIIEARSLDSGIYLCSATGSAQQAYTLEVLVS~KI
4138
gi~13872813~ 1683 ------------ WAWPWGTCESC~KGTQT~LC -----------P~ F 1715
4990 5000 5010 5020 5030 5040
IO
NOV9 3179 -----------------------------E-~LQAGQPLRAS~~L'.LPDGSLWLN--- 3206
gi~14575679~ 4679 G YCDGAETQMQVCNE P~~H KWATW S,,,~ACSVSCG 'Q~ CSDPV~~_~P~YGGR
4738
gi~185479431 2688 G YCDGAETQMQVCNE ' P;IFi KWATW ~~,,,AJCSVSCG _'Q~
CSDP~,PYGGR 2747
gi~17568539~ 4139 IT TPGVLTPSSGSKFSLP '~ YPDPII '15LNGNDIKD ENG I GT~HIEKAE
4198
IS gi~175685411 4139 IT TPGVLTPSSGSKFSLP ~YPDPII 1,,:?~LNGNDIKD ENGH,I
GT~~'.rHIEKAE 4198
gi~13872813~ 1716 G YCDGAETQMQVCNE P~ KWATW ~ACSVSCG 'Q' CSDP~PYGGR 1775
5050 5060 5070 5080 5090 5100
NOV9 3206 - ----- '~GDAGYD . -----------LLGSAT ' L ------ 3236
V ~~;
gi ~ 14575679 I 4739 ---C G~sD~DFC~~P ~~ ~ ~ SGWGTCSRTCNGGQ ~RRT ~P ----- 4788
gi ~ 18547943 ~ 2748 ~----C G~~~~'D1(QDFC~TDP T G ~ ~ SGWGTCSRTCNGGQ ~R'~.'RT
, P------ 2797
gi~17568539~ 4199 e'RHLIY CAKNDAGA~~"'LEF~ QTIU ~KISTSGNRYINGSEGTETVI
°IESESSEF 4258
gi ~ 17568541 ~ 4199 ~RHLI C~'I'AI~NDAGA~.'?~'LEF ~ QTIV KISTSGNRYINGSEGTETVI
IESESSEF 4258
gi~13872813~ 1776 ----C G~D~~DFC~IDP ~T~G~W,~~WSGWGTCSRTCNGGQM~'yIR~RT~P------
1825
5110 5120 5130 5140 5150 5160
NOV9 3236 -HAI S'HFRD ___________ _________ __________ -VF ~. ~ 3257
r ~ ,~, r r
3~ gi ~ 14575679 ~ 4788 -PPS CG ~SQIQRCNTD1~CP~D~GSWGSWHS~ QCiTS,C GEKT~ IiP ~
4847
gi~18547943~ 2797 -PPS ' CG ~SQIQRCNTD CP~GSWGSWHSQC~~C GEKT ~ 'HP ~ 2856
gi~17568539~ 4259 SWS PLLPSNLIFSEDYKL3~KI~t,(STRLSDQGE~, CTATQT ~GV ~ 4318
gi~17568541~ 4259 SWS PLLPSN~LIFSEDYKL~KISTRLSDQGE'~ CTS ~ ATQ~T GV 4318
gi ~ 13872813 ~ 1825 -PPS~G~ACGG~SQIQRCNTDCP~ST,DGSWGSWHS~J~QC~~~EKT;HP~ 1884
NOV9 3257 3292
giI14575679~ 4848
gi~7.8547943~ 2857 4900
2909
gi~17568539~ 4319 4378
gi~17568541~ 4319 4378
gi~13872813~ 1885 1937
5230 5240 5250 5260 5270 5280
.,..I
NOV9 3293 ----- 3345
gi~145756791 4901 ----- 4953
gi~18547943~ 2910 ----- 2962
gi~17568539~ 4379 TGIPE 4438
gi~17568541~ 4379 TGIPE 4438
gi~13872813~ 1938 ----- 1990
5290 5300 5310 5320 5330 5340
NOV9 3345 3400
gi~14575679~ 4953 5008
gi~18547943~ 2962 3017
gi~17568539~ 4439 4498
gi~17568541~ 4439 4498
gi~13872813~ 1990 2045
5350 5360 5370 5380 5390 5400
NOV9 3400 ------- F'~E ' yV ~1 FQ . L(PSFLRCi S~:Q ~ PQ ~Q . Q 3452
a r ...-
gi ~ 14575679 ~ 5008 ------- Y'T D Iv ~ ~A ~L I ~ IISIPYT TUF L3Qi ~F E<T 5060
giI185479431 3017 _______ ,~X':T D ~~ ~ Y'AY 'L I~ I',SIPYT T'~F yQil
~,Q~ ~F T~ 3069
giI17568539~ 4499 PSDRPAPI:; CDE~ICG ~KI~TEYMID~GD P~DNPQLLP
KDVEDSSLNGSIAYRC~PGP 4558
gi~17568541~ 4499 PSDRPAPI,, CDE~CG KI~TEYMID'GD P~ NPQLLP KDVEDSSLNGSIAYRC.
PGP 4558
gi113872813~ 2045 _______ yT ~~~ ~ v ~;AY 'L I~ I~SIPYT 'T''()F~_Qi~RMOFmE~T~
2097
5170 5180 5190 5200 5210 5220
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5410 5420 5430 5440 5450 5460
L, ~ ~ L,. . . I . I . I . I . . . I . . . . I . . . I . . . I . . I . . . I.
NOV9 3453 . ~ TS ~'~PE ~ . Q~.,iAT,~QAENEV . 'E ------ . E n Q n- n S 3505
gi I 14575679 ~ 5061 H~ S'ClIE Y~VQI IHAI~~SKG1~RSNQ 'S ----- ~ P ~-y ' 5113
gi~185479431 3070 H~ SUE D~QI~~T~ ~~HA~"~SKG~1RSNQ 'S ----- ~ P ~-W ~ 3122
gi1175685391 4559 R~ RTVLL~ P~FI~KP TT IGAIVEL S~1AGPPHPTI AKD KLIE~S~F I,~~,
4618
gi 1175685411 4559 R~ RTVLL~P~FI'U'KP TT~IGATVEL3 Sn~p,'AGPPHPTI AKD
KLIE~SIfiF Ii1 4618
gi 113872813 I 2098 H~ S~E~DY.~TQI~T~ ~I'HA~~'SKGRSNQ~S~e----- ~ P ~-~ ' 2150
5470 5480 5490 5500 5510 5520
NOV9 3506 3565
gi1145756791 5114 5172
gi1185479431 3123 3181
gi1175685391 4619 4677
gi1175685411 4619 4677
gi1138728131 2151 2209
5530 5540 5550 5560 5570 5580
.I. .~. ..I....I....I....I....I....I....I....I....I....I
NOV9 3566 '~xiPD .P _______________-________________________________ 3576
gi1145756791 5173 ~ S ~RTS ~i---------------------- Cf~~IECQESPCHQ~CF1~ 5209
gi~18547943~ 3182 ~ S ~RTS~ T~----------------------- C~~IECQESPCHQ'CF3218
gi1175685391 4678 ERNQAYS LTWE ~ ~PMPKNLAGIHFMNNGSLVILDTS L~GLELY(~'CKV 'R 3
4737
gi117568541~ 4678 ERNQAYS LTWE ~ PMPKNLAGIHFMNNGSLVILDTS L~G~LELY'CKV 'RF~
4737
V V ~
gi I 13872813 ~ 2210 ~t~(1R~S 'RTS-----------------------
CE~7~IECQESFSPCHQ~'yCF2246
5590 5600 5610 5620 5630 5640
30 ....I....I....I....I....I....1....1....1....1....1....1....1
NOV9 3576 -___________________________________________________________ 3576
gi1145756791 5210 5262
gi~185479431 3219 3271
gi~17568539~ 4738 4797
35 gi1175685411 4738 4797
gi1138728131 2247 2299
5650 5660 5670 5680 5690 5700
.I.. .I... I . .I. .I. .I ...I. ..I. .1....1....I...
NOV9 3577 ADGAG- E~'n L.LDD~. ~.'. PGGv '.S ~'~------------------ 3616
gi114575679i 5263 TKAEG ~W ICC7 T f1 vT RSS,' ~~ __________________ 5304
gii185479431 3272 ~TKAE'GT yI~KT~Q ~~ RGSG,..' ----------------- 3313
gi1175685391 4798 ~LPNNLVL~:~YDANSIGKAFDDTLNVYG------------------- 4838
gi1175685411 4798 ~tLPNNLVL~ICYD~ V~~ ANSIGKAFDDTLNVYE FLPLTGFEGSGINIDDS 4857
gi 113872813 I 2300 TKAE~tGT~T~~ .-, T,~IR~I~RGS----------------- 2341
5710 5720 5730 5740 5750 5760
....I.
NOV9 3616 ------ 3665
50 gi1145756791 5304 ------ 5353
gi~185479431 3313 ------ 3362
gi1175685391 4838 ----GS 4894
gi1175685411 4858 SNAGGS 4917
gi1138728131 2341 ------ 2390
5770 5780 5790 5800 5810 5820
NOV9 3666 3718
gi1145756791 5354 5413
gi118547943~ 3363 3422
gi117568539~ 4895 4952
gi117568541~ 4918 4975
gi113872813~ 2391 2450
5830 5840 5850 5860 5870 5880
.. .I.. .1....I., .~. .~. ..~L. .I. .I. .I. .~. .I. .I
NOV9 3718 -- ' W ~P FIR--QG Tn n RVRL.~ E .~~ L.' ~~LP~u.' 3773
gi1145756791 5414 RTI'KT ~ SEA--S~DT ~I~ ENT~3 t F ~ I 'P' Q THE ~ ~T 5471
i 18547943 3423 RTI'KT ' SEA SDT ~I~ ENT v F ~ I P Q TH ~T 3480
gi1175685391 4953 TG-E FAMNPHTRI En_L~AFYQP F I Y G._,~ 'L~E ~Ee~-- 5009
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gi~17568541~ 4976TG-E
FAMNPHTRI
E~
~
FYQP
F
I
YD
G
~
L
E
--
5032
r
gi 7.3872813 2451RTI~'yKT L ~F ~ ~THT~S
~ ~ Z 2508
SEA P~Q
S~IDT~2
~
ENT~)A
~
5890
5900
5910
5920
5930
5940
NOV9 3774 3830
gi~14575679~ 5472 5531
gi~18547943~ 3481 3540
gi~17568539~ 5010 5059
gi117568541~ 5033 5082
gi~13872813) 2509 2568
5950
5960
5970
5980
5990
6000
NOV9 3831 3882
gi~14575679~ 5531 5582
gi~18547943~ 3540 3591
gi~17568539) 5060 5118
gi117568541~ 5083 5141
gi~13872813~ 2568 2619
6010 6020 6030 6040 6050
L, . . ~ ~...v., ~ . . I . . . . I . . . I .
~~ ~ 7 ~ i -
NOV9 3883 .~PLRAGL ~T ~GL1~LT " P------- .~~~ .,ltL ~ .P ~ 3931
gi~14575679~ 5583 E-NL T'P RE~ T ' '~~ YS~=-GTIE'~~ T ~~Y ' 5636
V~ i1
gi~185479431 3592 ~ E-NL T~P RE~'T ' ~~S YS,~, -GTIE ~ T IyY ~ 3645
gi1175685391 5119 ' ~AVKRGHAQ G H ~~H DH~,~,TNELHAPK~L GQ ~~ 5175
gi~17568541~ 5142 T~AVKRGHAQ ' GH~K~~H DH~TNELHAPIt~ ~L GQ ~5198
gi ~ 13872813 ~ 2620. ~ E NLI~eV~T P RE~TS~YS ' -
GTIEiY~T~~1~Y ' 2673
The NOV9 Clustal W alignment shown in Table 9E was modified to begin at amino
residue 4080. The data in Table 9E includes all of the regions overlapping
with the NOV9
35 protein sequences.
The presence of identifiable domains in the protein disclosed herein was
determined by
searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and
then
determining the Interpro number by crossing the domain match (or numbers)
using the
Interpro website (http:www.ebi.ac.uk/interpro~. Table 9F lists the domain
description from
40 DOMAIN analysis results against NOV9.
Table 9F Domain
Anal sis
of NOV9
Model Region of Score (bits) E value
Homology
ig 99-157 46.50 6.1e-10
ig 192-251 38.00 2.1e-07
ig 286-344 41.90 1.4e-08
ig 380-438 32,10 1.3e-05
ig 473-531 45.90 8.9e-10
ig 565-615 35.50 1.2e-06
ig 648-706 43.80 4e-09
ig 740-800 39.10 1e-07
ig 833-891 27.10 0.00041
ig 981-1039 42,90 7.1e-09
ig 1075-1133 28.90 0.00012
ig 1168-1226 30.80 3.2e-05
ig 1264-1322 39.60 7.2e-08
ig 1362-1420 43.40 5.2e-09
ig 1473-1531 29.40 8.2e-05
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ig 1568-1626 35.70 1e-06
ig 1654-1712 _ 1.5e-09
45.20
ig 1749-1807 43.80 3.8e-09
ig 1841-1899 43.00 6.8e-09
ig 1934-1994 41.40 2e-OS
ig 2030-2088 49.90 5.8e-11
ig 2123-2177 49.60 6.8e-11
ig 2212-2268 37.50 3e-07
ig 2303-2361 28.60 0.00014
ig 2394-2459 28.20 0.00019
ig 2492-2552 32.90 7.2e-06
ig 2585-2643 22.80 0.0081
ig 2676-2733 37.50 3.1e-07
ig 2766-2824 43.10 6.3e-09
ig 2857-2913 44.10 3e-09
ig 2947-3012 18.00 0.22
ig 3162-3219 34.70 2.1e-06
EGF 3504-3539 38.30 1.8e-07
EGF 3589-3626 13.80 1.3
EGF 3632-3667 33.30 5,4e-06
EGF 3739-3773 38.40 1.6e-07
Consistent with other known members of the Hemicentin Precursor-like family of
proteins, NOV9 has, for example, thirty-three immunoglobulin (ig) signature
sequences and
four epidermal growth factor (EGF) signature sequences, as well as homology to
other
members of the Hemicentin Precursor-like Protein Family. NOV9 nucleic acids,
and the
encoded polypeptides, according to the invention are useful in a variety of
applications and
contexts. For example, NOV9 nucleic acids and polypeptides can be used to
identify proteins
that are members of the Hemicentin Precursor-like Protein Family. The NOV9
nucleic acids
and polypeptides can also be used to screen for molecules, which inhibit or
enhance NOV9
activity or function. Specifically, the nucleic acids and polypeptides
according to the
invention may be used as targets for the identification of small molecules
that modulate or
inhibit, e.g., cellular activation, cellular differentiation, and signal
transduction. These
molecules can be used to treat, e.g., Cardiovascular diseases,
Hyperparathyroidism,
Hypoparathyroidism, Lymphedema, Allergies as well as other diseases, disorders
and
conditions..
In addition, various NOV9 nucleic acids and polypeptides according to the
invention
are useful, ifater alia, as novel members of the protein families according to
the presence of
domains and sequence relatedness to previously described proteins. For
example, the NOV9
nucleic acids and their encoded polypeptides include structural motifs that
are characteristic of
proteins belonging to the Hemicentin Precursor-like Protein Family.
Hemicentrin is an extracellular matrix protein with a modular sturcture. Like
NOV9,
the hemicentrin structure includes many immunoglobulin domains flanked by EGF
domains.
The protein is likely involved in cellular differentiation of epithelial
tissue.
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The NOV9 nucleic acids and polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and diagnostic
applications in the
mediation of cardiac, immune and endocrine physiology. As such, the NOV9
nucleic acids
and polypeptides, antibodies and related compounds according to the invention
may be used to
treat cardiovascular, immune, and endocrine disorders, e.g., Cardiovascular
diseases,
Hyperparathyroidism, Hypoparathyroidism, Lymphedema, Allergies as well as
other diseases,
disorders and conditions.
The NOV9 nucleic acids and polypeptides are useful for detecting specific cell
types.
For example, expression analysis has demonstrated that a NOV9 nucleic acid is
expressed in
Adipose, Thyroid, Colon, Lymph node, Bone, Myometrium, Prostate, Testis,
Aorta, Vein.
Additional utilities for NOV9 nucleic acids and polypeptides according to the
invention are disclosed herein.
NOV10
A NOV10 polypeptide has been identified as a Selectin-like protein. The novel
NOV10
nucleic acid sequences maps to the chromosome 9. Two alternative novel NOV10,
NOVlOa
and NOVlOb, nucleic acids and encoded polypeptides are provided.
NOVlOa
A NOV10 variant is NOVlOa (alternatively referred to herein as CG94661-Ol),
which
includes the 1268 nucleotide sequence (SEQ m N0:19) shown in Table 10A. A NOV
1 Oa
ORF begins with a ATG initiation codon at nucleotides 145-147 and ends with a
TGA codon
at nucleotides 871-873. Putative untranslated regions upstream from the
initiation codon and
downstream from the termination codon are underlined in Table 10A, and the
start and stop
codons are in bold letters.
Table 10A. NOVlOa Nucleotide Sequence (SEQ ID N0:19)
GCGGCCGCCACCCTCCGTGGCAAGGCGAGGCCCCGGGGGCGGGCCGGGGTCACCACGCCTGTCCCAG
GGAACCGCACAGACGGTACTCACCCTTCTTGCGATGATGTGAGATGATAAAATGCCTACATGATGAG
ATGAAGTGAGATGAAAAACATAGGCCTTGTGATGGAATGGGAAATTCCAGAGATAATTTGCACGTGC
GCTAAGCTGCGGCTACCCCCGCAAGCAACCTTCCAAGTCCTTCGTGGCAATGGTGCTTCCGTGGGGA
CCGTGCTCATGTTCCGCTGCCCCTCCAACCACCAGATGGTGGGGTCTGGGCTCCTCACCTGCACCTG
GAAGGGGAGCATCGCTGAGTGGTCTTCAGGGTCCCCAGTGTGCAAACTGGTGCCACCACACGAGACC
TTTGGCTTCAAGGTGGCCGTGATCGCCTCCATTGTGAGCTGTGCCATCATCCTGCTCATGTCCATGG
CCTTCCTCACCTGCTGCCTCCTCAAGTGCGTGAAGAAGAGCAAGCGGCGGCGCTCCAACAGGTCAGC
CCAGCTGTGGTCCCAGCTGAAAGATGAGGACTTGGAGACGGTGCAGGCCGCATACCTTGGCCTCAAG
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CACTTCAACAAACCCGTGAGCGGGCCCAGCCAGGCGCACGACAACCACAGCTTCACCACAGACCATG
GTGAGAGCACCAGCAAGCTGGCCAGTGTGACCCGCAGCGTGGACAAGGACCCTGGGATCCCCAGAGC
TCTAAGCCTCAGTGGCTCCTCCAGCTCACCCCAAGCCCAGGTGATGGTGCACATGGCAAACCCCAGA
CAGCCCCTGCCTGCCTCTGGGCTGGCCACAGGAATGCCACAACAGCCCGCAGCATATGCCCTAGGGT
GACCACGCAGTGAGGCTGGTGCCCATGCTCCACACTGGGAGGCCAGGCTGACCCCACCAGCCAGTCA
GCTACAACTCCACATCAACTCCACATGCGCCCAGCTCGAGACTGATGAGTGGAATCAGCTTCCAGGT
GTAGGGACCCCTTGAGGGGCCGAGCTGACATCCAAGGCTGAGGACCCCAGTGGGGAGTGTTCTGTTC
CGGCATATCCTGGCCGTAACGATTTTTATAGTTATGGACTACTTGAAACCACTACTGAGGGTAATTT
ACTAGCTGTGGCCTCCCACTAACTAGCATTCCTTTAAAGAGACTGGGAAATGTTTTAAGCAAATCTA
GTTTTGTATAATAAAATAAGAAAATAGCAATAAACTTCTTTTCAGCAACTACF,~~e~AAAAAAA
The NOV 10a polypeptide (SEQ ID N0:20) encoded by SEQ ID N0:19 is 242 amino
acid residues in length and is presented using the one-letter amino acid code
in Table l OB. The
Psort profile for the NOVlOa predicts that this peptide is likely to be
localized at the plasma
membrane with a certainty of 0.7000.
Table 10B. NOVlOa protein sequence (SEQ ID N0:20)
MKDTIGLVMEWEIPEIICTCAKLRLPPQATFQVLRGNGASVGTVLMFRCPSNHQMVGSGLLTCTWKGS
IAEWSSGSPVCKLVPPHETFGFKVAVIASIVSCAIILLMSMAFLTCCLLKCVKKSKRRRSNRSAQLW
SQLKDEDLETVQAAYLGLKHFNKPVSGPSQAHDNHSFTTDHGESTSKLASVTRSVDKDPGIPRALSL
SGSSSSPQAQVMVHMANPRQPLPASGLATGMPQQPAAYALG
NOVlOb
Alternatively, a NOV 10 variant is the novel NOV l Ob (alternatively referred
to herein
as CG94661-02), which includes the 887 nucleotide sequence (SEQ TD N0:21)
shown in
Table l OC. NOV l Ob was created by polymerase chain reaction (PCR) using the
primers
detailed in Example 1, Table 17. Primers were designed based on ih silico
predictions of the
full length or some portion (one or more exons) of the cDNA/protein sequence
of the
invention. The PCR product derived by exon linking, covering the entire open
reading frame,
was cloned into the pCR2.1 vector from Invitrogen to provide clone
143260::COR100348691 extn.698976.C20.
The NOVlOb ORF begins with a Kozak consensus ATG initiation colon at
nucleotides 72-74 and ends with a TGA colon at nucleotides 1958-1960. Putative
untranslated
regions upstream from the initiation colon and downstream from the termination
colon are
underlined in Table l OC, and the start and stop colons are in bold letters.
Table IOC. NOVlOb Nucleotide Sequence (SEQ ID N0:21)
GCACAGACGGTACTCACCCTTCTTGCGATGATGTGAGATGATAAAATGCCTACATGATGAGATGAAG
TGAGATGAAAAACATAGGCCTTGTGATGGAATGGGAAATTCCAGAGATAATTTGCATGTGCGCTAAG
CTGCGGCTACCCCCGCAAGCAACCTTCCAAGTCCTTCGTGGCAATGGTGCTTCCGTGGGGACCGTGC
TCATGTTCCGCTGCCCCCCCAACCACCAGATGGTGGGGTCTGGGCTCCTCACCTGCACCTGGAAGGG
GAGCATCGCTGAGTGGTCTTCAGGGTCCCCAGTGTGCAAACTGGTGCCACCACACGAGACCTTTGGC
TTCAAGGTGGCCGTGATCGCCTCCATTGTGAGCTGTGCCATCATCCTGCTCATGTCCATGGCCTTCC
TCACCTGCTGCCTCCTCAAGTGCGTGAAGAAGAGCAAGCGGCGGCGCTCCAACAGGTCAGCCCAGCT
GTGGTCCCAGCTGAAAGATGAGGACTTGGAGACGGTGCAGGCCGCATACCTTGGCCTCAAGCACTTC
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AACAAACCCGTGAGCGGGCCCAGCCAGGCGCACGACAACCACAGCTTCACCACAGACCATGGTGAGA
GCACCAGCAAGCTGGCCAGTGTGACCCGCAGCGTGGACAAGGACCCTGGGATCCCCAGAGCTCTAAG
CCTCAGTGGCTCCTCCAGCTCACCCCAAGCCCAGGTGATGGTGCACATGGCAAACCCCAGACAGCCC
CTGCCTGCCTCTGGGCTGGCCACAGGAATGCCACAACAGCCCGCAGCATATGCCCTAGGGTGACCAC
GCAGTGAGGCTGGTGCCCATGCTCCACACTGGGAGGCCAGGCTGACCCCACCAGCCAGTCAGCTACA
ACTCCACATCAACTCC
Variant sequences of NOVlOb are included in Example 3, Table 22. A variant
sequence can include a single nucleotide polymorphism (SNP). A SNP can, in
some instances,
be referred to as a "cSNP" to denote that the nucleotide sequence containing
the SNP
originates as a cDNA.
The NOV l Ob protein (SEQ ID N0:22) encoded by SEQ ID NO:21 is 242 amino acid
residues in length and is presented using the one-letter code in Table l OD.
The Psort profile
for NOV l Ob predicts that this sequence is likely to be localized at the
plasma membrane with
a certainty of 0.7000.
Table 10D. NOVlOb protein sequence (SEQ ID N0:22)
MKNIGLVMEWEIPEIICMCAKLRLPPQATFQVLRGNGASVGTVLMFRCPPNHQMVGSGLLTCTWKGS
IAEWSSGSPVCKLVPPHETFGFKVAVIASIVSCAIILLMSMAFLTCCLLKCVKKSKRRRSNRSAQLW
SQLKDEDLETVQAAYLGLKHFNKPVSGPSQAHDNHSFTTDHGESTSKLASVTRSVDKDPGIPRALSL
SGSSSSPQAQVMVHMANPRQPLPASGLATGMPQQPAAYALG
NOV10 Clones
Unless specifically addressed as NOVlOa or NOVlOb, any reference to NOV10 is
assumed to encompass all variants. NOVlOa differs from NOVlOb at amino acid
position 18
(T>M) and amino acid position 50 (S>P) as shown in Tables 10B and l OD.
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 10E.
Table 10E. Pat results for NOV10
Smallest
Sum
eading igh Prob
equences Frame ScoreP(N)
producing
High-scoring
Segment
Pairs:
>patp:AAM93054Human digestive system antigen +1 210 7.2e-17
>patp:AAR05494Endothelial leukocyte adhesion +1 113 0.0016
molecule-1
>patp:AAR08116Endothelial leucocyte adhesion +1 l13 0.0016
molecule-1
>patp:AAW18839E-selectin +1 113 0.0016
>patp:AAW46733Endothelial leukocyte adhesion +1 113 0.0016
molecule-1
hi a BLAST search of public sequence databases, it was found, for example,
that the
NOVlOa nucleic acid sequence of this invention has 438 of 447 bases (97%)
identical to a
gb:GENBANI~-m:HSM802384~acc:AL137623.1 mRNA from Homo sapieras (cDNA
DKFZp434J1812 (from clone DKFZp434J1812)). The full amino acid sequence of the
protein
of the invention was found to have 110 of 139 amino acid residues (79%)
identical to, and 123
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of 139 amino acid residues (88%) similar to, the 269 amino acid residue
ptnr:SPTREMBL-
ACC:Q9D176 protein from Mus musculus (1700017I11R1K PROTEIN).
Similarly, it was found, for example, the NOV l Ob nucleic acid sequence of
this
invention has 438 of 447 bases (97%) identical to a gb:GENBANK-
ID:HSM802384~acc:AL137623.1 mRNA from Homo Sapiens (cDNA DKFZp434J1812 (from
clone DKFZp434J1812)). The full amino acid sequence of the protein of the
invention was
found to have 108 of 138 amino acid residues (78%) identical to, and 121 of
138 amino acid
residues (87%) similar to, the 269 amino acid residue ptnr:SPTREMBL-ACC:Q9D176
protein
from Mus musculus (1700017I11RIK PROTEIN.
Additional BLAST results are shown in Table 10F.
Table 10F. BLAST
results for
NOV10
Gene Index/ Protein/ OrganismLength Identity PositivesExpect
Identifier as
>gi~15779059~gb~ASimilar to RIKEN255 192/225 192/225 e-101
AH14601.1~AAH1460cDNA 1700017I11 (85%) (85%)
1(BC014601) gene
[Homo Sapiens]
>gi~12834785~dbj~Sushi domain 269 130/246 142/246 8e-56
BAB23043.1~(AK003(SCR repeat) (52%) (56%)
860) containing
protein~data
source:Pfam,
source
key:PF00084,
evidence:ISS-put
ative
[Mus musculus]
>gi~128505441dbjlSushi domain 170 71/102 77/102 6e-35
BAB28764.1I(AK013(SCR repeat) (69%) (74%)
276) containing
protein-.data
source:Pfam,
source
key:PF00084,
evidence:ISS-put
ative
[Mus musculus]
>gi~128389761dbjlSushi domain 149 55/73 61/73 2e-26
'
BAB24394.1~(AK006(SCR repeat) (75%) (83%)
068) containing
protein-data
source:Pfam,
source
key:PF00084,
evidence:ISS-.put
ative
[Mus musculus]
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>gi~7494498~pir~~scavenger 2043 30/87 42/87 2e-04
T18524 receptor (34%) (47%)
cysteine-rich
protein homolog
srcrm2 - Geodia
cydonium
A multiple sequence alignment is given in Table 1 OG, with the NOV 10 protein
of the
invention being shown on line 1, in a ClustalW analysis comparing NOV10 with
related
protein sequences disclosed in Table IOF.
Table 10G. Information for the ClustalW proteins:
1) > NOVlOa; SEQ ID N0:20
2) > NOV l Ob; SEQ ID N0:22
3) > gi~1577905/ similar to RTKFN cDNA 1700017I11 gene [Homo Sapiens]; SEQ ID
N0:77
4) > gig 1283478/ Sushi domain (SCR repeat) containing protein-data source:
Pfam, source key:
PF00084, evidence:ISS-putative [Mus musculus]; SEQ ID N0:78
5) > gig 1285054/ Sushi domain (SCR repeat) containing protein-data source:
Pfam, source key:
PF00084, evidence:ISS-putative [Mus musculus]; SEQ ID N0:79
6) > gi~1283897/ Sushi domain (SCR repeat) containing protein-data source:
Pfam, source key:
PF00084: ISS-putative [Mus nausculus]; SEQ ID N0:80
7) > gi~7494498/ scavenger receptor cysteine-rich protein homolog srcrm2-
Geodia cydohiu~ra; SEQ ll~
NO:81
1610
1620
1630
1640
1650
NOVlOa ___ IGLVMEWEIP--_____________________EIIC
RL
~ v
NOVlOb -- NIGLVMEWEIP-----------------------EIIC
RL
K
.
gi~ 1577905--- WAAATLRGKARPRGRA--------- GNRT
RL
R TT
P
gi~ 1283478--- RTSATLRGRARPRWRA--------- VNQT Q
R TT P
P
V
V
~S gi~ 1285054--- RTSATLRGRARPRWRA--------- VNQT
' Q
R TT P
P
gi~ 1283897--- RTSATLRGRAR.PRWRA--------- VNQT
Q
IR TT P
P
gi1 7494498GRYTQDTGWIACEDGYQPTEGAADVLCTED PACSVS
P
TWSRT QV
1660
1670
1680
1690
1700
NOVlOa ~ F~ -__________________________________________
NOVlOb v F~ -__________________________________________
gi~1577905 'v Fv -_____________________________-____________
gi~1283478 ~~v.~L~ ___________________________________________
giI1285054 ~ 7 -__________________________________________
~~ L~
gi~1283897 ~~,.'~~1IL~ -__________________________________________
gi~7494498 SLSSPDTIPGLWSLVCDKGYTYDASSDGDFSWCGLDGEWNSTLG
1710
1720
1730
1740
1750
NOVlOa _________,___________ L. .
NOVlOb _____________________ L. .p
gi~ 1577905--___________________ L.
gi1 1283478--__________________ T L I
v n
g7.I 1285054--__________________ T L I 1
gi1 1283897--___________________ T L I '
gi1 7494498TCKLVLCPAYSFNVTTNLRVSLTQ S TT T S 'FH SVI
S
1760
1770
1780
1790
1800
.
.
NOVlOa T IAE ~L
'
NOVlOb T IAE ~L ' . '
'
gi~ 1577905T IAE ~L
gi~ 1283478 TVD ' '
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gi~1285054 TVD '
gi~1283897 TVD ~ --VPSSC~CPW
gi17494498 S S ---T GTV'H SRE " TKCPTLTISDHVTAS SETTINTVVS~
1810 1820 1830 1840 1850
.)
NOVlOa ------ S ' 'S ~SAQ .SQLKDED -ET ~~~Y G
NOVlOb ------ S ' 'S 'SAQ SQLKDED -ETi~~ y G
giI1577905 ------ S ' 'S 'SAQ SQLKDED -ET ~' Y G
1O gi~1283478 ------ Q~ E " TAQ YQLRGED -ET s~ Y G
giI1285054 ------ Q~ E " ~-------YSG-- -MSF~KLC
gi~1283897 PSS-__________p~SSSVC'KMSG--_______G_________ T
gi~7494498 TCDNGYFLKGDKI~E~LSTGVWNGTAPTCS~PNSCPSLI~SDH~TiSSTD
15 1860 1870 1880 1890 1900
NOVlOa LKHFN-------------------KPVSGP ~ NHSF TDHGESTSKL
NOVlOb LKHFN-------------------KPVSGP ~ DNHSF TDHGESTSKL
g1~1577905 LKHFN-------------------KPVSGP t DNHSF TDHGESTSKL
2O gi~1283478 LKGHI~NSSSVGGGNGGPSGGGGKPGIQH ~ DNHSF TDPGD-IREQ
gi~1285054 VR-__-__-________-_______LLITSC~ PWG--____________
gi~1283897 GR-________________________ GTS-___-__-________
gi~7494498 TRINAVVTFTCD--DDRYTLNGNKIIACQSTGVWNGTAP~CKEIPTCPEL
25 1910 1920 1930 1940 1950
NOVlOa ASVT KD~GIPR----------------------------ALSLSGS
NOVlOb ASVT DKD~GIPR----------------------------ALSLSGS
gi~1577905 ASVT TLD'GIPR----------------------------ALSLSGS
3O gi11283478 AGVTH DKD' TFR--------------------------MGTPGPGGC
giI1285054 ____- ___________________________________________
gi~1283897 -______________________-________________,_________
gi~7494498 TPSSHVIPST~DNSVGAEVSFQCEDGYTLQGEKKITCLPTQKWSANPPSC
35 1960 1970 1980 1990 2000
NOVlOa S S~QAQ PRQPLP---AS T PQQPAAYALG---------
NOVlOb S S'QAQ PRQPLP---AS T PQQPAAYALG---------
gi'1577905 S S'QAQ PRQPLP---AS T PQQPAAYALG-________
40 gi11283478 S S'GTYVMVHAL ---------S P PGRPKVYLPG--------
gi~1285054 _____-___-_______-________________________________
gi~1283897 ___________,______________________________________
g1~7494498 G~TSQPLSNND~GSGTKVGPIVG~IG~ILVIVLIIVATAILFWKLSS
The NOV 10 Clustal W alignment shown in Table l OF was modified to begin at
amino
residue 1600 and end at amino acid residue 2000. The data in Table 1 OF
includes all of the
regions overlapping with the NOV 10 protein sequences.
The NOV 10 Clustal W alignment shown in Table l OG was modified to begin at
amino
residue 1601. The data in Table l OG includes all of the regions overlapping
with the NOV 10
protein sequences.
The presence of identifiable domains in the protein disclosed herein was
determined by
searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and
then
determining the Interpro number by crossing the domain match (or numbers)
using the
Interpro website (http:www.ebi.ac.uklinterpro~. Table lOH lists the domain
description from
DOMAIN analysis results against NOV10.
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Table lOH
Domain Anal
sis of NOV10
Model Region of Score (bits) E value
Homology
Sushi domain 19-78 15.8 0.0075
(SCR repeat)
Consistent with other known members of the Selectin-like family of proteins,
NOV 10
has, for example, a Sushi domain (SCR repeat) signature sequences and homology
to other
members of the Selectin-like Protein Family. NOV 10 nucleic acids, and the
encoded
polypeptides, according to the invention are useful in a variety of
applications and contexts.
For example, NOV10 nucleic acids and polypeptides can be used to identify
proteins that are
members of the Selectin-like Protein Family. The NOV 10 nucleic acids and
polypeptides can
also be used to screen for molecules, which inhibit or enhance NOV 10 activity
or function.
Specifically, the nucleic acids and polypeptides according to the invention
may be used as
targets for the identification of small molecules that modulate or inhibit,
e.g., cellular adhesion
and signal transduction. These molecules can be used to treat, e.g.,
Cardiovascular diseases,
Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects,
Aortic stenosis,
Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus
arteriosus , Pulmonary
stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases,
Tuberous
I5 sclerosis, Scleroderma, Obesity, Transplantation, Von Hippel-Lindau (VHL)
syndrome,
Cirrhosis, Transplantation, Diabetes, Autoimmune disease, Renal artery
stenosis, lilterstitial
nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus
erythematosus,
Renal tubular acidosis, IgA nephropathy, Hypercalceimia, Lesch-Nyhan syndrome,
Systemic
lupus erythematosus, Autoimmune disease, Asthma, Emphysema, Scleroderma,
allergy as
well as other diseases, disorders and conditions.
In addition, various NOV 10 nucleic acids and polypeptides according to the
invention
are useful, inter alia, as novel members of the protein families according to
the presence of
domains and sequence relatedness to previously described proteins. For
example, the NOV 10
nucleic acids and their encoded polypeptides include structural motifs that
are characteristic of
proteins belonging to the Selectin-like Protein Family.
The NOV 10 nucleic acids and polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and diagnostic
applications in the
mediation of cardiac and immune physiology. As such, the NOV10 nucleic acids
and
polypeptides, antibodies and related compounds according to the invention may
be used to
treat cardiovascular and immune disorders, e.g., Cardiovascular diseases,
Cardiomyopathy,
Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis,
Atrial septal defect
(ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary
stenosis, Subaortic
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stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis,
Scleroderma,
Obesity, Transplantation, Von Hippel-Lindau (VHL) syndrome, Cirrhosis,
Transplantation,
Diabetes, Autoixnmune disease, Renal artery stenosis, Interstitial nephritis,
Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus,
Renal tubular
acidosis, IgA nephropathy, Hypercalceimia, Lesch-Nyhan syndrome, Systemic
lupus
erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy as
well as
other diseases, disorders and conditions.
The NOV 10 nucleic acids and polypeptides are useful for detecting specific
cell types.
For example, expression analysis has demonstrated that a NOV 10 nucleic acid
is expressed in
Heart, Thyroid, Parotid Salivaxy glands, Liver, Colon, Ascending Colon, Bone
Marrow,
Peripheral Blood, Lymphoid tissue, Spleen, Lymph node, Tonsils, Thymus,
Cerebellum,
Spinal Chord, Cervix, Mammary glandBreast, Ovary, Placenta, Uterus,
Oviduct/LTterine
Tube/Fallopian tube, Vulva, Prostate, Testis, Lung, Kidney, Kidney Cortex,
Retina, Skin.
Additional utilities for NOV 10 nucleic acids and polypeptides according to
the
invention are disclosed herein.
NOV11
A NOV 11 polypeptide has been identified as a Nucleax Protein-like protein
(also
referred to as CG94325-O1). The disclosed novel NOV11 nucleic acid (SEQ ID
NO:23) of
8670 nucleotides is shown in Table 11A. The novel NOV 11 nucleic acid
sequences maps to
the chromosome 15.
An ORF begins with an ATG initiation codon at nucleotides 204-206 and ends
with a
TAA codon at nucleotides 7152-7154. A putative untranslated region and/or
downstream
from the termination codon is underlined in Table 11A, and the start and stop
codons are in
bold letters.
Table 11A. NOVll Nucleotide Sequence (SEQ ID N0:23)
ACGCGTAGAGCCGCTTTGCGCGTGCGCATCACCTAGGCGGTTAGATTTGAATACTTCACTGAGGCGA
GCCGGGCGTTGTGAGCGGACTGCTAGAGGCGGCTGTCTGTTTCCGCTCTAAGGAAACTCAGAGCGTG
TGGACCCCAAACAAGTCTGCGCAAAATTTGTCGAGGAGGTTTGCCGCGGCAGAAAAGTTTTCTTCAA
AAATGGATGGGGTGTCTTCAGAGGCTAATGAAGAAAATGACAATATAGAGAGACCTGTTAGAAGACG
GCATTCTTCAATATTGAAACCCCCAAGGAGTCCTCTTCAGGACCTCAGAGGTGGGAATGAAAGAGTT
CAGGAATCCAATGCTTTGAGAAATAAGAAAAACTCTCGTCGAGTCAGCTTTGCAGATACTATAAAGG
TATTCCAGACGGAGTCTCATATGAAAATAGTGAGAAAGTCAGAAATGGAAGAAACAGAAACAGGAGA
AAATCTTCTTTTGATACAGAATAAGAAATTAGAAGATAATTACTGTGAAATTACTGGGATGAACACA
TTGCTTTCTGCTCCCATTCATACCCAGATGCAACAGAAGGAGTTTTCAATTATAGAACATACCCGTG
AAAGGAAACATGCAAATGACCAGACAGTCATTTTTTCAGATGAAAACCAGATGGACCTGACATCAAG
$1
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TCACACTGTAATGATTACCAAAGGCCTTTTAGATAATCCCATAAGTGAAAAGTCCACCAAGATAGAT
ACCACATCATTTCTAGCTAATTTAAAGCTTCACACCGAGGACTCAAGAATGAAAAAAGAAGTAAATT
TTTCCGTGGATCAAAACACTTCTTCAGAAAATAAAATAGATTTCAATGACTTCATAAAAAGATTGAA
AACAGGAAAATGTAGTGCTTTTCCTGATGTGCCTGATAAAGAAAATTTTGAGATACCTATTTATTCC
AAGGAACCGAACAGTGCCTCTTCTACACATCAAATGCATGTATCTCTTAAGGAAGATGAAAATAACA
GTAATATTACTAGGCTCTTTAGAGAAAAAGATGATGGGATGAATTTCACCCAGTGTCATACAGCCAA
TATTCAGACATTGATTCCCACATCCAGTGAGACCAACTCACGGGAATCTAAAGGTAATGATATTACA
ATTTATGGCAATGACTTTATGGACTTGACATTTAACCACACTTTGCAGATCTTACCTGCAACAGGTA
ATTTTTCTGAAATAGAAAATCAAACTCAGAATGCCATGGATGTAACAACAGGTTATGGAACTAAAGC
TTCAGGAAATAAAACAGTTTTTAAGAGTAAACAAAATACTGCTTTTCAAGACCTTTCCATAAACTCT
GCAGACAAAATACATATTACCAGAAGTCATATTATGGGGGCAGAAACTCACATAGTCTCACAGACTT
GTAATCAGGATGCCAGAATATTAGCCATGACCCCAGAATCTATATATTCTAATCCATCTATTCAAGG
TTGTAAGACTGTTTTCTATTCTAGTTGTAATGATGCCATGGAAATGACCAAATGTCTCTCAAATATG
AGAGAGGAGAAAAATTTGCTAAAGCATGACAGTAATTATTCTAAAATGTATTGCAATCCAGATGCTA
TGTCTTCTCTCACAGAGAAAACTATTTATTCCGGAGAGGAGAACATGGACATTACCAAGAGTCATAC
AGTTGCAATAGATAATCAAATTTTTAAACAAGATCAATCAAATGTGCAAATAGCAGCTGCACCAACA
CCCGAAAAAGAAATGATGCTCCAAAATCTTATGACCACATCAGAAGATGGGAAAATGAATGTAAATT
GTAACTCAGTTCCTCATGTATCTAAGGAAAGAATACAGCAGAGCCTGTCAAATCCTTTGTCTATTTC
ATTGACTGATAGAAAGACTGAACTCTTATCAGGTGAAAATACGGATTTGACTGAAAGTCACACAAGT
AACTTAGGAAGTCAGGTTCCTCTTGCAGCTTATAATCTAGCACCGGAGAGTACCAGTGAATCTCACT
CTCAGAGCAAAAGCTCTTCAGATGAATGTGAAGAAATTACCAAAAGTCGTAATGAACCATTTCAGCG
ATCAGACATAATAGCCAAAAACAGCTTAACCGACACCTGGAACAAAGACAAAGATTGGGTTTTGAAG
ATTTTGCCCTACCTTGATAAAGATTCTCCTCAGTCAGCTGATTGTAATCAGGAGATAGCAACAAGCC
ATAATATAGTCTACTGTGGTGGAGTTCTTGATAAACAAATAACTAATAGAAATACAGTATCATGGGA
ACAATCTTTGTTTTCTACCACAAAGCCATTATTTTCATCAGGACAGTTCTCTATGAAAAATCATGAT
ACTGCTATAAGTAGTCATACAGTGAAATCTGTACTAGGCCAGAATTCTAAACTGGCTGAGCCACTGA
GGAAAAGTTTAAGCAATCCCACACCTGACTATTGCCATGACAAGATGATTATATGTTCAGAGGAAGA
GCAAAATATGGATCTAACAAAGAGCCACACTGTCGTCATTGGATTTGGTCCTTCTGAACTACAAGAA
CTTGGTAAAACTAATTTAGAACACACTACTGGCCAGCTAACAACAATGAACAGACAGATAGCTGTAA
AAGTTGAAAAATGTGGTAAAAGTCCCATAGAAAAAAGTGGAGTGCTTAAATCTAACTGTATTATGGA
TGTGTTAGAGGACGAAAGTGTACAGAAACCTAAATTTCCAAAGGAAAAGCAAAATGTCAAAATTTGG
GGAAGGAAAAGTGTTGGTGGACCAAAAATTGATAAGACTATTGTATTTTCAGAAGACGATAAGAATG
ATATGGATATCACTAAGAGTTATACAATAGAAATAAACCATAGACCTTTATTAGAGAAACGTGATTG
TCATTTGGTGCCATTGGCAGGAACTTCTGAAACTATTTTATATACATGTGGGCAGGATGACATGGAG
ATCACTAGAAGTCACACAACTGCCTTAGAATGTAAAACTGTCTCACCAGATGAAATAACTACTAGGC
CTATGGACAAAACTGTAGTGTTTGTAGATAATCATGTTGAACTAGAAATGACAGAGTCCCATACTGT
TTTCATTGACTACCAAGAAAAGGAAAGAACAGACAGACCTAACTTTGAACTATCCCAAAGGAAAAGC
CTAGGAACACCAACAGTGATATGTACTCCTACTGAGGAGAGTGTTTTCTTTCCAGGAAATGGTGAAA
GTGACCGTCTAGTAGCAAATGACAGCCAGCTAACCCCTCTGGAGGAATGGTCTAATAATAGGGGCCC
TGTAGAGGTAGCTGATAACATGGAATTGTCTAAATCAGCCACTTGCAAAAACATCAAAGATGTACAA
AGTCCTGGATTTCTGAATGAACCTCTATCAAGCAAAAGTCAGAGAAGAAAAAGCCTTAAGCTAAAAA
ATGACAAGACCATTGTATTTTCAGAGAATCATAAAAATGATATGGATATTACCCAGAGTTGTATGGT
GGAAATAGATAACGAAAGTGCCCTGGAGGATAAAGAGGACTTCCATTTGGCAGGGGCTTCTAAAACT
ATTTTGTATTCATGTGGGCAGGATGACATGGAGATCACTAGGAGTCACACAACTGCCTTAGAATGTA
AAACTCTCCTGCCAAACGAAATAGCTATTAGGCCCATGGACAAAACCGTATTGTTCACAGATAATTA
CAGTGATCTGGAAGTCACCGATTCCCATACTGTTTTCATTGACTGTCAAGCCACAGAGAAAATACTT
GAAGAAAACCCTAAATTTGGAATAGGAAAAGGAAAAAACTTGGGTGTTTCCTTTCCTAAGGATAATA
GCTGTGTTCAAGAAATCGCTGAAAAACAAGCACTGGCTGTAGGAAACAAAATAGTTCTTCACACCGA
GCAAAAGCAACAACTCTTTGCTGCTACTAATAGAACTACTAATGAAATCATCAAATTTCATAGTGCT
GCTATGGATGAAAAGGTCATAGGGAAAGTTGTAGACCAGGCCTGTACATTGGAAAAAGCGCAAGTTG
AAAGCTGTCAGTTAAATAATAGAGATAGAAGAAATGTGGACTTTACAAGTAGTCATGCAACTGCTGT
TTGTGGATCCAGTGATAATTATTCCTGTTTACCAAATGTTATTTCCTGTACTGATAATTTGGAGGGT
AGTGCCATGCTCTTATGTGATAAAGATGAGGAAAAAGCCAATTATTGCCCAGTGCAAAATGATCTTG
CTTATGCAAATGATTTTGCCAGTGAATATTACTTGGAATCTGAGGGACAGCCTCTCTCTGCTCCTTG
TCCTTTGTTAGAGAAGGAAGAAGTTATTCAAACCAGTACCAAAGGACAGTTAGACTGTGTTATAACA
CTGCACAAAGATCAAGATCTGATTAAGGATCCACGAAATCTATTGGCTAATCAAACTTTAGTATATA
GTCAAGATCTGGGGGAGATGACTAAACTTAATTCAAAGCGAGTATCTTTTAAGCTTCCAAAGGATCA
AATGAAAGTCTATGTTGATGACATTTATGTTATTCCTCAGCCTCATTTCTCAACCGACCAACCTCCA
TTACCTAAAAAAGGACAGAGTAGTATCAATAAAGAAGAAGTAATACTGTCTAAAGCTGGAAATAAGA
GTTTAAATATTATAGAAAATTCCTCTGCACCCATATGTGAAAACAAGCCCAAAATACTCAATAGTGA
GGAATGGTT'T'GCTGCAGCCTGTAAAAAAGAACTGAAGGAAAATATTCAAACAACTAACTATAATACA
GCTCTAGATTTCCACAGTAACTCAGACGTAACTAAGCAAGTCATTCAAACTCATGTCAATGCTGGAG
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AAGCACCAGATCCTGTAATTACATCTAATGTTCCATGTTTTCATAGTATCAAACCAAATCTGAATAA
TTTGAATGGAAAAACTGGAGAGTTTTTAGCCTTTCAAACTGTTCATCTACCACCCCTTCCAGAGCAA
TTACTTGAATTAGGAAATAAGGCACACAATGATATGCATATAGTGCAAGCTACAGAAATACATAATA
TTAACATAATCTCCAGCAATGCTAAAGATAGTAGAGATGAGGAAAATAAAAAGTCTCATAATGGAGC
TGAAACCACCTCTCTACCGCCAAAGACAGTTTTTAAAGATAAAGTAAGGAGATGTTCTTTGGGAATC
TTTTTGCCTAGATTGCCCAACAAGAGAAATTGTAGTGTCACTGGTATTGATGACCTGGAACAGATTC
CAGCAGACACAACTGATATAAATCACTTAGAAACTCAGCCGGTCTCTAGCAAAGATTCAGGCATTGG
ATCTGTTGCAGGTAAACTGAACCTAAGTCCTTCTCAATATATAAATGAGGAAAATCTTCCTGTATAT
CCTGATGAGATCAATTCTTCAGACTCTATTAACATAGAAACTGAGGAAAAGGCCTTGATTGAGACAT
ACCAAAAAGAGATTTCACCATATGAAAATAAAATGGGAAAAACTTGCAATAGCCAAAAAAGAACGTG
GGTACAAGAAGAAGAAGATATTCATAAGGAGAAAAAAATCAGAAAAAATGAGATTAAGTTTAGTGAT
ACGACACAAGATCGGGAGATTTTTGATCACCATACTGAAGAGGATATAGATAAAAGTGCTAACAGTG
TATTGATAAAAAACCTGAGCAGGACCCCATCTAGTTGCAGCAGCTCTCTGGATTCAATCAAGGCTGA
TGGGACCTCTCTGGACTTCAGCACTTACCGCAGTAGTCAAATGGAATCACAGTTTCTCAGAGATACT
ATTTGTGAAGAGAGCTTGAGGGAGAAACTCCAAGATGGGAGAATAACAATAAGGGAGTTCTTTATAC
TTCTCCAGGTCCACATCTTGATACAGAAACCCCGACAGAGCAATCTCCCAGGCAATTTTACTGTAAA
CACACCACCTACTCCAGAAGACCTGATGTTAAGTCAATATGTTTACCGACCCAAGATACAGATTTAT
AGAGAAGATTGTGAGGCTCGTCGCCAAAAGATTGAAGAATTAAAGCTTTCTGCATCGAACCAAGATA
AGCTGTTGGTTGATATAAATAAGAACCTGTGGGAAAAAATGAGACACTGCTCTGACAAAGAGCTGAA
GGCCTTTGGAATTTATCTTAACAAAATAAAGTCATGTTTTACCAAGATGACTAAAGTCTTCACTCAC
CAAGGAAAAGTGGCTCTGTATGGCAAGCTGGTGCAGTCAGCTCAGAATGAGAGGGAGAAACTTCAAA
TAAAGATAGATGAGATGGATAAAATACTTAAGAAGATCGATAACTGCCTCACTGAGATGGAAACAGA
AACTAAGAATTTGGAGGATGAAGAGAAAAACAATCCTGTGGAAGAATGGGATTCTGAAATGAGAGCT
GCAGAAAAAGAATTGGAACAGCTGAAAACTGAAGAAGAGGAGCTTCAAAGAAATCTCTTAGAACTGG
AGGTACAAAAAGAGCAGACCCTTGCTCAAATAGACTTTATGCAAAAACAAAGAAATAGAACTGAAGA
GCTACTGGATCAGTTGAGCTTGTCTGAGTGGGATGTCGTTGAGTGGAGTGATGATCAAGCTGTATTC
ACCTTTGTTTATGACACGATACAACTCACCATCACCTTTGAAGAGTCAGTTGTTGGTTTCCCTTTCC
TGGACAAGCGTTATAGGAAGATTGTTGATGTCAATTTTCAATCTCTGTTAGATGAGGATCAAGCTCC
TCCTTCCTCCCTTTTAGTTCATAAGCTTATTTTCCAGTACGTTGAAGAAAAGGAATCCTGGAAGAAG
ACATGTACAACCCAGCATCAGTTACCCAAGATGCTTGAAGAATTCTCACTGGTAGTGCACCATTGCA
GACTCCTTGGAGAGGAGATTGAGTATTTAAAGAGATGGGGACCAAATTATAACCTAATGAACATAGA
TATTAATAAT.AATGAATTGAGACTTTTATTCTCTAGCTCCGCAGCATTTGCAAAGTTTGAAATAACT
TTGTTTCTCTCAGCCTATTATCCATCTGTACCATTACCTTCCACCATTCAGAATCACGTTGGGAACA
CTAGCCAAGATGATATTGCTACCATTCTATCTAAAGTGCCACTGGAGAACAACTACCTGAAGAATGT
AGTCAAGCAAATTTACCAAGATCTGTTTCAGGACTGCCATTTCTACCACTAGACCCTTGGACCACCA
TTGGAACAACCAAGCAGAATGTACTTGATATTATTTCAGGGTCCCATTGCTGTTCAGCCTTTGTTTT
TACGTCATTACAAGCTGAGTAAAATTCCTTCTGATGATGTTATAGTTAATCTGTATGTTTTTTATAT
CTCTGCAGAATGATGGTGATGAAGTCTGGATGGTAGGCCTCATAGCCTACTATCAACTTACTCATCT
TTGTACCAAAGGTTTAAGTAATAGGACACTTAGGAAAAATGTCTCCTAACTAAACTAGTGCTTTCTG
CTTTAGTACAAGCCCTAAGGATTAACTTAAGTATAAGAAGTGTTATCACTGACAAGAACATTAGCCA
TTTTCCCATAACTAGATAGAGCTATGATTTTTTAGGTTGCCTGGCTTCTGCCTAGCAGATATTTCTG
GAGTAGAAATGTATCTGTCTACAAACTATTATCCTTTTTCTCCGTTACTAAAATGCTATTAAGAGAA
AGTAGGGCTGGGTGTGAGCCACCACACCCAGCAATGTTTTCTTAATAAGTATAGTTTTTCTAGGGAA
AGTTAATTCATTTTTGTCTAGTACATATATGTAAATATATTAATGTTGTTTTTGTGTTTGTGATGTA
GTAAGGAGATGTACATAGAAATTCATTGAGGTATATAGATACTCATCTGTCTAGGCAGTTCCCAATT
TTCTGAAGAATGTTTTACAGCAAAATTTTCTATTTTCTTTTATTAAATAGTGACACGTCAAACAATG
TCACATCCAAAACACTAGTTTCATCAATTTCTAGCAGTAATAATAGACTTGCTGTAAGTATTGTTTT
CTGATGCCATACCCTTGTCATACATATTATTAAATGACCAATATTATGTATGAAGTAGACAAAAAAA
TTTACTCAAACTTCATTCAAATCCTAATTGTGATAATTTTTGTTTTATATTTAATTATAAACCAAAA
TACATTTGCATTTTTAAGCTAATTTGTCTCAAAATTTTGCTTTATATTTTTGGATCAGGTTAAAGTC
CTGTGGATCCCCTGAATGTTATTGTCCCTCTTGATTGGTTTTTACTTCTGAGCTATACGTCAAAAGA
CACATAAGCTTCAAAAGTCAAGACAAACCTCATTTGCCATAAAAATCAAGATATAGATGTTCTGTTC
CGTAAACTCCTTGAAAAACATTTTAAAGTCATCAATATGATCTGTTTCCCATGAAACTTAAGTTAGC
TTTCTTATTGGAGTTATTTCTTTTCTGTAAGTCTGAAAAGTAGAGATTTTGTTTTACGCATTTTAGT
AACCTGCAACAACCAACTCTAAAAA.AGATTTGGCTTGTAATGACGGTCTCTGCTTTTTTGGGTTTGG
AGTACACAATTGTAATATTTACTTAGTTATTTGTGTTTTTCTTTGTTCAAGGTATTGACTAGTTTCA
TAAATTTTTTGCAAGTTTTTCTTTCATTGGTTGGAAAGCAGATTACATTTTGCACTATTAAAATAAG
TTTATTACTTTP,AAAP~AAGTCGACG
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Variant sequences of NOV11 are included in Example 3, Table 23. A variant
sequence
can include a single nucleotide polymorphism (SNP). A SNP can, in some
instances, be
referred to as a "cSNP" to denote that the nucleotide sequence containing the
SNP originates
as a cDNA.
The NOV 11 protein (SEQ m N0:24) encoded by SEQ m N0:23 is 2316 amino acid
residues in length and is presented using the one-letter amino acid code in
Table 11B. Psort
analysis predicts the NOV 11 protein of the invention to be localized at the
nucleus with a
certainty of 0.8800.
Table 11B. Encoded NOVll protein sequence (SEQ ID N0:24)
MDGVSSEANEENDNIERPVRRRHSSILKPPRSPLQDLRGGNERVQESNALRNKKNSRRVSFADTI
KVFQTESHMKIVRKSEMEETETGENLLLIQNKKLEDNYCEITGMNTLLSAPIHTQMQQKEFSIIE
HTRERKHANDQTVIFSDENQMDLTSSHTVMITKGLLDNPISEKSTKIDTTSFLANLKLHTEDSRM
KKEVNFSVDQNTSSENKIDFNDFIKRLKTGKCSAFPDVPDKENFEIPIYSKEPNSASSTHQMHVS
LKEDENNSNITRLFREKDDGMNFTQCHTANIQTLIPTSSETNSRESKGNDITIYGNDFMDLTFNH
TLQILPATGNFSEIENQTQNAMDVTTGYGTKASGNKTVFKSKQNTAFQDLSINSADKIHITRSHI
MGAETHIVSQTCNQDARILAMTPESIYSNPSIQGCKTVFYSSCNDAMEMTKCLSNMREEKNLLKH
DSNYSKMYCNPDAMSSLTEKTIYSGEENMDITKSHTVAIDNQIFKQDQSNVQIAAAPTPEKEMML
QNLMTTSEDGKMNVNCNSVPHVSKERIQQSLSNPLSISLTDRKTELLSGENTDLTESHTSNLGSQ
VPLAAYNLAPESTSESHSQSKSSSDECEEITKSRNEPFQRSDIIAKNSLTDTWNKDKDWVLKILP
YLDKDSPQSADCNQEIATSHNIVYCGGVLDKQITNRNTVSWEQSLFSTTKPLFSSGQFSMKNHDT
AISSHTVKSVLGQNSKLAEPLRKSLSNPTPDYCHDKMIICSEEEQNMDLTKSHTWIGFGPSELQ
ELGKTNLEHTTGQLTTMNRQIAVKVEKCGKSPIEKSGVLKSNCIMDVLEDESVQKPKFPKEKQNV
KIWGRKSVGGPKIDKTIVFSEDDKNDMDITKSYTIEINHRPLLEKRDCHLVPLAGTSETILYTCG
QDDMEITRSHTTALECKTVSPDEITTRPMDKTVVFVDNHVELEMTESHTVFIDYQEKERTDRPNF
ELSQRKSLGTPTVICTPTEESVFFPGNGESDRLVANDSQLTPLEEWSNNRGPVEVADNMELSKSA
TCKNIKDVQSPGFLNEPLSSKSQRRKSLKLKNDKTIVFSENHKNDMDITQSCMVEIDNESALEDK
EDFHLAGASKTILYSCGQDDMEITRSHTTALECKTLLPNEIAIRPMDKTVLFTDNYSDLEVTDSH
TVFIDCQATEKILEENPKFGIGKGKNLGVSFPKDNSCVQEIAEKQALAVGNKIVLHTEQKQQLFA'
ATNRTTNEIIKFHSAAMDEKVIGKVVDQACTLEKAQVESCQLNNRDRRNVDFTSSHATAVCGSSD
NYSCLPNVISCTDNLEGSAMLLCDKDEEKANYCPVQNDLAYANDFASEYYLESEGQPLSAPCPLL
EKEEVIQTSTKGQLDCVITLHKDQDLIKDPRNLLANQTLVYSQDLGEMTKLNSKRVSFKLPKDQM
KVYVDDIYVIPQPHFSTDQPPLPKKGQSSINKEEVILSKAGNKSLNIIENSSAPICENKPKILNS
EEWFAAACKKELKENIQTTNYNTALDFHSNSDVTKQVIQTHVNAGEAPDPVITSNVPCFHSIKPN
LNNLNGKTGEFLAFQTVHLPPLPEQLLELGNKAHNDMHIVQATEIHNINIISSNAKDSRDEENKK
SHNGAETTSLPPKTVFKDKVRRCSLGIFLPRLPNKRNCSVTGIDDLEQIPADTTDINHLETQPVS
SKDSGIGSVAGKLNLSPSQYINEENLPVYPDEINSSDSINIETEEKALIETYQKEISPYENKMGK
TCNSQKRTWVQEEEDIHKEKKIRKNEIKFSDTTQDREIFDHHTEEDIDKSANSVLIKNLSRTPSS
CSSSLDSIKADGTSLDFSTYRSSQMESQFLRDTICEESLREKLQDGRITIREFFILLQVHILIQK
PRQSNLPGNFTVNTPPTPEDLMLSQYVYRPKIQIYREDCEARRQKIEELKLSASNQDKLLVDINK
NLWEKMRHCSDKELKAFGIYLNKIKSCFTKMTKVFTHQGKVALYGKLVQSAQNEREKLQIKIDEM
DKILKKIDNCLTEMETETKNLEDEEKNNPVEEWDSEMRAAEKELEQLKTEEEELQRNLLELEVQK
EQTLAQIDFMQKQRNR'T'EELLDQLSLSEWDVVEWSDDQAVFTFVYDTIQLTITFEESVVGFPFLD
KRYRKIVDVNFQSLLDEDQAPPSSLLVHKLIFQYVEEKESWKKTCTTQHQLPKMLEEFSLVVHHC
RLLGEEIEYLKRWGPNYNLMNIDINNNELRLLFSSSAAFAKFEITLFLSAYYPSVPLPSTIQNHV
GNTSQDDIATILSKVPLENNYLKN~TVKQIYQDLFQDCHFYH
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 11 C.
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Table 11C. results
Pat fox NOVll
Smallest
Sum
eading igh Prob
equences igh-scoringegment Frame ScoreP(N)
producing Pairs:
>patp:AAW8839.8Humantestis +1 2444 1.7e-253
secreted
protein
dol5 4
>patp:AAU71933Humanbone marrowtissuepolypeptide#11+1 2444 1.7e-253
>patp:AAU71961Humanbone marrowtissuepolypeptide#39+1 2444 1.7e-253
>patp:AAU71933Humanbone marrowtissuepolypeptide#11+1 2444 1.7e-253
>patp:AAU71961Humanbone marrowtissuepolypeptide#39+l 2444 1.7e-253
In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 5584 of 5584 bases (100%)
identical to a
gb:GENBANK-m:AB046790~acc:AB046790.1 mRNA from Homo Sapiens (mRNA for
KTA_A_1570 protein, partial cds). The full amino acid sequence of the protein
of the invention
was found to have 1790 of 1793 amino acid residues (99%) identical to, and
1792 of 1793
amino acid residues (99%) similar to, the 1833 amino acid residue
ptnr:SPTREMBL-
ACC:Q9NR92 protein from Horno sapieras (AF15Q14 PROTEIN).
NOV11 also has homology to the proteins shown in the BLASTP data in Table 11D.
Table 11D. BLAST
results for
NOV11
Gene Index/ Protein/ OrganismLength Identity PositivesExpect
Identifier (aa) (%) (%)
giI18308012~gb~AAL6AF15q14 isoform2316 2316/23162316/23160.0
2
7803.1~AF461041 [Homo Sapiens] (100%) (100%)
1(A
F461041)
gi~9966807~refINPAF15q14 protein1833 1790/17931792/17930.0
0
_ [Homo Sapiens] (99%) (99%)
65113.1~(NM_020380)
gi~14749154Iref~XP_AF15q14 protein1833 1789/17931791/17930.0
031524.1~(XM [Homo Sapiens] (99%) (99%)
031524
giI10047205~dbj~BABKIAA1570 protein1360 1360/13601360/13600.0
13396.1~(AB046790)[Homo Sapiens] (100%) (100%)
gi~14749150~ref~XPsimilar to 915 900/900 900/900 0.0
_ KIAA1570 protein (100%) (100%)
012461.3
(XM 012461) [Homo Sapiens]
A multiple sequence alignment is given in Table 11E, with the NOV11 protein
being
shown on line 1 in Table 11E in a ClustalW analysis, and comparing the NOV11
protein with
the related protein sequences shown in Table 11D. This BLASTP data is
displayed graphically
in the ClustalW in Table 11E.
Table 11E. ClustalW Analysis of NOVll
1) > NOVl 1; SEQ JD N0:24
2) > gi~18308012~/ AF15q14 isoform 2 [Homo Sapiens]; SEQ ID N0:82
3) > gi~9966807~/ AF15q14 protein [Homo Sapiens]; SEQ ID N0:83
4) > gi~14749154~/ AF15q14 protein [Homo Sapiens]; SEQ >D N0:84
5) > gi~10047205~/ KIAA1570 protein [Homo Sapiens]; SEQ >D N0:85
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6) > gi~14749150~/ similar to KIAA1570 protein [Hoyno Sapiens]; SEQ >D N0:~6
10 20 30 40 50 60
.(. .( . .(..(. .~. .(..(
.(.
NOV11 1 1 r 1 ~ 60
gi(18308012(1 1 1 1 ~ 60
gi(9966807(1 1 1 1 ~ 60
gi(14749154(1 1 1 1 ' ~ 60
gi(10047205(1 _____ ______ _______ ____ ___ ________ _________
______ _____ _______
1
gi(14749150(1 _____ ______ _______ ____ ___ ________ _________
______ _____ _______
1
70 80 90 100 110 120
( ( ( ( ( ( ( ( ( ' ( (
NOV11 61 12
0
gi(18308012(61 120
gi(9966807(6l
120
gi(14749154(61
120
gi(1004720511 _____ ______ _______ ____ ___ ________ _________
______ _____ _______
1
gi(14749150~1 _____ ______ _______ ____ ___ ________ __________
_____ _____ _______
1
130 140 150 160 170 180
( (
( . (. .I. .( I. y .( ( (
.~ .
NOV11 121 180
gi~183080121121 180
gi~9966807~121
180
gi(14749154(121 180
gi~10047205(1 _____ ______ _______ ____ ___ ________ __________
_____ _____ _______
1
gi(14749150~1 _____ ______ _______ ____ ____ _______ __________
_____ _____ _______
1
190 200 210 220 230 240
( ( ( ( ( ( ( ( ( ( ( (
NOV11 181 240
gi(18308012(181 240
gi~9966807(181 240
gi(14749154(181 240
gi(10047205(1 _____ ______ _______ ____ ____ _______ __________
_____ _____ _______
1
gi(14749150(1 _____ ______ _______ ____ ____ _______ __________
_____ _____ _______
1
40 250 260 270 280 290 300
( . ( .( y .(.I ~ .( y y
.
NOV11 241 300
gi(183080121 241 300
gi(9966807( 241
300
45 gi(14749154( 241 300
giI10047205( 1 _____ ______ _______ ____ ___ ________
__________ _____ _____ _______
1
gi(147491501 1 _____ ______ _______ ____ ____ _______
__________ _____ _____ _______
1
310 320 330 340 350 360
50 (
NOV11 301 360
gi(18308012( 301 360
gi(9966807( 301
360
gi(14749154( 301 360
gi(10047205~ 1 _____ ______ _______ ____ ____ _______
__________ _____ _____ _______
1
gi(14749150( 1 _____ ______ _______ ____ ____ _______
__________ _____ _____ _______
1
370 380 390 400 410 420
( .( .( ( .(.. (
60 NOV11 361 420
gi(18308012(361
420
gi(9966807(361
420
gi(14749154(361
420
g1(10047205(1 1
6$ _____ ______ _______ ____ ____ _______ __________
_____ _____ _______
giI14749150(1 1
430 440 450 460 470 480
( ( ( ( (
NOV11 421 .( . . ~.. . . .(. .(..(. . . .(..
.(. . 1 . . r 1 . . .. 480
8~ i1'
~6
1 1
1 v v 1 I v
1 1 I
1 1 1 1 I 1
. y~ 11 1 1I'Il1 / '1 1 1 1
1
1 1 1 ! 1 1
1 r o v I Ir r 1 a 1
~ -
11 1 1 1 r
1 ,~ I 11 1 1
1 9~ I 11 1 1
11 ~ I ~ 1W 1 1
~1
1 11 11 1 1
a 1 r 1 11 y 1
1 1 11 I 1 1
1 11
v 1 11 I v v
.(. .(.. .(. (. . .(.
.(. . .( .(. .(..
.~.
.(
r 1 1 v 1. v IvI 1
1 1 1 1 . 1 , 1
1 1 1 1
1 1 1 1
W 11 1 y1.
~
1 11 1 v ~~i1
11 1 1
11 1 11
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02/085922
gi(18308012( 421 m 1 1 SI '1' 480
gi(9966807( 42l v . 1 1 1 1 SI 1 480
gi (14749154( 421 . 1 1 ~ 1 1 1 1 480
gi(10047205( 1 ______ _____ ___________ ____ ________ ____
_____ _____ ____ ____ 1
_____
gi(14749150( 1 ______ _____ ___________ ____ ________ ________
_____ _____ ____ ____ 1
490 500 510 520 530 540
NOV11 481 540
gi(18308012( 481 540
gi (9966807( 481 540
gi(14749154( 481 540
gi(10047205( 1 ______ ______ __________ ____ ________ ________
_____ _____ ____ ____ 1
gi(14749150( 1 ______ ______ __________ ____ ________ ________
_____ _____ ____ ____ 1
550 560 570 580 590 600
( (
(. .( .( (. .(. .(. . ( .(
NOV11 541 Iv . 600
T
gi(18308012( 541 I TI i . 600
gi(9966807( 541 1 TI
600
gi(14749154( 541 1 ' ' 600
gi(10047205( 1 -_____ ______ __________ ____ ________
________ _____ _____ ____ ____ 1
gi(14749150( 1 ______ ______ __________ ____ ________
________ _____ _____ ____ ____ 1
610 620 630 640 650 660
(..( ~ ~. .( . ~ ~..( . (
NOV11 601 660
gi(18308012~ 601 660
gi(9966807( 601 660
30 gi(14749154( 601 660
gi(10047205( 1 ______ ______ __________ ____ ________
________ _____ _____ ____ _
gi(14749150( 1 ______ ______ __________ ____ ________
________ _____ _____ ____ ____ 1
670 680 690 700 710 720
35
NOV11 661 720
gi(18308012( 661 720
gi(9966807( 661 720
gi(14749154( 661 720
40 gi(10047205( 1 ______ ______ __________ ____ ________
________ _____ _____ ____ ___
- 1
gi(14749150( 1 ______ ______ __________ ____ ________
________ _____ _____ ____ ____ 1
730 740 750 760 770 780
(. .( .(. ( .( ( (. ( .(. .
.(. . .(
45 NOV11 721 780
giI18308012( 721 780
gi~9966807( 721 780
gi(14749154( 721 780
gi(10047205( 1 ______ ______ __________ ____ ________ ________
_____ _____ ____ ____ 1
gi(14749150( 1 ______ ______ __________ ____ ________ ________
_____ _____ ____ ____ 1
790 800 810 820 830 840
( (
( .~ .(. ( (. ( ( ~. .(
.~.
NOV11 781 840
5$ gi(18308012( 781 840
gi(9966807( 781 840
gi(14749154( 781 840
gi(10047205( 1 ______ ______ __________ ____ ________ ________
_____ _____ ____ ____ 1
gi(14749150( 1 ______ ______ __________ ____ ________ ________
_____ _____ ____ ____ 1
60
850 860 870 880 890 900
( ~ ( ( ( (
NOV11 841 .. .(. .( . .~. .(. . . . . . . . .(.. 900
.( . . . .
gi(18308012( 841 900
65 gi(9966807( 841 900
gi(14749154( 841 900
gi(10047205( 1 ______ ______ __________ ____ ________ ________
_____ _____ ____ ____ 1
gi(14749150( 1 ______ ______ __________ ____ ________ ________
_____ _____ ____ ____ 1
910 920 930 940 950 960
g7
r ~ 1 1 1 1 1
~I' v v
1 1 1 1 1 0 1
~ 0
1 1 1 0 1 1
r
1 1 1 1 1 1 1
0
.(. .(. . . (.. . .(..
.~ .(. . .(. .(. .(
.(.
.(
1 v v 1 ~ I 1
n
1 1 1 1 1
1 ~ 1 1 1
1 1 1 1
1 1 11
1
1 11 1
1 1 1
1
1 11 1
1 1
1 t' 1
~ '
1 '1 1 1
'
~ r 1
1 1
1 1 1 1 1
1 1
11
1 111 1 1
1
1 111 1 1
1
1 111 1 1
1
<IMG>
<IMG>
CA 02443770 2003-10-15
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02/085922
giI10047205~ 845r v ~ ~~ v v' 904
~ ~ v
~
gi~14749150~ 768~ ' ' ~ '~ ~ v 827
~ v v
~
1870 0
1880
1890
1900
1910
192
S y
.y
~
~
y
.
~.
y
y
~.
~
y
r
-
NOV11 1861 ~~ ~ ~ ~ '~ ~ 1920
~
gi~183080121 1861 ~~ ~ ~ ~ ~ ~ 1920
~
gi~9966807~ 1817_________________yL
S_____________G_______________ Q I 1832
S
gi~14749154~ 1817-________________~~L .S_-
___________G_______________ Q I 1832
S
gi~10047205~ 905 ~~ ~ ~~ ~ ~ ~ 964
gi~14749150~ 828 ~~ ~ ~~ ~ ~ ~ 887
1930 0
1940
1950
1960
1970
198
IS NOV11 1921. ~ LLSASNQDKLLVDINKNLWEKMRHCSDKELICAFGIYLNKIKSCFTK
1980
t
gi~18308012~ 1921 ~ LLSASNQDKLLVDINKNLWEKMRHCSDKELKAFGIYLNKIKSCFTK
1980
~
a
gi~9966807~ 1833T-__________________________________________________________
1833
gi~14749154~ 1833T-__________________________________________________________
1833
gi~10047205~ 965 ~ LLSASNQDKLLVDINKNLWEKMRHCSDKELKAFGIYLNKIKSCFTK
1024
~
gi~14749150~ 888 ~ y8____________________________________________
903
v
1990 0
2000
2010
2020
2030
204
NOV11 1981MT 2040
T-~
AL'~'G
QSAQNEREKLQIKIDEMDKILKKIDNCLTEMETETKNLEDEE
ZS gi~18308012~ 1981MT T ~Q~.~L"~'GQSAQNEREKLQIKIDEMDKILKKIDNCLTEMETETKNLEDEE
2040
gi~9966807~ 1833___________________________________________________________
- 1833
gi~14749154~ 1833___________________________________________________________
- 1833
gi~10047205~ 1025MTK T LGKQSAQNEREKLQIKIDEMDKILKKIDNCLTEMETETKNLEDEE
1084
G
gi~14749150~ 903~ ~ ~ 915
L~~"""T 1~C~
I'
________________________________________
30
2050
2060
2070
2080
2090
2100
NOV11 2041KNNPVEEWDSEMRAAEKELEQLKTEEEELQRNLLELEVQKEQTLAQIDFMQKQRNRTEEL
2100
gi~18308012~ 2041KNNPVEEWDSEMRAAEKELEQLKTEEEELQRNLLELEVQKEQTLAQIDFMQKQRNRTEEL
2100
3S gi~9966807~ 1833-
___________________________________________________________
1833
gi~14749154~ 1833____________________________________________________________
1833
gi~10047205~ 1085KNNPVEEWDSEMRAAEKELEQLKTEEEELQRNLLELEVQKEQTLAQIDFMQKQRNRTEEL
1144
gi~14749150~ 915___________________________________________________________
- 915
40 2110 0
2120
2130
2140
2150
216
NOV11 2101LDQLSLSEWDWEWSDDQAVFTFVYDTIQLTITFEESWGFPFLDKRYRKIWVNFQSLL
2160
gi~183080121 2101LDQLSLSEWDWEWSDDQAVFTFVYDTIQLTITFEESWGFPFLDKRYRKIVDVNFQSLL
2160
gi~9966807~ 1833-___________________________________________________________
1833
4S gi~14749154~
1833____________________________________________________________
1833
gi~10047205~ 1145LDQLSLSEWDWEWSDDQAVFTFVYDTTQLTITFEESWGFPFLDKRYRKIVDVNFQSLL
1204
gi~14749150~ 915____________________________________________________________
915
2170 0
2180
2190
2200
2210
222
50
NOV11 2161DEDQAPPSSLLVHKLIFQYVEEKESWKKTCTTQHQLPKMLEEFSLWHHCRLLGEEIEYL
2220
gi.~18308012~ 2161DEDQAPPSSLLVHKLIFQYVEEKESWKKTCTTQHQLPKMLEEFSLWHHCRLLGEEIEYL
2220
gi~9966807~ 1833-___________________________________________________________
1833
gi~14749154, 1833____________________________________________________________
1833
SS gi~10047205~
1205DEDQAPPSSLLVHKLIFQYVEEKESWKKTCTTQHQLPKMLEEFSLWHHCRLLGEEIEYL
1264
gi~14749150~ 915____________________________________________________________
915
2230 0
2240
2250
2260
2270
228
C7ONOV11
2221KRWGPNYNLMNIDINNNELRLLFSSSAAFAKFEITLFLSAYYPSVPLPSTIQNHVGNTSQ
2280
gi I183080121
2221KRWGPNYNLMNIDINNNELRLLFSSSAAFAKFEITLFLSAYYPSVPLPSTIQNHVGNTSQ
2280
giI9966807~ 1833____________________________________________________________
1833
gi~14749154~ 1833____________________________________________________________
1833
gi.'10047205~
1265KRWGPNYNLMNIDINNNELRLLFSSSAAFAKFEITLFLSAYYPSVPLPSTIQNHVGNTSQ
1324
6S gi~14749150~
915____________________________________________________________
915
2290
2300
2310
....~....~....~....~....~....~....~.
NOV11 2281DDIATILSKVPLENNYLKNWKQIYQDLFQDCHFYH
2316
gi~18308012~ 2281DDIATILSKVPLENNYLKNWKQIYQDLFQDCHFYH
2316
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giI9966807~ 1833 ____________________________________ 1833
gi~14749154~ 1833 ____________________________________ 1833
gi~100472051 1325 DDIATILSKVPLENNYLKNWICQIYQDLFQDCHFYH 1360
gi~14749150~ 915 ____________________________________ 915
NOV 11 nucleic acids, and the encoded polypeptides, according to the invention
are
useful in a variety of applications and contexts. For example, NOV 11 nucleic
acids and
polypeptides can be used to identify proteins that are members of the Nuclear
Protein-like
Protein Family. The NOV 11 nucleic acids and polypeptides can also be used to
screen for
molecules, which inhibit or enhance NOV11 activity or function. Specifically,
the nucleic
acids and polypeptides according to the invention may be used as targets for
the identification
of small molecules that modulate or inhibit, e.g., cellular activation,
cellular replication, and
signal transduction. These molecules can be used to treat, e.g., Von Hippel-
Lindau (VHL)
syndrome, Cirrhosis, Transplantation, Hemophilia, hypercoagulation, Idiopathic
thrombocytopenic purpura, autoimmume disease, allergies, immunodeficiencies,
transplantation, Graft vesus host, Cardiovascular diseases, Von Hippel-Lindau
(VHL)
syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia,
Parkinson's
disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome,
Multiple
sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders,
Addiction, Anxiety,
Pain, Neuroprotection, Systemic lupus erythematosus , Autoimmune disease,
Asthma,
Emphysema, Scleroderma, allergy, as well as other diseases, disorders and
conditions.
In addition, various NOV 11 nucleic acids and polypeptides according to the
invention
are useful, inter olio, as novel members of the protein families according to
the presence of
sequence relatedness to previously described proteins. The NOV 11 nucleic
acids and
polypeptides, antibodies and related compounds according to the invention will
be useful in
therapeutic and diagnostic applications in the mediation of cardiac, immune,
and nerve
physiology. As such, the NOV 11 nucleic acids and polypeptides, antibodies and
related
compounds according to the invention may be used to treat cardiovascular,
immune, and
nervous system disorders, e.g., Von Hippel-Lindau (VHL) syndrome, Cirrhosis,
Transplantation, Hemophilia, hypercoagulation, Idiopathic thrombocytopenic
purpura,
autoimmume disease, allergies, immunodeficiencies, transplantation, Graft
vesus host,
Cardiovascular diseases, Von Hippel-Lindau (VHL) syndrome , Alzheimer's
disease, Stroke,
Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease,
Cerebral palsy,
Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia,
Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain,
Neuroprotection, Systemic
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lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma,
allergy, as
well as other diseases, disorders and conditions.
The NOV11 nucleic acids and polypeptides are useful for detecting specific
cell types.
For example, expression analysis has demonstrated that a NOV 11 nucleic acid
is expressed in
Adipose, Aorta, Artery, Coronary Artery, Umbilical Vein, Thyroid, Liver, Small
Intestine,
Duodenum, Colon, Ascending Colon, Bone Marrow, Lymph node, Tonsils, Thymus,
Cartilage, Muscle, Brain, Cervix, Uterus, Vulva, Prostate, Testis, Lmig,
Bronchus, Urinary
Bladder, Kidney, Skin, Epidermis, Dermis.
Additional utilities for NOV 11 nucleic acids and polypeptides according to
the
invention are disclosed herein.
NOV12
A NOV 12 polypeptide has been identified as a Plasma Membrane Protein-like
protein
(also referred to as CG94282-O1). The disclosed novel NOV12 nucleic acid (SEQ
ID N0:25)
of 8811 nucleotides is shown in Table 12A. The novel NOV 12 nucleic acid
sequences maps
to the chromosome 12.
An ORF begins with an ATG initiation codon at nucleotides 1-3 and ends with a
TAG
codon at nucleotides 4378-4380. A putative untranslated region and/or
downstream from the
termination codon is underlined in Table 12A, and the start and stop, codons
are in bold letters.
Table 12A. NOV12 Nucleotide Sequence (SEQ ID N0:25)
ATGCTGTTCAAGCTCCTGCAGAGACAAACCTATACCTGCCTGTCCCACAGGTATGGGCTCTACGTGTGCTTCTT
GGGCGTCGTTGTCACCATCGTCTCCGCCTTCCAGTTCGGAGAGTGGGTAGAAGCCAGGGATCCTGCCAAACATC
CTATAGTGCACAGGACAGCCCCTACAACAAAGAATCATCCAGCCCAAAATGTCGATAGTGCTGAAGTTGAGAAA
TCCGGAATTAGAAGGGGCAAGAATGGCTGCAGGGCAGTTAGTCTACAGGACTGGCCTGGGACTAGAGGATGTGC
CAATTTCACCTTCGCCTTCTGCCATGATTGTAAGTTTTCTGAGGTCTCCCAGAAACGCTTCCTGTACATCCTGC
AGAACTGTCATTGGTTAACTGATTGGGGTTGGACTTGGTTGGCTCTGCTCCACGGGTCTCTCATCCTCCAGGGA
CCAGCCAGCGAACCTGGTTGTGTTCTTCTCAAGGCAAAGGTGGTTCTGGAATGGAGCCGAGATCAATACCATGT
TTTGTTTGATTCCTATAGAGACAATATTGCTGGAAAGTCCTTTCAGAATCGGCTTTGTCTGCCCATGCCGATTG
ACGTTGTTTACACCTGGGTGAATGGCACAGATCTTGAACTACTGAAGGAACTACAGCAGGTCAGAGAACAGATG
GAGGAGGAGCAGAAAGCAATGAGAGAAATCCTTGGGAAAAACACAACGGAACCTACTAAGAAGAGTGAGAAGCA
GTTAGAGTGTTTGCTAACACACTGCATTAAGGTGCCAATGCTTGTCCTGGACCCAGCCCTGCCAGCCAACATCA
CCCTGAAGGACCTGCCATCTCTTTATCCTTCTTTTCATTCTGCCAGTGACATTTTCAATGTTGCAAAACCAAAA
AACCCTTCTACCAATGTCTCAGTTGTTGTTTTTGACAGTACTAAGGATGGGACATTGCTCACTCAGAAGGTGAC
TTTTGAGTGGAAATGTGAAGAAGGTGAGGTAGCCAGCAATGCGAATATCTGGGGAAAGACTGATCTGGGTTCCC
CCAGGAGGCCTTTGCCATGGCCTGTGGCCCTGGAGCCACCTAGGGCTCAGCTCAGCTCTGCCCTACAGATTCTC
ACTAGGCCACGGGTATCTCAGGACAGAGCCAACACAAGTTATGAAATTAAACTAGACACACCCCTTCTTCGAGG
TTACGCCAAGCCAGTGCCTGGGCCTGAAACTGGCCTGCAGCCCCTCAGCTTCGCCCACTGCCTTCCGACCCTGG
ACCTTCGCAAAGTGAACGAGCTTCGGGACTTCGTGAAAATGTATAAGCAGGATCCGAGCATTCTGCATACCAAG
GAAACGTGCTTTCTGAGGGAGCAGGTGGAGAGCATGGGGGAAAGCTATTATAAATCAGAAGAAAATATCAAGGA
ATTAAAAACAGGTAGTAAGAAGGTGGAGGAAAACATAAGCACAGACGAACTATCAAGTGAGGAAAGTGATCTAG
AAATTGATAACGAAGCTGTGATTGAACCAGACACTGATTCCCCTCAAGAAATGGGAGATGGAGAGGCCAGTGTA
GCGCTTCTAAAACTGAATAACCCCAAGGATTTTCAAGAATTGAATAAGCAAACTAAGAAGAACATGACCATTGA
TGGAAAAGAACTGACCATAAGTCCTGCATATTTATTATGGGATCTGAGCGCCATCAGCCAGTCTAAGCAGGATG
AAGACATCTCTGCCAGTCGTTTTGAAGATAACGAAGAACTGAGGTACTCATTGCGATCTATCGAGAGGCATGCA
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CCATGGGTTCGGAATATTTTCATTGTCACCAACGGGCAGATTCCATCCTGGCTGAACCTTGACAATCCTCGAGT
GACAATAGTAACACACCAGGATGTTTTTCGAAATTTGAGCCACTTGCCTACCTTTAGTTCACCTGCTATTGAAA
GTCACATTCATCGCATCGAAGGGCTGTCCCAGAAGTTTATTTACCTAAATGATGATGTCATGTTTGGGAAGGAT
GTCTGGCCAGATGATTTTTACAGTCACTCCAAAGGCCAGAAGGTTTATTTGACATGGCCTGTGCCAAACTGTGC
CGAGGGCTGCCCAGGTTCCTGGATTAAGGATGGCTATTGTGACAAGGCTTGTAATAATTCAGCCTGCGATTGGG
ATGGTGGGGATTGCTCTGGAAACAGTGGAGGGAGTCGCTATATTGCAGGAGGTGGAGGTACTGGGAGTATTGGA
GTTGGACAGCCCTGGCAGTTTGGTGGAGGAATAAACAGTGTCTCTTACTGTAATCAGGGATGTGCGAATTCCTG
GCTCGCTGATAAGTTCTGTGACCAAGCATGCAATGTCTTGTCCTGTGGGTTTGATGCTGGCGACTGTGGGCAAG
AAAACTCAGACTCAAAGAATAGGAAAACAGAGGAAAAATGCCCAGTT TCATGTTTCTGTTT
TTTCCTCTAGATCATTTTCATGAATTGTATAAAGTGATCCTTCTCCCAAACCAGACTCACTATATTATTCCAAA
AGGTGAATGCCTGCCTTATTTCAGCTTTGCAGAAGTAGCCAAAAGAGGAGTTGAAGGTGCCTATAGTGACAATC
CAATAATTCGACATGCTTCTATTGCCAACAAGTGGAAAACCATCCACCTCATAATGCACAGTGGAATGAATGCC
ACCACAATACATTTTAATCTCACGTTTCAAAATACAAACGATGAAGAGTTCAAAATGCAGATAACAGTGGAGGT
GGACACAAGGGAGGGACCAAAACTGAATTCTACAGCCCAGAAGGGTTACGAAAATTTAGTTAGTCCCATAACAC
TTCTTCCAGAGGCGGAAATCCTTTTTGAGGATATTCCCAAAGAAAAACGCTTCCCGAAGTTTAAGAGACATGAT
GTTAACTCAACAAGGAGAGCCCAGGAAGAGGTGAAAATTCCCCTGGTAAATATTTCACTCCTTCCAAAAGACGC
CCAGTTGAGTCTCAATACCTTGGATTTGCAACTGGAACATGGAGACATCACTTTGAAAGGATACAATTTGTCCA
AGTCAGCCTTGCTGAGATCATTTCTGATGAACTCACAGCATGCTAAAATAAAAAATCAAGCTATAATAACAGAT
GAAACAAATGACAGTTTGGTGGCTCCACAGGAAAAACAGGTTCATAAAAGCATCTTGCCAAACAGCTTAGGAGT
GTCTGAAAGATTGCAGAGGTTGACTTTTCCTGCAGTGAGTGTAAAAGTGAATGGTCATGACCAGGGTCAGAATC
CACCCCTGGACTTGGAGACCACAGCAAGATTTAGAGTGGAAACTCACACCCAAAAAACCATAGGCGGAAATGTG
ACAAAAGAAAAGCCCCCATCTCTGATTGTTCCACTGGAAAGCCAGATGACAAAAGAAAAGAAAATCACAGGGAA
AGAAAAAGAGAACAGTAGAATGGAGGAAAATGCTGAAAATCACATAGGCGTTACTGAAGTGTTACTTGGAAGAA
AGCTGCAGCATTACACAGATAGTTACTTGGGCTTTTTGCCATGGGAGAAAAAAAAGTATTTCCAAGATCTTCTC
GACGAAGAAGAGTCATTGAAGACACAATTGGCATACTTCACTGATAGCAAAAATACTGGGAGGCAACTAAAAGA
TACATTTGCAGATTCCCTCAGATATGTAAATAAAATTCTAAATAGCAAGTTTGGATTCACATCGCGGAAAGTCC
CTGCTCACATGCCTCACATGATTGACCGGATTGTTATGCAAGAACTGCAAGATATGTTCCCTGAAGAATTTGAC
AAGACGTCATTTCACAAAGTGCGCCATTCTGAGGATATGCAGTTTGCCTTCTCTTATTTTTATTATCTCATGAG
TGCAGTGCAGCCACTGAATATATCTCAAGTCTTTGATGAAGTTGATACAGATCAATCTGGTGTCTTGTCTGACA
GAGAAATCCGAACACTGGCTACCAGAATTCACGAACTGCCGTTAAGTTTGCAGGATTTGACAGGTCTGGAACAC
ATGCTAATAAATTGCTCAAAAATGCTTCCTGCTGATATCACGCAGCTAAATAATATTCCACCAACTCAGGAATC
CTACTATGATCCCAACCTGCCACCGGTCACTAAAAGTCTAGTAACAAACTGTAAACCAGTAACTGACAAAATCC
ACAAAGCATATAAGGACAAAAACAAATATAGGTTTGAAATCATGGGAGAAGAAGAAATCGCTTTTAAAATGATT
CGTACCAACGTTTCTCATGTGGTfiGGCCAGTTGGATGACATAAGAAAAAACCCfiAGGATCTCACTCTGTTGTCC
AAGCTGGAATGCAGTAATGCAAACATGGCTCACTGTAGCCTCGACCTCGTGGGCTCAAGCAATCCTCCCACCTC
AGCCTCCTGACTAGTGGAACCACAGACATGAGCTGCTGCACCCAGCTAAAATGGAGTATTTTTAATTTCTGGGT
CTTTTAAATGCATTTGGAGGTCTfifiAGTTTTACCTCACTGAAATTAGGATTTTAATTATAAATAATCAAAGATG
TGAACCTTACAGACATTTTAAAGCCATTATATTTTTTCTATAAACCCTGTTCTCGTTTGGAGGAGAAAGAAATT
GGAATTTTCP~AAA~1AAATAAAAATACCTTTAACACCTATTTAGTGTCTTTAGTAATCCAGTAAAATACTTGATT
TTTTACTAAATGTTTCCCACAAGCCAAGCAAACCATAAGCTACAATAATAAfiTACCTAGCGTACAGCCCTCTTT
GCATATGCTGTTCCCTCCACTTGAAGTGTACTGTTTAATTTCTTAAAATAACTTTAGCTTTTAAGAACCAATTT
TGATGGGAGTACAGACTTCCCCCATTTTCTTGATGAGTTCTCTCCGTCATGTGTAGTAATAATGTGAGAATTTG
CAGTTTTTAGTTGTAGCCTATACTTTTAGGTCTTTGTGCCAATTTGAAAGTfiATTGGGTTAGAGTATTCATAGA
CATTTTCATGGTACTTAAAGGGACAGGGGTTTAGTAAAAAGACACATGGCAAGCCAGGCTTTTTCCACAGTTTG
CCAGGCCCAGCTGCCTCTTGTGTACCTGAACAGATTTTATCATTAACCCTTGTTTATGTTGTTTTGTTTTATTT
CGACGAAGGCTTATTTTAAGTCAGGCATGGAAAACTAGACTTCAGACTGACTTCAGCTTTAAGGACATGTTTAT
CCCGTTAACAGGGAGTCTGGGATAGACAATCTCCAGGCTTTGTTTTTCTCTGAATTTCTTAGCTCTGCTTGTGA
TGGCTTCATCATCAGGCCACAGACCATTAACACATTCTAGAACTTTAACATTGGTTAAATAATACCATCTAATA
GCCTGTCTTCAGCATTTCCCCAGTTGCCTCCAAATGCCCTTCATAGCTGTTCTCTGCCTCTGTTTGTTTTTAAT
CCAAGATACACTCAAGGCTCATATATTAGGTTGACATAGCTCTTTAGTATCCTTTAATTTAAAGCAGTCTCCAG
GTTTAGAGAAAGATGAATGAGCTTTCACATACCCCTCACTTGTCTTCTTCAGAAGTGTAGGCTACAACTAAAAC
TTCCTTCTTCAGAAGGAAGACAAGTGATTTATATTTATTTACTTCCATTTCTATTTGACCTTGTTTCATCTAAA
CACACCCACTCCACCACTGCTACCTGATTAATATTAAGTGAATCTCAAACATTGTATCATTTTAGCTCCACGTT
TTTTGTATGTATCTCCAAAATATAAAGATTCTTAAAAATATAACCACAATACCATTATCACCCTAAAAAAATCA
ATAATGATTCCTTAATATGACCAACTACTTTGTCAATGTACACCTTTCACTCCTCTTAACTTTCATAAAGACTT
ATGTGTTTTTTTGGTTTTTAAGTTTGTTGGTTTGAAGTTAAATCTATGGGTTTTCCCTCCATCTCTCTTTTTTT
AACCGTATAATTTTTTGCATGTGTATATGAATAAATCTGATTATAGATTCTATAGCTATCTTACACTTTGTCCC
TCTCTGATTGAATCCTAGTTAACAAGTTTCTATGTCTCTTGTATTTCCCATAAATTGGTAGTTGGATCTGAAGG
CTTTATCAGGTTTGTTTGATTTTTTTTTTTTTAATTTTGGCGAATCTACTTCAAAAGTATTGGCCTACCTACAA
GCCACTTTAATGGGCCCTTAGTTTAGTGACCTTTGCCTTGAAAGGAACTTGAAACAAGCAAGGAAGCACCACTG
TAATCTGCTTTTTTGCCAGAACTGTAGCATCTTACAGCTTGGTTAGAGACATAGTAAGCAGAAATTATCAAATT
CATATAATCTGTAGCTATAAGGCACTGTCTCTCTCTCTCAATTATTTACATGATTTTTCTTTGTAATATAACTA
TCATTTCAGAGAACTTGGTTTTGATTTTTTTTTTTTAATCTTTTTGAGACAGAGTCTCGCTTTATCACCCAGGC
TGGAGTGCAGTGGTGCAATCfiAAAGATTGCTGACTGCAACCTCTGCCTCCCGAGTTCAGCAATTCTAGTGCCTC
AGCCTCTCGAGTAGCTGGGATTACAGGCATGCCACCACACCCGGCTAATTTTTTTGTATTTTTAGTAAAGACAG
GGTTTCACCATGTTGGCTAGGCTGGTCTCAAATTTTTGACCTCAAGTAATCAGCCTACCTTGATCTCCCAAAGT
GCTGGGATTACAGGCATGAGCCACCATGCATGGCCTTCAGAGAACTTGGTTTTAGGTACTTACGGATTGTCTTT
CTTTTTTTTCCTCACTGCAGCCTCTCCCTCCCAGGTTCAAGCGATTCTCCTACCTCAGCTTCCTGAAGAGCTGG
GACCACAGGAAGTTTGTTTGCCTGAATGACAACATTGACCACAATCATAAAGATGCTCAGACAGTGAAGGCTGT
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TCTCAGGGACTTCTATGAATCCATGTTCCCCATACCTTCCCAATTTGAACTGCCAAGAGAGTATCGAAACCGTT
TCCTTCATATGCATGAGCTGCAGGAATGGAGGGCTTATCGAGACAAATTGAAGTTTTGGACCCATTGTGTACTA
GCAACATTGATTATGTTTACTATATTCTCATTTTTTGCTGAGCAGTTAATTGCACTTAAGCGGAAGATATTTCC
CAGAAGGAGGATACACAAAGAAGCTAGTCCCAATCGAATCAGAGTATAGAAGATCTTCATTTGAAAACCATCTA
CCTCAGCATTTACTGAGCATTTTAAAACTCAGCTTCACAGAGATGTCTTTGTGATGTGATGCTTAGCAGTTTGG
CCCGAAGAAGGAAAATATCCAGTACCATGCTGTTTTGTGGCATGAATATAGCCCACTGACCAGGAATTATTTAA
CCAACCCACTGAAAACTTGTGTGTTG_AGCAGCTCTGAACTGATTTTACTTTTAAAGAATTTGCTCATGGACCTG
TCATCCTTTTTATAAAAAGGCTCACTGACAAGAGACAGCTGTTAATTTCCCACAGCAATCATTGCAGACTAACT
TTATTAGGAGAAGCCTATGCCAGCTGGGAGTGATTGCTAAGAGGCTCCAGTCTTTGCATTCCAAAGCCTTTTGC
TAAAGTTTTGCACTTTTTTTTTTTCATTTCCCATTTTTAAGTAGTTACTAAGTTAACTAGTTATTCTTGCTTCT
GAGTATAACGAATTGGGATGTCTAAACCTATTTTTATAGATGTTATTTAAATAATGCAGCAATATCACCTCTTA
TTGACAATACCTAAATTATGAGTTTTATTAATATTTAAGACTGTAAATGGTCTTAAACCACTAACTACTGAAGA
GCTCAATGATTGACATCTGAAATGCTTTGTAATTATTGACTTCAGCCCCTAAGAATGCTATGATTTCACGTGCA
GGTCTAATTTCAAAGGGCTAGAGTTAGTACTACTTACCAGATGTAATTATGTTTTGGAAATGTACATATTCAAA
CAGAAGTGCCTCATTTTAGAAATGAGTAGTGCTGATGGCACTGGCACATTACAGTGGTGTCTTGTTTAATACTC
ATTGGTATATTCCAGTAGCTATCTCTCTCAGTTGGTTTTTGATAGAACAGAGGCCAGCAAACTTTCTTTGTAAA
AGGCTGGTTAGTAAATTATTGCAGGCCACCTGTGTCTTTGTCATACATTCTTCTTGCTGTTGTTTAGTTTGTTT
TTTTTCAAACAACCCTCTAAAAATGTAAAAACCATGTTTAGCTTGCAGCTGTACAAAAACTGCCCACCAGCCAG
ATGTGACCCTCAGGCCATCATTTGCCAATCACTGAGAATTAGTTTTTGTTGTTGTTGTTGTTGTTGTTTTTGAG
ACAGAGTCTCTCTCTGTTGCCCAGGCTGGAGTGCAGTGGCGCAATCTCAGCTCACTGCAACCTCCGCCTCCCGG
GTTCAAGCAGTTCTGTCTCAGCCTTCTGAGTAGCTGGGACTACAGGTGCATGCCACCACACCCTGCTAATTTTT
GTATTTTTAGTAGAGACGGGGGTTCCACCATATTGGTCAGGCTTATCTTGAACTCCTGACCTCAGGTGATCCAC
CTGCCTCTGCCTCCCAAAGTGCTGAGATTACAGGCATAAGCCAGTGCACCCAGCCGAGAATTAGTATTTTTATG
TATGGTTAAACCTTGGCGTCTAGCCATATTTTATGTCATAATACAATGGATTTGTGAAGAGCAGATTCCATGAG
TAACTCTGACAGGTATTTTAGATCATGATCTCAACAATATTCTTCCAAAATGGCATACATCTTTTGTACAAAGA
ACTTGAAATGTAAATACTGTGTTTGTGCTGTAAGAGTTGTGTATTTCAAAAACTGAAATCTCATAAAAAGTTAA
ATTTT
Variant sequences of NOV12 are included in Example 3, Table 24. A variant
sequence
can include a single nucleotide polymorphism (SNP). A SNP can, in some
instances, be
referred to as a "cSNP" to denote that the nucleotide sequence containing the
SNP originates
as a cDNA.
The NOV 12 protein (SEQ ID N0:26) encoded by SEQ TLS N0:25 is 1459 amino acid
residues in length and is presented using the one-letter amino acid code in
Table 12B. Psort
analysis predicts the NOV 12 protein of the invention to be localized at the
plasma membrane
with a certainty of 0.6500.
Table 12B. Encoded NOV12 protein sequence (SEQ ID NO:26)
MLFKLLQRQTYTCLSHRYGLYVCFLGVWTIVSAFQFGEWVEARDPAKHPIVHRTAPTTKNHPAQ
NVDSAEVEKSGIRRGKNGCRAVSLQDWPGTRGCANFTFAFCHDCKFSEVSQKRFLYILQNCHWLT
DWGWTWLALLHGSLILQGPASEPGCVLLKAKWLEWSRDQYHVLFDSYRDNIAGKSFQNRLCLPM
PIDVVYTWVNGTDLELLKELQQVREQMEEEQKAMREILGKNTTEPTKKSEKQLECLLTHCIKVPM
LVLDPALPANITLKDLPSLYPSFHSASDIFNVAKPKNPSTNVSVWFDSTKDGTLLTQKVTFEWK
CEEGEVASNANIWGKTDLGSPRRPLPWPVALEPPRAQLSSALQILTRPRVSQDRANTSYEIKLDT
PLLRGYAKPVPGPETGLQPLSFAHCLPTLDLRKVNELRDFVKMYKQDPSILHTKETCFLREQVES
MGESYYKSEENIKELKTGSKKVEENISTDELSSEESDLEIDNEAVIEPDTDSPQEMGDGEASVAL
LKLNNPKDFQELNKQTKKNMTIDGKELTISPAYLLWDLSAISQSKQDEDISASRFEDNEELRYSL
RSIERHAPWVRNIFIVTNGQIPSWLNLDNPRVTIVTHQDVFRNLSHLPTFSSPAIESHIHRIEGL
SQKFIYLNDDVMFGKDVWPDDFYSHSKGQKVYLTWPVPNCAEGCPGSWIKDGYCDKACNNSACDW
DGGDCSGNSGGSRYIAGGGGTGSIGVGQPWQFGGGINSVSYCNQGCANSWLADKFCDQACNVLSC
GFDAGDCGQENSDSKNRKTEEKCPVKKKKIMFLFFPLDHFHELYKVILLPNQTHYIIPKGECLPY
FSFAEVAKRGVEGAYSDNPIIRHASIANKWKTIHLIMHSGMNATTIHFNLTFQNTNDEEFKMQIT
VEVDTREGPKLNSTAQKGYENLVSPITLLPEAEILFEDIPKEKRFPKFKRHDVNSTRRAQEEVKI
94
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PLVNISLLPKDAQLSLNTLDLQLEHGDITLKGYNLSKSALLRSFLMNSQHAKIKNQAIITDETND
SLVAPQEKQVHKSILPNSLGVSERLQRLTFPAVSVKVNGHDQGQNPPLDLETTARFRVETHTQKT
IGGNVTKEKPPSLIVPLESQMTKEKKITGKEKENSRMEENAENHIGVTEVLLGRKLQHYTDSYLG
FLPWEKKKYFQDLLDEEESLKTQLAYFTDSKNTGRQLKDTFADSLRYVNKILNSKFGFTSRKVPA
HMPHMIDRIVMQELQDMFPEEFDKTSFHKVRHSEDMQFAFSYFYYLMSAVQPLNTSQVFDEVDTD
QSGVLSDREIRTLATRIHELPLSLQDLTGLEHMLINCSKMLPADTTQLNNIPPTQESYYDPNLPP
VTKSLVTNCKPVTDKIHKAYKDKNKYRFEIMGEEEIAFKMIRTNVSHWGQLDDIRKNPRISLCC
PSWNAVMQTWLTVASTSWAQAILPPQPPD
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 12C.
Table 12C. results
Patp for NOV12
Smallest
Sum
eading igh Prob
equences Frame Score P(N)
producing
High-scoring
Segment
Pairs:
>patp:ABB30279Peptide#2930 encodedby breast cell+1 1900 7.5e-196
>patp:AAM56268Human single +l 1900 7.5e-196
brain exon
expressed probe
>patp:AAM16457Peptide#2891 encodedby probe +1 1900 7.5e-196
>patp:AAM28952Peptide#2989 encodedby probe +1 1900 7.5e-196
>patp:AAM04186Peptide#2868 encodedby probe +1 1900 7.5e-196
In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 6444 of 6447 bases (99%) identical
to a
gb:GENBANK-m:AB033034~acc:AB033034.1 mRNA from Homo sapiens (mRNA for
I~A_A1208 protein, partial cds). The full amino acid sequence of the protein
of the invention
was found to have 663 of 663 amino acid residues (100%) identical to, and 663
of 663 amino
acid residues (100%) similar to, the 663 amino acid residue ptnr:SPTREMBL-
ACC:Q9ULL2
protein from Homo sapiehs (KIAA1208 PROTEIN).
NOV 12 also has homology to the proteins shown in the BLASTP data in Table
12D.
Table 12D. BLASTresults
for
NOV12
Gene Index/ Protein/ OrganismLength Identity PositivesExpect
Identifier (aa) (~)
gi~6382022~dbj~BAA8KIAA1208 protein663 663/663 663/663 0.0
6522.1~(AB033034)[Homo sapiens] (1000 (1000
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gi~16551459~dbjIBABunnamed protein847 585/613 585/613 0.0
71102.1~(AK056137)product (95%) (95%)
[Homo sapiensl
gi~2137411~pir~~I49hypothetical 384 277/400 307/400 e-142
528 protein (69%) (76%)
[Mus musculus]
gi~11360271~pir~~T5hypothetical 248 134/137 135/l37 2e-73
0618 protein (97%) (97%)
DKFZp762B226.1
[Homo sapiens]
gi~7303923~gb~AAF58CG8027 gene 652 84/l55 114/155 9e-49
967.1~(AE003834)product (54%) (73%)
[Drosophila
melanogaster]
A multiple sequence alignment is given in Table 12E, with the NOV 12 protein
being
shown on line 1 in Table 12E in a ClustalW analysis, and comparing the NOV 12
protein with
the related protein sequences shown in Table 12D. This BLASTP data is
displayed graphically
in the ClustalW in Table 12E.
Table 12E. ClustalW Analysis of NOV12
1) > NOV12; SEQ >D N0:26
2) > gi~6382022~/ KIAA1208 protein [Homo sapiens]; SEQ m NO:87
3) > gi~16551459~/ unnamed protein product [Homo sapieras]; SEQ m N0:88
4) > gi~2137411 ~/ hypothetical protein - mouse (fragment); SEQ >D N0:89
5) > gi~11360271~1 hypothetical protein DKFZp762B226.1 - human (fragment); SEQ
II? N0;90
6) > gi~7303923~/ CG8027 gene product [Drosoplaila naelanogaster]; SEQ II7
N0:91
10 20 30 40 50 60
NOV12 1 ----MLFKLLQRQTYTCLSHRYGLWCFLGWVTIVSAFQFGEWVEARDPAKHPIVHRTA 56
gi~63820221 1 RKTEEKCPVKKKKIMFLFFPLDHFHELYKVILLPNQTHYIIPKGECLP------------ 48
gi~165514591 1 ----MLFKLLQRQTYTCLSHRYGLWCFLGVWTIVSAFQF------------------- 37
gi~2137411~ 1 ____________________________________________________-_______ 1
giI113602711 1 ____________________________________________________________ 1
gi~7303923~ 1 ____________________________________________________________ 1
70 80 90 100 110 120
2S NOV12 57 PTTKNHPAQNVDSAEVEKSGIRRGKNGCRAVSLQDWPGTRGCANFTFAFCHDCKFSEVSQ 116
gi~63820221 48 ___________________________________________________________
- 48
gi~16551459~ 37 _______________________G____________________________________
3g
gi121374111 1 ____________________________________________________________ 1
gi~11360271~ 1 ____________________________________________________________ 1
gi~7303923~ 1 ___________-________________________________________________ 1
130 140 150 160 170 180
NOV12 117 KRFLYILQNCHWLTDWGWTWLALLHGSLILQGPASEPGCVLLKAKWLEWSRDQYHVLFD 176
giI63820221 48 ____________________________________________YFSFAEVA--KRGVEG 62
gi~16551459~ 38 --------------------------------------------EWLEWSRDQYHVLFD 54
g I I ______ 1
a. 2137411 1 ______________________________________________________
gi~11360271~ 1 ___________________________________________________________
' - 1
giI73039231 1 _______________________________________-____________________ 1
190 200 210 220 230 240
NOV12 177 SYRDNIAGKSFQNRLCLPMPIDWYTWVNGTDLELLKELQQVREQMEEEQKAMREILGKN 236
gi~6382022~ 63 AYSDNPIIRHASIANKWKTIHLIMHSGMNATTIHFNLTFQNT-NDEEFKMQITVEVDTRE
121
4S gi1165514591 55 SYRDNIAGKSFQNRLCLPMPIDWYTWVNGTDLELLKELQQVREQMEEEQKAMREILGKN
114
gi~2137411~ 1 ____________________________________________________________ 1
gi~11360271~ 1 __________________________________________________pTRPVFDEVD 10
gi17303923~ 1 ____________________________________________________________
1
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250 260 270 280 290 300
NOV12 237 TTI.~~PTKKSE~QL C LTHCI LVLDPALP~TT;fr~~L'SLYPSFHSASDIFNV~ ~P 296
S giI63820221 122 GP~GLNST~.Q~GY PITL~.,~------- E~~ELFyT~--------------- ,E''
157
gi ~ 16551459 ~ 115 TT~PTKKSE 'QL C T.~'1~HCI ' VLDPALP~~TI~ ~L
~SLYPSFHSASDIFNV P 174
v L
gi12137411~ 1 - ~-~-ILT~!'~ Y S PVTP~~_- ____QiD~~PFEv ~ _______________ ~E 32
gi ~ 11360271 ~ 1l TD~'~SGVL~D~EIRT V'~!RIHE~ ---- ---~T.iST~QyT---------------
---- 40
gi~73039231 1 ---MEQLRLC~SRR IiALTMTGG---------LC~~WV~;AN------------------ 30
310 320 330 340 350 360
NOV12 297 NPSTNVS .STDGTLLTQKVTFEWKCE~.'GEVASNANIWGKTDLGSPRRPLPWPVAL 356
gi~63820221 158 R--_______ PKF______________H~S-________________________ 169
LS gi~16551459~ 175 NPSTNVS STUD-------------VE~AHS-------G-----------------
197
gi~2137411~ 33 R-________ PKIR______________H~A-________________________ 44
gi~11360271~ 40 _________________G_____________'.____________________________
41
gi~7303923~ 30 ____________________________________________________________
370 380 390 400 410 420
NOV12 357 EPPRAQLSSALQILTRPRVSQDRANTSYEIKLDTPLLRGYAKPVPGPETGLQPLSFAHCL 416
gi~6382022~ 169 ______________________________-_______________________~R____
171
gi1165514591 197 ---------LLKGNSRQTVWRGYLTT------D--------KEVPG-LVLMQDLAFLSGF
233
ZS gi~2137411~ 44 ______________________________________________________TG____
46
gi~11360271~ 41 ____________________________________________________________
41
gi~7303923~ 30 ___________________________________________________________
- 30
430 440 450 460 470 480
NOV12 417 PTLDLRKVNELRDFVKMYKQDPSILHTKETCFLREQVESMGESYYKSEENTKELKTGSIL476
gi~6382022~ 171 ___________________________________________________________172
gi116551459~ 234 P-_____________pTFK--________________ET__N__________QLKT-
__246
gi~21374111 46 ___________________________________________________________47
3S gi~11360271~ 41
____________________________________________________________ 41
giI7303923~ 30 ____________________________________________________________ 30
490 500 510 520 530 540
NOV12 477 VE~ ISTDELSSEESDLE~DNEA'~'IEPDTDSPQEMGDGEASi~F'~- KLNNPKDFQE ..~T
536
gi I 6382022 ~ 173 AQ E--------------~~CIPI,~----------------I~ P~CDAQLSLNT
~L~L 202
gi~165514591 247 LP ----LSS-----K~t'T~LLQI~Y-----S-------EASE KLNNPKDFQE ~T
284
gi121374111 48 FQ E______________~IP~-______________~~E P,EAQVRLS vLvL 77
gi~11360271~ 41 __________________~~~L-_______________ __CSNILPADITQ ' IP 64
4S gi~73039231 30 --------------------YLSA--------------------EGQTGGFSSACTAI
50
~..:
550 560 570 580 590 600
NOV12 537 KN ~. KELTI P~ DL SQ LCQ~DISA'~~FEDN~ L~; . RS'~EiAPWV 595
S0 gi I 6382022 I 203 ~HGDIj --YN~ K~ R-- F I~HAIKNQ~i;II- TD TND -VAPf-----
250
gi l 16551459 l 285 ~KN ~ KELT,I PY DL 5~'Q~DISA~FEDN~ L RSrI~E,~iFiAPWV 343
gi ~ 2137411 I 78 RGD' --YNN~.~ KR-- F.iG'~'~yLDT ~IKPQ --T~ T~fiG E'V'P~-----
124
gi ~ 11360271 ~ 65 PTQESYY~.7PNLPP~TICLV'TN-=-=-CKPVTyIHKAY~----~3 ~ RFEIG~----
- 110
SS g1 ~ 7303923 I 51 DAVYTW~V~?'1~- --SDPFED- - --
IR'T~F~DKYDP~FDDK~~RST.tEEiAAWI 100
610 620 630 640 650 660
~..v. ~ . . ~,"
NOVl2 596 RNIFIVTNGQIPSWLNL~YNPRVTISTTHQDVFRNLSH PTFSS"'I~ESH.I~R~EGLSQK~I 655
gi ~ 6382022 I 250 -----------------KQVHKS:~~-LPNSLGVSE QRL'I'EF" ' SV
GHI~QGQNPP., 292
E)0 gi ~ 16551459 I 344 RNIFIVTNGQIPSWLNL~NPRVTITHQDVFRNLSH
PT~FSS"IES~R~E~GLS~",'?KQI, 403
gi~21374111 124 ------------- --E"NPSHR---RPHGFAGEHRSER~IT ETVT~KG~ HALNPPP
164
gi~113602711 110 ------------- --EIAFK-------------IRT,t~VSH~GQiDKNP~. 140
gi I 7303923 I 101
RHVYIVTNGQIPSWLDLSYERVTV~~pHEVLAPDPDQ~PT~SS~31ETFL~t~PKLS~~I~ 160
6S 670 680 690 700 710 720
NOV12 656 Y ~ 'VMFGKDVWPD~FY~HSKGQI~''V~'YLTWP~' CAEGC~ KDGYC~I~ACNNSACDW 715
gi~6382022~ 293 D ETTARFRVETHT''~;,,'3KTIGGNVTKKPPSL~ -----LE QI~TK- ~KKIT-----
G 338
gi I 16551459 ~ 404 Y ~ VMFGKDVWPDpFYwaHSKG'QKV[l"LTWP~ ' CAEGC
~KDGYC~'JK'ACNNSACDW 463
gi~21374111 165 ET~i'CARL-----APTLGVTVS.'fO~NLSPL~ -----8E H~iPK- ~---------
201
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giI11360271~ 141 C ~ IDHN--- ~ ~ Q"TVKAVLRD3k"'YESF ------- -- --------- 169
giI73039231 161 YI~FLGAPLYP~LY~'EAEGV~VQAWGCALDC~I~I'YGDGAC~RHCNIDACQF 220
730 740 750 760 770 780
NOV12 716 DGGC GNSGGRYIAGGGGTGSIGVGQPWQFGGGI S~SYCNQGCANSW ~FCDAC 775
gi~6382022~ 339 KE RMEENE---------------------- H1;G------VTE LG1~KLQHYT 369
gi~165514591 464 DGGT7C GNSGGSRYIAGGGGTGSIGVGQPWQFGGGI S'~t~/,SYCNQGCANSW
~FCD'~~A'C 523
gi~2137411~ 201 - SDRAEG------------------------- VlP------VKEL~PG~'tRCSII 226
l~ gi~11360271~ 169
___.'________________________________Ip'________SQFEpPREYRNFL 185
giI7303923~ 221 DGG~C~ETGPA--------------------DAHVIPPSKEVLEVQPAA'V'PQSRVH~tFP
260
790 800 810 820 830 840
IS NOV12 776 NVLSC ~.AGDCGENSDSKNRKTEEKCPVKKKKIMFLFFPLD .YKVILLP~t~THY 835
gi~63820221 370 DSY-L LPWEKK--------------------------- Q~ LDEEESLKT-Q- 397
gi1165514591 524 NVLSC AGDCG----------------------------D~ YKVILLPTHY 555
gi~2137411~ 227 Q--- CPGKKIC-----------------------------~ Q~7 LDAEESLKT-Q-
251
gi~11360271~ 186 H-__________ '._______________________________
QEWRAYR~;7IC___ 199
gi~7303923~ 261 QMGLQKLFRRSSANFKDVMRHRN----------'-------VSTL RRIVERF~T~AKL
302
850 860 870 880 890 900
NOV12 836 IIPKGEC P FA~VA~ G'~TGA~'S~ :PIIRHASIANKWT~I~L ~ GMN I'IHFNL 895
gi~6382022~ 397 ------- ~'D~~NT ,QL_i~DTF ~ -----------LL KFGF RKVPA 439
gi ~ 16551459 ~ 556 IIPKGEC P FVA G~,GA~~~ PIIRHASIANKWY'~L GMN '.~'IHFNL 615
gi ~ 2137411 ~ 251 ------ D ~CHT Q',~.yDT~.~' y-- -L( L KFGF RKVPA 293
gi I 11360271 I 199 ------- K.~'WT'HCVLATLIFTI~3.~'~ ------------- - ---- 7----
- 218
gi~7303923~ 303 MSLN--PELETSk~'"PQTTQRHGLRKyDFKSSTD2YSHSLIAT~~RAYGFKARHVLA 360
30 _.~:.,
910 920 930 940 950 960
":.~,.~ ...~ ..) ..~....~. ~ .~. ~ ~ .v
NOV12 896 TFQT BEEF ~ eIT 'CDT GP ~LTSTAQGY VSPTLLPEAEILFD,IPKEn~~ 955
gi I 6382022 I 440 HMPMI ~R-I ~E~Q~~FP;~''t, FD ~T FH;V~iHS
FAF~aYFYYLMSAVP~sNISE,~ 498
35 gi ~ 16551459 ~ 616 TFQT BEEF ~ITV'C~DT GP ~L~STAQ~~GY
VSPTLLPEAEILF:DI'PKE~ 675
gi ~ 2137411 ~ 294 HMPMI ~R-I ~E'~.cQpt~FP~ FD ~Tc~SFIi~~V~'t'HS
NIFAFi~YFYYLMSAV~P~.rI~TIS'352
gi ~ 11360271 ~ 218 ------- FFAQIP.~jKR'~I_FP~2RRIH~CEASPNR~V------- - --- 248
gi I 7303923 I 361 HVGFLI~K
DI'EAQRRFHt~(;~TLDTAHQF3~2APT~1~,,YAFAYYSFLMSET~SVE'rI~ 419
40 970 980 990 1000 ' 1010 1020
NOV12 956 P'~FKRHD TRR,A,QE.~ICIPL SL.'KDALSLNTLD~:iQLEHG DTLKGYNLSK 1012
gi~6382022~ 499 DVDTD--Q GVLSD IRTLAT HE 'LSLDLTGLEHLINCS ICMLPADITQLN 553
gi~165514591 676 PFKRHD TRR'AQE ~CIPL SL 'KDA~LSLNTLD1.~.~r~,QLEHG DkTLKGYNLSK
732
gi~2137411~ 353 HVDTD--Q GVLDRF~TLATE2 HD ~LTCI------------ --------- 384
gi~11360271~ 248 -____________.____.__________________________________-__-__
- 248
gi~7303923~ 420 DFDTDG--~ATWDRTFLTR~YQP~LDWSAMRYFEEz'~VQNCTRNLGT~HLKVDTVEH 477
1030 1040 1050 1060 1070 1080
NOV12 1013 ALLRSFLMNSQHAKIKNQAITD-ETNDSLVAPQE VHKSILPNSLGVSERLQR'~C.tTFP 1071
gi~6382022~ 554 ~IPPTQESYYDPNLPPVTKS~~V;TN-CKPVTDKIHKAY~KNKYRFEIMGEEEIAFKIRT
612
gi.~16551459~ 733 kALLRSFLMNSQHAKIKNQA,STD-
ETNDSLVAPQEIC~1HKSILPNSLGVSERLQR~TFP 791
gi~2137411~ 384 .______-______,_____________________________________________
384
gi~11360271~ 248 __________________-_________________________________________
248
gi~7303923~ 478 TLVYERYEDSNLPTITRDLRCPLLAEALAANFAV1PKYNFHVSPKRTSHSNFM~FLTS 537
1090 1100 1110 1120 1130 1140
CO NOV12 1072 A.'~IS'V G ~QG~7NP'LDLETTARFRVETHTQKTIGGNVTICKPPSLI 'LESQMTKEKK
1131
gi~6382022~ 613 NVS GQL~ I~ 'RISLCCPSWNAVMQTWLTVASTSWA~øAILPPQP'D--------- 663
gi~16551459~ 792 A~~GH~GNP'LDLETTARFRVETHTQKTIGGNVTIC~F.yKPPSLI 'LESQMT----
847
gi~2137411~ 384 _____-______________________________-_______________________
384
gi~11360271~ 248 ____________________________________________________________
248
GS gi~7303923~ 538
N~'~',EV~ESLQRLf~RIVQRKFNCINDNLDANRGEDNEMVRHLLDFYLSFFQRRSKFELPPQ 597
1150 1160 1170 1180 1190 1200
NOV12 1132 ITGKEKENSRMEENAENHIGVTEVLLGRKLQHYTDSYLGFLPWEKKKYFQDLLDEEESLK 1191
giI6382022~ 663 __________-_________________________________________________
663
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gi116551459~ 847 ____________________________________________________________
847
gi~2137411~ 384 __________________________________________-_____________-___
384
gi~11360271~ 248 __________________________________,_________________________
248
gi~7303923~ 598 YR1VRFESWRDFQRWKRRKR.AVLVIGYGVSLLLWCLLRFMCHHKAKLVRRCVQRL-----
652
The NOV 12 Clustal W alignment shown in Table 12E was modified to end at amino
residue 1200. The data in Table 1E includes all of the regions overlapping
with the NOV12
protein sequences.
NOV12 nucleic acids, and the encoded polypeptides, according to the invention
are
useful in a variety of applications and contexts. For example, NOV12 nucleic
acids and
polypeptides can be used to identify proteins that are members of the Plasma
Membrane
Protein-like Protein Family. The NOV12 nucleic acids and polypeptides can also
be used to
screen for molecules, which inhibit or enhance NOV12 activity or function.
Specifically, the
nucleic acids and polypeptides according to the invention may be used as
targets for the
identification of small molecules that modulate or inhibit, e.g., cellular
activation and signal
transduction. These molecules can be used to treat, e.g., Diabetes,Von Hippel-
Lindau (VHL)
syndrome , Pancreatitis, Obesity, Cardiomyopathy, Atherosclerosis,
Hypertension, Congenital
heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventriculax
(A-V) canal defect,
Ductus arteriosus , Pulmonary stenosis, Subaortic stenosis, Ventricular septal
defect (VSD),
valve diseases, Tuberous sclerosis, Sclerodenna, Obesity, Transplantation, Von
Hippel-Lindau
(VHL) syndrome, Cirrhosis, Transplantation, Von Hippel-Lindau (VHL) syndrome ,
Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's
disease,
Huntington°s disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome,
Multiple sclerosis,
Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction,
Anxiety, Pain,
Neuroprotection as well as other diseases, disorders and conditions.
In addition, various NOV 12 nucleic acids and polypeptides according to the
invention
are useful, inter alia, as novel members of the protein families according to
the sequence
relatedness to previously described proteins. The NOV 12 nucleic acids and
polypeptides,
antibodies and related compounds according to the invention will be useful in
therapeutic and
diagnostic applications in the mediation of cardiac, nerve, and immune
physiology. As such,
the NOV12 nucleic acids and polypeptides, antibodies and related compounds
according to the
invention may be used to treat cardiovascular, immune, and nervous system
disorders, e.g.,
Diabetes,Von Hippel-Lindau (VHL) syndrome, Pancreatitis, Obesity,
Cardiomyopathy,
Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis,
Atrial septal defect
(ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus , Pulmonary
stenosis, Subaortic
stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis,
Scleroderma,
Obesity, Transplantation, Von Hippel-Lindau (VHL) syndrome, Cirrhosis,
Transplantation,
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Von Hippel-Lindau (VHL) syndrome , Alzheimer's disease, Stroke, Tuberous
sclerosis,
hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy,
Epilepsy, Lesch-
Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies,
Behavioral
disorders, Addiction, Anxiety, Pain, Neuroprotection as well as other
diseases, disorders and
conditions.
The NOV12 nucleic acids and polypeptides are useful for detecting specific
cell types.
For example, expression analysis has demonstrated that a NOV 12 nucleic acid
is expressed in
Pancreas, Uterus, Epidermis, Heart, Coronary Artery, Adrenal GlandlSuprarenal
gland,
Pancreas, Parathyroid Gland, Salivary Glands, Liver, Small Intestine, Bone
Marrow,
Peripheral Blood, Lymphoid tissue, Lymph node, Cartilage, Brain, Hypothalamus,
Spinal
Chord, Mammary glandBreast, Uterus, Prostate, Testis, Lung, Kidney, Epidermis,
Hair
Follicle.
Additional utilities for NOV 12 nucleic acids and polypeptides according to
the
invention are disclosed herein.
NOV13
A NOV13 polypeptide has been identified as a BHLH Factor MATH6-like protein
(also referred to as CG94399-O1). The disclosed novel NOV13 nucleic acid (SEQ
ID N0:27)
of 2244 nucleotides is shown in Table 13A. The novel NOV 13 nucleic acid
sequences maps
to the chromosome 2.
An ORF begins with an ATG initiation codon at nucleotides 105-107 and ends
with a
TGA codon at nucleotides 1062-1064. A putative untranslated region and/or
downstream
from the termination codon is underlined in Table 13A, and the start and stop
codons are in
bold letters.
Table 13A. NOV13 Nucleotide Sequence (SEQ ID N0:27)
ACGCGTGAAGGGCGGGCGAAGCGGGAGAGCCAGAGACTCCTCGGCGCTGAGCGCGGCGGCGGCCCGG
GCAGCCCCACGCCCCTGCCTCGCGCGCCGCCCGCGCCATGAAGCACATCCCGGTCCTCGAGGACGGG
.CCGTGGAAGACCGTGTGCGTGAAGGAGCTGAACGGCCTTAAGAAGCTCAAGCGGAAAGGCAAGGAGC
CGGCGCGGCGCGCGAACGGCTATAAAACTTTCCGACTGGACTTGGAAGCGCCCGAGCCCCGCGCCGT
AGCCACCAACGGGCTGCGGGACAGGACCCATCGGCTGCAGCCGGTCCCGGTACCGGTCCGGTGCCAG
TCCCAGTGGCGCCGGCCGTTCCCCCAAGAGGGGGCACGGACACAGCCGGGGAGCGCGGGGGCTCTCG
GGCGCCCGAGGTCTCCGACGCGCGGAAACGTGCTTCGCCCTAGGCGCAGTGGGGCCAGGACTCCCCA
CGCCGCCGCCGCCGCCGCCTCCTGCGCCCCAGAGCCAGGCACCTGGGGGCCCAGAGGCACAGCCTTT
CGGGAGCCGGGTCTGCGTCCTCGCATCTTGCTGTGCGCACCGCCCGCGCCCCGCGCCGTCAGCACCC
CCAGCACCGCCAGCGCCCCCGGAGTCCACTGTGCGCCCTGGCCCCCGACGCGCCCCGGGGAAAGTTC
CTACTCGTCAATTTCACACGTAATTTACAATAACCACCAGGATTCCTCCGCGTCGCCTAGGAAACGA
CCGGGCGAAGCGACTGCCGCCTCCTCCGAGATCAAAGCCCTGCAGCAGACCCGGAGGCTCCTGGCGA
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ACGCCAGGGAGCGGACGCGGGTGCACACCATCAGCGCAGCCTTCGAGGCGCTCAGGAAGCAGGTGCC
GTGCTACTCATATGGGCAGAAGCTGTCCAAACTGGCCATCCTGAGGATCGCCTGTAACTACATCCTG
TCCCTGGCGCGGCTGGCTGACCTTGACTACAGTGCCGACCACAGCAACCTCAGCTTCTCCGAGTGTG
TGCAGCGCTGCACCCGCACCCTGCAGGCCGAGGGACGTGCCAAGAAGCGCAAGGAGTGACTGGCTGC
AGGCAAGACCAAGGCCACCACTGTGGGCCCTCCTTCCAGTCAGGCCTGAGGACAAGGTGAGCTCGCT
GAGTCCAGCCTCGTGGTCTTCTCCAAGATGGCGCCCCACTTGGAGCCTACAGCCTCTCAGGGTCGGA
TCGGAGCACGCCTGCCTCCCTCTCCCCTCCGCCCTCACCCAGCCAATCCGAGGCTGCTTCGCACGTT
GCCCTCTGCCTGGTGGGGAGGGGAGAGCTCAGCCCCCGACTCACTCAGACCCCAAGGCCCACTGTCC
AGCTGCAGAAATTCGTTGCCAAAGATTGGACAGAGACACCGAAGGAAATGGGGTGGTGAAACCCCAC
AGCGAAAAGCCACACCGTTGCTCTGTGACTTTTGCTCCTCCTGTTGCCTGAGCCCCATCTCAAGCCA
AAGATGAGTCAGTGGTTCTGCTAGGAACTCATGGAATGGATGGGCATTTGATGACCCCTGGGGGTCA
TCTTGGCCCTCTGACCTGGTGCTCTCTCTCCACTGGGCCTTGTGCTGGTTGAGTGCAAGACAAGCCT
TAGGGGCTGTGAGAGGGAGGCTGGGGTGCCTGGGCGGGGCTGGGAGTGGGACCTGAGATCCCTGCCC
ACTCTCTCCCCTTCATTGGCTTGCCCAGGCCACTGGCCCCAGTTCTCAGTGTCCCTTGGGGTCCAGG
CTCCTTGGGCCCTAAGCATCACCAGAAGGGAGTAAGCAGGGAGAGAAGCAATATTACTCCCTCCCCT
ACACCAGGGACTTGCCCCAGGGCGGCTACCTATGGGTCTTTGCTTCCCCAGCCAGCCTCTCCTCACT
GTGACCCACCCCCATGGGCCCCCGTCCCAGGCAGCCAGCACCATGGGCAGGCCCTGCCATGGACAGA
AAAAGAGTTTTTCTCTTGTTCAGCCTGCACGTGGCCTGAGGAAGGAGTAGAGGCTGGGTTGGCTGGA
GCCGTCCTACTGGGCAAGATGGCGCCCCACTTGGAGGGCGGTGGTCTGTTACAGGGTGTGCAGGGGC
AGAGAAGGAAGGGACCAGGGGACTGGGCCAGTATGTGGAGGATGGGGCCTGCGTGTTCAAAGCCAAG
GCCCGCCCCTTCCTTGTGCTCAAATGGCCAAAGCTGTTCACGTCTGTGCTCAACCATCTGCTTCAAA
TTGAAGTAAAAGCCCCAAAATGTCAAGAAA.AAA
Variant sequences of NOV13 are included in Example 3, Table 25. A variant
sequence
can include a single nucleotide polymorphism (SNP). A SNP can, in some
instances, be
referred to as a "cSNP" to denote that the nucleotide sequence containing the
SNP originates
as a cDNA.
The NOV 13 protein (SEQ m N0:28) encoded by SEQ m N0:27 is 319 amino acid
residues in length and is presented using the one-letter amino acid code in
Table 13B. Psort
analysis predicts the NOV 13 protein of the invention to be localized at the
nucleus with a
certainty of 0.7000.
Table 13B. Encoded NOV13 protein sequence (SEQ ID N0:28)
MKHIPVLEDGPWKTVCVKELNGLKKLKRKGKEPARRANGYKTFRLDLEAPEPRAVATNGLRDRTH
RLQPVPVPVRCQSQWRRPFPQEGARTQPGSAGALGRPRSPTRGNVLRPRRSGARTPHAAAAAASC
APEPGTWGPRGTAFREPGLRPRILLCAPPAPRAVSTPSTASAPGVHCAPWPPTRPGESSYSSISH
VIYNNHQDSSASPRKRPGEATAASSEIKALQQTRRLLANARERTRVHTISAAFEALRKQVPCYSY
GQKLSKLAILRIACNYILSLARLADLDYSADHSNLSFSECVQRCTRTLQAEGRAKKRKE
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 13C.
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Table 13C. Patp results
for NOV13
Smallest
Sum
eadingigh Prob
equences Frame Score P(N)
producing
High-scoring
Segment
Pairs:
>patp:AAM93743Humanpolypeptide, SEQ ID NO: +1 1197 2.3e-121
3717
>patp:AAB95274Humanprotein sequence SEQ ID +1 1197 2.3e-121
N0:17476
>patp:AAU16607Humannovel secreted protein, +1 534 4.2e-51
Seq TD 1560
>patp:ABG00300Novelhuman diagnostic protein +1 334 6.8e-33
#291
>patp:ABG00300Novelhuman diagnostic protein +1 334 6.8e-33
#291
In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 372 of 657 bases (56%) identical
to a
gb:GENBANK-ID:HSBBICP4A~acc:L14320.1 mRNA from Bovine herpesvirus 1 (Bovine
herpesvirus type 1 early-intermediate transcription control protein (BICP4)
gene, complete
cds). The full amino acid sequence of the protein of the invention was found
to have 238 of
322 amino acid residues (73%) identical to, and 244 of 322 amino acid residues
(75%) similar
to, the 322 amino acid residue ptnr:TREMBLNEW-ACC:BAB39468 protein from Mus
musculus (BHLH FACTOR MATH6)..
NOV 13 also has homology to the proteins shown in the BLASTP data in Table
13D.
Table 13D. BLAST
results for
NOV13
Gene Indeac/ Protein/ OrganismLength Identity PositivesEacpect
Identifier (aa) (%) (%)
giI14249530Iref~NPhypothetical 321 246/321 246/321 2e-93
_ protein FLJ14708 (76%) (76%)
116216.11(NM
032827
[Homo sapiens]
gi~i3383235~dbj~BABbHLH factor 322 233/329 240/329 3e-86
Math6
39468.1~(AB049066)[Mus musculus] (70%) (72%)
gi~17864454Iref~NPnet [Drosophila365 55/97 76/97 5e-21
_ melanogaster] (56%) (77%)
524820.1~(NM
080081
gi~7296271~gb~AAF51CG11450 gene 261 55/97 76/97 2e-20
562.11 (AE003590)product (56%) (77%)
[Drosophila
melanogaster]
gi~18858289Iref~NPatonal homolog 325 36/82 51/82 2e-10
2a
_ [Danio rerio] (43%) (61%)
571891.1I(NM
131816
A multiple sequence alignment is given in Table 13E, with the NOV13 protein
being
shown on line I in Table 13E in a ClustalW analysis, and comparing the NOV13
protein with
the related protein sequences shoum in Table 13D. This BLASTP data is
displayed graphically
in the ClustalW in Table 13E.
Table I3E. CIustalW Analysis of NOVI3
1) > NOV13; SEQ m N0:28
2) > gi~14249530~/ hypothetical protein FLJ14708 [Homo Sapiens]; SEQ m N0:92
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3) > gi~13383235~/ bHLH factor Math6 [Mus rrausculus]; SEQ ZD N0:93
4) > gi~ 17864454/ net [Dr~osoplaila rraelanogaster]; SEQ m N0:94
5) > gi~7296271~/ CG11450 gene product [Dr-osophila nrelanogaster~]; SEQ m
N0:95
6) > gi~18858289~/ atonal homolog 2a [Danio rerio]; SEQ m N0:96
10 20 30 40 50 60
.1... ~... .I
NOV13 1 __________________________ ' CHI 'LiE~GPKTVCVL'~' ___ ~ 3' ' 28
1~ gi1142495301 1 -------------------------- HI~~T~E~GP~KTVCVLi~==-- ~ ~ ~ 28
gi1133832351 l --------------------------- HI~ ~1E~GP~KTVCVK~#L -- ~ 28
gi~17864454~ 1 MSFAAMANTNTEKLYMQLSASELSAII ~DS~ S ~RDAGFCSASSE"~c~'EGGDDLVVHAv
60
gi172962711 1 ______________________________________________________'=_____ 1
gi1188582891 1 -----------------------MLTLPFED~_~,M~TQ~'GANFPCV~-------D~yG 30
_.
70 80 90 100 110 120
I....I....J. .~.. .L... .~....~....~
NOV13 29 ~F~ANGYKTFRLW,~APE4P-----RAVAT R',t'~RTHRL' ~ PVRCQSQWRRP 83
a V V
~ r V V
gi ~ 14249530 ~ 29 'TtANGYKTFRL~Z~~APP-----RAVA ~ Rx?RTHRLf~ ~ ~ PVPVPVPV ~ 83
2~ gi113383235~ 29 ~ GYKTFRLy.P~LGATVSTTAAT~ R'RT---~'F~IATPVPA'V ~ 85
gi1178644541 61 S SPDI~PKGTDSADSKP~LALVRNKRKSSEPFKV~TTPNSKS ' ~PSSASMN~T ~L
120
gi172962711 1 __________________.________________________ ~PSSASMNT ~L 16
gi ~ 18858289 1 3l NK~mFEEETLSHVMD~DF~SED-----EDEREQDNGLPRR ~RKKKMTK~RVDR 85
130 140 150 160 170 180
.1...,1... ~ . .1 .I ~.. 1'.
NOVl3 ~ 84 FPQEGARTQPGSAGAG~~R --PT GNLRPRI~SGAR~..,,PHAAAAA ---ASCAPEPGT 136
gi ~ 14249530 ~ 84 VPPRG ~'~;~1GERGGS ~E~"SDAR ~CFALGA'~'GPGLP~~~,~P'P ~PPP----
APQ~QAPG- 138
gi1133832351 86 VPPGG L~~~,REFRGI'., ~E~ZDAR ~GFALGT~~GPGLPP~P~P------ASQ LAPG-
138
3~ gi ~ 17864454 ~ 121 KKRIR S~~DSAVV~,'TP ~DSPPPNSC~PSTI~F~LQHEI ~
IYVRHPGV~TLHRS 180
gi ~ 7296271 ~ 17 KKRIRY~S~~~,DSAVVTP'ADSPPPNSC,PSTLr~LQHEI ~ IYVRHPGVfI'TLHRS
76
gi~18858289~ 86 VKVRRMEAN~RERNRI~HGLNNLDSLE7KV~',PCYSLC~TQKLKIETLR-----
LAKNYIWAL 140
190 200 210 220 230 240
..
NOV13 137 WGPRGTAF~EPG~RPRIL PP'i1PRAVTP'u~TASAPG--VHCAPW~PTRP.~ .I 194
gi1142495301 138 -GPEAQPF~EPGPRPRIL~ PPi~1, ~ PyiIPP~,~PPAPP-E TV~P ~PTRP' I
196
gi1133832351 138 -DPEAHSF~EQ RPRIL PP~i1 ~TQi~PL~~PPAAPQE PV,I2P ~PTRP I 197
gi1178644541 181 LAAHPEQLEPL TTKKQ DQiI ~KIE~F~~iLLIGKQP K~TLKERTQ TS FL 240
4O gi172962711 77 LAAHPEQLEPL VTTKKQ DQ~ KIE~F~LLIGKQP ITLKERTQ T'S FL 136
gi1188582891 141 SEILSTGI~PDL TFVQT KGLQ~TT~1'LV;~,;GCLQLN---ARNFT~DQISF GR~
197
250 260 270 280 290 300
1.~.~..1.. .I....1 . .1....1 .1. ..~.. .1. .1....1. .1
NOV13 195 HVIY~HQ~S ~PRKRP ET~~SSEI ~Q T ~ L~ ~~~~ ~~ ~Q 254
v i w v
gi ~ 14249530 1 197 HVIY~HQrS PRKRP E;T ~SSEI ~Q~T~ ~ 'L~ ~' ~ ~ ~ ~~CQ 256
gi ~ 133832351 198 HVIY~'1HP~5 PRKRP ET ~STEI I QT' ~ ''L~ ~ ~ ~ ~I~Q 257
w a
gi1178644541 241 EASL~~3~E-~LNL~.~K~!GLAPISRPHQHQRNY T~E:y.E~ ~~ ~ ~~ T~~A 299
gi172962711 137 EASL~S~E-~LKGLAPISRPHQHQRNY T~E '~E~ ~~ ~ ~ ~'~' T ~A 195
gi (18858289 l 198 PYESVYSTYI3~P~WWTPS~P~VD~1VKPFR~FNYCSSYE-FY SVSPECG'I'PQ
GPLSP 256
310 320 330 340 350 360
NOV13 255 310
gi1142495301 257 312
gi1133832351 258 313
gi1178644541 300 355
gi172962711 196 251
gi1188582891 257 316
370
NOV13 311 . . ~'. 1 ~ ~ '~~I 319
gi1142495301 313 ~ ~- 321
gi1133832351 314 ~ ~- 322
gi1178644541 356 ~2 ~E 365
gi172962711 252 ~ ~;E 261
gi~188582891 317 ~DELNTFHN- 325
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The presence of identifiable domains in the protein disclosed herein was
determined by
searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and
then
determining the Interpro number by crossing the domain match (or numbers)
using the
Interpro website (http:www.ebi.ac.uk/interpro~. Table 13F lists the domain
description from
DOMAIN analysis results against NOV 13.
Table 13F
Domain Anal
sis of NOV13
Model Region of Score (bits) E value
Homology
Helix-loop- 234-280 55.8 4.0e-09
helix domain
Helix-loop- 229-281 61.4 1.9e-14
helix DNA-
binding domain
(HLH)
. Consistent with other known members of the BHLH Factor MATH6-like family of
proteins, NOV 13 has, for example, a Helix-loop-helix domain and a Helix-loop-
helix DNA
binding domain (HLH) signature sequence as well as homology to other members
of the
BHLH Factor MATH6-like Protein Family. NOV 13 nucleic acids, and the encoded
polypeptides, according to the invention are useful in a variety of
applications and contexts.
For example, NOV 13 nucleic acids and polypeptides can be used to identify
proteins that are
members of the BHLH Factor MATH6-like Protein Family. The NOV 13 nucleic acids
and
polypeptides can also be used to screen for molecules, which inhibit or
enhance NOV13
activity or function. Specifically, the nucleic acids and polypeptides
according to the
invention may be used as targets for the identification of small molecules
that modulate or
inhibit, e.g., cellular activation, cellular replication, and signal
transduction. These molecules
can be used to treat, e.g., Diabetes,Von Hippel-Lindau (VHL) syndrome ,
Pancreatitis,
Obesity, Inflammatory bowel disease, Diverticular disease, Von Hippel-Lindau
(VHL)
syndrome , Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia,
Parkinson's
disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome,
Multiple
sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders,
Addiction, Anxiety,
Pain, Neuroprotection, Systemic lupus erythematosus , Autoimmune disease,
Asthma,
Emphysema, Scleroderma, allergy as well as other diseases, disorders and
conditions.
In addition, various NOV13 nucleic acids and polypeptides according to the
invention
are useful, inter alia, as novel members of the protein families according to
the presence of
domains and sequence relatedness to previously described proteins. For
example, the NOV13
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nucleic acids and their encoded polypeptides include structural motifs that
are characteristic of
proteins belonging to the BHLH Factor MATH6-like Protein Family.
A number of eukaryotic proteins, probably sequence specific DNA- binding
proteins
that act as transcription factors belong to this family. They share a
conserved domain that is
formed of two amphipathic helices joined by a variable length linker region
that could form a
loop (Littlewood and Evan, Pf°otein Prof. 2: 621-702 (1995).) This
'helix-loop-helix' (HLH)
domain mediates protein dimerization and has been found in a large variety of
proteins
(Garrell and Campuzano, Bioassays 13: 493-498 (1991); Kato and Dang, FASEB J.
6: 3065-
72 (1992).) Most of these proteins have an short basic region adjacent to the
HLH domain that
specifically binds to DNA. They are referred as basic helix-loop-helix
proteins (bHLH), and
are classified in two groups: class A (ubiquitous) and class B (tissue-
specific). The HLH
proteins lacking the basic domain (Emc, Id) function as negative regulators
since they form
heterodimers, but fail to bind DNA. The hairy-related proteins (hairy, E(spl),
deadpan) also
repress transcription although they can bind DNA. The proteins of this
subfamily act together
with co-repressor proteins, like groucho, through their C-terminal motif WRPW.
MATH6 (moue, et al., Genes to Cells 6: 977-86 (2001)) is a distant homolog of
D~osophila proneuronal gene Atonal. Murine expression is higest in developing
nervous
system (ventricular zone and mantle layer, spinal cord, dorsal root ganglia).
MATH6 is
expressed by neuronal precursor cells and designated neurons, e.g., cerebellar
Purkinje cells.
The closest mammalian homolog to MATH6 is NeuroD. NeuroD point mutations and
NeuroD gene knockout animals have severe diabetes and die perinatally. The
NeuroD
knockout anmals lack beta-Islet cells and could not be rescued with insulin
administration.
Also, the NeuroD knockout animals are deaf due to a loss of inner ear sensory
neurons.
The NOV13 nucleic acids and polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and diagnostic
applications in the
mediation of metabolism as well as nerve and immune physiology. As such, the
NOV 13
nucleic acids and polypeptides, antibodies and related compounds according to
the invention
may be used to treat metabolic, hearing, nervous system, immune disorders,
e.g.,
Diabetes,Von Hippel-Lindau (VHL) syndrome , Pancreatitis, Obesity,
inflammatory bowel
disease, Diverticular disease, Von Hippel-Lindau (VHL) syndrome , Alzheimer's
disease,
Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's
disease,
Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-
telangiectasia,
Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain,
Neuroprotection, Systemic
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lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma,
allergy as
well as other diseases, disorders and conditions.
The NOV13 nucleic acids and polypeptides are useful for detecting specific
cell types.
For example, expression analysis has demonstrated that a NOV 13 nucleic acid
is expressed in
Pancreas, Umbilical Vein, Small Intestine, Cartilage, Synovium/Synovial
membrane, Brain,
Placenta, Oviduct/LTterine Tube/Fallopian tube, Lung, Brain, Uterus.
Additional utilities for NOV 13 nucleic acids and polypeptides according to
the
invention are disclosed herein.
NOV14
A NOV 14 polypeptide has been identified as a Putative Protein-Tyrosine
Phosphatase-
like protein (also referred to as CG94366-O1). The disclosed novel NOV14
nucleic acid (SEQ
ID N0:29) of nucleotides is shown in Table 14A. The novel NOV 14 nucleic acid
sequences
maps to the chromosome 22.
An ORF begins with an ATG initiation codon at nucleotides 248-250 and ends
with a
TAA codon at nucleotides 1679-1681. A putative untranslated region and/or
downstream
from the termination codon is underlined in Table 14A, and the start and stop
codons are in
bold letters.
Table 14A. NOV14 Nucleotide Sequence (SEQ ID N0:29)
ATTGAGTTTGAAATAACTGCCACCACAAAGTCTGTCACACATTGAGACTGAGGTCATAATAAAGAGG
TTTACTTAAATAGGGAAGCATTACTATTTTCCCCCGCCTAAGATTTTGGTTGTCGCCATATAAATCC
TCATTTCTAATAAAGAGAAAAAGACATTCCAGGTTCCAATAGTGCTATACACATGAATAGTCAGAAA
TTAATTGGTTTCTGTCTAGAATAATGAAAAGTAATTTTTCCAAAATATGAATTCAGAATTAAGTCTC
CTCTCTGACTGTTTTCTCTTATCATCCGCTAGTCCACAGACAAACGAATTTAAAGGAGCAACCGAGG
AGGCACCTGCGAAAGAAAGCCCACACACAGGTGAATTTAAAGGAGCAGCCCTGGTGTCACCTATCAG
TAAAAGAATGTTAGAACGACTTTCCAAGTTTGAAGTTGGAGATGCTGAAAATGTTGCTTCATATGAC
AGCAAGATTAAGAAAATTGTTCATTCAATTGTGTCATCCTTTGCAGTTGGGATATTTGGAGTTTTCC
TGATCTTGTTGGATGTGGCTCTGATCTTTGCTGACCTAATTTTCACTGATAGCAAAGTTTATATTCC
TTTGGAGTATCGTTCTATTTCTCTAGCTATTGCTTTATTTTTTCTCATGGATGTTCTTCTTCGAGTA
TTTGTAGAAAGGAGACAGCATTATTTTTCTGATTTACTTAACATTTTAGATACTGCCGTTACTGTGA
TTATTCTGCTGGTTGATGTCGTTTACATTTTTTTTGACGTTAAGTTTCTTAAGGATATTCCCAGATG
GACACGTTTATTTCGACTTCTACGACTTATCATTCTGATAAGAGTTTTTCGTCTGGCTCATCTAAAA
AGACAACTTGGAAAGCTGATAAGAAGGCTGGTAAGTAGGNGATACGAAAGGGATGGATTTGACCTAG
ACCTCACTTATATTACAGAACGTATTGTCGCTATGTCATTTCCATCTTCGGGAGGCCAGTCTTTCTA
TCGGAATCCAATTAAGGAAGTCGTACAGTTTCTAGACAAGAAACATCCAAACCACTATCGAGTCTAC
AATCTATGCAGTGAAAGAGCTTATGATCCTAAGCACTTCCATAATAGGGTCAGTAGAATCATGATCG
ATGATCATAATGTCCCCACTCTAAGGGAGATGGTAGCATTCTCCAAGGAAGTGTTGGAGTGGATGGC
TCAAGATTCTGAAAACATCGTAGTGATTCACTGTAAAGGAGGCAAAGGTAGAACCGGAACTATGGTT
TGTGCCTGCCTGATTGCCAGTGAAATATTTTTAACTGCAGAGGAAAGATTGTACTATTTTGGAGAAC
GGCGAACAGATAAAACCAATGGCACTAAATATCAGGGAGTAGAAACTCCTTCTCAGAATAGATATGT
TGGATATTTTGCACAAGTGAAACATAGCTACAACTGGAATCTCCCTCCAAGAAAAACACTGTTTATA
AAAAGATTAGTTATTTATTCGATTCATGGTAAGTGTTTAGATCTAAAAGTCCAAATAGTAATGAAGA
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AAAAGATTGTCTTTTCCTGCACTTCCTTAAACAGTTGTCGGGTAAGAGAAAACATGGAAACAGACAG
GGTAATAATTGATGTGTTCAACTGTCCACCTCTGTATGATGATGTGAAAGTGCAATTTTTTTTTTCT
TTTTAGGATTTTCCTAAATACTATCACAACTACCCTTTTTTCTTCTGGTTTAACACATCTTTAATAC
AAAATAACAGGCTTTATCTACAAAGAAATGAATTGGATAATCTTCATAAACAAAAAACATGGAAAAT
TTATCAACCAGAATATGCAGTAGAGATATATTTTGATGAGAAATGACTTAAGTTATGTTGTAACTGG
TAGCTGATTAAGTATAGTTCCCTGCACCCCTTCTGGGAAAGAATTATGTTCTTTCTAACCCTGCCAC
ATAGTTATATGTTCTAAATCTTCCTTGCTGGTACATCTATATTGATATATGTATACACATGTTCTTT
ATAAATCTATTAAATATATACAGATAAA
The NOV14 protein (SEQ ID N0:30) encoded by SEQ )D N0:29 is 477 amino acid
residues in length and is presented using the one-letter amino acid code in
Table 14B. Psort
analysis predicts the NOV 14 protein of the invention to be localized at the
plasma membrane
with a certainty of 0.6000.
Table 145. Encoded NOV14 protein sequence (SEQ ID N0:30)
MNSELSLLSDCFLLSSASPQTNEFKGATEEAPAKESPHTGEFKGAALVSPISKRMLERLSKFEVG
DAENVASYDSKIKKIVHSIVSSFAVGIFGVFLILLDVALIFADLIFTDSKVYIPLEYRSISLAIA
LFFLMDVLLRVFVERRQHYFSDLLNILDTAVTVIILLVDVVYIFFDVKFLKDIPRWTRLFRLLRL
IILIRVFRLAHLKRQLGKLIRRLVSRXYERDGFDLDLTYITERIVAMSFPSSGGQSFYRNPIKEV
VQFLDKKHPNHYRVYNLCSERAYDPKHFHNRVSRIMIDDHNVPTLREMVAFSKEVLEWMAQDSEN
IVVIHCKGGKGRTGTMVCACLIASEIFLTAEERLYYFGERRTDKTNGTKYQGVETPSQNRYVGYF
AQVKHSYNWNLPPRKTLFIKRLVIYSIHGKCLDLKVQIVMKKKIVFSCTSLNSCRVRENMETDRV
IIDVFNCPPLYDDVKVQFFFSF
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 14C.
Table 14C. Pat results for NOV14
Smallest
Sum
eading igh Prob
Sequences Score P(N)
producing
High-scoring
Segment
Pairs: Frame
>patp:AAG67459Amino acid sequence of a human +1 1895 2.5e-195
polypeptide
>patp:AAG67638Amino acid sequence of a human +1 1895 2.5e-195
protein
>patp:AAB73230Human phosphatase AA493915_h +1 574 1.8e-111
>patp:AAW34402Protein encoded by gene IMAGE +1 473 1.2e-44
clone 264611
>patp:AAY07450Human TS10q23.3 gene bases 453-2243+1 473 1.2e-44
In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 1105 of 1427 bases (77%) identical
to a
gb:GENBANK-m:AF007118~acc:AF007118.1 mRNA from Homo Sapiens (putative tyrosine
phosphatase mRNA, complete cds). The full amino acid sequence of the protein
of the
invention was found to have 369 of 462 amino acid residues (79%) identical to,
and 402 of
462 amino acid residues (87%) similar to, the 551 amino acid residue
ptnr:SWISSNEW-
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ACC:P56180 protein from Ho~rao Sapiens (PUTATIVE PROTEIN-TYROSINE
PHOSPHATASE TPTE (EC 3.1.3.48)).
NOV14 also has homology to the proteins shown in the BLASTP data in Table 14D.
Table 14D. BLAST
results for
NOV14
Gene Index/ Protein/ OrganismLength Identity PositivesExpect
Identifier (aa) (%) (~)
gi~7019559~ref~NPtransmembrane 551 369/462 402/462 0.0
0
_ phosphatase (79%) (86%)
37447.1I(NM 013315)with
tensin homology;
tensin, putative
protein-tyrosine
phosphatase
[Homo Sapiens]
gi~16166555~ref~XPsimilar to 551 367/462 401/462 0.0
_ transmembrane (79%) (86%)
055073.1~(XM_055073
phosphatase
with
tensin homology
[Homo Sapiens]
gi~18640756~refINPsimilar to 445 316/462 343/462 e-156
_ PUTATIVE PROTEIN- (68%) (73%)
570141.1I(NM
130785
TYROSINE
PHOSPHATASE
TPTE
[Homo sapien]
gi~14787415~emb~CACtyrosine 664 275/432 336/432 e-141
44243.11(AJ311311)phosphatase (63%) (77%)
isoform A [Mus
musculus]
gi~14787417~emb~CACtyrosine 645 275/432 336/432 e-141
44244.1~(AJ311312)phosphatase (63%) (77%)
isoform B [Mus
musculus]
A multiple sequence alignment is given in Table 14E, with the NOV 14 protein
being
shown on line 1 in Table 14E in a ClustalW analysis, and comparing the NOV14
protein with
the related protein sequences shown in Table 14D. This BLASTP data is
displayed graphically
in the ClustalW in Table 14E.
Table 14E. ClustalW Analysis of NOV14
1) > NOV14; SEQ ID N0:30
2) > gi~7019559~/ transmembrane phosphatase with tensin homology; tensin,
putative protein-tyrosine
phosphatase Homo sapieras]; SEQ ID N0:97
3) > gi~16166555~/ similar to transmembrane phosphatase with tensin
homology [Homo sapiefts]; SEQ ID N0:98
. 4) > gi~18640756~/ similar to PUTATIVE PROTEIN-TYROSINE PHOSPHATASE TPTE;
similar to
transmembrane phosphatase with tensin homology; tensin, putative protein-
tyrosine phosphatase
[Flomo Sapiens]; SEQ ID N0:99
5) > gi~14787415~/ tyrosine phosphatase isoform A.[Mus musculus]; SEQ ID
NO:100
6) > gi~14787417~/ tyrosine phosphatase isoform B [Mus musculus]; SEQ ID
NO:101
10 20 30 40 50 60
..
NOV14 1 _________________________________________________
gi~7019559~ 1 _________________________________________________ ~SELSLLS
--~I E~PDPTD
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gi~16166555~ 1 ___________________________________________________ i E PDPTD 9
gi~186407561 1 __________________________________________________ __
- i E P-- 5
gi~147874151 1 MYGEKKSHLYLWMEHYGYDMPANIYKMYSQPSRKTDDANKKVSVSASRTIKI~ TGYDT 60
gi1147874171 1 -------------MHFGNINSTNWF------FRDKKHQNKKVSVSASRTI TGYDT 41
70 80 90 100 110 120
w
NOV14 9 -DCFL~S- ~SASP~ ' ~. ~~~ G---------------- ~~L 48
giI70195591 10 LAGVI'I!ELGPI7SPv S ~ ... ________________ .. 52
gi~16166555~ 10 LAGIIELGP,SP~ S ~ ~~~ ---------------- ~~, ~ 52
gi~18640756~. 5 ______________~ T ________________ ..L 34
gi1147874151 61 NEQ~ T~.iITNG;SLSYP~ I.~YS,~~,S'YAD~ISTKA~,'' SSVYDPGGASSSTTLY
LNSL E 120
gi~14787417~ 42 NEQTITNGSLSYPII~S~YAD~ISTKA~S. ,s SSVYDPGGASSSTTLY LNSL~E 101
130 140 150 160 170 180
NOV14 49 ________________________________________________ ~ .'E~'t 58
gi~70195591 53 ________________________________________________ _~g 62
gi~16166555~ 53 ________________________________________________ S n'' 62
gi~18640756~ 35 ________________________________________________ ___'. 39
gi1147874151 121 KEIITQGESALLRDKEATSELKIPSTLQTQTSMSTNTLSLSDLSSDYQEEQ" ~C 180
gi~14787417~ 102 KEIITQGESALLRDKEATSELKIPSTLQTQTSMSTNTLSLSDLSSDYQEEQ;., CI~Q
161
190 200 210 220 230 240
NOV14 59 ~ ~ ~ FEV ~ A~ . .~K~ . . I=HS3; F~1V I ~ ~ ~ I<F ~ ~ . n 116
gi I 16166555 ~ 63 ~ 'FEVW--AB:NV DK~~.'C, , 'IVHS~ ~ F~G~ ~ nT Il;n ~ ~~.i
120
gi~18640756~ 39 ~-__ _______=__ ____'_'.___R__.______ ~ T I,~~ ~ ~~ 65
i 14787415 181 LYD7WERTt~IQ F GI~Z ' ~ I ~ F ! n ~I 240
gi I 14787417 I 162 ?LYD~7~ERTIQW~~~yF~GII I ~ F ~ ~ ~If I 221
250 260 270 280 290 300
NOV14 117 176
gi~7019559~ 121 180
gi~16166555~ 121
180
giI186407561 66 94
gi~14787415~ 241 300
gi~14787417~ 222 281
310 320 330 340 350 360
NOV14 177 233
giI7019559~ 181 240
gi~161665551 181 240
giI186407561 94 145
gi~147874151 301 360
341
gif14787417~ 282
370 380 390 400 410 420
NOV14 234 293
gi~7019559~ 241
55 gi~16166555~ 241 300
300
gi~18640756~ 146 205
420
gi~14787415~ 361
401
gi~14787417~ 342
60 430 440 450 460 470 480
NOV14 294 353
360
gi~7019559~ 301
gi~161665551 301
gi~18640756~ 206 360
265
gi~14787415~ 421 480
461
gi~14787417~ 402
490 500 510 520 530 540
70 ..
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NOVl4 354 413
gi~7019559~ 361 420
420
gi~16166555~ 361
giI186407561 266 325
gi114787415~ 481 540
gi~14787417~ 462 521
550 560 570 580 590 600
NOV14 414 469
476
gi~7019559~ 421
gi~161665551 421 476
gi~186407561 326 381
gi~14787415~ 541 600
gi~14787417~ 522 581
610 620 630 640 650 660
NOV14 470 ~~~. F~________F___-____-___-_______________________________ 477
gi~7019559~ 477 ~ Y ~T ~ S L E ~ L Q ~, PSD ~ TL 536
gi~161665551 477 t ~T n S L Ei ~ L Q PSD ~ ~L 536
.k
gi I 18640756 ~ 382 ~ S ~ ~ P F F C ' nP 'Q W~ PPE ~ I=L 441
gi114787415~ 601 LSP ~~P F F~R~T ~P~TW~GE ~ D 660
gi ~ 14787417 ~ 582 LSP ' ~ P F''.~!~!~ ~ T P ~TW GE ~ 641
670
NOV14 477 --------------- 477
giI7019559~ 537 KMTSSDWAGSD 551
30 gi~16166555~ 537 KMTSSDWAGSD 551
gi~18640756~ 442 i~K----------- 445
gi1147874151 661 ---------- 664
gi~147874171 642 ---------- 645
The presence of identifiable domains in the protein disclosed herein was
determined by
searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and
then
determining the Interpro number by crossing the domain match (or numbers)
using the
Interpro website (http:www.ebi.ac.uklinterpro~. Table 14F lists the domain
description from
DOMAIN analysis results against NOV14.
Table 14F
Domain Anal
sis of NOV14
Model Region of Score (bits) E value
Homology
Dual 231-378 -48.8 3.2
specificity
phosphatase,
catalytic
doma
Consistent with other known members of the Putative Protein-Tyrosine
Phosphatase-
like family of proteins, NOV 14 has, for example, a dual specificity protein
phosphatase
signature sequence and homology to other members of the Putative Protein-
Tyrosine
Phosphatase-like Protein Family. NOV14 nucleic acids, and the encoded
polypeptides,
according to the invention are useful in a variety of applications and
contexts. For example,
NOV 14 nucleic acids and polypeptides can be used to identify proteins that
are members of
the Putative Protein-Tyrosine Phosphatase-like Protein Family. The NOV 14
nucleic acids and
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polypeptides can also be used to screen for molecules, which inhibit or
enhance NOV14
activity or function. Specifically, the nucleic acids and polypeptides
according to the
invention may be used as targets for the identification of small molecules
that modulate or
inhibit, e.g., cellular activation, cellular replication, and signal
transduction. These molecules
can be used to treat, e.g., Cardiovascular diseases, cystitis, incontinence as
well as other
diseases, disorders and conditions
W addition, various NOV 14 nucleic acids and polypeptides according to the
invention
are useful, iyater alia, as novel members of the protein families according to
the presence of
domains and sequence relatedness to previously described proteins. For
example, the NOV 14
nucleic acids and their encoded polypeptides include structural motifs that
are characteristic of
proteins belonging to the Putative Protein-Tyrosine Phosphatase-like Protein
Family.
Cellular processes involving growth, differentiation, transformation and
metabolism
are often regulated in part by protein phosphorylation and dephosphorylation.
The protein
tyrosine phosphatases (PTPs), which hydrolyze the phosphate monoesters of
tyrosine residues,
all share a common active site motif and are classified into 3 groups. These
include the
receptor-like PTPs, the intracellular PTPs, and the dual-specificity PTPs,
which can
dephosphorylate at serine and threonine residues as well as at tyrosines.
Diamond et al. (1994)
described a PTP from regenerating rat liver that is a member of a fourth
class. The gene, which
they designated Prll, was one of many immediate-early genes. Overexpression of
Prll in
stably transfected cells resulted in a transformed phenotype, which suggested
that it may play
some role in tumorigenesis. By using an ih vitro prenylation screen, Cates et
al. (1996)
isolated 2 human cDNAs encoding PRL1 homologs, designated PTP(CAAXl) and
PTP(CAAX2)(PRL2), that are farnesylated i~a vitro by mammalian
farnesyl:protein
transferase. Overexpression of these PTPs in epithelial cells caused a
transformed phenotype
in cultured cells and tumor growth in nude mice. The authors concluded that
PTP(CAAX1)
and PTP(CAAX2) represent a novel class of isoprenylated, oncogenic PTPs. Peng
et al.
(1998) reported that the human PTP(CAAX1) gene, or PRL1, is composed of 6
exons and
contains 2 promoters. The predicted mouse, rat, and human PRLl proteins are
identical. Zeng
et al. (1998) determined that the human PRLl and PRL2 proteins share 87% amino
acid
sequence identity.
The NOV14 nucleic acids and polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and diagnostic
applications in the
mediation of cardiac and renal physiology. As such, the NOV 14 nucleic acids
and
polypeptides, antibodies and related compounds according to the invention may
be used to
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treat cardiovascular and urogenital system disorders, e.g., Cardiovascular
diseases, cystitis,
incontinence as well as other diseases, disorders and conditions. The NOV 14
nucleic acids
and polypeptides are useful for detecting specific cell types. For example,
expression analysis
has demonstrated that a NOV14 nucleic acid is expressed in Urinary bladder.
Additional utilities fox NOV 14 nucleic acids and polypeptides according to
the
invention are disclosed herein.
NOV15
A NOV15 polypeptide has been identified as a Leucine Rich Repeat (LRR)-like
protein (also referred to as CG95387-02). The disclosed novel NOV15 nucleic
acid (SEQ m
N0:31) of 3136 nucleotides is shown in Table 15A. The novel NOV15 nucleic acid
sequences maps to the chromosome 19.
An ORF begins with an ATG initiation codon at nucleotides 330-332 and ends
with a
TAA codon at nucleotides 2331-2333. A putative untranslated region and/or
downstream
from the termination codon is underlined in Table 15A, and the start and stop
codons are in
bold letters.
Table 15A. NOV15 Nucleotide Sequence (SEQ ID N0:31)
ACTCCTGACCTAAAGTGATCCACTCGCCTTGGCCTCCCAAAGTGCTAGGATTACAGCCCTCATTCTC
TTTTGCTCCTCAGGTGACACAGGACAAGATCATCTGTCTACCCAATCATGAGCTCCAGGAGAACTTA
TCAGAGGCCCCGTGCCAGCAATTGCTGCCTCGGGGGATCCCTGAGCAGATTGGGGCCCTGCAGGAGG
TTAAAGGCCTTAAGAACAATTTGGACCTGCAGCAATACAGCTTTATTAACCAGCTGTGTTATGAGAC
GGCCCTGCACTGGTATGCCAAGTACTTCCCTTACCTCGTGGTCATTCACACACTCATCTTCATGGTC
TGCACCAGTTTCTGGTTCAAGTTCCCTGGCACCAGCTCCAAGATTGAACACTTCATCTCCATCCTGG
GCAAGTGTTTCGACTCTCCATGGACCACCAGGGCCCTATCCGAGGTCTCCGGGGAGAACCAGAAGGG
CCCAGCAGCCACCGAACGGGCTGCGGCATCCATAGTGGCCATGGCAGGGACCGGGCCGGGGAAGGCA
GGGGAGGGTGAGAAGGAGAAAGTGCTGGCGGAACCGGAGAAGGTGGTGACCGAGCCTCCAGTTGTCA
CCCTGTTGGACAAGAAGGAGGGTGAGCAAGCCAAAGCCCTGTTTGAGAAGGTGAAGAAGTTCCGCAT
GCACGTGGAAGAGGGCGACATCCTGTACACCATGTACATCCGACAGACGGTGCTGAAAGTGTGTAAG
TTCCTGGCCATCCTGGTCTACAACCTGGTCTATGTGGAGAAGATCAGTTTCCTGGTGGCCTGTAGGG
TGGAGACGTCAGAGGTCACGGGCTACGCCAGCTTCTGCTGCAACCACACCAAGGCCCACCTCTTCTC
CAAGCTGGCCTTCTGTTACATCTCCTTTGTGTGCATCTACGGACTTACCTGCATCTACACGCTCTAC
TGGCTCTTCCACCGGCCCCTCAAGGAGTACTCCTTCCGTTCCGTGCGGGAGGAGACTGGCATGGGGG
ACATTCCTGACGTCAAGAATGACTTCGCCTTCATGCTGCACCTCATCGATCAGTACGACTCCCTCTA
CTCCAAGCGCTTCGCCGTCTTCCTGTCCGAGGTCAGCGAAAGCCGTCTAAAGCAGCTCAATCTCAAC
CACGAGTGGACCCCCGAGAAGCTTCGACAGAAGCTGCAGCGCAATGCCGCGGGCCGGCTGGAGCTGG
CCCTCTGCATGCTGCCGGGTCTGCCCGACACCGTCTTTGAGCTCAGTGAGGTGGAGTCACTCAGGCT
GGAGGCCATCTGCGATATCACCTTCCCCCCGGGGCTGTCACAGCTGGTGCACTTGCAGGAGCTCAGC
TTGCTCCACTCGCCCGCCAGGCTACCCTTCTCCTTGCAGGTCTTCCTGCGGGACCACCTGAAGGTGA
TGCGCGTCAAATGCGAGGAGCTCCGCGAGGTGCCGCTTTGGGTGTTTGGGCTGCGGGGCTTGGAGGA
GCTGCACCTGGAGGGGCTTTTCCCCCAGGAGCTAGCTCGGGCAGCCACCCTGGAGAGCCTCCGGGAG
CTGAAGCAGCTCAAGGTGTTGTCCCTCCGGAGCAACGCCGGGAAGGTGCCAGCCAGTGTGACCGACG
TTGCTGGCCACCTGCAGAGGCTCAGCCTGCACAACGATGGGGCCCGTCTGGTTGCCCTGAACAGCCT
CAAGAAGCTGGCGGCATTGCGGGAGCTGGAGCTGGTGGCCTGCGGGCTGGAGCGCATCCCCCATGCA
GTGTTCAGCCTGGGTGCGCTGCAGGAACTTGACCTCAAGGACAACCACCTGCGCTCCATCGAGGAAA
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TCCTCAGCTTCCAGCACTGCCGGAAGCTGGTCACGCTCAGGCTGTGGCACAACCAGATCGCCTACGT
CCCTGAGCACGTGCGGAAGCTCAGGAGCCTGGAGCAGCTCTACCTCAGCTACAACAAGCTGGAGACC
CTGCCCTCCCAGCTCGGCCTGTGCTCAGGCCTCCGTCTGCTGGATGTGTCCCACAATGGGCTACACT
CCCTGCCACCCGAGGTGGGCCTCCTGCAGAACCTACAGCACCTGGCCCTCTCCTACAATGCCCTGGA
GGCCCTGCCCGAAGAGCTCTTCTTCTGCCGCAAGCTGCGGACGTTGCTTCTGGGCGACAACCAACTG
AGCCAGCTCTCGCCCCACGTGGGTGCCCTCAGAGCCCTCAGCCGCCTGGAGCTCAAAGGCAACCGCT
TAGAGGCGCTGCCAGAAGAACTTGGCAACTGTGGGGGGCTCAAGAAGGCGGGGCTCCTGGTGGAAGA
CACGCTTTACCAGGGTCTGCCGGCAGAAGTGCGGGACAAGATGGAGGAGGAATGAAGCTGGGGTGGG
GCCGTTTTAGGTAGAGCCTTAAAAATGCTTCTGCCCTGGAATCTCAACCATTATCTTCCAAGATAGG
AAGCCAAGTGGGTCTAGGCCAGGAGATGGGGGGGGGCGGGGGCAGCTGTGTCATCTTTCTGGGGCC_C
AGGAGGATCTGGGCTGGTTTGTCTGGGGAGACAGACAGGATGTTGTGGAGGTGGGGTGGAACCTGGT
ATGGAGGGATTAACTCAGTCATGGCATTCTCCGACCAAAACCACACCTGTGTCTCTGGCAGGCTGGC
TGGCCTTGCTCCCATCCCTAGAACTGCTGCCTCTCCCTGGATATTCCAGCTCAATTAGTGCCACATA
TGGGGGAAACGACACATCCCAGTGGGATTTCCAACACTCCCCCTCCCCATGCAACAAAGCAACTTAC
TTCTGGAGTTCTCTCCCAAGGAGAGGACACAGACACAGTTGTTTGCTGTGTTATATGTTAGCTCCGA
ACAATGGTTCTCATTTGGCTAAGCATCAAAATCACCTAGGGAGCCGGTGCAAAACAAAATATCCCAG
TCCCCTCCCCTGAAACACTGACTCAGGAGGTTTGGTTGGGGGCCAGGAGTCTGTTCCTAAATATTCC
AGGTAGTTCTGGTGCAGGTAAGTGGCCCTGAGACAGTATGTTGGGAAATGCTGACGTAAAGGTATCA
GGGCCGGGCGCTGTGGCTCATGACTATAATCCCAGCTGTTTGAGAGGCCAATGCAGGAGGATGGTTG
AGCTCAGGAGTTCGAGATCAGCCTGGGTAACATAGCGAGACCCCACCTCTGCCA
Variant sequences of NOV 15 are included in Example 3, Table 26. A variant
sequence
can include a single nucleotide polymorphism (SNP). A SNP can, in some
instances, be
referred to as a "cSNP" to denote that the nucleotide sequence containing the
SNP originates
as a cDNA.
The NOV 15 protein (SEQ m NO:32) encoded by SEQ m N0:31 is 667 amino acid
residues in length and is presented using the one-letter amino acid code in
Table 15B. Psort
analysis predicts the NOV15 protein of the invention to be localized at the
plasma membrane
with a certainty of 0.7900.
Table 15B. Encoded NOV15 protein sequence (SEQ ID N0:32)
MVCTSFWFKFPGTSSKIEHFISILGKCFDSPWTTRALSEVSGENQKGPAATERAAASIVAMAGTG
PGKAGEGEKEKVLAEPEKWTEPPVVTLLDKKEGEQAKALFEKVKKFRMHVEEGDILYTMYIRQT
VLKVCKFLAILVYNLVYVEKISFLVACRVETSEVTGYASFCCNHTKAHLFSKLAFCYISFVCIYG
LTCIYTLYWLFHRPLKEYSFRSVREETGMGDIPDVKNDFAFMLHLIDQYDSLYSKRFAVFLSEVS
ESRLKQLNLNHEWTPEKLRQKLQRNAAGRLELALCMLPGLPDTVFELSEVESLRLEAICDITFPP
GLSQLVHLQELSLLHSPARLPFSLQVFLRDHLKVMRVKCEELREVPLWVFGLRGLEELHLEGLFP
QELARAATLESLRELKQLKVLSLRSNAGKVPASVTDVAGHLQRLSLHNDGARLVALNSLKKLAAL
RELELVACGLERIPHAVFSLGALQELDLKDNHLRSIEEILSFQHCRKLVTLRLWHNQIAYVPEHV
RKLRSLEQLYLSYNKLETLPSQLGLCSGLRLLDVSHNGLHSLPPEVGLLQNLQHLALSYNALEAL
PEELFFCRKLRTLLLGDNQLSQLSPHVGALRALSRLELKGNRLEALPEELGNCGGLKKAGLLVED
TLYQGLPAEVRDKMEEE
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 15C.
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Table 15C. Patp results
for NOV15
Smallest
Sum
eading igh Prob
equences igh-scoring Segment Pairs:Frame Score P(N)
producing
>patp:AAY70473HumanCNAP-1 +l 2147 5.0e-222
>patp:AAG75413Humancolon cancer antigen protein+1 1882 6.0e-194
>patp:AAM41692Humanpolypeptide SEQ ID NO 6623+1 1878 1.6e-193
>patp:AAU20426Humansecreted protein, Seq ID +1 1835 5.8e-189
No 418
>patp:AAB92855Humanprotein sequence SEQ ID +1 1795 1.0e-184
N0:11424
In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 2227 of 2228 bases (99%) identical
to a
gb:GENBANI~-ID:AK.027073~acc:AK027073.1 mRNA from Ho~rao Sapiens (cDNA:
FLJ23420 fis, clone HEP22352). The full amino acid sequence of the protein of
the invention
was found to have 444 of 444 amino acid residues (100%) identical to, and 444
of 444 amino
acid residues (100%) similar to, the 444 amino acid residue ptnr:SPTREMBL-
ACC:Q9HSH8
protein from Ho~ao sapiefzs (CDNA: FLJ23420 FIS, CLONE HEP22352).
NOV 15 also has homology to the proteins shown in the BLASTP data in Table
15D.
Table 15D. BLAST
results for
NOV15
Gene Index/ Protein/ OrganismLength Identity PositivesExpect
Identifier (aa) (%) (%)
gi~13376597~ref~NPhypothetical 444 444/444 444/444 0.0
_ protein FLJ23420 (100%) (100%)
079337.1~(NM_025061
[Homo Sapiens]
gi~14150009~ref~NPhypothetical 708 404/667 520/667 0.0
_ protein (60%) (77%)
115646.1~(NM_032270
DKFZp586J1119
[Homo Sapiens]
gi~19343671~gb~AAH2Similar to 708 404/671 526/671 0.0
5473.1 (BC025473)hypothetical (60%) (78%)
protein
DKFZp586J1119
[Mus musculus]
gi~7243272~dbj~BAA9KIAA1437 protein811 367/666 84/666 0.0
2675.1 (AB037858)[Homo Sapiens] (55%) (72%)
gi~8922442Iref~NPhypothetical 682 345/673 470/673 0.0
0
_ protein FLJ10470 (51%) (69%)
60573.1I(NM~018103)
[Homo Sapiens]
A multiple sequence alignment is given in Table 1 SE, with the NOV 15 protein
being
shown on line 1 in Table 15E in a ClustalW analysis, and comparing the NOV 15
protein with
the related protein sequences shown in Table 15D. This BLASTP data is
displayed graphically
in the ClustalW in Table 15E.
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Table 15E. ClustalW Analysis of NOV15
1) > NOV15; SEQ ID NO:32
2) > gig 13376597/ hypothetical protein FLJ23420 [Homo Sapiens]; SEQ )D N0:102
3) > gig 14150009/ hypothetical protein DKFZp586J1119 [Hofno Sapiens]; SEQ >D
N0:103
4) > gi~19343671~/ Similar to hypothetical protein DKFZp586J1119 [Mus
musculus]; SEQ ID N0:104
5) > gi~7243272~/ KIAA1437 protein [Homo sapierts]; SEQ ID NO:105
6) > gi~8922442~/ hypothetical protein FLJ10470 [Homo Sapiens]; SEQ >D N0:106
130 140 150 160 170 180
.1....1....I ..I . .I .1,....1....I. .I. .I. ..1....I
NOV15 l -------------- -'S~GT~~~I ~ ' '~ V~G~ 43
gi1133765971 1 _____________________________.______________________________ 1
v n ~ v V
gi1141500091 27 WYAKYFPYLVLIHTLVF~L GS ~ I ~ ' '~ ~; 86
gi1193436711 27 WYAKYFPYLVLTHTLVF~ G,S t~ ~ ~ ~ " . 86
gi172432721 121 WFAKYFPYLVLLHTLIF' R~' L ~ ~ '~ T~~ 180
gi189224421 1 ______________ y"~,,5 ~ ~K?~C ~ ~ ~ 'tC 43
190 200 210 220 230 240
..1....1....1....1 [....1....I.. I ...I... 1....1....I
NOV15 44 Q~CGPAATERAAASIVAMAG'GPGA------GEGEKEKVLEP T PPWTL ~ 97
_~
gi1133765971 1 ___________________ ______ __ __________ ___________ 1
gi1141500091 87 EKDNRKNNN1NRSNTIQ-~GP G----- STSQSL~ IP F ~KSTAG ~ 138
gi I 193436711 87 E'EKDNRKNNMNRSGTIQ-~S.'GP G----- N~,r'X~RSQSL ~IP F~tKSAAG ~
138
gi 17243272 I 181 ~ ~PKPAFSKMNGSMDKKSS'I~,VSE~--VEATVPMIiQRT-- ~S'~RI
G~VC7RSETG ~ 236
gi I 8922442 I 44 y, E<ENKQRITGAQTLPKHVS~"SSD~GSPSASTP~;NKTGF IFS P~~VPSMTI ~
103
250 260 270 280 290 300
.I. .I. .I. .I....I....I,....1....1....1....1....1....1
NOV15 98 ~ ~ ~ ' ~ L T1~T'~L~CI:i'VONLVYVEICTS~L~ 157
gi1133765971 1 __________________________ __________ ___ _________.______ 1
gi1141500091 139 ~' ' 'L ~ L '~ L I IiI SAL SI~VQ T~ 198
gi1193436711 139 ~~'~ '~ ~ 'L ~ L w .L I LiI VSAL.SK~Q T~,1~~1,~,-~, 198
gi172432721 237 ~~ ' 'T ~ RL '~ TI I I'L I;C TVYY ~~ VD 296
gi I 8922442 I 104 ~ ~ ~ S ~LiI R~i~~~~ ~T I<F IiC~TANF 1~ ~S E 163
310 320 330 340 350 360
I....I....I....I....I ..I. 1....1....1....1....1....1
NOV15 158 R~TSES~C~TES~FC~IiSF~C~~T~~~X~P~T~RS 217
gi1133765971 1 ___________________________________=________________________ 1
gi1141500091 199 N~ltD3 S T 'T S AFC hCF S~ T ~ 'S Y 258
gi I 19343671 I 199 = ~~D , IQ. S T- ' S SFC ~:~1CF ST L Y'S Y 258
gi I 7243272 I 297 TTI~S~, RTE P 'T KI SF 1S1 IF I L ,S K S 356
gi I 8922442 I 164 KPF~VVIiI EVE T '~L LIS ~SICI L F RIP K 223
370 400
380 410
390 420
NOV15 218 277
gi1133765971 1 54
gi1141500091 259 318
gi1193436711 259 318
gi172432721 357 416
gi189224421 224
283
$5 430 460
440 470
450 480
NOV15 278 337
gi1133765971 55
114
gi1141500091 319 378
gi1193436711 319 378
gi17243272) 417 476
gi189224421 284 343
490 520
500 530
510 540
~I ~~I~
~. .I~
I~ .I~~~~I~
~I ~I
. ~
I
I~~~-I .
~I~ .
.
.
NOV15 338 L SP~RLtPF~LQ _ _ _ _ ' ELFPQ 397
C~E~ L' L ~
F
~
gi 115 BL~SP~R~.iPF,LQV ' , RC,E''1~'' L'~V'L G
E~LFPQ ~L ~ 174
I E'
13376597
I
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gi ~ 14150009 ~ 379 QCS HSL~ ~S n' ~ 'P S SHI3I~438
gi ~ 19343671 ~ 379 QCS ~THS~L KE S n ' L'P ' S SHiIISKNS~ 438
gi ~ 8922442 ~ 344 C~CP~ ~EQT~F~E"H~T T~I~I ~L I L ~T y I~' 403
550 560 570 580 590 600
NOV15 398 457
gi~13376597~ 175 234
gi~14150009~ 439 498
gi~19343671~ 439 498
gi~7243272~ 537
596
gi~8922442~ 404 463
1$ 610 620 630 640 650 660
NOV15 458 517
gi~13376597~ 235 294
gi~14150009~ 499 558
gi~19343671~ 499 558
gi~7243272~ 597 656
gi~8922442~ 464 523
670 680 690 700 710 720
NOV15 518 577
gi~13376597~ 295 354
gi~14150009~ 559 618
gi~19343671~ 559 618
gi~7243272~ 657 716
gi~89224421 524 583
730 740 750 760 770 780
NOV15 578 637
gi~13376597~ 355 414
gi~141500091 6l9
678
gi~19343671~ 619
678
gi~7243272~ 717
gi~8922442~ 584 76
643
NOV15 638 -------- 667
gi113376597~ 415 -------- 444
gi~14150009~ 679 -------- 708
gi~193436711 679 -------- 708
gi~7243272~ 777 KEQA---- 811
gi~8922442~ 644 NIPFANGI 682
790 800 B10
. ,I I'.
f f~,
G v . ~ ~; ~ T Y~ ~''E-
r y~7
~ ~ ~ , ' SD ' Q I~T
~ 'S~j ' ~~_
PL ~ ~P ~W~
L x... QI
The NOV15 Clustal W alignment shown in Table 15E was modified to begin at
amino
residue 121. The data in Table 15E includes all of the regions overlapping
with the NOV 15
protein sequences.
The presence of identifiable domains in the protein disclosed herein was
determined by
searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and
then
determining the Interpro number by crossing the domain match (or numbers)
using the
Interpro website (http:www.ebi.ac.ukfinterpron. Table 15F lists the domain
description from
DOMAIN analysis results against NOV15.
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Table 15F
Domain Anal
sis of NOV15
Model Region of Score (bits) E value
Homology
LRR 454-476 5.6 1.5e+02
LRR 477-498 10.8 26
LRR 502-524 11.9 16
LRR 525-547 18.8 0.13
LRR 548-570 15.9 0.99
LRR 571-593 13.9 4
LRR 594-616 12.3 12
LRR 617-639 9.4 42
Consistent with other known members of the LRR-like family of proteins, NOV 15
has,
for example, eight Leucine Rich Repeat (LRR) signature sequences and homology
to other
members of the LRR-like Protein Family. NOV15 nucleic acids, and the encoded
polypeptides, according to the invention axe useful in a variety of
applications and contexts.
For example, NOV 15 nucleic acids and polypeptides can be used to identify
proteins that are
members of the LRR-like Protein Family. The NOV 15 nucleic acids and
polypeptides can
also be used to screen for molecules, which inhibit or enhance NOV15 activity
or function.
Specifically, the nucleic acids and polypeptides according to the invention
may be used as
targets for the identification of small molecules that modulate or inhibit,
e.g., cellular
activation, cellular replication, and signal transduction. These molecules can
be used to treat,
e.g., cancer, trauma, regeneration (ifz vitro and izz vivo),
viral/bacterial/parasitic infections,
Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's
disease,
Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple
sclerosis,
ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction,
anxiety, pain,
neurodegeneration, Von Hippel-Lindau (VHL) syndrome, cirrhosis,
transplantation,
Hirschsprung's disease , Crohn's Disease, appendicitis, osteoporosis,
hypercalceimia, arthritis,
ankylosing spondylitis, scoliosis, systemic lupus erythematosus, autoimmune
disease,
xerostomia as well as other diseases, disorders and conditions.
In addition, various NOV15 nucleic acids and polypeptides according to the
invention
are useful, hater alia, as novel members of the protein families according to
the presence of
domains and sequence relatedness to previously described proteins. For
example, the NOV15
nucleic acids and their encoded polypeptides include structural motifs that
are characteristic of
proteins belonging to the LRR-like Protein Family.
LRR Proteins are a family of proteins characterized by a structural motif rich
in leucine
residues. They are either transmembrane or secreted proteins and are involved
in protein-
protein interactions. Members of this family have been implicated in
extracellular matrix
assembly and cellular growth. In addition, several proteins belonging to this
family, such as
slit, Toll and robo have been shown to mediate key roles in central nervous
system
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development and organogenesis in Drosophila. Vertebrate orthologs of these
proteins have
also been shown to have similar roles in the CNS as well as other organ
systems like kidney..
LRRs are relatively short motifs (22-28 residues in length) found in a variety
of
cytoplasmic, membrane and extracellular proteins. Although these proteins are
associated with
widely different functions, a common property involves protein-protein
interaction. Little is
known about'the 3D structure of LRRs, although it is believed that they can
form amphipathic
structures with hydrophobic surfaces capable of interacting with membranes.
Irz vitro studies
of a synthetic LRR from Drosophila Toll protein have indicated that the
peptides form gels by
adopting beta-sheet structures that form extended filaments (Packman et al.
FEBS Lett.1991;
291: 87-91). These results are consistent with the idea that LRRs mediate
protein-protein
interactions and cellular adhesion. Other functions of LRR-containing proteins
include, for
example, binding to enzymes and vascular repair.
The NOV15 nucleic acids and polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and diagnostic
applications in the
mediation of cardiac, immune, and nerve physiology. As such, the NOV 15
nucleic acids and
polypeptides, antibodies and related compounds according to the invention may
be used to
treat cardiovascular, nervous, and immune system disorders, e.g., cancer,
trauma, regeneration
(ih vitro and in vivo), viral/bacterial/parasitic infections, Alzheimer's
disease, stroke, tuberous
sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral
palsy, epilepsy,
Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia,
leukodystrophies, behavioral
disorders, addiction, anxiety, pain, neurodegeneration, Von Hippel-Lindau
(VHL) syndrome,
cirrhosis, transplantation, Hirschsprung's disease, Crohn's Disease,
appendicitis, osteoporosis,
hypercalceimia, arthritis, ankylosing spondylitis, scoliosis, systemic lupus
erythematosus,
autoimmune disease, xerostomia as well as other diseases, disorders and
conditions.
The NOV15 nucleic acids and polypeptides are useful for detecting specific
cell types.
For example, expression analysis has demonstrated that a NOV 15 nucleic acid
is expressed in
Coronary Artery, Parotid Salivary glands, Liver, Colon, Bone,
Synovium/Synovial membrane,
and Brain.
Additional utilities for NOV 15 nucleic acids and polypeptides according to
the
invention are disclosed herein.
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NOV16
A NOV16 polypeptide has been identified as a RhoGEF-like protein (also
referred to
as CG95419-02). The disclosed novel NOV16 nucleic acid (SEQ B7 N0:33) of 5372
nucleotides is shown in Table 16A. The novel NOV16 nucleic acid sequences maps
to the
chromosome 5.
An ORF begins with an ATG initiation codon at nucleotides 61-63 and ends with
a
TAA codon at nucleotides 5179-5181. A putative untranslated region and/or
downstream
from the termination codon is underlined in Table 16A, and the start and stop
codons are in
bold letters.
Table 16A. NOV16 Nucleotide Sequence (SEQ ID N0:33)
CCATGGGGCCTCCTGCAATAACTTCTCTTGTTTATTATTTTCATTGCAGATGCGAAAGCCATGGAGT
TGAGCTGCAGCGAAGCACCTCTTTACCAGGGGCAGATGATGATCTATGCGAAGTTTGACAAAAATGT
GTATCTTCCTGAAGATGCTGAGTTTTACTTTACTTATGACGGATCTCATCAGCGACATGTCATGATT
GCAGAGCGCATCGAGGATAACGTTCTCCAGTCCAGCGTCCCAGGCCATGGGCTTCAGGAGACGGTGA
CGGTATCTGTGTGCCTCTGCTCGGAAGGTTACTCTCCGGTGACCATGGGCTCTGGCTCAGTGACCTA
CGTGGACAACATGGCTTGCAGGCTGGCTCGTCTGCTGGTGACGCAGGCCAATCGCCTCACAGCCTGC
AGCCACCAGACCCTGCTGACCCCATTTGCCTTGACGGCAGGAGCACTGCCTGCCTTGGATGAGGAGC
TCGTGCTGGCTCTGACCCATCTGGAATTGCCTCTAGAGTGGACTGTGTTGGGAAGTTCTTCACTTGA
AGTATCTTCTCACAGAGAATCTCTTCTACACCTGGCTATGAGATGGGGCCTGGCTAAACTTTCCCAG
TTCTTCTTGTGTCTCCCGGGGGGAGTCCAGGCCTTGGCTTTACCCAACGAAGAGGGTGCCACACCAT
TAGACTTAGCTTTACGTGAAGGACACTCCAAGCTGGTGGAAGACGTCACAAGTTTTCAGGGCAGATG
GTCCCCAAGCTTCTCCCGAGTGCAGCTCAGTGAAGAAGCCTCCTTGCATTACATTCACTCATCGGAA
ACGCTGACCCTGACCCTGAACCACACAGCCGAGCATTTGTTGGAGGCAGATATTAAACTCTTCCGGA
AATACTTTTGGGATAGAGCCTTTCTTGTCAAGGCCTTTGAGCAAGAAGCCAGGCCAGAGGAAAGAAC
AGCTATGCCCTCCAGCGGTGCAGAAACTGAAGAAGAGATTAAGAATTCAGTGTCCAGCAGATCAGCA
GCCGAAAAGGAAGATATAAAGCGTGTCAAAAGCCTGGTGGTTCAACACAATGAACATGAAGACCAGC
ACAGCCTAGATTCTAGATCGCTCCTTCGATATCCTAAAAAATCCAAGCCGCCCTCGACATTGCTTGC
TGCAGGCCGGCTTTCAGACATGCTGAATGGAGGTGATGAAGTCTACGCTAACTGTATGGTGATTGAT
CAGGTTGGTGATTTGGATATCAGCTATATTAATATAGAGGGAATCACTGCCACTACCAGCCCTGAAT
CCAGAGGTTGCACTCTGTGGCCTCAGAGCAGCAAACACACCCTTCCTACAGAAACCAGTCCCAGTGT
GTACCCACTTAGTGAAAATGTCGAAGGGACAGCACACACTGAAGCCCAGCAGTCCTTCATGTCACCA
TCAAGTTCGTGTGCTTCCAACTTGAATCTTTCTTTTGGTTGGCATGGATTTGAAAAGGAACAAAGTC
ATCTAAAGAAAAGAAGTTCTAGCCTTGATGCCTTGGACGCCGACAGTGAAGGGGAAGGGCATTCTGA
GCCATCCCACATCTGTTACACTCCAGGGTCTCAGAGCTCCTCAAGAACTGGGATTCCTAGTGGGGAT
GAATTGGACTCTTTTGAGACTAACACTGAACCGGATTTTAATATCTCCAGGGCTGAATCCCTTCCTC
TATCAAGTAATCTACAGTTGAAGGAATCACTGCTTTCTGGAGTTCGCTCACGTTCTTATTCTTGCTC
GTCACCCAAAATTTCTTTAGGAAAAACTCGTTTGGTGCGTGAATTAACAGTATGCAGTTCAAGTGAA
GAGCAAAAAGCTTACAGCTTATCGGAGCCACCAAGAGAAAACAGGATTCAGGAAGAAGAATGGGATA
AATACATCATACCTGCCAAATCAGAGTCTGAAAAATATAAAGTGAGTCGAACTTTCAGTTTCCTCAT
GAATAGGATGACTAGCCCTCGGAATAAATCAAAGACAAAAAGCAAGGATGCCAAAGATAAAGAGAAG
CTGAATCGACATCAGTTTGCCCCAGGAACATTCTCTGGGGTTCTGCAGTGTTTGGTTTGTGATAAAA
CACTCCTGGGGAAAGAGTCACTGCAGTGTTCTAGTTGTAATGCAAATGTGCACAAAGGTTGTAAAGA
TGCTGCGCCTGCATGCACCAAGAAATTCCAAGAGAAATATAACAAGAACAAACCACAGACCATCCTT
GGAAGTTCTTCATTTAGAGACATCCCACAGCCTGGTCTCTCCTTGCACCCTTCTTCCTCCGTGCCTG
TTGGATTGCCGACTGGAAGGAGGGAGACTGTGGGACAGGTCCATCCATTGTCCAGAAGTGTTCCAGG
TACCACCTTGGAAAGCTTCAGGAGGTCAGCCACATCCTTGGAGTCTGAGAGTGACAATAACAGCTGC
AGAAGCAGGTCTCATTCTGATGAGCTGCTACAGTCCATGGGCTCTTCTCCCTCTACAGAGTCTTTCA
TAATGGAAGATGTTGTGGATTCTTCTCTGTGGAGTGACCTCAGCAGTGATGCCCAGGAGTTTGAAGC
AGAATCTTGGAGTCTTGTGGTGGATCCCTCATTTTGTAATAGGCAGGAGAAGGATGTCATCAAAAGA
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CAGGATGTCATTTTTGAGCTAATGCAAACAGAGATGCATCACATCCAGACCCTGTTCATCATGTCTG
AGATCTTCAGGAAAGGCATGAAAGAGGAGCTGCAGCTGGACCACAGCACCGTGGATAAAATTTTCCC
CTGTTTAGATGAGTTGCTTGAAATCCACAGGCATTTCTTCTACAGTATGAAGGAACGAAGGCAGGAA
TCAAGTGCTGGCAGCGACAGGAATTTTGTGATCGACCGAATTGGAGATATTTTGGTACAACAGTTTT
CAGAAGAAAATGCAAGTAAAATGAAGAAAATATATGGAGAATTCTGTTGCCATCATAAAGAAGCTGT
TAACCTCTTTAAAGAACTCCAGCAGAATAAAAAGTTTCAGAATTTTATTAAGCTCCGAAATAGTAAT
CTTTTGGCTCGACGCCGAGGAATTCCAGAATGCATTCTGTTGGTCACTCAGCGTATTACAAAATACC
CTGTCTTGGTGGAAAGGATATTGCAGTACACAAAGGAAAGAACTGAGGAACATAAAGACTTACGCAA
AGCCCTTTGCTTAATTAAAGACATGATTGCAACAGTGGATTTAAAAGTCAATGAATATGAGAAAAAC
CAAAAATGGCTTGAGATCCTAAATAAGATTGAAAACAAAACATACACGAAGCTCAAAAATGGACATG
TGTTTAGGAAGCAGGCACTGATGAGTGAAGAAAGGACTCTGTTATATGATGGCCTTGTTTACTGGAA
AACTGCTACAGGTCGTTTCAAAGATATCCTAGCTCTACTTCTAACTGATGTGCTGCTCTTTTTACAA
GAAAA21GACCAGAAATACATCTTTGCAGCCGTTGATCAGAAGCCATCAGTTATTTCCCTTCAAAAGC
TTATTGCTAGAGAAGTTGCTAATGAGGAGAGAGGAATGTTTCTGATCAGTGCTTCATCTGCTGGTCC
TGAGATGTATGAAATTCACACCAATTCCAAGGAGGAACGCAATAACTGGATGAGACGGATCCAGCAG
GCTGTAGAAAGTTGTCCTGAAGAAAAAGGGGGAAGGACAAGTGAATCTGATGAAGACAAGAGGAAAG
CTGAAGCCAGAGTGGCCAAAATTCAGCAATGTCAAGAAATACTCACTAACCAAGACCAACAAATTTG
TGCGTATTTGGAGGAGAAGCTGCATATCTATGCTGAACTTGGAGAACTGAGCGGATTTGAGGACGTC
CATCTAGAGCCCCACCTCCTTATTAAACCTGACCCAGGCGAGCCTCCCCAGGCAGCCTCATTACTGG
CAGCAGCACTGAAAGAAGCTGAGAGCCTACAAGTTGCAGTGAAGGCCTCACAGATGGGCGCCGTGAG
TCAATCATGTGAGGACAGTTGTGGAGACTCTGTCTTGGCGGACACACTCAGTTCTCATGATGTACCA
GGATCACCGACTGCCTCATTAGTCACAGGAGGGAGAGAAGGAAGAGGCTGTTCGGATGTGGATCCCG
GGATCCAGGGTGTGGTAACCGACTTGGCCGTCTCTGATGCAGGGGAGAAGGTGGAATGTAGAAATTT
TCCAGGTTCTTCACAATCAGAGATTATACAAGCCATACAGAATTTAACCCGTCTCTTATACAGCCTT
CAGGCCGCCTTGACCATTCAGGACAGCCACATTGAGATCCACAGGCTGGTTCTCCAGCAGCAGGAGG
GCCTGTCTCTCGGCCACTCTATCCTCCGAGGCGGCCCCTTGCAGGACCAGAAGTCTCGCGACGCGGA
CAGGCAGCATGAGGAGCTGGCCAATGTGCACCAGCTTCAGCACCAGCTCCAGCAGGAGCAGCGGCGC
TGGCTGCGCAGGTGTGAGCAGCAGCAGCGGGCGCAGGCGACCAGGGAGAGCTGGCTGCAGGAGCGGG
AGCGGGAGTGCCAGTCGCAGGAGGAGCTGCTGCTGCGGAGCCGGGGCGAGCTGGACCTCCAGCTCCA
GGAGTACCAGCACAGCCTGGAGCGGCTGAGGGAGGGCCAGCGCCTGGTGGAGAGGGAGCAGGCGAGG
ATGCGGGCCCAGCAGAGCCTGCTGGGCCACTGGAAGCACGGCCGGCAGAGGAGCCTGCCCGCGGTGC
TCCTTCCGGGTGGCCCCGAGGTAATGGAACTTAATCGATCTGAGAGTTTATGTCATGAAAACTCATT
CTTCATCAATGAAGCTTTAGTACAAATGTCATTTAACACTTTCAACAAACTGAATCCGTCAGTTATC
CATCAGGATGCCACTTACCCTACAACTCAATCTCATTCTGACTTGGTGAGGACTAGTGAACATCAAG
TAGACCTCAAGGTGGACCCTTCTCAGCCTTCGAATGTCAGTCACAAACTGTGGACAGCCGCTGGTTC
CGGCCATCAGATACTTCCTTTCCAAGAAAGCAGCAAGGATTCTTGTAAAAATGATTTGGACACCTCC
CACACTGAGTCCCCAACCCCCCATGACTCAAATTCACACCGCCCTCAACTGCAGGCGTTTATAACAG
AAGCAAAGCTAAATCTACCGACAAGGACAATGACCAGACAAGATGGGGAAACTGGAGATGGAGCCAA
AGAAAATATTGTTTACCTCTAATTGTGTTGTCATTTTTCCAAACAAAACAAAACACTGGCACTTTTG
GGAGAAACTTTTTGTCTCCATTCCTTATGTATGTGTGATTGTCTGTGTCCAAATTGCTTTAAGAATA
ATATTTAATATTTCCTGGAAGCTCATTTTTTTGGCATGAGTCTAATTAAATTATTGAAAGCCAAAAA
The NOV16 protein (SEQ 1D N0:34) encoded by SEQ 1D NO:33 is 1706 amino acid
residues in length and is presented using the one-letter amino acid code in
Table 16B. Psort
analysis predicts the NOV16 protein of the invention to be localized in the
cytoplasm with a
certainty of 0.4500.
Table 16B. Encoded NOV16 protein sequence (SEQ TD N0:34)
MELSCSEAPLYQGQMMIYAKFDKNVYLPEDAEFYFTYDGSHQRHVMIAERIEDNVLQSSVPGHGL
QETVTVSVCLCSEGYSPVTMGSGSVTYVDNMACRLARLLVTQANRLTACSHQTLLTPFALTAGAL
PALDEELVLALTHLELPLEWTVLGSSSLEVSSHRESLLHLAMRWGLAKLSQFFLCLPGGVQALAL
PNEEGATPLDLALREGHSKLVEDVTSFQGRWSPSFSRVQLSEEASLHYIHSSETLTLTLNHTAEH
LLEADIKLFRKYFWDRAFLVKAFEQEARPEERTAMPSSGAETEEEIKNSVSSRSAAEKEDIKRVK
SLVVQHNEHEDQHSLDSRSLLRYPKKSKPPSTLLAAGRLSDMLNGGDEVYANCMVIDQVGDLDIS
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YINIEGITATTSPESRGCTLWPQSSKHTLPTETSPSVYPLSENVEGTAI~TEAQQSFMSPSSSCAS
NLNLSFGWHGFEKEQSHLKKRSSSLDALDADSEGEGHSEPSHICYTPGSQSSSRTGIPSGDELDS
FETNTEPDFNISRAESLPLSSNLQLKESLLSGVRSRSYSCSSPKISLGKTRLVRELTVCSSSEEQ
KAYSLSEPPRENRIQEEEWDKYIIPAKSESEKYKVSRTFSFLMNRMTSPRNKSKTKSKDAKDKEK
LNRHQFAPGTFSGVLQCLVCDKTLLGKESLQCSSCNANVfiKGCKDAAPACTKKFQEKYNKNKPQT
ILGSSSFRDIPQPGLSLHPSSSVPVGLPTGRRETVGQVHPLSRSVPGTTLESFRRSATSLESESD
NNSCRSRSHSDELLQSMGSSPSTESFIMEDVVDSSLWSDLSSDAQEFEAESWSLVVDPSFCNRQE
KDVIKRQDVIFELMQTEMHHIQTLFIMSEIFRKGMKEELQLDHSTVDKIFPCLDELLEIHRHFFY
SMKERRQESSAGSDRNFVIDRIGDILVQQFSEENASKMKKIYGEFCCHHKEAVNLFKELQQNKKF
QNFIKLRNSNLLARRRGIPECILLVTQRITKYPVLVERILQYTKERTEEHKDLRKALCLIKDMIA
TVDLKVNEYEKNQKWLEILNKIENKTYTKLKNGHVFRKQALMSEERTLLYDGLVYWKTATGRFKD
ILALLLTDVLLFLQEKDQKYIFAAVDQKPSVISLQKLIAREVANEERGMFLISASSAGPEMYEIH
TNSKEERNNWMRRIQQAVESCPEEKGGRTSESDEDKRKAEARVAKIQQCQEILTNQDQQICAYLE
EKLHIYAELGELSGFEDVHLEPHLLIKPDPGEPPQAASLLAAALKEAESLQVAVKASQMGAVSQS
CEDSCGDSVLADTLSSHDVPGSPTASLVTGGREGRGCSDVDPGIQGVVTDLAVSDAGEKVECRNF
PGSSQSEIIQAIQNLTRLLYSLQAALTIQDSHIEIHRLVLQQQEGLSLGHSILRGGPLQDQKSRD
ADRQHEELANVHQLQHQLQQEQRRWLRRCEQQQRAQATRESWLQERERECQSQEELLLRSRGELD
LQLQEYQHSLERLREGQRLVEREQARMRAQQSLLGHWKHGRQRSLPAVLLPGGPEVMELNRSESL
CHENSFFINEALVQMSFNTFNKLNPSVIHQDATYPTTQSHSDLVRTSEHQVDLKVDPSQPSNVSH
KLWTAAGSGHQILPFQESSKDSCKNDLDTSHTESPTPHDSNSHRPQLQAFITEAKLNLPTRTMTR
QDGETGDGAKENIVYL
A search against the Patp database, a proprietary database that contains
sequences
published in patents and patent publications, yielded several homologous
proteins shown in
Table 16C.
Table 16C. Pat results
for NOV16
Smallest
Sum
eading igh Prob
equences Frame Score P(N)
producing
High-scoring
Segment
Pairs:
>patp:ABB44551Humanwound healing related polypeptide+1 1470 2.8e-150
>patp:AAW93941Humanbrx protein +1 1436 1.5e-148
>patp:ABG05537Novelhuman diagnostic protein +1 1436 1.5e-148
#5528
>patp:ABG05537Novelhuman diagnostic protein +1 1436 1.5e-148
#5528
>patp:ABG15870Novelhuman diagnostic protein +1 1447 7.5e-148
#15861
In a BLAST search of public sequence databases, it was found, for example,
that the
nucleic acid sequence of this invention has 4339 of 5274 bases (82%) identical
to a
gb:GENBANI~-m:MMU73199~acc:U73199.1 mRNA from Mus musculus (Rho-guanine
nucleotide exchange factor mRNA, complete cds). The full amino acid sequence
of the protein
of the invention was found to have 1350 of 1670 amino acid residues (80%)
identical to, and
1460 of 1670 amino acid residues (87%) similar to, the 1693 amino acid residue
ptnr:SWISSPROT-ACC:P97433 protein fromMus n2usculus (RHO-GUANINE
NUCLEOTIDE EXCHANGE FACTOR (RHOGEF) (RIP2)).
NOV16 also has homology to the proteins shown in the BLASTP data in Table 16D.
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Table 16D. BLAST
results for
NOV16
Gene Index/ Protein/ OrganismLength Identity PositivesExpect
Identifier (aa) (~) (~)
gi~7106395~ref~NPRho interacting1693 1350/16741460/16740.0
0
_ protein 2; Rho (80~) (86~)
36156.1~(NM_012026)
specific exchange
factor
[Mus musculus]
gi~18602674~ref~XPhypothetical 669 668/669 668/669 0.0
_ protein FLJ21817 (99~) (99~)
016989.5~(XM
016989
similar to Rhoip2
[Homo Sapiens]
gi~10438441~dbj~BABunnamed protein669 669/669 669/669 0.0
15243.1~(AK025816)product (1000 (1000
[Homo Sapiens]
gi~15341761~gb~AAH1hypothetical 615 609/613 609/613 0.0
2946.1~AAH12946(BCOprotein FLJ21817 (99~) (99~)
12946) similar to Rhoip2
[Homo Sapiens]
gi~17437752~ref~XPsimilar to Rho 590 290/292 291/292 e-170
_ interacting (99$) (99~)
068710.1~(XM
068710
_ protein 2; Rho
specific exchange
factor
[Homo Sapiens]
A multiple sequence alignment is given in Table 16E, with the NOV 16 protein
being
shown on line 1 in Table 16E in a ClustalW analysis, and comparing the NOV 16
protein with
the related protein sequences shown in Table 16D. This BLASTP data is
displayed graphically
in the ClustalW in Table 16E.
Table 16E. ClustalW Analysis of NOV16
1) > NOV16; SEQ >D NO:34
2) > gi~7106395~/ Rho interacting protein 2; Rho specific exchange factor [Mus
musculus]; SEQ >D
N0:107
3) > gig 18602674/ hypothetical protein FLJ21817 similar to Rhoip2 [Homo
sapieras]; SEQ >D NO:108
4) > gig 10438441 ~/ unnamed protein product [Horno Sapiens]; SEQ ID N0:109
5) > gi~15341761~/ hypothetical protein FLJ21817 similar to Rhoip2 [Homo
Sapiens]; SEQ ID NO:110
6) > gig 17437752/ similar to Rho interacting protein 2; Rho specific exchange
factor [Homo Sapiens];
SEQ ID NO:111
'
10 20 30 40 50 60
NOV16 1 MELSCSEAPLYQGQMMIYAKFDKNVYLPEDAEFYFTYDGSHQRHVMIAERIEDNVLQSSV 60
giI71063951 1 -MELSCSEVPLYGQKTVYAKFGKNVYLPEDAEFYFVYGGSHQRHWIADRVQDNVLQSSI 59
gip8sozs74~ 1 ____________________________________________________________ 1
gi~10438441~ 1 ____________________________________________________________ 1
gi~15341761~ 1 ____________________________________________________________ 1
gi~17437752~ 1 ____________________________________________________________ 1
70 80 90 100 110 120
NOV16 61 PGHGLQETVTVSVCLCSEGYSPVTMGSGSVTYVDNMACRLARLLVTQANRLTACSHQTLL 120
gi~71063951 60 PGHWLQETVTVSVCLCSEGYSPVTMGSGSVTYVDNMACRLARLLVTQADRLTACSHQTLL
119
gi~18602674) 1 ____________________________________________________________ 1
gi~10438441~ 1 ____________________________________________________________ 1
gi~15341761~ 1 ____________________________________________________________ 1
gi~17437752~ 1 ____________________________________________________________ 1
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130 140 150 160 170 7.80
NOVl6 121
TPFALTAGALPALDEELVLALTHLELPLEWTVLGSSSLEVSSHRESLLHLAMRWGLAKLS180
gi~71063951120 TPFALTVEALPALDEELVLALTQLELPLGWTVLGNSSLEVSLHRESLLHLAVRWALPKLF179
$ giI18602674~1
____________________________________________________________1
gi~10438441~1 ____________________________________________________________
1
gi~15341761~1 ____________________________________________________________1
gi~17437752~1 1
1~ 190 200 210 220 230 240
NOV16 181
QFFLCLPGGVQALALPNEEGATPLDLALREGHSKLVEDVTSFQGRWSPSFSRVQLSEEAS240
gi~7106395~ 180
HFLLCLPGGVKALKLPNEEATTPLDLALQGGHSTLVEDITNFQGSHSPGFSRLRLNEEAT239
gi~18602674~ 1
____________________________________________________________1
IS gi~104384411 1
____________________________________________________________1
gi1153417611 1
____________________________________________________________1
gi~17437752~ 1
____________________________________________________________1
250 260 270 280 290 300
NOV16 241
LHYIHSSETLTLTLNHTAEHLLEADTKLFRKYFWDRAFLVKAFEQEARPEERTAMPSSGA300
giI71063951240 LQFVHSSETLTLTVNHTAEHLLEADIKLFRKYFWDRAFLVKALEQEAKTEKATMPSGAAE299
gi~18602674)1 ____________________________________________________________1
gi~10438441~l ____________________________________________________________1
gi~15341761~1 ____________________________________________________________1
gi~17437752)1 ____________________________________________________________1
310 320 330 340 350 360
3O NOV16 301
ETEEEIKNSVSSRSAAEKEDIKRVKSLWQHNEHEDQHSLDSRSLLRYPKKSKPPSTLLA360
gi~7106395~300 TEEEVRNLESGRSPSEEEEDAKSIKSQVDGPSEHEDQDRLALDRSFDGLKKSKHVPASLA359
gi~18602674~1 ____________________________________________________________1
gi~10438441~1 ____________________________________________________________1
gi~1534176111 ____________________________________________________________1
gi~17437752~1 ____________________________________________________________1
370 380 390 400 410 420
NOV16 361
AGRLSDMLNGGDEVYANCMVIDQVGDLDISYINIEGITATTSPESRGCTLWPQSSKHTLP420
4O giI71063951360
AGQLSDVLNGGDEVYANCMVIDQVGDLDINYINLEGLSTHTSPESGRSMLGPQACMHTLP419
gi~18602674~1 ____________________________________________________________1
gi~1043844111 ____________________________________________________________1
gi~15341761~1 ____________________________________________________________1
gi~17437752~1 ____________________________________________________________1
45
430 440 450 460 470 480
NOV16 421
TETSPSVYPLSENVEGTAHTEAQQSFMSPSSSCASNLNLSFGWHGFEKEQSHLKKRSSSL480
gi~7106395~420 PDTSPCGRPLIENSEGTLDAAASQSFVTPSSSRTSNLNLSFGLHGFEKEQSHLKKRSSSL479
gi~1860267411 ____________________________________________________________1
gi~10438441~1 ____________________________________________________________1
gi~15341761~1 ____________________________________________________________1
gi~17437752~1 ____________________________________________________________1
55 490 500 510 520 530 540
NOV16 481
DALDADSEGEGHSEPSHICYTPGSQSSSRTGIPSGDELDSFETNTEPDFNISRAESLPLS540
gi ~71063951480 DALVADSEGEGGSEPPICYAVGSQSSPRTG-
LPSGDELDSFETNTEPDCNISRTESLSLS538
gi ~1860267411 ____________________________________________________________1
gi ~10438441~1 ____________________________________________________________1
gi ~15341761~1 ____________________________________________________________1
gi ~17437752~1 ____________________________________________________________1
550 560 570 580 590 600
65
NOV16 541
SNLQLKESLLSGVRSRSYSCSSPKISLGKTRLVRELTVCSSSEEQKAYSLSEPPRENRIQ600
gi I7106395~539
STLHSKESLLSGIRSRSYSCSSPKISSGKSRLVRDFTVCSTSEEQRSYSFQEPPGEKRIQ598
gi ~1860267411 ____________________________________________________________1
gi I10438441~1 ____________________________________________________________1
gi ~15341761~1 ____________________________________________________________1
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gi~17437752~ 1 ____________________________________________________________ 1
610 620 630 640 650 660
S NOV16
601EEEWDKYITPAKSESEKYKVSRTFSFLMNRMTSPRNKSKTKSKDAKDKEKLNRHQFAPGT660
gi~7106395~
599EEEWDEWIPAKSESEKYKVSRTFSFLMNRMTSPRNKSKMKNKDTKEKEKMNRHQFVPGT658
gi~186026741 1
____________________________________________________________1
gi~10438441~ 1
____________________________________________________________1
giI15341761~ 1
____________________________________________________________1
gi117437752~ 1
____________________________________________________________1
670 680 690 700 710 720
NOV16
661FSGVLQCLVCDKTLLGKESLQCSSCNANVHKGCKDAAPACTKKFQEKYNKNKPQTILGSS720
IS
gi~71063951659FSGVLQCSGCDKTLLGKESLQCANCKANTHKGCKDAVPPCTKKFQEKYNKNKPQSILGSS718
gi~1860267411 ____________________________________________________________1
gi~1043844111 ____________________________________________________________1
gi~15341761)1 ____________________________________________________________1
gi~17437752~1 ____________________________________________________________1
730 740 750 760 770 780
NOV16
721SFRDIPQPGLSLHPSSSVPVGLPTGRRETVGQVHPLSRSVPGTTLESFRRSATSLESESD780
giI7106395~719SVRDVPAPGLSLHPSSSMPIGLPAGRKEFAAQVHPLSRSVPGTTLESFRR--AVTSLESE776
2S giI1860267411
____________________________________________________________1
gi ~10438441~1 ____________________________________________________________1
gi~1534176111 ____________________________________________________________1
gi~17437752~1 ____________________________________________________________1
790 800 810 820 830 840
NOV16 781NNSCRSRSHSDELLQSMGSSPSTESFIMEDVVDSSLWSDLSSDAQEFEAESWSLWDPSF840
giI71063951777GDSWRSRSHSDELFQSMGSSPSTESFMMEDWDSSLWIDLSSDAQEFEAESWSLWDPSF836
gi~1860267411 ____________________________________________________________1
3S gi~10438441~1
____________________________________________________________1
gi~15341761~1 ____________________________________________________________1
gi~17437752~1 ____________________________________________________________1
850 860 870 880 890 900
NOV16
841CNRQEKDVIKRQDVIFELMQTEMHHIQTLFIMSEIFRKGMKEELQLDHSTVDKIFPCLDE900
gi~71063951837CSRQEKDVIKRQDVIFELMQTEVHHIQTLLIMSEVFRKGMKEELQLDHSTVDKIFPCLDE896
gi~18602674~1 ____________________________________________________________1
gi~1043844111 ____________________________________________________________1
4S gi~1534176111
____________________________________________________________1
gi~1743775211 ____________________________________________________________1
910 920 930 940 950 960
.)
SO NOV16
901LLEIHRHFFYSMKERRQESSAGSDRNFVIDRIGDILVQQFSEENASKMKKIYGEFCCHHK960
giI71063951897LLETHRHFFFSMKERRQESCAGSDRNFVINQIGDILVQQFSEENASKMKRIYGEFCSHHK956
gi~18602674~1 ____________________________________________________________1
gi~10438441~1 ____________________________________________________________1
gi115341761~1 ____________________________________________________________
SS gi~17437752~1 ____________________________________________________________
1
970 980 990 1000 1010 1020
NOV16
961EAVNLFKELQQNKKFQNFIKLRNSNLLARRRGIPECILLVTQRITKYPVLVERILQYTKE1020
C70gi~7106395~957EAMSLFKELQQNKKFQNFIKIRNSNLLARRRGIPECILLVTQRITKYPVLVERILQYTKE10
16
gi~1860267411 ____________________________________________________________1
gi~10438441~1 ____________________________________________________________1
gi~15341761~1 ____________________________________________________________1
gi~17437752~1 ____________________________________________________________
1
6S
1030 1040 . 1050 1060 1070 1080
.
'
'
a 1077
r rv .n
NOV16 1021 RTEEHKDLRKALCLIKD
~ --a ~
~
i a 1073
giI71063951 1017 RTEEHRDLCKALGLIKD ~ ~ --a ~
70 gi~18602674~ 1 ---------------- ~ --a ~ a 40
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gi~10438441~ 1 ________________ . , __~ 1 ~ 40
gi~7.5341761~ 1 ________________ . , __~ 1 ~ 40
gi~17437752~ 1 ------------------PDNQNPT CSGET~~ P EGGS---- PAS RCES 38
1090 1100 1110 1120 1130 1140
NOV16 1078 1137
gi~71063951 1074 1133
gi~186026741 41 100
giI10438441~ 41 100
gi~15341761~ 41 100
gi117437752~ 39 94
1150 1160 1170 1180 1190 1200
NOV16 1138 1197
gi~7106395~ 1134 1193
gi~18602674~ 101 160
gi~10438441~ 101 160
gi115341761~ 101 160'
gi117437752~ 95 148
1210 1220 1230 1240 1250 1260
NOV16 1198 ' 1.1i~- ~ ~. ~ 11 1 ~m 111 s~~ ~~~~ . 1-- 1256
gi~7106395~ 1194 ' 1 ~ ~ ~' ~ 11 1 SPm 111 1T ~ 1 1252
gi~18602674~ 161 1 1~'- ~ ~' ~ 11 1 1111 ~ ~ 1 219
gi~10438441~ 161 1 1 ' ~ ~~ ~ 11 1 1111 ~ 1 219
gi~15341761~ 161 ' 1 1 '- ~ ~~ ~ 11 1 1111 ~ ~ 1 219
gi ~ 17437752 ~ 149 L QSL ~LNIFLPVT~I~QIKFGMFG,- ------FVTIVMSVCKVG~TKE~ 202
1270 1280 1290 1300 1310 1320
~ y
NOV16 1257 ~ 1316
giI7106395~ 1253 1312
gi~18602674~ 220 279
gi~10438441~ 220 279
gi~15341761~ 220 279
gi~17437752~ 203 252
1330 1340 1350 1360 1370 1380
NOV16 1317 . 1375
gi~7106395~ 1313 1371
gi~186026741 280 338
gi~10438441~ 280 338
gi~15341761~ 280 338
gi117437752~ 253 311
1390 1400 1410 1420 1430 1440
NOV16 1376 1434
gi~7106395~ 1372 1430
gi~186026741 339 397
gi~10438441~ 339 397
gi~15341761~ 339 397
gi~174377521 312 351
1450 1460 1470 1480 1490 1500
NOV16 1435 1494
gi~7106395~ 1431 1490
giI186026741 398 457
gi~104384411 398 457
gi~15341761~ 398 457
gi~174377521 351 399
1510 1520 1530 1540 1550 1560
..
NOV16 1495 1~1 1 ~1 1 1~~~ ~11~ ~1 .' 1554
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.1. .1..... . 1 . 1 1 1 ~1
. i 1 11 ~
~
.1. .1.. . 1 . 1 E 1 P
. S P T'
.1. ..l....~.'~a'a1 ' . 1 1
a a 1
.1. .1..... . 1 . 1 . 1 1 1
. 1
.1. .1..... 1 . 1 . 1 1 1
1
GQ.".~.:. T- PAEFYFTYD HQRH I-- BRIE..,.QS,'r'"'LPG
~'AKFD _ 1
_
_
_
_
_
_
VY
1 s 1 1 1 r 1 1
1
QI E T 1 1 1 1 -- S 1 1
1
1 aee .1 1 1 ' 1 a - 1 1
1 . a
1 1 1 1 1 1 1
1
1 1 1 1 1 1 1
1
G~QEE,,, CLCS- YSPVTGS SVTYVD CRLiRLLVTQA1~ RLTAC QT,'C.e
IiT
CA 02443770 2003-10-15
550
17
17
17
$ ~ I 56
1570 1580 1590 1600 1610 1620
NOV16 1555 ~ ' ~ ~~~ -- ~ ~ -m 1611
giI71063951 1551 ~FGH L S ~P ~ C,'~PW~~--- ~LE F~ S-PT 1606
gi~18602674~ 518 ~ ~ ~~~ - ~ ~ -~ 574
giI10438441~ 518 ~ t ~s~ - ~ ~ -~ 574
gi~15341761~ 518 ~ ~ ~ ~ ~~~ -- ~ ~F ~ -~ 574
gi ~ 17437752 ~ 457 KLFRKYFW~3 ~, LV'KAFEPARPEERTAts'~PS
GAET~:EARLK2VLT~KQFL~I'~'LG PDA 516
. ..
1630 1640 1650 1660 1670 1680
y ~~~ .y ~~~ ~~. ~~.~ ~~. y
~r vr --w n ~~~ ~ ~ ~v v
NOV16 1612 ~~~ ~~1~~S ~ ~ o~~~ --~ vTSHT P'~ ~~~ H 1668
giI7106395~ 1607 rI~I ~~P -STG-P ~RPAL~ ~ Y~ r yFQS S~Q~ QR 1664
gi ~ 18602674 ~ 575 ~ ~ ~ ~ ~S ~'~ . ~ w. r ~ - r ~TPH~' P'~ ~ ~ 631
gi I 10438441 ~ 575 ~ ~ ~ v ~S ~ ~ ~ ~ o ~ --~ ~'SFi':G' P'T~ ~ H 631
gi~15341761~ 575 ~ L ~~ ~~L ~~ ~ ~ ~ --GN---------------- 615
gi~17437752~ 517 L ~LDLP~QTAL ~PREFAFT ~LWVLAT y,;GAT~YPAAQ~IYILTT'~'V~R 576
1690 1700 1710
NOV16 1669 (~ ~Q~FI~rE~,~ rLNLPTRT ~QDGETGDGAKENIVYL 1706
gi~71063951 1665 W~PATDTHNR~ TKSSDGGW ~ RCWRRG--------- 1693
giI186026741 632 Q~FI~E' LNLPTRT ~QDGETGDGAKENIVYL 669
gi~10438441~ 632 Q ~ FI E LNLPTRT ~QDGETGDGAKENIVYL 669
gi~15341761~ 615 ______________________________________ 615
gi~17437752~ 577 F~~KED~RCARG-_________________-_____ 590
The presence of identifiable domains in the protein disclosed herein was
determined by
searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and
then
determining the Interpro number by crossing the domain match (or numbers)
using the
Interpro website (http:www.ebi.ac.uk/interpro~. Table 16F lists the domain
description from
DOMAIN analysis results against NOV 16.
Tab le 16F Domain
Anal sis
of NQV16
Model Region of Score (bits) E value
Homology
Phorbol 654-700 40.1 4.9e-08
esters/diacylglycerol
binding dom (DAG
PE-
bind)
PHD-finger (PHD) 666-703 1.4 0.06
Phorbol 654-698 46.2 2.0e-05
esters/diacylglycerol
binding domain
(DAG
PE-bind)
Cl, Protein kinase654-698 45.8 2.0e-05
C
conserved region
1
RhoGEF domain 854-1044 78.4 1.5e-19
(RhoGEF)
bZIP transcription1022-1048 5.0 7.1
factor (bZIP)
PH domain (PH) 1088-1189 40.9 4.5e-10
WO 02/085922 PCT/US02/11634
gi~7106395~ 1491 1
gi~18602674~ 458
5
gi~10438441~ 458 5
gi~153417611 458 5
gi 17437752 399 4
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Consistent with other known members of the subunit family of proteins, NOV 16
has,
for example, a RhoGEF signature sequence and homology to other members of the
RhoGEF-
like Protein Family. NOV 16 nucleic acids, and the encoded polypeptides,
according to the
invention are useful in a variety of applications and contexts. For example,
NOV 16 nucleic
acids and polypeptides can be used to identify proteins that are members of
the RhoGEF-like
Protein Family. The NOV16 nucleic acids and polypeptides can also be used to
screen for
molecules, which inhibit or enhance NOV16 activity or function. Specifically,
the nucleic
acids and polypeptides according to the invention may be used as targets for
the identification
of small molecules that modulate or inhibit, e.g., cellular activation,
cellular replication, and
signal transduction. These molecules can be used to treat, e.g., cancer,
trauma, regeneration
(ifa vitro and in vivo), viral/bacterial/parasitic infections, diabetes, Von
Hippel-Lindau (VHL)
syndrome, pancreatitis, obesity, anemia , bleeding disorders, scleroderma,
transplantation,
hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura,
immunodeficiencies,
graft versus host disease, Alzheimer's disease, stroke, tuberous sclerosis,
hypercalceimia,
Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-
Nyhan syndrome,
multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral
disorders, addiction,
anxiety, pain, neurodegeneration, cirrhosis, transplantation,
adrenoleukodystrophy , congenital
adrenal hyperplasia, hemophilia, hypercoagulation, idiopathic thrombocytopenic
purpura,
autoimmme disease, allergies, immunodeficiencies, transplantation, graft
versus host disease,
lyrnphedema , allergies, immunodeficiencies, osteoporosis, hypercalceimia,
arthritis,
ankylosing spondylitis, scoliosis, tendinitis, systemic lupus erythematosus,
autoimmune
disease, asthma, emphysema, scleroderma, ARDS, endometriosis, fertility,
hyperthyroidism,
hypothyroidism, diabetes, autoimmune disease, renal artery stenosis,
interstitial nephritis,
glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus,
renal tubular
acidosis, IgA nephropathy, hypercalceimia, psoriasis, actinic keratosis,
tuberous sclerosis,
acne, hair growth/loss, alopecia, pigmentation disorders, endocrine disorders
as well as other
diseases, disorders and conditions.
In addition, various NOV 16 nucleic acids and polypeptides according to the
invention
are useful, ifate~ alia, as novel members of the protein families according to
the presence of
domains and sequence relatedness to previously described proteins. For
example, the NOV 16
nucleic acids and their encoded polypeptides include structural motifs that
are characteristic of
proteins belonging to the RhoGEF-like Protein Family.
GEF (Guanine nucleotide exchange factor) for Rho/Rac/Cdc42-like GTPases is
also
called Dbl-homologous (DH) domain. It appears that PH (pleckstrin homology)
domains
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invariably occur C-terminal to RhoGEF/DH domains. Although the exact function
of PH
domains is unclear, several choices include binding to the beta/gamma subunit
of
heterotrimeric G proteins, binding to lipids, e.g. phosphatidylinositol-4,5-
bisphosphate,
binding to phosphorylated Ser/Thr residues, attachment to membranes by an
unknown
mechanism. The DAG PE-binding domain binds two zinc ions; the ligands of these
metal ions
are probably the six cysteines and two histidines that are conserved in this
domain and can
regulate signal transduction by the PKC family of kinases.
NOV 16 belongs to the guanine nucleotide exchange factor family of proteins
which
play a signficant role in signal transduction. The guanine nucleotide exchange
factor (GEF)
domain that regulates GTP binding protein signaling. The GEF domain regulates
positively the
signaling cascades that utilize GTP-binding proteins (such as those of the ras
superfamily) that
function as molecular switches in fundamental events such as signal
transduction, cytoskeleton
dynamics and intracellular trafficking. An example of a protein containing GEF
and PH
domains is FGD1 (faciogenital dyplasia protein) Experiments have shown that
the GEF and
(PH) domains of FGDl can bind specifically to the Rho family GTPase Cdc42Hs
and
stimulates the GDP-GTP exchange of the isoprenylated form of Cdc42Hs. The GEF
domain of
FGD1 has also been shown to activate 2 kinases involved in cell proliferation;
the Jun NH2-
terminal kinase and the p70 S6 kinase (~heng et al.; J. Biol. Chem 1996 Dec
27;271(52):33169-72). Thus, NOV16 polypeptide may play an important role in
normal
development as well as disease. This class of molecules (GEFs) is also being
considered as a
good drug target as the guanine nucleotide exchange factor RasGRP is a high -
affinity target
for diacylglycerol and phorbol esters and is bound by bryostatin 1, a compound
currently in
cliW cal trials (Lorenzo et al.; Mol. Pharmacol 2000 May; 57(5):840-6). The
homolog of
RhoGEF, DRhoGEF2 fail to gastrulate due to a defect in cell shape changes
required for tissue
invagination and the mRNA is found throughout oogenesis and embryogenesis
(Barrett et al.;
Cell 1997; 91(7):905-15; Werner et al.; Gene 1997; 187(1):107-14). RhoGEF also
interacts
with c-Jun amino-terminal kinase (JNK) interacting protein-1 (JIP-1). JIP-1
might function as
a scaffold protein by complexing specific components of the JNK signaling
pathway, namely
JNK, mitogen-activated protein kinase kinase 7, and mixed lineage kinase 3
(Meyer et al.; J
Biol Chem 1999; 274(49):35113-8).
The NOV 16 nucleic acids and polypeptides, antibodies and related compounds
according to the invention will be useful in therapeutic and diagnostic
applications in the
mediation of blood and nerve physiology. As such, the NOV 16 nucleic acids and
polypeptides, antibodies and related compounds according to the invention may
be used to
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treat blood and nervous system disorders, e.g., cancer, trauma, regeneration
(in vitro and in
vivo), viral/bacterial/parasitic infections, diabetes, Von Hippel-Lindau (VHL)
syndrome,
pancreatitis, obesity, anemia , bleeding disorders, scleroderma,
transplantation, hemophilia,
hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies,
graft versus
host disease, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia,
Parkinson's
disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome,
multiple
sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders,
addiction, anxiety,
pain, neurodegeneration, cirrhosis, transplantation, adrenoleukodystrophy ,
congenital adrenal
hyperplasia, hemophilia, hypercoagulation, idiopathic thrombocytopenic
purpura, autoimmune
disease, allergies, immunodeficiencies, transplantation, graft versus host
disease, lymphedema
allergies, immunodeficiencies, osteoporosis, hypercalceirriia, arthritis,
ankylosing spondylitis,
scoliosis, tendinitis, systemic lupus erythematosus, autoimmune disease,
asthma, emphysema,
scleroderma, ARDS, endometriosis, fertility, hyperthyroidism, hypothyroidism,
diabetes,
autoimmune disease, renal artery stenosis, interstitial nephritis,
glomerulonephritis, polycystic
kidney disease, systemic lupus erythematosus, renal tubulax acidosis, IgA
nephropathy,
hypercalceimia, psoriasis, actinic keratosis, tuberous sclerosis, acne, hair
growth/loss,
alopecia, pigmentation disorders, endocrine disorders as well as other
diseases, disorders and
conditions.
The NOV16 nucleic acids and polypeptides are useful for detecting specific
cell types.
For example, expression analysis has demonstrated that a NOV 16 nucleic acid
is expressed in
Adipose, Umbilical Vein, Pancreas, Thymus, Brain, Lung, Kidney, Adrenal
Gland/Suprarenal
gland, Peripheral Blood, Lymph node, Cartilage, Mammary gland/Breast, Uterus,
Prostate,
Trachea, Cochlea, I~ermis, Heart, Aorta, Coronary Artery, Thyroid, Liver,
Bone, Bone
Marrow, Spinal Cord, Cervix, and Retina.
Additional utilities for NOV16 nucleic acids and polypeptides according to the
invention are disclosed herein.
NOVX Nucleic Acids and Polypeptides
One aspect of the invention pertains to isolated nucleic acid molecules that
encode
NOVX polypeptides or biologically active portions thereof. Also included in
the invention are
nucleic acid fragments sufficient for use as hybridization probes to identify
NOVX-encoding
nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR primers for the
amplification and/or mutation of NOVX nucleic acid molecules. As used herein,
the term
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"nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or
genomic
DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using
nucleotide analogs, and derivatives, fragments and homologs thereof. The
nucleic acid
molecule may be single-stranded or double-stranded, but preferably is
comprised double-
s stranded DNA.
An NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a
"mature" form of a polypeptide or protein disclosed in the present invention
is the product of a
naturally occurring polypeptide or precursor form or proprotein. The naturally
occurring
polypeptide, precursor or proprotein includes, by way of nonlimiting example,
the full-length
gene product, encoded by the corresponding gene. Alternatively, it may be
defined as the
polypeptide, precursor or proprotein encoded by an ORF described herein. The
product
"mature" form arises, again by way of nonlimiting example, as a result of one
or more
naturally occurring processing steps as they may take place within the cell,
or host cell, in
which the gene product arises. Examples of such processing steps leading to a
"mature" form
of a polypeptide or protein include the cleavage of the N-terminal methionine
residue encoded
by the initiation colon of an ORF, or the proteolytic cleavage of a signal
peptide or leader
sequence. Thus a mature form arising from a precursor polypeptide or protein
that has
residues 1 to N, where residue 1 is the N-terminal methionine, would have
residues 2 through
N remaining after removal of the N-terminal methionine. Alternatively, a
mature form arising
from a precursor polypeptide or protein having residues 1 to N, in which an N-
terminal signal
sequence from residue 1 to residue M is cleaved, would have the residues from
residue M+1 to
residue N remaining. Further as used herein, a "mature" form of a polypeptide
or protein may
arise from a step of post-translational modification other than a proteolytic
cleavage event.
Such additional processes include, by way of non-limiting example,
glycosylation,
myristoylation or phosphorylation. In general, a mature polypeptide or protein
may result
from the operation of only one of these processes, or a combination of any of
them.
The term "probes", as utilized herein, refers to nucleic acid sequences of
variable length,
preferably between at least about 10 nucleotides (nt), 100 nt, or as many as
approximately,
e.g., 6,000 nt, depending upon the specific use. Probes are used in the
detection of identical,
similar, or complementary nucleic acid sequences. Longer length probes axe
generally
obtained from a natural or recombinant source, are highly specific, and much
slower to
hybridize than shorter-length oligomer probes. Probes may be single- or double-
stranded and
designed to have specificity in PCR, membrane-based hybridization
technologies, or ELISA-
like technologies.
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The term "isolated" nucleic acid molecule, as utilized herein, is one, wluch
is separated
from other nucleic acid molecules which are present in the natural source of
the nucleic acid.
Preferably, an "isolated" nucleic acid is free of sequences which naturally
flank the nucleic
acid (i. e., sequences located at the 5'- and 3'-termini of the nucleic acid)
in the genomic DNA
of the organism from which the nucleic acid is derived. For example, in
various embodiments,
the isolated NOVX nucleic acid molecules can contain less than about 5 kb, 4
kb, 3 kb, 2 kb, 1
kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic
acid molecule in
genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g.,
brain, heart, liver,
spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA
molecule, can
be substantially free of other cellular material or culture medium when
produced by
recombinant techniques, or of chemical precursors or other chemicals when
chemically
synthesized.
A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having
the
nucleotide sequence SEQ ID NOS:1, 3, S, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,
27, 29, 31, and
33, or a complement of this aforementioned nucleotide sequence, can be
isolated using
standard molecular biology techniques and the sequence information provided
herein. Using
all or a portion of the nucleic acid sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11,
13, 15, 17, 19,
21, 23, 25, 27, 29, 31, and 33 as a hybridization probe, NOVX molecules can be
isolated using
standard hybridization and cloning techniques (e.g., as described in Sambrook,
et al., (eds.),
MOLECULAR CLONING: A LABORATORY MANUAL 2"d Ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, 199; and Ausubel, et al., (eds.), CURRENT
PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
A nucleic acid of the invention can be amplified using cDNA, mRNA or
alternatively,
genomic DNA, as a template and appropriate oligonucleotide primers according
to standard
PCR amplification techniques. The nucleic acid so amplified can be cloned into
an
appropriate vector and characterized by DNA sequence analysis. Furthermore,
oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by
standard
synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked
nucleotide
residues, which oligonucleotide has a sufficient number of nucleotide bases to
be used in a
PCR reaction. A short oligonucleotide sequence may be based on, or designed
from, a
genomic or cDNA sequence and is used to amplify, confirm, or reveal the
presence of an
identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise portions of a nucleic acid sequence having about 10
nt, 50 nt, or
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100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment
of the
invention, an oligonucleotide comprising a nucleic acid molecule less than 100
nt in length
would further comprise at least 6 contiguous nucleotides SEQ ID NOS:1, 3, 5,
7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, and 33, or a complement thereof.
Oligonucleotides may be
chemically synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention
comprises a
nucleic acid molecule that is a complement of the nucleotide sequence shown in
SEQ ll~
NOS:1, 3, 5, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, or a
portion of this
nucleotide sequence (e.g., a fragment that can be used as a probe or primer or
a fragment
encoding a biologically-active portion of an NOVX polypeptide). A nucleic acid
molecule
that is complementary to the nucleotide sequence shown SEQ ID NOS:1, 3, 5, 7,
9, 1 l, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, or 33 is one that is sufficiently
complementary to the nucleotide
sequence shown SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,
29, 31, or 33 that
it can hydrogen bond with little or no mismatches to the nucleotide sequence
shown SEQ m
NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, thereby
forming a stable
duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen
base
pairing between nucleotides units of a nucleic acid molecule, and the term
"binding" means
the physical or chemical interaction between two polypeptides or compounds or
associated
polypeptides or compounds or combinations thereof. Binding includes ionic, non-
ionic, van
der Waals, hydrophobic interactions, and the like. A physical interaction can
be either direct
or indirect. Indirect interactions may be through or due to the effects of
another polypeptide or
compound. Direct binding refers to interactions that do not take place
through, or due to, the
effect of another polypeptide or compound, but instead are without other
substantial chemical
intermediates.
Fragments provided herein are defined as sequences of at least 6 (contiguous)
nucleic
acids or at least 4 (contiguous) amino acids, a length sufficient to allow for
specific
hybridization in the case of nucleic acids or for specific recognition of an
epitope in the case of
amino acids, respectively, and are at most some portion less than a full
length sequence.
Fragments may be derived from any contiguous portion of a nucleic acid or
amino acid
sequence of choice. Derivatives are nucleic acid sequences or amino acid
sequences formed
from the native compounds either directly or by modification or partial
substitution. Analogs
are nucleic acid sequences or amino acid sequences that have a structure
similar to, but not
identical to, the native compound but differs from it in respect to certain
components or side
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chains. Analogs may be synthetic or from a different evolutionary origin and
may have a
similar or opposite metabolic activity compared to wild type. Homologs are
nucleic acid
sequences or amino acid sequences of a particular gene that are derived from
different species.
Derivatives and analogs may be full length or other than full length, if the
derivative or analog
contains a modified nucleic acid or amino acid, as described below.
Derivatives or analogs of
the nucleic acids or proteins of the invention include, but are not limited
to, molecules
comprising regions that are substantially homologous to the nucleic acids or
proteins of the
invention, in various embodiments, by at least about 70%, 80%, or 95% identity
(with a
preferred identity of 80-95%) over a nucleic acid or amino acid sequence of
identical size or
when compared to an aligned sequence in which the alignment is done by a
computer
homology program known in the art, or whose encoding nucleic acid is capable
of hybridizing
to the complement of a sequence encoding the aforementioned proteins under
stringent,
moderately stringent, or low stringent conditions. See e.g. Ausubel, et al.,
CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and
below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or
variations thereof, refer to sequences characterized by a homology at the
nucleotide level or
amino acid level as discussed above. Homologous nucleotide sequences encode
those
sequences coding for isofonns of NOVX polypeptides. Isoforms can be expressed
in different
tissues of the same organism as a result of, for example, alternative splicing
of RNA.
Alternatively, isoforms can be encoded by different genes. In the invention,
homologous
nucleotide sequences include nucleotide sequences encoding for an NOVX
polypeptide of
species other than humans, including, but not limited to: vertebrates, and
thus can include, e.g.,
frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous
nucleotide
sequences also include, but are not limited to, naturally occurring allelic
variations and
mutations of the nucleotide sequences set forth herein. A homologous
nucleotide sequence
does not, however, include the exact nucleotide sequence encoding human NOVX
protein.
Homologous nucleic acid sequences include those nucleic acid sequences that
encode
conservative amino acid substitutions (see below) in SEQ ID NOS:1, 3, 5, 7, 9,
11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, and 33, as well as a polypeptide possessing NOVX
biological
activity. Various biological activities of the NOVX proteins are described
below.
An NOVX polypeptide is encoded by the open reading frame ("ORF") of an NOVX
nucleic acid. An ORF corresponds to a nucleotide sequence that could
potentially be translated
into a polypeptide. A stretch of nucleic acids comprising an ORF is
uninterrupted by a stop
codon. An ORF that represents the coding sequence for a full protein begins
with an ATG
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"start" codon and terminates with one of the three "stop" codons, naively,
TAA, TAG, or
TGA. For the purposes of this invention, an ORF may be any part of a coding
sequence, with
or without a start codon, a stop codon, or both. For an ORF to be considered
as a good
candidate for coding for a bona fzde cellular protein, a minimum size
requirement is often set,
e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
The nucleotide sequences determined from the cloning of the human NOVX genes
allows for the generation of probes and primers designed for use in
identifying and/or cloning
NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX
homologues
from other vertebrates. The probe/primer typically comprises substantially
purified
oligonucleotide. The oligonucleotide typically comprises a region of
nucleotide sequence that
hybridizes under stringent conditions to at least about 12, 25, S0, 100, 150,
200, 250, 300, 350
or 400 consecutive sense strand nucleotide sequence SEQ m NOS:1, 3, 5, 7, 9,
11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, or 33; or an anti-sense strand nucleotide sequence
of SEQ m
NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33; or of a
naturally occurring
mutant of SEQ m NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,
and 33.
Probes based on the human NOVX nucleotide sequences can be used to detect
transcripts or genomic sequences encoding the same or homologous proteins. In
various
embodiments, the probe further comprises a label group attached thereto, e.g.
the label group
can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-
factor. Such
probes can be used as a part of a diagnostic test kit for identifying cells or
tissues which mis-
express an NOVX protein, such as by measuring a level of an NOVX-encoding
nucleic acid in
a sample of cells from a subject e.g., detecting NOVX mRNA levels or
determining whether a
genomic NOVX gene has been mutated or deleted.
"A polypeptide having a biologically-active portion of an NOVX polypeptide"
refers
to polypeptides exhibiting activity similar, but not necessarily identical to,
an activity of a
polypeptide of the invention, including mature forms, as measured in a
particular biological
assay, with or without dose dependency. A nucleic acid fragment encoding a
"biologically-
active portion of NOVX" can be prepared by isolating a portion SEQ m NOS:1, 3,
5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, that encodes a polypeptide
having an NOVX
biological activity (the biological activities of the NOVX proteins are
described below),
expressing the encoded portion of NOVX protein (e.g., by recombinant
expression in vitro)
and assessing the activity of the encoded portion of NOVX.
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NOVX Nucleic Acid and Polypeptide Variants
The invention further encompasses nucleic acid molecules that differ from the
nucleotide sequences shown in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25, 27,
29, 31, and 33 due to degeneracy of the genetic code and thus encode the same
NOVX
proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS:1, 3,
5, 7, 9, 1 l,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33. In another embodiment, an
isolated nucleic acid
molecule of the invention has a nucleotide sequence encoding a protein having
an amino acid
sequence shown in SEQ m NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,
28, 30, 32, or 34.
In addition to the human NOVX nucleotide sequences shown in SEQ 1T7 NOS:1, 3,
5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, it will be
appreciated by those skilled in
the art that DNA sequence polymorphisms that lead to changes in the amino acid
sequences of
the NOVX polypeptides may exist within a population (e.g., the human
population). Such
genetic polymorphism in the NOVX genes may exist among individuals within a
population
due to natural allelic variation. As used herein, the terms "gene" and
"recombinant gene" refer
to nucleic acid molecules comprising an open reading frame (ORF) encoding an
NOVX
protein, preferably a vertebrate NOVX protein. Such natural allelic variations
can typically
result in 1-5% variance in the nucleotide sequence of the NOVX genes. Any and
all such
nucleotide variations and resulting amino acid polymorphisms in the NOVX
polypeptides,
which are the result of natural allelic variation and that do not alter the
functional activity of
the NOVX polypeptides, are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding NOVX proteins from other species,
and
thus that have a nucleotide sequence that differs from the human SEQ m NOS:1,
3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 are intended to be within
the scope of the
invention. Nucleic acid molecules corresponding to natural allelic variants
and homologues of
the NOVX cDNAs of the invention can be isolated based on their homology to the
human
NOVX nucleic acids disclosed herein using the human cDNAs, or a portion
thereof, as a
hybridization probe according to standard hybridization techniques under
stringent
hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the
invention is at least 6 nucleotides in length and hybridizes under stringent
conditions to the
nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3,
5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33. In another embodiment, the
nucleic acid is at
least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides
in length. In yet
another embodiment, an isolated nucleic acid molecule of the invention
hybridizes to the
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coding region. As used herein, the term "hybridizes under stringent
conditions" is intended to
describe conditions for hybridization and washing under which nucleotide
sequences at least
60% homologous to each other typically remain hybridized to each other.
Homologs (i. e., nucleic acids encoding NOVX proteins derived from species
other
than human) or other related sequences (e.g., paralogs) can be obtained by
low, moderate or
high stringency hybridization with all or a portion of the particular human
sequence as a probe
using methods well knovcni in the art for nucleic acid hybridization and
cloning.
As used herein, the phrase "stringent hybridization conditions" refers to
conditions
under which a probe, primer or oligonucleotide will hybridize to its target
sequence, but to no
other sequences. Stringent conditions are sequence-dependent and will be
different in
different circumstances. Longer sequences hybridize specifically at higher
temperatures than
shorter sequences. Generally, stringent conditions are selected to be about 5
°C lower than the
thermal melting point (Tm) for the specific sequence at a defined ionic
strength and pH. The
Tm is the temperature (under defined ionic strength, pH and nucleic acid
concentration) at
which 50% of the probes complementary to the target sequence hybridize to the
target
sequence at equilibrium. Since the target sequences are generally present at
excess, at Tm,
SO% of the probes are occupied at equilibrium. Typically, stringent conditions
will be those in
which the salt concentration is less than about 1.0 M sodium ion, typically
about 0.01 to 1.0 M
sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least
about 30°C for short
probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about
60°C for longer
probes, primers and oligonucleotides. Stringent conditions may also be
achieved with the
addition of destabilizing agents, such as formainide.
Stringent conditions are known to those skilled in the art and can be found in
Ausubel,
et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, rohrl Wiley & Sons,
N.Y.
(1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at
least about 65%,
70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain
hybridized to each other. A non-limiting example of stringent hybridization
conditions are
hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH
7.5), 1 mM
EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm
DNA
at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at
50°C. An isolated
nucleic acid molecule of the invention that hybridizes under stringent
conditions to the
sequences SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,
and 33,
corresponds to a naturally-occurring nucleic acid molecule. As used herein, a
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"naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule
having a
nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the
nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9,
11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, and 33, or fragments, analogs or derivatives
thereof, under
conditions of moderate stringency is provided. A non-limiting example of
moderate
stringency hybridization conditions are hybridization in 6X SSC, SX Denhardt's
solution,
0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by
one or more
washes in 1X SSC, 0.1% SDS at 37°C. Other conditions of moderate
stringency that may be
used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993,
CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990;
GENE
TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid
molecule
comprising the nucleotide sequences SEQ )D NOS:1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25,
27, 29, 31, and 33, or fragments, analogs or derivatives thereof, under
conditions of low
stringency, is provided. A non-limiting example of low stringency
hybridization conditions
are hybridization in 35% formamide, SX SSC, 50 mM Tris-HCl (pH 7.5), 5 mM
EDTA,
0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%
(wt/vo1) dextran sulfate at 40°C, followed by one or more washes in 2X
SSC, 25 mM Tris-HCl
(pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low
stringency that may
be used are well known in the art (e.g., as employed for cross-species
hybridizations). See,
e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
John Wiley
& Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY
MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA
78:
6789-6792.
Conservative Mutations
In addition to naturally-occurring allelic variants of NOVX sequences that may
exist in
the population, the skilled artisan will further appreciate that changes can
be introduced by
mutation into the nucleotide sequences SEQ ID NOS:1, 3, 5, 7, 9, 1 l, 13, 15,
17, 19, 21, 23,
25, 27, 29, 31, and 33, thereby leading to changes in the amino acid sequences
of the encoded
NOVX proteins, without altering the functional ability of said NOVX proteins.
For example,
nucleotide substitutions leading to amino acid substitutions at "non-
essential" amino acid
residues can be made in the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24,
26, 28, 30, 32, or 34. A "non-essential" amino acid residue is a residue that
can be altered
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from the wild-type sequences of the NOVX proteins without altering their
biological activity,
whereas an "essential" amino acid residue is required for such biological
activity. For
example, amino acid residues that are conserved among the NOVX proteins of the
invention
are predicted to be particularly non-amenable to alteration. Amino acids for
which
conservative substitutions can be made are well-known within the art.
Another aspect of the invention pertains to nucleic acid molecules encoding
NOVX
proteins that contain changes in amino acid residues that are not essential
for activity. Such
NOVX proteins differ in amino acid sequence from SEQ ID NOS:2, 4, 6, 8, 10,
12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, and 34 yet retain biological activity. In one
embodiment, the
isolated nucleic acid molecule comprises a nucleotide sequence encoding a
protein, wherein
the protein comprises an amino acid sequence at least about 45% homologous to
the amino
acid sequences SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,
30, 32, and 34.
Preferably, the protein encoded by the nucleic acid molecule is at least about
60% homologous
to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and
34; more
preferably at least about 70% homologous SEQ ID NOS:2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22,
24, 26, 28, 30, 32, or 34; still more preferably at least about 80% homologous
to SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34; even
more preferably at
least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, 28,
30, 32, or 34; and most preferably at least about 95% homologous to SEQ ID
NOS:2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
An isolated nucleic acid molecule encoding an NOVX protein homologous to the
protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,
32, or 34 can be
created by introducing one or more nucleotide substitutions, additions or
deletions into the
nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31,
and 33, such that one or more amino acid substitutions, additions or deletions
are introduced
into the encoded protein.
Mutations can be introduced into SEQ ID NOS:1, 3, S, 7, 9, 1 l, 13, 15, 17,
19, 21, 23,
25, 27, 29, 31, and 33 by standard techniques, such as site-directed
mutagenesis and
PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions
are made at
one or more predicted, non-essential amino acid residues. A "conservative
amino acid
substitution" is one in which the amino acid residue is replaced with an amino
acid residue
having a similar side chain. Families of amino acid residues having similar
side chains have
been defined within the art. These families include amino acids with basic
side chains (e.g.,
lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged
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polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Thus, a predicted
non-essential amino acid residue in the NOVX protein is replaced with another
amino acid
residue from the same side chain family. Alternatively, in another embodiment,
mutations can
be introduced randomly along all or part of an NOVX coding sequence, such as
by saturation
mutagenesis, and the resultant mutants can be screened for NOVX biological
activity to
identify mutants that retain activity. Following mutagenesis of SEQ ID NOS:1,
3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, the encoded protein can be
expressed by any
recombinant technology known in the art and the activity of the protein can be
determined.
The relatedness of amino acid families may also be determined based on side
chain
interactions. Substituted amino acids may be fully conserved "strong" residues
or fully
conserved "weak" residues. The "strong" group of conserved amino acid residues
may be any
one of the following groups: STA, NEQK, NHQK, NDEQ, QIiRK, MILV, MTLF, HY,
FYW,
wherein the single letter amino acid codes are grouped by those amino acids
that may be
substituted for each other. Likewise, the "weak" group of conserved residues
may be any one
of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK,
HFY, wherein the letters within each group represent the single letter amino
acid code.
In one embodiment, a mutant NOVX protein can be assayed for (i) the ability to
form
protein:protein interactions with other NOVX proteins, other cell-surface
proteins, or
biologically-active portions thereof, (ii) complex formation between a mutant
NOVX protein
and an NOVX ligand; or (iii) the ability of a mutant NOVX protein to bind to
an intracellular
target protein or biologically-active portion thereof; (e.g. avidin proteins).
In yet another embodiment, a mutant NOVX protein can be assayed for the
ability to
regulate a specific biological function (e.g., regulation of insulin release).
Antisense Nucleic Acids
Another aspect of the invention pertains to isolated antisense nucleic acid
molecules
that are hybridizable to or complementary to the nucleic acid molecule
comprising the
nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31,
and 33, or fragments, analogs or derivatives thereof. An "antisense"nucleic
acid comprises a
nucleotide sequence that is complementary to a ".sense" nucleic acid encoding
a protein (e.g.,
complementary to the coding strand of a double-stranded cDNA molecule or
complementary
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to an mRNA sequence). In specific aspects, antisense nucleic acid molecules
are provided that
comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or
500 nucleotides
or an entire NOVX coding strand, or to only a portion thereof. Nucleic acid
molecules
encoding fragments, homologs, derivatives and analogs of an NOVX protein of
SEQ m
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, or
antisense nucleic acids
complementary to aii NOVX nucleic acid sequence of SEQ m NOS:1, 3, 5, 7, 9,
11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, and 33, are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a
"coding
region" of the coding strand of a nucleotide sequence encoding an NOVX
protein. The term
"coding region" refers to the region of the nucleotide sequence comprising
codons which are
translated into amino acid residues. In another embodiment, the antisense
nucleic acid
molecule is antisense to a "noncoding region" of the coding strand of a
nucleotide sequence
encoding the NOVX protein. The term "noncoding region" refers to 5' and 3'
sequences which
flank the coding region that are not translated into amino acids (i.e., also
referred to as 5' and
3' untranslated regions).
Given the coding strand sequences encoding the NOVX protein disclosed herein,
antisense nucleic acids of the invention can be designed according to the
rules of Watson and
Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be
complementary
to the entire coding region of NOVX mRNA, but more preferably is an
oligonucleotide that is
antisense to only a portion of the coding or noncoding region of NOVX mRNA.
For example,
the antisense oligonucleotide can be complementary to the region surrounding
the translation
start site of NOVX mRNA. An antisense oligonucleotide can be, for example,
about 5, 10, 15,
20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid
of the invention
can be constructed using chemical synthesis or enzymatic ligation reactions
using procedures
known in the art. For example, an antisense nucleic acid (e.g., an antisense
oligonucleotide)
can be chemically synthesized using naturally-occurring nucleotides or
variously modified
nucleotides designed to increase the biological stability of the molecules or
to increase the
physical stability of the duplex formed between the antisense and sense
nucleic acids (e.g.,
phosphorothioate derivatives and acridine substituted nucleotides can be
used).
Examples of modified nucleotides that can be used to generate the antisense
nucleic
acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylinethyl) uracil, 5-
carboxymethylaminomethyl-
2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-
galactosylqueosine,
inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-
dimethylguanine,
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2-methyladeiune, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
adenine,
7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,
pseudouracil,
queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-
methyluracil,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-
thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively, the
antisense nucleic acid can be produced biologically using an expression vector
into which a
nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from the
inserted nucleic acid will be of an antisense orientation to a target nucleic
acid of interest,
described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered to a
subject or generated ih situ such that they hybridize with or bind to cellular
mRNA and/or
genomic DNA encoding an NOVX protein to thereby inhibit expression of the
protein (e.g., by
inhibiting transcription and/or translation). The hybridization can be by
conventional
nucleotide complementarity to form a stable duplex, or, for example, in the
case of an
antisense nucleic acid molecule that binds to DNA duplexes, through specific
interactions in
the major groove of the double helix. An example of a route of administration
of antisense
nucleic acid molecules of the invention includes direct injection at a tissue
site. Alternatively,
antisense nucleic acid molecules can be modified to target selected cells and
then administered
systemically. For example, for systemic administration, antisense molecules
can be modified
such that they specifically bind to receptors or antigens expressed on a
selected cell surface
(e.g., by linking the antisense nucleic acid molecules to peptides or
antibodies that bind to cell
surface receptors or antigens). The antisense nucleic acid molecules can also
be delivered to
cells using the vectors described herein. To achieve sufficient nucleic acid
molecules, vector
constructs in which the antisense nucleic acid molecule is placed under the
control of a strong
pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention is an
oc-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms
specific
double-stranded hybrids with complementary RNA in which, contrary to the usual
/3-units, the
strands run parallel to each other. See, e.g., Gaultier, et al., 1987. Nucl.
Acids Res. I5:
6625-6641. The antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (See, e.g., moue, et al. 1987. Nucl. Acids Res. 15:
6131-6148) or a
chimeric RNA-DNA analogue (See, e.g., moue, et al., 1987. FEBSLett. 215: 327-
330.
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Ribozymes and PNA Moieties
Nucleic acid modifications include, by way of non-limiting example, modified
bases,
and nucleic acids whose sugar phosphate backbones are modified or derivatized.
These
modifications are carried out at least in part to enhance the chemical
stability of the modified
nucleic acid, such that they may be used, for example, as antisense binding
nucleic acids in
therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are
capable of
cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described
in
Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically
cleave NOVX
mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme
having
specificity for an NOVX-encoding nucleic acid can be designed based upon the
nucleotide
sequence of an NOVX cDNA disclosed herein (i.e., SEQ m NOS:1, 3, 5, 7, 9, 11,
13, 15, 17,
19, 21, 23, 25, 27, 29, 31, and 33). For example, a derivative of a
Tetrahynaena L-19 IVS
RNA can be constructed in which the nucleotide sequence of the active site is
complementary
to the nucleotide sequence to be cleaved in an NOVX-encoding mRNA. See, e.g.,
U.S. Patent
4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al. NOVX mRNA
can also be
used to select a catalytic RNA having a specific ribonuclease activity from a
pool of RNA
molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, NOVX gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of the NOVX nucleic acid
(e.g., the NOVX
promoter and/or enhancers) to form triple helical structures that prevent
transcription of the
NOVX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6:
569-84; Helene,
et al. 1992. Ann. N. Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-
15.
In various embodiments, the NOVX nucleic acids can be modified at the base
moiety,
sugar moiety or phosphate backbone to improve, e.g., the stability,
hybridization, or solubility
of the molecule. For example, the deoxyribose phosphate backbone of the
nucleic acids can
be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996.
Bioorg Med
Chem 4: 5-23. As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic
acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is
replaced by
a pseudopeptide backbone and only the four natural nucleobases are retained.
The neutral
backbone of PNAs has been shown to allow for specific hybridization to DNA and
RNA under
conditions of low ionic strength. The synthesis of PNA oligomers can be
performed using
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standard solid phase peptide synthesis protocols as described in Hyrup, et
al., 1996. supra;
Perry-O'Keefe, et al., 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.
PNAs of NOVX can be used in therapeutic and diagnostic applications. For
example,
PNAs can be used as antisense or antigene agents for sequence-specific
modulation of gene
expression by, e.g., inducing transcription or translation arrest or
inhibiting replication. PNAs
of NOVX can also be used, for example, in the analysis of single base pair
mutations in a gene
(e.g., PNA directed PCR clamping; as artificial restriction enzymes when used
in combination
with other enzymes, e.g., S1 nucleases (See, Hyrup, et al., 1996.supra); or as
probes or primers
for DNA sequence and hybridization (See, Hyrup, et al., 1996, supra; Perry-
O'Keefe, et al.,
1996. supra).
In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their
stability or cellular uptake, by attaching lipophilic or other helper groups
to PNA, by the
formation of PNA-DNA chimeras, or by the use of liposomes or other techniques
of drug
delivery known in the art. For example, PNA-DNA chimeras of NOVX can be
generated that
may combine the advantageous properties of PNA and DNA. Such chimeras allow
DNA
recognition enzymes (e.g., RNase H and DNA polyrnerases) to interact with the
DNA portion
while the PNA portion would provide high binding affinity and specificity. PNA-
DNA
chimeras can be linked using linkers of appropriate lengths selected in terms
of base stacking,
number of bonds between the nucleobases, and orientation (see, Hyrup, et al.,
1996. supra).
The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et
al., 1996.
supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA
chain can
be synthesized on a solid support using standard phosphoramidite coupling
chemistry, and
modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-
thymidine
phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g.,
Mag, et al.,
1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise
manner
to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment.
See, e.g.,
Finn, et al., 1996. supra. Alternatively, chimeric molecules can be
synthesized with a 5' DNA
segment and a 3' PNA segment. See, e.g., Petersen, et al., 1975. Bioorg. Med.
Chem. Lett. 5:
1119-11124.
In other embodiments, the oligonucleotide may include other appended groups
such as
peptides (e.g., for targeting host cell receptors in vivo), or agents
facilitating transport across
the cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl. Acad. Sci.
U.S.A. 86:
6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT
Publication No.
W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO
89/10134). In
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addition, oligonucleotides can be modified with hybridization triggered
cleavage agents (see,
e.g., I~rol, et al., 1988. BioTechniques 6:958-976) or intercalating agents
(see, e.g., Zon, 1988.
Phar~n. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated
to another
molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a
transport agent, a
hybridization-triggered cleavage agent, and the like.
NOVX Polypeptides
A polypeptide according to the invention includes a polypeptide including the
amino
acid sequence of NOVX polypeptides whose sequences are provided in SEQ ID
NOS:2, 4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. The invention also
includes a mutant or
variant protein any of whose residues may be changed from the corresponding
residues shown
in SEQ 1D NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or
34 while still
encoding a protein that maintains its NOVX activities and physiological
functions, or a
functional fragment thereof.
In general, an NOVX variant that preserves NOVX-like function includes any
variant
in which residues at a particular position in the sequence have been
substituted by other amino
acids, and further include the possibility of inserting an additional residue
or residues between
two residues of the parent protein as well as the possibility of deleting one
or more residues
from the parent sequence. Any amino acid substitution, insertion, or deletion
is encompassed
by the invention. In favorable circumstances, the substitution is a
conservative substitution as
defined above.
One aspect of the invention pertains to isolated NOVX proteins, and
biologically-
active portions thereof, or derivatives, fragments, analogs or homologs
thereof. Also provided
are polypeptide fragments suitable for use as immunogens to raise anti-NOVX
antibodies. Tn
one embodiment, native NOVX proteins can be isolated from cells or tissue
sources by an
appropriate purification scheme using standard protein purification
techniques. In another
embodiment, NOVX proteins are produced by recombinant DNA techniques.
Alternative to
recombinant expression, an NOVX protein or polypeptide can be synthesized
chemically
using standard peptide synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active
portion thereof
is substantially free of cellulax material or other contaminating proteins
from the cell or tissue
source from which the NOVX protein is derived, or substantially free from
chemical
precursors or other chemicals when chemically synthesized. The language
"substantially free
of cellular material" includes preparations of NOVX proteins in which the
protein is separated
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from cellular components of the cells from which it is isolated or
recombinantly-produced. In
one embodiment, the language "substantially free of cellular material"
includes preparations of
NOVX proteins having less than about 30% (by dry weight) of non-NOVX proteins
(also
referred to herein as a "contaminating protein"), more preferably less than
about 20% of
non-NOVX proteins, still more preferably less than about 10% of non-NOVX
proteins, and
most preferably less than about 5% of non-NOVX proteins. When the NOVX protein
or
biologically-active portion thereof is recombinantly-produced, it is also
preferably
substantially free of culture medium, i.e., culture medium represents less
than about 20%,
more preferably less than about 10%, and most preferably less than about 5% of
the volume of
the NOVX protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes
preparations of NOVX proteins in which the protein is separated from chemical
precursors or
other chemicals that are involved in the synthesis of the protein. In one
embodiment, the
language "substantially free of chemical precursors or other chemicals"
includes preparations
of NOVX proteins having less than about 30% (by dry weight) of chemical
precursors or
non-NOVX chemicals, more preferably less than about 20% chemical precursors or
non-NOVX chemicals, still more preferably less than about 10% chemical
precursors or
non-NOVX chemicals, and most preferably less than about 5% chemical precursors
or
non-NOVX chemicals.
Biologically-active portions of NOVX proteins include peptides comprising
amino
acid sequences sufficiently homologous to or derived from the amino acid
sequences of the
NOVX proteins (e.g., the amino acid sequence shown in SEQ m NOS:2, 4, 6, 8,
10, 12, 14,
16, 18, 20, 22, 24, 26, 28, 30, 32, or 34) that include fewer amino acids than
the full-length
NOVX proteins, and exhibit at least one activity of an NOVX protein.
Typically, biologically-
active portions comprise a domain or motif with at least one activity of the
NOVX protein. A
biologically-active portion of an NOVX protein can be a polypeptide which is,
for example,
10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions of the
protein are deleted,
can be prepared by recombinant techniques and evaluated for one or more of the
functional
activities of a native NOVX protein.
Tn an embodiment, the NOVX protein has an amino acid sequence shown SEQ m
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. In
other embodiments, the
NOVX protein is substantially homologous to SEQ m NOS:2, 4, 6, 8, 10, 12, 14,
16, 18, 20,
22, 24, 26, 28, 30, 32, or 34, and retains the functional activity of the
protein of SEQ ~
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NOS:2, 4, 6, 8, 10, 12, I4, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, yet
differs in amino acid
sequence due to natural allelic variation or mutagenesis, as described in
detail, below.
Accordingly, in another embodiment, the NOVX protein is a protein that
comprises an
amino acid sequence at least about 45% homologous to the amino acid sequence
SEQ m
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, and
retains the functional
activity of the NOVX proteins of SEQ m NOS:2, 4, 6, 8, 10, 12, I4, 16, 18, 20,
22, 24, 26, 28,
30, 32, or 34.
Determining Homology Between Two or More Seguences
I O To determine the percent homology of two amino acid sequences or of two
nucleic
acids, the sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced
in the sequence of a first amino acid or nucleic acid sequence for optimal
aligmnent with a
second amino or nucleic acid sequence). The amino acid residues or nucleotides
at
corresponding amino acid positions or nucleotide positions are then compared.
When a
position in the first sequence is occupied by the same amino acid residue or
nucleotide as the
corresponding position in the second sequence, then the molecules are
homologous at that
position (i.e., as used herein amino acid or nucleic acid "homology" is
equivalent to amino
acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity
between two sequences. The homology may be determined using computer programs
known
in the art, such as GAP software provided in the GCG program package. See,
Needleman and
Wunsch, 1970. JMoI Bi~l 48: 443-453. Using GCG GAP software with the following
settings
for nucleic acid sequence comparison: GAP creation penalty of S.0 and GAP
extension
penalty of 0.3, the coding region of the analogous nucleic acid sequences
referred to above
exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%,
95%, 98%, or
99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NOS: I,
3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.
The term "sequence identity" refers to the degree to which two polynucleotide
or
polypeptide sequences are identical on a residue-by-residue basis over a
particular region of
comparison. The term "percentage of sequence identity" is calculated by
comparing two
optimally aligned sequences over that region of comparison, determining the
number of
positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or T,
in the case of
nucleic acids) occurs in both sequences to yield the number of matched
positions, dividing the
number of matched positions by the total number of positions in the region of
comparison (i. e.,
the window size), and multiplying the result by 100 to yield the percentage of
sequence
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identity. The term "substantial identity" as used herein denotes a
characteristic of a
polynucleotide sequence, wherein the polynucleotide comprises a sequence that
has at least 80
percent sequence identity, preferably at least 85 percent identity and often
90 to 95 percent
sequence identity, more usually at least 99 percent sequence identity as
compared to a
reference sequence over a comparison region.
Chimeric and Fusion Proteins
The invention also provides NOVX chimeric or fusion proteins. As used herein,
an
NOVX "chimeric protein" or "fusion protein" comprises an NOVX polypeptide
operatively-
linked to a non-NOVX polypeptide. An "NOVX polypeptide" refers to a
polypeptide having
an amino acid sequence corresponding to an NOVX protein SEQ m NOS:2, 4, 6, 8,
10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34), whereas a "non-NOVX
polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a protein that is
not substantially
homologous to the NOVX protein, e.g., a protein that is different from the
NOVX protein and
that is derived from the same or a different organism. Within an NOVX fusion
protein the
NOVX polypeptide can correspond to all or a portion of an NOVX protein. In one
embodiment, an NOVX fusion protein comprises at least one biologically-active
portion of an
NOVX protein. In another embodiment, an NOVX fusion protein comprises at least
two
biologically-active portions of an NOVX protein. In yet another embodiment, an
NOVX
fusion protein comprises at least three biologically-active portions of an
NOVX protein.
Within the fusion protein, the term "operatively-linked" is intended to
indicate that the NOVX
polypeptide and the non-NOVX polypeptide are fused in-frame with one another.
The
non-NOVX polypeptide can be fused to the N-terminus or C-terminus of the NOVX
polypeptide.
In one embodiment, the fusion protein is a GST-NOVX fusion protein in which
the
NOVX sequences are fused to the C-terminus of the GST (glutathione S-
transferase)
sequences. Such fusion proteins can facilitate the purification of recombinant
NOVX
polypeptides.
In another embodiment, the fusion protein is an NOVX protein containing a
heterologous signal sequence at its N-terminus. In certain host cells (e.g.,
mammalian host
cells), expression and/or secretion of NOVX can be increased through use of a
heterologous
signal sequence. ,
In yet another embodiment, the fusion protein is an NOVX-immunoglobulin fusion
protein in which the NOVX sequences are fused to sequences derived from a
member of the
immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins of the
invention
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can be incorporated into pharmaceutical compositions and administered to a
subject to inhibit
an interaction between an NOVX ligand and an NOVX protein on the surface of a
cell, to
thereby suppress NOVX-mediated signal transduction i:2 vivo. The NOVX-
immunoglobulin
fusion proteins can be used to affect the bioavailability of an NOVX cognate
ligand.
Inhibition of the NOVX ligand/NOVX interaction may be useful therapeutically
for both the
treatment of proliferative and differentiative disorders, as well as
modulating (e.g. promoting
or inhibiting) cell survival. Moreover, the NOVX-immunoglobulin fusion
proteins of the
invention can be used as imrnunogens to produce anti-NOVX antibodies in a
subject, to purify
NOVX ligands, and in screening assays to identify molecules that inhibit the
interaction of
NOVX with an NOVX ligand.
An NOVX chimeric or fusion protein of the invention can be produced by
standard
recombinant DNA techniques. For example, DNA fragments coding for the
different
polypeptide sequences are Iigated together in-frame in accordance with
conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini for
ligation, restriction
enzyme digestion to provide for appropriate termini, filling-in of cohesive
ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and enzymatic
ligation. In
another embodiment, the fusion gene can be synthesized by conventional
techniques including
automated DNA synthesizers. Alternatively, PCR amplification of gene fragments
can be
carried out using anchor primers that give rise to complementary overhangs
between two
consecutive gene fragments that can subsequently be annealed and reamplified
to generate a
chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN
MOLECULAR
BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are
commercially
available that already encode a fusion moiety (e.g., a GST polypeptide). An
NOVX-encoding
nucleic acid can be cloned into such an expression vector such that the fusion
moiety is linked
in-frame to the NOVX protein.
NOVX A~onists and Antagonists
The invention also pertains to variants of the NOVX proteins that function as
either
NOVX agonists (i.e., mimetics) or as NOVX antagonists. Variants of the NOVX
protein can
be generated by mutagenesis (e.g., discrete point mutation or truncation of
the NOVX protein).
An agonist of the NOVX protein can retain substantially the same, or a subset
of, the
biological activities of the naturally occurring form of the NOVX protein. An
antagonist of
the NOVX protein can inhibit one or more of the activities of the naturally
occurring form of
the NOVX protein by, for example, competitively binding to a downstream or
upstream
member of a cellular signaling cascade which includes the NOVX protein. Thus,
specific
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biological effects can be elicited by treatment with a variant of limited
function. In one
embodiment, treatment of a subj ect with a variant having a subset of the
biological activities
of the naturally occurring form of the protein has fewer side effects in a
subj ect relative to
treatment with the naturally occurring form of the NOVX proteins.
Variants of the NOVX proteins that function as either NOVX agonists (i.e.,
mimetics)
or as NOVX antagonists can be identified by screening combinatorial libraries
of mutants
(e.g., truncation mutants) of the NOVX proteins for NOVX protein agonist or
antagonist
activity. In one embodiment, a variegated library of NOVX variants is
generated by
combinatorial mutagenesis at the nucleic acid level and is encoded by a
variegated gene
library. A variegated library of NOVX variants can be produced by, for
example,
enzymatically ligating a mixture of synthetic oligonucleotides into gene
sequences such that a
degenerate set of potential NOVX sequences is expressible as individual
polypeptides, or
alternatively, as a set of larger fusion proteins (e.g., for phage display)
containing the set of
NOVX sequences therein. There are a variety of methods which can be used to
produce
libraries of potential NOVX variants from a degenerate oligonucleotide
sequence. Chemical
synthesis of a degenerate gene sequence can be performed in an automatic DNA
synthesizer,
and the synthetic gene then ligated into an appropriate expression vector. Use
of a degenerate
set of genes allows for the provision, in one mixture, of all of the sequences
encoding the
desired set of potential NOVX sequences. Methods for synthesizing degenerate
oligonucleotides are well-known within the art. See, e.g., Narang, 1983.
Tetrahedron 39: 3;
Itakura, et al., 1984. Ayauu. Rev. Bioehem. 53: 323; Itakura, et al., 1984.
Scieyace 198: 1056;
Ike, et al., 1983. Nucl. Acids Res. 11: 477.
Polypeutide Libraries
In addition, libraries of fragments of the NOVX protein coding sequences can
be used
to generate a variegated population of NOVX fragments for screening and
subsequent
selection of variants of an NOVX protein. In one embodiment, a library of
coding sequence
fragments can be generated by treating a double stranded PCR fragment of an
NOVX coding
sequence with a nuclease under conditions wherein nicking occurs only about
once per
molecule, denaturing the double stranded DNA, renaturing the DNA to form
double-stranded
DNA that can include sense/antisense pairs from different nicked products,
removing single
stranded portions from reformed duplexes by treatment with S 1 nuclease, and
ligating the
resulting fragment library into an expression vector. By this method,
expression libraries can
be derived which encodes N-terminal and internal fragments of various sizes of
the NOVX
3 S proteins.
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Various techniques are known in the art for screening gene products of
combinatorial
libraries made by point mutations or truncation, and for screening cDNA
libraries for gene
products having a selected property. Such techniques are adaptable for rapid
screening of the
gene libraries generated by the combinatorial mutagenesis of NOVX proteins.
The most
widely used techniques, which are amenable to high throughput analysis, for
screening large
gene libraries typically include cloning the gene library into replicable
expression vectors,
transforming appropriate cells with the resulting library of vectors, and
expressing the
combinatorial genes under conditions in which detection of a desired activity
facilitates
isolation of the vector encoding the gene whose product was detected.
Recursive ensemble
mutagenesis (REM), a new technique that enhances the frequency of functional
mutants in the
libraries, can be used in combination with the screening assays to identify
NOVX variants.
See, e.g., Arkin and Yourvan, 1992. Pnoc. Natl. Acad. Sci. USA 89: 7811-7815;
Delgrave, et
al., 1993. Protein Engineering 6:327-331.
Anti-NOVX Antibodies
Also included in the invention are antibodies to NOVX proteins, or fragments
of
NOVX proteins. The term "antibody" as used herein refers to immunoglobulin
molecules and
imrnunologically active portions of immunoglobulin (Ig) molecules, i.e.,
molecules that
contain an antigen binding site that specifically binds (immunoreacts with) an
antigen. Such
antibodies include, but are not limited to, polyclonal, monoclonal, chimeric,
single chain, Fab,
Fab~ and F~ab~>a fragments, and an Fab expression library. In general, an
antibody molecule
obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD,
which differ
from one another by the nature of the heavy chain present in the molecule.
Certain classes
have subclasses as well, such as IgGI, IgG2, and others. Furthermore, in
humans, the light
chain may be a kappa chain or a lambda chain. Reference herein to antibodies
includes a
reference to all such classes, subclasses and types of human antibody species.
An isolated NOVX-related protein of the invention may be intended to serve as
an
antigen, or a portion or fragment thereof, and additionally can be used as an
immunogen to
generate antibodies that immunospecifically bind the antigen, using standard
techniques for
polyclonal and monoclonal antibody preparation. The full-length protein can be
used or,
alternatively, the invention provides antigenic peptide fragments of the
antigen for use as
immunogens. An antigenic peptide fragment comprises at least 6 amino acid
residues of the
amino acid sequence of the full length protein and encompasses an epitope
thereof such that an
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antibody raised against the peptide forms a specific immune complex with the
full length
protein or with any fragment that contains the epitope. Preferably, the
antigenic peptide
comprises at least 10 amino acid residues, or at least 15 amino acid residues,
or at least 20
amino acid residues, or at least 30 amino acid residues. Preferred epitopes
encompassed by
the antigenic peptide are regions of the protein that are located on its
surface; commonly these
are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by
the
antigenic peptide is a region of NOVX-related protein that is located on the
surface of the
protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human
NOVX-related
protein sequence will indicate which regions of a NOVX-related protein are
particularly
hydrophilic and, therefore, are likely to encode surface residues useful for
targeting antibody
production. As a means for targeting antibody production, hydropathy plots
showing regions
of hydrophilicity and hydrophobicity may be generated by any method well known
in the art,
including, for example, the Kyte Doolittle or the Hopp Woods methods, either
with or without
Fourier transformation. See, e.g., Hopp and Woods, 1981, Py~oc. Nat. Acad.
Sci. USA 78:
3824-3828; I~yte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each of which
is incorporated
herein by reference in its entirety. Antibodies that are specific for one or
more domains within
an antigenic protein, or derivatives, fragments, analogs or homologs thereof,
are also provided
herein.
A protein of the invention, or a derivative, fragment, analog, homolog or
ortholog
thereof, may be utilized as an immunogen in the generation of antibodies that
immmospecifically bind these protein components.
Various procedures known within the art may be used for the production of
polyclonal or
monoclonal antibodies directed against a protein of the invention, or against
derivatives,
fragments, analogs homologs or orthologs thereof (see, for example,
Antibodies: A Laboratory
Manual, Harlow and Lane, 1988, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor,
NY, incorporated herein by reference). Some of these antibodies are discussed
below.
Polyclonal Antibodies
For the production of polyclonal antibodies, various suitable host animals
(e.g., rabbit,
goat, mouse or other mammal) may be immunized by one or more injections with
the native
protein, a synthetic variant thereof, or a derivative of the foregoing. An
appropriate
immunogenic preparation can contain, for example, the naturally occurring
immunogenic
protein, a chemically synthesized polypeptide representing the immunogenic
protein, or a
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recombinantly expressed immunogenic protein. Furthermore, the protein may be
conjugated
to a second protein known to be immunogenic in the mammal being immunized.
Examples of
such immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum
albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation
can further
include an adjuvant. Various adjuvants used to increase the immunological
response include,
but are not limited to, Freund's (complete and incomplete), mineral gels
(e.g., aluminum
hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols,
polyanions,
peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such
as Bacille
Calmette-Guerin and Corynebacterium parvum, or similar immmlostimulatory
agents.
Additional examples of adjuvants which can be employed include MPL-TDM
adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can
be
isolated from the mammal (e.g., from the blood) and further purified by well
known
techniques, such as affinity chromatography using protein A or protein G,
which provide
primarily the IgG fraction of immune serum. Subsequently, or alternatively,
the specific
antigen which is the target of the immunoglobulin sought, or an epitope
thereof, may be
immobilized on a column to purify the immune specific antibody by
immunoaffinity
chromatography. Purification of immunoglobulins is discussed, for example, by
D. Wilkinson
(The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14,
No. 8 (April 17,
2000), pp. 25-28).
Monoclonal Antibodies
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as
used herein, refers to a population of antibody molecules that contain only
one molecular
species of antibody molecule consisting of a unique light chain gene product
and a unique
heavy chain gene product. In particular, the complementarity determining
regions (CDRs) of
the monoclonal antibody are identical in all the molecules of the population.
MAbs thus
contain an antigen binding site capable of immunoreacting with a particular
epitope of the
antigen characterized by a unique binding affiuty for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those
described by I~ohler and Milstein, Nature, 256:495 (1975). In a hybridoma
method, a mouse,
hamster, or other appropriate host animal, is typically immunized with an
immunizing agent to
elicit lymphocytes that produce or are capable of producing antibodies that
will specifically
bind to the immunizing agent. Alternatively, the lymphocytes can be immunized
in vitro.
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The immunizing agent will typically include the protein antigen, a fragment
thereof or
a fusion protein thereof. Generally, either peripheral blood lymphocytes are
used if cells of
human origin are desired, or spleen cells or lymph node cells are used if non-
human
mammalian sources are desired. The lymphocytes are then fused with an
immortalized cell
line using a suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell
(Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press,
(1986) pp.
59-103). Immortalized cell lines are usually transformed mammalian cells,
particularly
myeloma cells of rodent, bovine and human origin. Usually, rat or mouse
myeloma cell lines
are employed. The hybridoma cells can be cultured in a suitable culture medium
that
preferably contains one or more substances that inhibit the growth or survival
of the unfused,
immortalized cells. For example, if the parental cells lack the enzyme
hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the
hybridomas
typically will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which
substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high
level expression of antibody by the selected antibody-producing cells, and are
sensitive to a
medium such as HAT medium. More preferred immortalized cell lines are murine
myeloma
lines, which can be obtained, for instance, from the Salk Institute Cell
Distribution Center, San
Diego, California and the American Type Culture Collection, Manassas,
Virginia. Human
myeloma and mouse-human heteromyeloma cell lines also have been described for
the
production of human monoclonal antibodies (Kozbor, J. Inanaunol., 133:3001
(1984); Brodeur
et al., MONOCLONAL ANTIBODY PRODUCTION TECHNIQUES AND APPLTCATIONS, MarCel
Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be
assayed for
the presence of monoclonal antibodies directed against the antigen.
Preferably, the binding
specificity of monoclonal antibodies produced by the hybridoma cells is
determined by
immunoprecipitation or by an ira vitf°o binding assay, such as
radioimmunoassay (RIA) or
enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are
known in
the art. The binding affinity of the monoclonal antibody can, for example, be
determined by
the Scatchard analysis of Munson and Pollard, Anal. BioclZefn., 107:220
(I980). Preferably,
antibodies having a high degree of specificity and a high binding affinity for
the target antigen
are isolated.
After the desired hybridoma cells are identified, the clones can be subcloned
by
limiting dilution procedures and grown by standard methods. Suitable culture
media for this
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purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640
medium.
Alternatively, the hybridoma cells can be grown in vivo as ascites in a
mammal.
The monoclonal antibodies secreted by the subclones can be isolated or
purified from the
culture medium or ascites fluid by conventional immunoglobulin purification
procedures such
as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis,
dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as
those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal
antibodies of
the invention can be readily isolated and sequenced using conventional
procedures (e.g., by
using oligonucleotide probes that are capable of binding specifically to genes
encoding the
heavy and light chains of marine antibodies). The hybridoma cells of the
invention serve as a
preferred source of such DNA. Once isolated, the DNA can be placed into
expression vectors,
which are then transfected into host cells such as simian COS cells, Chinese
hamster ovary
(CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin
protein, to
obtain the synthesis of monoclonal antibodies in the recombinant host cells.
The DNA also
can be modified, for example, by substituting the coding sequence for human
heavy and light
chain constant domains in place of the homologous marine sequences ((J.S.
Patent No.
4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to
the
immunoglobulin coding sequence all or part of the coding sequence for a non-
immunoglobulin
polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the
constant
domains of an antibody of the invention, or can be substituted for the
variable domains of one
antigen-combiung site of an antibody of the invention to create a chimeric
bivalent antibody.
Humanized Antibodies
The antibodies directed against the protein antigens of the invention can
further
comprise humanized antibodies or human antibodies. These antibodies are
suitable for
administration to humans without engendering an immune response by the human
against the
administered immunoglobulin. Humanized forms of antibodies are chimeric
immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or
other antigen-
binding subsequences of antibodies) that are principally comprised of the
sequence of a human
immunoglobulin, and contain minimal sequence derived from a non-human
immunoglobulin.
Humanization can be performed following the method of Winter and co-workers
(Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);
Verhoeyen et al.,
Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences
for the
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corresponding sequences of a human antibody. (See also U.S. Patent No.
5,225,539.) In some
instances, Fv framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies can also comprise
residues which
are found neither in the recipient antibody nor in the imported CDR or
framework sequences.
In general, the humanized antibody will comprise substantially all of at least
one, and typically
two, variable domains, in which all or substantially all of the CDR regions
correspond to those
of a non-human immunoglobulin and all or substantially all of the framework
regions are
those of a human immunoglobulin consensus sequence. The humanized antibody
optimally
also will comprise at least a portion of an immunoglobulin constant region
(Fc), typically that
of a human immunoglobulin (Jones et al., 1986; Riechrnann et al., 1988; and
Presta, Curr. Op.
Struct. Biol., 2:593-596 (1992)).
Human Antibodies
Fully human antibodies relate to antibody molecules in which essentially the
entire
sequences of both the light chain and the heavy chain, including the CDRs,
arise from human
genes. Such antibodies are termed "human antibodies", or "fully human
antibodies" herein.
Human monoclonal antibodies can be prepared by the trioma technique; the human
B-cell
hybridoma technique (see Kozbor, et al., 1983 Itnmunol Today 4: 72) and the
EBV hybridoma
technique to produce human monoclonal antibodies (see Cole, et al., 1985 In:
MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human
monoclonal
antibodies may be utilized in the practice of the present invention and may be
produced by
using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:
2026-2030) or
by transforming human B-cells with Epstein Barn Virus ih vitro (see Cole, et
al., 1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional
techniques,
including phage display libraries (Hoogenboom and Winter, J: Mol. Biol.,
227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can
be made by
introducing human immunoglobulin loci into transgenic animals, e.g., mice in
which the
endogenous immunoglobulin genes have been partially or completely inactivated.
Upon
challenge, human antibody production is observed, which closely resembles that
seen in
humans in all respects, including gene rearrangement, assembly, and antibody
repertoire. This
approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806;
5,569,825;
5,625,126; 5,633,425; 5,661,016, and in Marks et al. (BiolTechn.ology 10, 779-
783 (1992));
Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-I3
(1994));
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Fishwild et al,( Nature Biotechnology 14, 845-51 (1996)); Neuberger (Natune
Biotechnology
14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. IynmurZOl. 13 6S-93
(1995)).
Human antibodies may additionally be produced using transgenic nonhuman
animals
which are modified so as to produce fully human antibodies rather than the
animal's
endogenous antibodies in response to challenge by an antigen. (See PCT
publication
W094/02602). The endogenous genes encoding the heavy and light imrnunoglobulin
chains in
the nonhuman host have been incapacitated, and active loci encoding human
heavy and light
chain immunoglobulins are inserted into the host's genome. The human genes are
incorporated, for example, using yeast artificial chromosomes containing the
requisite human
DNA segments. An animal which provides all the desired modifications is then
obtained as
progeny by crossbreeding intermediate transgenic animals containing fewer than
the full
complement of the modifications. The preferred embodiment of such a nonhuman
animal is a
mouse, and is termed the Xenomouse~ as disclosed in PCT publications WO
96/33735 and
WO 96/34096. This animal produces B cells which secrete fully human
immunoglobulins.
The antibodies can be obtained directly from the animal after immunization
with an
immunogen of interest, as, for example, a preparation of a polyclonal
antibody, or alternatively
from immortalized B cells derived from the animal, such as hybridomas
producing
monoclonal antibodies. Additionally, the genes encoding the immunoglobulins
with human
variable regions can be recovered and expressed to obtain the antibodies
directly, or can be
further modified to obtain analogs of antibodies such as, for example, single
chain Fv
molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse,
lacking expression of an endogenous immunoglobulin heavy chain is disclosed in
U.S. Patent
No. 5,939,598. It can be obtained by a method including deleting the J segment
genes from at
least one endogenous heavy chain locus in an embryonic stem cell to prevent
rearrangement of
the locus and to prevent formation of a transcript of a rearranged
immunoglobulin heavy chairr
locus, the deletion being effected by a targeting vector containing a gene
encoding a selectable
marker; and producing from the embryonic stem cell a transgenic mouse whose
somatic and
germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is
disclosed
in U.S. Patent No. 5,916,771. It includes introducing an expression vector
that contains a
nucleotide sequence encoding a heavy chain into one mammalian host cell in
culture,
introducing an expression vector containing a nucleotide sequence encoding a
light chain into
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another mammalian host cell, and fusing the two cells to form a hybrid cell.
The hybrid cell
expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a
clinically
relevant epitope on an immunogen, and a correlative method for selecting an
antibody that
binds immunospecifically to the relevant epitope with high affinity, are
disclosed in PCT
publication WO 99/53049.
Fab Fragments and Single Chain Antibodies
According to the invention, techniques can be adapted for the production of
single-chain antibodies specific to an antigenic protein of the invention (see
e.g., U.S. Patent
No. 4,946,778). In addition, methods can be adapted for the construction of
Fab expression
libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid
and effective
identification of monoclonal Fab fragments with the desired specificity for a
protein or
derivatives, fragments, analogs or homologs thereof. Antibody fragments that
contain the
idiotypes to a protein antigen may be produced by techniques known in the art
including, but
not limited to: (i) an F~ab')2 fragment produced by pepsin digestion of an
antibody molecule; (ii)
an Fab fragment generated by reducing the disulfide bridges of an F~~b~~2
fragment; (iii) an Fab
fragment generated by the treatment of the antibody molecule with papain and a
reducing
agent and (iv) F,, fragments.
Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that
have binding specificities for at least two different antigens. In the present
case, one of the
binding specificities is for an antigenic protein of the invention. The second
binding target is
any other antigen, and advantageously is a cell-surface protein or receptor or
receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the recombinant
production of bispecific antibodies is based on the co-expression of two
immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have different
specificities
(Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random
assortment of
immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a
potential
mixture of ten different antibody molecules, of which only one has the correct
bispecific
structure. The purification of the correct molecule is usually accomplished by
affinity
chromatography steps. Similar procedures are disclosed in WO 93/08829,
published 13 May
1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.
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Antibody variable domains with the desired binding specificities (antibody-
antigen
combining sites) can be fused to immunoglobulin constant domain sequences. The
fusion
preferably is with an immunoglobulin heavy-chain constant domain, comprising
at least part
of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-
chain constant
region (CH1) containing the site necessary for light-chain binding present in
at least one of the
fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired,
the
immunoglobulin light chain, are inserted into separate expression vectors, and
are co-
transfected into a suitable host organism. For further details of generating
bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210
(1986).
According to another approach described in WO 96/27011, the interface between
a pair
of antibody molecules can be engineered to maximize the percentage of
heterodimers which
are recovered from recombinant cell culture. The preferred interface comprises
at least a part
of the CH3 region of an antibody constant domain. In this method, one or more
small amino
acid side chains from the interface of the first antibody molecule are
replaced with larger side
chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to the
large side chains) are created on the interface of the second antibody
molecule by replacing
large amino acid side chains with smaller ones (e.g. alanine or threonine).
This provides a
mechanism for increasing the yield of the heterodimer over other unwanted end-
products such
as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody
fragments
(e.g. F(ab')z bispecific antibodies). Techniques for generating bispecific
antibodies from
antibody fragments have been described in the literature. For example,
bispecific antibodies .
can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985)
describe a
procedure wherein intact antibodies are proteolytically cleaved to generate
F(ab')2 fragments.
These fragments are reduced in the presence of the dithiol complexing agent
sodium arsenite
to stabilize vicinal ditluols and prevent intermolecular disulfide formation.
The Fab'
fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
One of the
Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB
derivative
to form the bispecific antibody. The bispecific antibodies produced can be
used as agents for
the selective immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and
chemically
coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-
225 (1992)
describe the production of a fully humanized bispecific antibody F(ab')a
molecule. Each Fab'
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fragment was separately secreted from E. coli and subjected to directed
chemical coupling in
vitro to form the bispecific antibody. The bispecific antibody thus formed was
able to bind to
cells overexpressing the ErbB2 receptor and normal human T cells, as well as
trigger the lytic
activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments
directly
from recombinant cell culture have also been described. For example,
bispecific antibodies
have been produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553
(1992). The leucine zipper peptides from the Fos and Jun proteins were linked
to the Fab'
portions of two different antibodies by gene fusion. The antibody homodimers
were reduced
at the hinge region to form monomers and then re-oxidized to form the antibody
heterodimers.
This method can also be utilized for the production of antibody homodimers.
The "diabody"
technology described by Hollinger et al., Proc. Natl. Acad. Sci. US'A 90:6444-
6448 (1993) has
provided an alternative mechanism for making bispecific antibody fragments.
The fragments
comprise a heavy-chain variable domain (VH) connected to a light-chain
variable domain (VL)
by a linker which is too short to allow pairing between the two domains on the
same chain.
Accordingly, the VH and VL domains of one fragment are forced to pair with the
complementary VL and VH domains of another fragment, thereby forming two
antigen-binding
sites. Another strategy for making bispecific antibody fragments by the use of
single-chain Fv
(sFv) dimers has also been reported. See, Gruber et al., J. Imrnunol. 152:5368
(1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific
antibodies can be prepared. Tutt et al., J. ImnZUnol. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least
one of
which originates in the protein antigen of the invention. Alternatively, an
anti-antigenic arm
of an immunoglobulin molecule can be combined with an arm which binds to a
triggering
molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3,
CD28, or B7); or
Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII
(CD16) so as
to focus cellular defense mechanisms to the cell expressing the particular
antigen. Bispecific
antibodies can also be used to direct cytotoxic agents to cells which express
a particular
antigen. These antibodies possess an antigen-binding arm and an arm which
binds a cytotoxic
agent or a radionuclide chelator, such as EOTLTBE, DPTA, DOTA, or TETA.
Another
bispecific antibody of interest binds the protein antigen described herein and
further binds
tissue factor (TF).
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Heteroconjugate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such
antibodies have, for example, been proposed to target immune system cells to
unwanted cells
(U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360;
WO
92/200373; EP 03089). It is contemplated that the antibodies can be prepared
in vitro using
known methods in synthetic protein chemistry, including those involving
crosslinking agents.
For example, irmnunotoxins can be constructed using a disulfide exchange
reaction or by
forming a thioether bond. Examples of suitable reagents for this purpose
include iminothiolate
and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.
Patent No.
4,676,980.
Effector Function Engineering
It can be desirable to modify the antibody of the invention with respect to
effector
function, so as to enhance, e.g., the effectiveness of the antibody in
treating cancer. For
example, cysteine residues) can be introduced into the Fc region, thereby
allowing interchain
disulfide bond formation in this region. The homodimeric antibody thus
generated can have
improved internalization capability and/or increased complement-mediated cell
killing and
antibody dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp
Med., 176: 1191-
1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric
antibodies with
enhanced anti-tumor activity can also be prepared using heterobifunctional
cross-linkers as
described in Wolff et al. Cancer Research, 53: 2560-2565 (1993).
Alternatively, an antibody
can be engineered that has dual Fc regions and can thereby have enhanced
complement lysis
and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-
230 (1989).
Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody
conjugated
to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an
enzymatically active
toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or
a radioactive
isotope (i.e., aradioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been described above. Enzymatically active toxins and fragments thereof that
can be used
include diphtheria A chain, nonbinding active fragments of diphtheria toxin,
exotoxin A chain
(from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
alpha-sarcin,
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Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPA, and
PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis
inhibitor,
gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes. A variety of
radionuclides are available for the production of radioconjugated antibodies.
Examples
S include magi, 131h t3yl~ 90~,~ and 186Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein-coupling agents such as N-succinimidyl-3-(2-
pyridyldithiol) propionate
(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as
dimethyl
adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes
(such as
glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)
hexanediamine), bis-
diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates
(such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as
1,5-difluoro-
2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as
described in
Vitetta et al., Science, 238: 1098 (1987). Caxbon-14-labeled 1-
isothiocyanatobenzyl-3-
methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating
agent for
conjugation of radionucleotide to the antibody. See W094/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such
streptavidin) for utilization in tumor pretargeting wherein the antibody-
receptor conjugate is
adminstered to the patient, followed by removal of unbound conjugate from the
circulation
using a clearing agent and then administration of a "ligand" (e.g., avidin)
that is in turn
conjugated to a cytotoxic agent.
In one embodiment, methods for the screening of antibodies that possess the
desired
specificity include, but are not limited to, enzyme-linked immunosorbent assay
(ELISA) and
other immunologically-mediated techniques known within the art. In a specific
embodiment,
selection of antibodies that are specific to a particular domain of an NOVX
protein is
facilitated by generation of hybridomas that bind to the fragment of an NOVX
protein
possessing such a domain. Thus, antibodies that axe specific for a desired
domain within an
NOVX protein, or derivatives, fragments, analogs or homologs thereof, are also
provided
herein.
Anti-NOVX antibodies may be used in methods known within the art relating to
the
localization and/or quantitation of an NOVX protein (e.g., for use in
measuring levels of the
NOVX protein within appropriate physiological samples, for use in diagnostic
methods, for
use in imaging the protein, and the like). In a given embodiment, antibodies
for NOVX
proteins, or derivatives, fragments, analogs or homologs thereof, that contain
the antibody
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derived binding domain, are utilized as pharmacologically-active compounds
(hereinafter
"Therapeutics").
An anti-NOVX antibody (e.g., monoclonal antibody) can be used to isolate an
NOVX
polypeptide by standard techniques, such as affinity chromatography or
immunoprecipitation.
An anti-NOVX antibody can facilitate the purification of natural NOVX
polypeptide from
cells and of recombinantly-produced NOVX polypeptide expressed in host cells.
Moreover,
an anti-NOVX antibody can be used to detect NOVX protein (e.g., in a cellular
lysate or cell
supernatant) in order to evaluate the abundance and pattern of expression of
the NOVX
protein. Anti-NOVX antibodies can be used diagnostically to monitor protein
levels in tissue
as part of a clinical testing procedure, e.g., to, for example, determine the
efficacy of a given
treatment regimen. Detectiomcan be facilitated by coupling (i.e., physically
linking) the
antibody to a detectable substance. Examples of detectable substances include
various
enzynes, prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent
materials, and radioactive materials. Examples of suitable enzymes include
horseradish
peroxidase, alkaline phosphatase, (3-galactosidase, or acetylcholinesterase;
examples of
suitable prosthetic group complexes include streptavidin/fiotin and
avidin/biotin; examples of
suitable fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example
of a luminescent material includes luminol; examples of bioluminescent
materials include
luciferase, luciferin, and aequorin, and examples of suitable radioactive
material include lash
i3ih ass or 3H.
NOVX Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors,
containing a nucleic acid encoding an NOVX protein, or derivatives, fragments,
analogs or
homologs thereof. As used herein, the term "vector" refers to a nucleic acid
molecule capable
of transporting another nucleic acid to which it has been linked. One type of
vector is a
"plasmid", which refers to a circular double stranded DNA loop into which
additional DNA
segments can be ligated. Another type of vector is a viral vector, wherein
additional DNA
segments can be ligated into the viral genome. Certain vectors are capable of
autonomous
replication in a host cell into which they are introduced (e.g., bacterial
vectors having a
bacterial origin of replication and episomal mammalian vectors). Other vectors
(e.g.,
non-episomal mammalian vectors) axe integrated into the genome of a host cell
upon
introduction into the host cell, and thereby are replicated along with the
host genome.
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Moreover, certain vectors are capable of directing the expression of genes to
which they are
operatively-linked. Such vectors are referred to herein as "expression
vectors". In general,
expression vectors of utility in recombinant DNA techniques are often in the
form of plasmids.
In the present specification, "plasmid" and "vector" can be used
interchangeably as the
plasmid is the most commonly used form of vector. However, the invention is
intended to
include such other forms of expression vectors, such as viral vectors (e.g.,
replication defective
retroviruses, adenoviruses and adeno-associated viruses), which serve
equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of
the
invention in a form suitable for expression of the nucleic acid in a host
cell, which means that
the recombinant expression vectors include one or more regulatory sequences,
selected on the
basis of the host cells to be used for expression, that is operatively-linked
to the nucleic acid
sequence to be expressed. Within a recombinant expression vector, "operably-
linked" is
intended to mean that the nucleotide sequence of interest is linked to the
regulatory
sequences) in a manner that allows for expression of the nucleotide sequence
(e.g., in an in
vitro transcription/translation system or in a host cell when the vector is
introduced into the
host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers
and other
expression control elements (e.g., polyadenylation signals). Such regulatory
sequences are
described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences
include
those that direct constitutive expression of a nucleotide sequence in many
types of host cell
and those that direct expression of the nucleotide sequence only in certain
host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by those skilled
in the art that the
design of the expression vector can depend on such factors as the choice of
the host cell to be
transformed, the level of expression of protein desired, etc. The expression
vectors of the
invention can be introduced into host cells to thereby produce proteins or
peptides, including
fusion proteins or peptides, encoded by nucleic acids as described herein
(e.g., NOVX
proteins, mutant forms of NOVX proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for
expression of
NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX proteins
can be
expressed in bacterial cells such as Eschey~ichia coli, insect cells (using
baculovirus expression
vectors) yeast cells or mammalian cells. Suitable host cells are discussed
further in Goeddel,
GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San
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Diego, Calif. (1990). Alternatively, the recombinant expression vector can be
transcribed and
translated iya vitro, for example using T7 promoter regulatory sequences and
T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in
Eschericlaia coli with vectors
containing constitutive or inducible promoters directing the expression of
either fusion or
non-fusion proteins. Fusion vectors add a number of amino acids to a protein
encoded therein,
usually to the amino terminus of the recombinant protein. Such fusion vectors
typically serve
three purposes: (i) to increase expression of recombinant protein; (ii) to
increase the solubility
of the recombinant protein; and (iii) to aid in the purification of the
recombinant protein by
acting as a ligand in affinity purification. Often, in fusion expression
vectors, a proteolytic
cleavage site is introduced at the junction of the fusion moiety and the
recombinant protein to
enable separation of the recombinant protein from the fusion moiety subsequent
to purification
of the fusion protein. Such enzymes, and their cognate recognition sequences,
include Factor
Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia
Biotech Inc; Smith and Johnson, 1988. Geue 67: 31-40), pMAL (New England
Biolabs,
Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) that fuse glutathione
S-transferase
(GST), maltose E binding protein, or protein A, respectively, to the target
recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc (Amrann et
al., (1988) Geyze 69:301-315) and pET 11 d (Studier et al., GENE EXPRESSION
TECHNOLOGY:
METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
One strategy to maximize recombinant protein expression in E. coli is to
express the
protein in a host bacteria with an impaired capacity to proteolytically cleave
the recombinant
protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY
185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to
alter the
nucleic acid sequence of the nucleic acid to be inserted into an expression
vector so that the
individual codons for each amino acid are those preferentially utilized in E.
coli (see, e.g.,
Wada, et al., 1992. Nucl. Acids Res. 20: 2111-211$). Such alteration of
nucleic acid
sequences of the invention can be carried out by standard DNA synthesis
techniques.
In another embodiment, the NOVX expression vector is a yeast expression
vector.
Examples of vectors for expression in yeast Sacclaaro~cyces cerivisae include
pYepSecl
(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz,
1982. Cell 30:
933-943), pJRY8f (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen
Corporation,
San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
Alternatively, NOVX can be expressed in insect cells using baculovirus
expression
vectors. Baculovirus vectors available for expression of proteins in cultured
insect cells (e.g.,
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SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:
2156-2165) and the
pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian
cells using a mammalian expression vector. Examples of mammalian expression
vectors
include pCDMB (Seed, 1987. Nature 329: 840) and pMT2PC (I~aufinan, et al.,
1987. EMBO
J. 6: 187-195). When used in mammalian cells, the expression vector's control
functions are
often provided by viral regulatory elements. For example, commonly used
promoters are
derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For
other suitable
expression systems for both prokaryotic and eukaryotic cells see, e.g.,
Chapters 16 and 17 of
Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable
of directing
expression of the nucleic acid preferentially in a particular cell type (e.g.,
tissue-specific
regulatory elements are used to express the nucleic acid). Tissue-specific
regulatory elements
are known in the art. Non-limiting examples of suitable tissue-specific
promoters include the
albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-
277),
lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Imyrl.unol. 43: 235-
275), in
particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J.
8: 729-733)
and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and
Baltimore, 1983.
Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament
promoter; Byrne and
Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific
promoters
(Edlund, et al., 1985. Scieyace 230: 912-916), and mammary gland-specific
promoters (e.g.,
milk whey promoter; U.S. Pat. No. 4,873,316 and European Application
Publication No.
264,166). Developmentally-regulated promoters are also encompassed, e.g., the
murine hox
promoters (I~essel and Gruss, 1990. Science 249: 374-379) and the ~-
fetoprotein promoter
(Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a
DNA
molecule of the invention cloned into the expression vector in an antisense
orientation. That
is, the DNA molecule is operatively-linked to a regulatory sequence in a
manner that allows
for expression (by transcription of the DNA molecule) of an RNA molecule that
is antisense to
NOVX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in
the
antisense orientation can be chosen that direct the continuous expression of
the antisense RNA
molecule in a variety of cell types, for instance viral promoters and/or
enhancers, or regulatory
sequences can be chosen that direct constitutive, tissue specific or cell type
specific expression
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of antisense RNA. The antisense expression vector can be in the form of a
recombinant
plasmid, phagemid or attenuated virus in which antisense nucleic acids are
produced under the
control of a high efficiency regulatory region, the activity of which can be
determined by the
cell type into which the vector is introduced. For a discussion of the
regulation of gene
expression using antisense genes see, e.g., Weintraub, et al., "Antisense RNA
as a molecular
tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a
recombinant
expression vector of the invention has been introduced. The terms "host cell"
and
"recombinant host cell" are used interchangeably herein. It is understood that
such terms refer
not only to the particular subject cell but also to the progeny or potential
progeny of such a
cell. Because certain modifications may occur in succeeding generations due to
either.
mutation or environmental influences, such progeny may not, in fact, be
identical to the parent
cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, NOVX
protein can
be expressed in bacterial cells such as E. coli, insect cells, yeast or
mammalian cells (such as
Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are
known to
those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional
transformation or transfection techniques. As used herein, the teens
"transformation" and
"transfection" are intended to refer to a variety of art-recognized techniques
for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate
or calcium
chloride co-precipitation, DEAF-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting host cells
can be found in
Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1989),
and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may integrate
the foreign DNA into their genome. In order to identify and select these
integrants, a gene that
encodes a selectable marker (e.g., resistance to antibiotics) is generally
introduced into the
host cells along with the gene of interest. Various selectable markers include
those that confer
resistance to drugs, such as G4I8, hygromycin and methotrexate. Nucleic acid
encoding a
selectable marker can be introduced into a host cell on the same vector as
that encoding
NOVX or can be introduced on a separate vector. Cells stably transfected with
the introduced
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nucleic acid can be identified by drug selection (e.g., cells that have
incorporated the
selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture, can
be used to produce (i. e., express) NOVX protein. Accordingly, the invention
further provides
methods for producing NOVX protein using the host cells of the invention. In
one
embodiment, the method comprises culturing the host cell of invention (into
which a
recombinant expression vector encoding NOVX protein has been introduced) in a
suitable
medium such that NOVX protein is produced. In another embodiment, the method
further
comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals
The host cells of the invention can also be used to produce non-human
transgenic
animals. For example, in one embodiment, a host cell of the invention is a
fertilized oocyte or
an embryonic stem cell into which NOVX protein-coding sequences have been
introduced.
Such host cells can then be used to create non-human transgenic animals in
which exogenous
NOVX sequences have been introduced into their genome or homologous
recombinant
animals in which endogenous NOVX sequences have been altered. Such animals are
useful
for studying the function and/or activity of NOVX protein and for identifying
and/or
evaluating modulators of NOVX protein activity. As used herein, a "transgenic
animal" is a
non-human animal, preferably a mammal, more preferably a rodent such as a rat
or mouse, in
which one or more of the cells of the animal includes a transgene. Other
examples of
transgenic animals include non-human primates, sheep, dogs, cows, goats,
chickens,
amphibians, etc. A transgene is exogenous DNA that is integrated into the
genome of a cell
from which a transgenic animal develops and that remains in the genome of the
mature
animal, thereby directing the expression of an encoded gene product in one or
more cell types
or tissues of the transgenic animal. As used herein, a "homologous recombinant
animal" is a
non-human animal, preferably a mammal, more preferably a mouse, in which an
endogenous
NOVX gene has been altered by homologous recombination between the endogenous
gene
and an exogenous DNA molecule introduced into a cell of the animal, e.g., an
embryonic cell
of the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing NOVX-
encoding
nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinj
ection, retroviral
infection) and allowing the oocyte to develop in a pseudopregnant female
foster animal. The
human NOVX cDNA sequences SEQ m NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27,
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29, 31, and 33 can be introduced as a transgene into the genome of a non-human
animal.
Alternatively, a non-human homologue of the human NOVX gene, such as a mouse
NOVX
gene, can be isolated based on hybridization to the human NOVX cDNA (described
further
supra) and used as a transgene. Intronic sequences and polyadenylation signals
can also be
included in the transgene to increase the efficiency of expression of the
transgene. A
tissue-specific regulatory sequences) can be operably-linked to the NOVX
transgene to direct
expression of NOVX protein to particular cells. Methods for generating
transgenic animals
via embryo manipulation and microinjection, particularly animals such as mice,
have become
conventional in the art and are described, for example, in U.S. Patent Nos.
4,736,866;
4,870,009; and 4,873,191; and Hogan, 1986. In: MANIPULATING TIC MousE EMBRYO,
Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are
used for
production of other transgenic animals. A transgenic founder animal can be
identified based
upon the presence of the NOVX transgene in its genome and/or expression of
NOVX mRNA
in tissues or cells of the animals. A transgenic founder animal can then be
used to breed
additional animals carrying the transgene. Moreover, transgenic animals
carrying a transgene-
encoding NOVX protein can further be bred to other transgenic animals carrying
other
transgenes.
To create a homologous recombinant anmal, a vector is prepared which contains
at
least a portion of an NOVX gene into which a deletion, addition or
substitution has been
introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The
NOVX gene can
be a human gene (e.g., the cDNA of SEQ ID NOS:1, 3, 5, 7, 9, 11; 13, 15, 17,
19, 21, 23, 25,
27, 29, 31, and 33), but more preferably, is a non-human homologue of a human
NOVX gene.
For example, a mouse homologue of human NOVX gene of SEQ m NOS:1, 3, 5, 7, 9,
1 l, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 can be used to construct a
homologous recombination
vector suitable for altering an endogenous NOVX gene in the mouse genome. In
one
embodiment, the vector is designed such that, upon homologous recombination,
the
endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a
functional protein;
also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous
recombination,
the endogenous NOVX gene is mutated or otherwise altered but still encodes
functional
protein (e.g., the upstream regulatory region can be altered to thereby alter
the expression of
the endogenous NOVX protein). In the homologous recombination vector, the
altered portion
of the NOVX gene is flanked at its 5'- and 3'-termini by additional nucleic
acid of the NOVX
gene to allow for homologous recombination to occur between the exogenous NOVX
gene
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earned by the vector and an endogenous NOVX gene in an embryonic stem cell.
The
additional flanking NOVX nucleic acid is of sufficient length for successful
homologous
recombination with the endogenous gene. Typically, several kilobases of
flanking DNA (both
at the 5'- and 3'-termini) are included in the vector. See, e.g., Thomas, et
al., 1987. Cell 51:
503 for a description of homologous recombination vectors. The vector is ten
introduced into
an embryonic stem cell line (e.g., by electroporation) and cells in which the
introduced NOVX
gene has homologously-recombined with the endogenous NOVX gene are selected.
See, e.g.,
Li, et al., 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a
mouse) to
form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND
EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp.
113-152.
A chimeric embryo can then be implanted into a suitable pseudopregnant female
foster animal
and the embryo brought to term. Progeny harboring the homologously-recombined
DNA in
their germ cells can be used to breed animals in which all cells of the animal
contain the
homologously-recombined DNA by germline transmission of the transgene. Methods
for
constructing homologous recombination vectors and homologous recombinant
animals are
described fixrther in Bradley, 1991. Curs. Opiya. Biotechhol. 2: 823-829; PCT
International
Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that
contain
selected systems that allow for regulated expression of the transgene. One
example of such a
system is the. cre/loxP recombinase system of bacteriophage P 1. For a
description of the
cre/loxP recombinase system, See, e.g., Lakso, et al., 1992. Pf~oc. Natl.
Aead. Sci. USA 89:
6232-6236. Another example of a recombinase system is the FLP recombinase
system of
Saccharomyces cerevisiae. See, O'Gorman, et al., 1991. Science 251:1351-1355.
If a cre/loxP
recombinase system is used to. regulate expression of the transgene, animals
containing
transgenes encoding both the Cre recombinase and a selected protein are
required. Such
animals can be provided through the construction of "double" transgenic
animals, e.g., by
mating two transgenic animals, one containing a transgene encoding a selected
protein and the
other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be
produced
according to the methods described in Wihnut, et al., 1997. Nature 385: 810-
813. In brief, a
cell (e.g., a somatic cell) from the transgenic animal can be isolated and
induced to exit the
growth cycle and enter Go phase. The quiescent cell can then be fused, e.g.,
through the use of
electrical pulses, to an enucleated oocyte from an animal of the same species
from which the
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quiescent cell is isolated. The reconstructed oocyte is then cultured such
that it develops to
morula or blastocyte and then transferred to pseudopregnant female foster
animal. The
offspring borne of this female foster animal will be a clone of the animal
from which the cell
(e.g., the somatic cell) is isolated.
Pharmaceutical Compositions
The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies (also
referred to herein as "active compounds") of the invention, and derivatives,
fragments, analogs
and homologs thereof, can be incorporated into pharmaceutical compositions
suitable for
administration. Such compositions typically comprise the nucleic acid
molecule, protein, or
antibody and a pharmaceutically acceptable carrier. As used herein,
"pharmaceutically
acceptable Garner" is intended to include any and all solvents, dispersion
media, coatings,
antibacterial and antifungal agents, isotonic and absorption delaying agents,
and the like,
compatible with pharmaceutical administration. Suitable Garners are described
in the most
recent edition of Remington's Pharmaceutical Sciences, a standard reference
text in the field,
which is incorporated herein by reference. Preferred examples of such carriers
or diluents
include, but are not limited to, water, saline, finger's solutions, dextrose
solution, and 5%
human serum albumin. Liposornes and non-aqueous vehicles such as fixed oils
may also be
used. The use of such media and agents for pharmaceutically active substances
is well known
in the art. Except insofar as any conventional media or agent is incompatible
with the active
compound, use thereof in the compositions is contemplated. Supplementary
active
compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible
with its
intended route of administration. Examples of routes of administration include
parenteral,
e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical),
transmucosal, and rectal administration. Solutions or suspensions used for
parenteral,
intradermal, or subcutaneous application can include the following components:
a sterile
diluent such as water for injection, saline solution, fixed oils, polyethylene
glycols, glycerine,
propylene glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;
chelating agents such
as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates
or phosphates,
and agents for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be
adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral
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preparation can be enclosed in ampoules, disposable syringes or multiple dose
vials made of
glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous
preparation of sterile injectable solutions or dispersion. For intravenous
administration,
suitable carriers include physiological saline, bacteriostatic water,
Cremophor ELTM (BASF,
Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the
composition must be
sterile and should be fluid to the extent that easy syringeability exists. It
must be stable under
the conditions of manufacture and storage and must be preserved against the
contaminating
action of microorganisms such as bacteria and fungi. The carrier can be a
solvent or
dispersion medium containing, for example, water, ethanol, polyol (for
example, glycerol,
propylene glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a coating
such as lecithin, by
the maintenance of the required particle size in the case of dispersion and by
the use of
surfactants. Prevention of the action of microorganisms can be achieved by
various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol, ascorbic
acid, thimerosal, and the like. In many cases, it will be preferable to
include isotonic agents,
for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride
in the
composition. Prolonged absorption of the injectable compositions can be
brought about by
including in the composition an agent which delays absorption, for example,
aluminum
monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound (e.g.,
an NOVX protein or anti-NOVX antibody) in the required amount in an
appropriate solvent
with one or a combination of ingredients enumerated above, as required,
followed by filtered
sterilization. Generally, dispersions are prepared by incorporating the active
compound into a
sterile vehicle that contains a basic dispersion medium and the required other
ingredients from
those enumerated above. In the case of sterile powders for the preparation of
sterile injectable
solutions, methods of preparation are vacuum drying and freeze-drying that
yields a powder of
the active ingredient plus any additional desired ingredient from a previously
sterile-filtered
solution thereof.
Oral compositions generally include an inert diluent or an edible carrier.
They can be
enclosed in gelatin capsules or compressed into tablets. For the purpose of
oral therapeutic
administration, the active compound can be incorporated with excipients and
used in the form
of tablets, troches, or capsules. Oral compositions can also be prepared using
a fluid carrier
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for use as a mouthwash, wherein the compound in the fluid Garner is applied
orally and
swished and expectorated or swallowed. Pharmaceutically compatible binding
agents, and/or
adjuvant materials can be included as part of the composition. The tablets,
pills, capsules,
troches and the Iike can contain any of the following ingredients, or
compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an excipient
such as starch or lactose, a disintegrating agent such as alginic acid,
Primogel, or corn starch; a
lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal
silicon dioxide; a
sweetening agent such as sucrose or saccharin; or a flavoring agent such as
peppermint,
methyl salicylate, or orange flavoring.
m
For administration by inhalation, the compounds are delivered in the form of
an
aerosol spray from pressured container or dispenser which contains a suitable
propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate to the
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art, and
include, for example, for transmucosal administration, detergents, bile salts,
and fusidic acid
derivatives. Transmucosal administration can be accomplished through the use
of nasal sprays
or suppositories. For transdermal admiiustration, the active compounds are
formulated into
ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with
conventional suppository bases such as cocoa butter and other glycerides) or
retention enemas
for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will
protect
the compound against rapid elimination from the body, such as a controlled
release
formulation, including implants and microencapsulated delivery systems.
Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods fox
preparation of
such formulations will be apparent to those skilled in the art. The materials
can also be
obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomal
suspensions (including Iiposomes targeted to infected cells with monoclonal
antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers. These can
be prepared
according to methods known to those skilled in the art, for example, as
described in U.S.
Patent No. 4,522,811.
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It is especially advantageous to formulate oral or parenteral compositions in
dosage
unit form for ease of administration and uniformity of dosage. Dosage unit
form as used
herein refers to physically discrete units suited as unitary dosages for the
subject to be treated;
each unit containing a predetermined quantity of active compound calculated to
produce the
desired therapeutic effect in association with the required pharmaceutical
carrier. The
specification for the dosage unit forms of the invention are dictated by and
directly dependent
on the unique characteristics of the active compound and the particular
therapeutic effect to be
achieved, and the limitations inherent in the art of compounding such am
active compound for
the treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and
used as
gene therapy vectors. Gene therapy vectors can be delivered to a subject by,
for example,
intravenous injection, local administration (see, e.g., U.S. Patent No.
5,32,470) or by
stereotactic injection (see, e.g., Chen, et al., 1994. P~oc. Natl. Acad. Sci.
USA 91: 3054-3057).
The pharmaceutical preparation of the gene therapy vector can include the gene
therapy vector
in an acceptable diluent, or can comprise a slow release matrix in which the
gene delivery
vehicle is imbedded. Alternatively, where the complete gene delivery vector
can be produced
intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can
include one or more cells that produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or
dispenser
together with instructions for administration.
Screening and Detection Methods
The isolated nucleic acid molecules of the invention can be used to express
NOVX
protein (e.g., via a recombinant expression vector in a host cell in gene
therapy applications),
to detect NOVX mRNA (e.g., in a biological sample) or a genetic lesion in an
NOVX gene,
and to modulate NOVX activity, as described further, below. In addition, the
NOVX proteins
can be used to screen drugs or compounds that modulate the NOVX protein
activity or
expression as well as to treat disorders characterized by insufficient or
excessive production of
NOVX protein or production of NOVX protein forms that have decreased or
aberrant activity
compared to NOVX wild-type protein (e.g.; diabetes (regulates insulin
release); obesity (binds
and transport lipids); metabolic disturbances associated with obesity, the
metabolic syndrome
X as well as anorexia and wasting disorders associated with chronic diseases
and various
cancers, and infectious disease(possesses anti-microbial activity) and the
various
dyslipidemias. In addition, the anti-NOVX antibodies of the invention can be
used to detect
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and isolate NOVX proteins and modulate NOVX activity..In yet a further aspect,
the invention
can be used in methods to influence appetite, absorption of nutrients and the
disposition of
metabolic substrates in both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening
assays
described herein and uses thereof for treatments as described, supna.
Screening Assays
The invention provides a method (also referred to herein as a "screening
assay") for
identifying modulators, i. e., candidate or test compounds or agents (e.g.,
peptides,
peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or
have a
stimulatory or iuubitory effect on, e.g., NOVX protein expression or NOVX
protein activity.
The invention also includes compounds identified in the screening assays
described herein.
In one embodiment, the invention provides assays for screening candidate or
test compounds
which bind to or modulate the activity of the membrane-bound form of an NOVX
protein or
polypeptide or biologically-active portion thereof. The test compounds of the
invention can be
obtained using any of the numerous approaches in combinatorial library methods
known in the
art, including: biological libraries; spatially addressable parallel solid
phase or solution phase
libraries; synthetic library methods requiring deconvolution; the "one-bead
one-compound"
library method; and synthetic library methods using affinity chromatography
selection. The
biological library approach is limited to peptide libraries, while the other
four approaches are
applicable to peptide, non-peptide oligomer or small molecule libraries of
compounds. S'ee,
e.g., Lam, 1997. AnticanceY Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has
a
molecular weight of less than about 5 kD and most preferably less than about 4
kD. Small
molecules can be, e.g., nucleic acids, peptides, polypeptides,
peptidomimetics, carbohydrates,
lipids or other organic or inorganic molecules. Libraries of chemical and/or
biological
mixtures, such as fungal, bacterial, or algal extracts, are known in the art
and can be screened
with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in
the art,
for example in: DeWitt, et al., 1993. P~oc. Natl. Acad. Sci. U.S.A. 90: 6909;
Erb, et al., 1994.
Proc. Natl. Acad. Sci. TLS'.A. 91: 11422; Zuckermaim, et al., 1994. J. Med.
Chezn. 37: 2678;
Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.
Izzt. Ed. Engl. 33:
2059; Carell, et al., 1994. Angew. Chezn. Int. Ed. Engl. 33: 2061; and Gallop,
et al., 1994. J.
Med. Clzem. 37: 1233.
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Libraries of compounds may be presented in solution (e.g., Houghten, 1992.
Bioteclzhiques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on
chips (Fodor,
1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409),
spores (Ladner,
U.S. Patent 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci.
USA 89:
1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin,
1990. Science
249: 404-406; Cwirla, et al., 1990. P~oc. Natl. Acad. Sci. USA. 87: 6378-6382;
Felici, 1991.
J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which
expresses a
membrane-bound form of NOVX protein, or a biologically-active portion thereof,
on the cell
surface is contacted with a test compound and the ability of the test compound
to bind to an
NOVX protein determined. The cell, for example, can of mammalian origin or a
yeast cell.
Determining the ability of the test compound to bind to the NOVX protein can
be
accomplished, for example, by coupling the test compound with a radioisotope
or enzymatic
label such that binding of the test compound to the NOVX protein or
biologically-active
portion thereof can be determined by detecting the labeled compound in a
complex. For
example, test compounds can be labeled with lash 3sS, 14C, or 3H, either
directly or indirectly,
and the radioisotope detected by direct counting of radioemission or by
scintillation counting.
Alternatively, test compounds can be enzymatically-labeled with, for example,
horseradish
peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label
detected by
determination of conversion of an appropriate substrate to product. In one
embodiment, the
assay comprises contacting a cell which expresses a membrane-bound form of
NOVX protein,
or a biologically-active portion thereof, on the cell surface with a known
compound which
binds NOVX to form an assay mixture, contacting the assay mixture with a test
compound,
and determining the ability of the test compound to interact with an NOVX
protein, wherein
determining the ability of the test compound to interact with an NOVX protein
comprises
determining the ability of the test compound to preferentially bind to NOVX
protein or a
biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell
expressing a membrane-bound form of NOVX protein, or a biologically-active
portion thereof,
on the cell surface with a test compound and determining the ability of the
test compound to
modulate (e.g., stimulate or inhibit) the activity of the NOVX protein or
biologically-active
portion thereof. Determining the ability of the test compound to modulate the
activity of
NOVX or a biologically-active portion thereof can be accomplished, for
example, by
determining the ability of the NOVX protein to bind to or interact with an
NOVX target
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molecule. As used herein, a "target molecule" is a molecule with which an NOVX
protein
binds or interacts in nature, for example, a molecule on the surface of a cell
which expresses
an NOVX interacting protein, a molecule on the surface of a second cell, a
molecule in the
extracellular milieu, a molecule associated with the internal surface of a
cell membrane or a
cytoplasmic molecule. An NOVX target molecule can be a non-NOVX molecule or an
NOVX protein or polypeptide of the invention. In one embodiment, an NOVX
target
molecule is a component of a signal transduction pathway that facilitates
transduction of an
extracellular signal (e.g. a signal generated by binding of a compound to a
membrane-bound
NOVX molecule) through the cell membrane and into the cell. The target, for
example, can be
a second intercellular protein that 'has catalytic activity or a protein that
facilitates the
association of downstream signaling molecules with NOVX.
Determining the ability of the NOVX protein to bind to or interact with an
NOVX
target molecule can be accomplished by one of the methods described above for
determining
direct binding. In one embodiment, determining the ability of the NOVX protein
to bind to or
interact with an NOVX target molecule can be accomplished by determining the
activity of the
target molecule. For example, the activity of the target molecule can be
determined by
detecting induction of a cellular second messenger of the target (i. e.
intracellular Ca2+,
diacylglycerol, IP3, etc.), detecting catalyticJenzymatic activity of the
target an appropriate
substrate, detecting the induction of a reporter gene (comprising an NOVX-
responsive
regulatory element operatively linked to a nucleic acid encoding a detectable
marker, e.g.,
luciferase), or detecting a cellular response, for example, cell survival,
cellular differentiation,
or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay
comprising
contacting an NOVX protein or biologically-active portion thereof with a test
compound and
determining the ability of the test compound to bind to the NOVX protein or
biologically-
active portion thereof. Binding of the test compound to the NOVX protein can
be determW ed
either directly or indirectly as described above. In one such embodiment, the
assay comprises
contacting the NOVX protein or biologically-active portion thereof with a
known compound
which binds NOVX to form an assay mixture, contacting the assay mixture with a
test
compound, and determining the ability of the test compound to interact with an
NOVX
protein, wherein determining the ability of the test compound to interact with
an NOVX
protein comprises determining the ability of the test compound to
preferentially bind to NOVX
or biologically-active portion thereof as compared to the known compound.
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In still another embodiment, an assay is a cell-free assay comprising
contacting NOVX
protein or biologically-active portion thereof with a test compound and
determining the ability
of the test compound to modulate (e.g. stimulate or inhibit) the activity of
the NOVX protein
or biologically-active portion thereof. Determining the ability of the test
compound to
modulate the activity of NOVX can be accomplished, for example, by determining
the ability
of the NOVX protein to bind to an NOVX target molecule by one of the methods
described
above for determining direct binding. In an alternative embodiment,
determining the ability of
the test compound to modulate the activity of NOVX protein can be accomplished
by
determining the ability of the NOVX protein further modulate an NOVX target
molecule. For
example, the catalytic/enzymatic activity of the target molecule on an
appropriate substrate
can be determined as described, supYa.
In yet another embodiment, the cell-free assay comprises contacting the NOVX
protein
or biologically-active portion thereof with a known compound which binds NOVX
protein to
form an assay mixture, contacting the assay mixture with a test compound, and
determining
the ability of the test compound to interact with an NOVX protein, wherein
determining the
ability of the test compound to interact with an NOVX protein comprises
determining the
ability of the NOVX protein to preferentially bind to or modulate the activity
of an NOVX
target molecule.
The cell-free assays of the invention are amenable to use of both the soluble
form or
the membrane-bound form of NOVX protein. In the case of cell-free assays
comprising the
membrane-bound form of NOVX protein, it may be desirable to utilize a
solubilizing agent
such that the membrane-bound form of NOVX protein is maintained in solution.
Examples of
such solubilizing agents include non-ionic detergents such as n-
octylglucoside,
n-dodecylglucoside, n-dodecylinaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton~ X-100, Triton° X-114, Thesit~,
Isotridecypoly(ethylene glycol ether)", N-dodecyl--N,N-dimethyl-3-ammonio-1-
propane
sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS),
or
3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it
may be desirable
to immobilize either NOVX protein ox its target molecule to facilitate
separation of complexed
from uncomplexed forms of one or both of the proteins, as well as to
accommodate
automation of the assay. Binding of a test compound to NOVX protein, or
interaction of
NOVX protein with a target molecule in the presence and absence of a candidate
compound,
can be accomplished in any vessel suitable for containing the reactants.
Examples of such
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vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In
one embodiment, a
fusion protein can be provided that adds a domain that allows one or both of
the proteins to be
bound to a matrix. For example, GST-NOVX fusion proteins or GST-target fusion
proteins
can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,
MO) or
glutathione derivatized microtiter plates, that are then combined with
the.test compound or the
test compound and either the non-adsorbed target protein or NOVX protein, and
the mixture is
incubated under conditions conducive to complex formation (e.g., at
physiological conditions
for salt and pH). Following incubation, the beads or microtiter plate wells
are washed to
remove any unbound components, the matrix immobilized in the case of beads,
complex
determined either directly or indirectly, for example, as described, supra.
Alternatively, the
complexes can be dissociated from the matrix, and the level of NOVX protein
binding or
activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the
screening assays of the invention. For example, either the NOVX protein or its
target
molecule can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated
NOVX protein or target molecules can be prepared from biotin-NHS
(N-hydroxy succinimide) using techniques well-known within the art (e.g.,
biotinylation kit,
Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well
plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein
or target
molecules, but which do not interfere with binding of the NOVX protein to its
taxget molecule,
can be derivatized to the wells of the plate, and unbound target or NOVX
protein trapped in
the wells by antibody conjugation. Methods for detecting such complexes, in
addition to those
described above for the GST-immobilized complexes, include immunodetection of
complexes
using antibodies reactive with the NOVX protein or target molecule, as well as
enzyme-linked
assays that rely on detecting an enzymatic activity associated with the NOVX
protein or target
molecule.
In another embodiment, modulators of NOVX protein expression are identified in
a
method wherein a cell is contacted with a candidate compound and the
expression of NOVX
mRNA or protein in the cell is determined. The level of expression of NOVX
mRNA or
protein in the presence of the candidate compound is compared to the level of
expression of
N~VX mRNA or protein in the absence of the candidate compound. The candidate
compound can then be identified as a modulator of NOVX mRNA or protein
expression based
upon this comparison. For example, when expression of NOVX mRNA or protein is
greater
(i.e., statistically significantly greater) in the presence of the candidate
compound than in its
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absence, the candidate compound is identified as a stimulator of NOVX mRNA or
protein
expression. Alternatively, when expression of NOVX mltNA or protein is less
(statistically
significantly less) in the presence of the candidate compound than in its
absence, the candidate.
compound is identified as an inhibitor of NOVX mIZNA or protein expression.
The level of
NOVX mIZNA or protein expression in the cells can be determined by methods
described
herein for detecting NOVX mRNA or protein.
In yet another aspect of the invention, the NOVX proteins can be used as "bait
proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent
No. 5,283,317;
Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. G'hem.
268: 12046-12054;
Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993.
Oncogene 8:
1693-1696; and Brent WO 94110300), to identify other proteins that bind to or
interact with
NOVX ("NOVX-binding proteins" or "NOVX-by") and modulate NOVX activity. Such
NOVX-binding proteins are also likely to be involved in the propagation of
signals by the
NOVX proteins as, for example, upstream or downstream elements of the NOVX
pathway.
The two-hybrid system is based on the modular nature of most transcription
factors, which
consist of separable DNA-binding and activation domains. Briefly, the assay
utilizes two
different DNA constructs. In one construct, the gene that codes for NOVX is
fused to a gene
encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
In the other
construct, a DNA sequence, from a library of DNA sequences, that encodes an
unidentified
protein ("prey" or "sample") is fused to a gene that codes for the activation
domain of the
known transcription factor. If the "bait" and the "prey" proteins are able to
interact, in vivo,
forming an NOVX-dependent complex, the DNA-binding and activation domains of
the
transcription.factor are brought into close proximity. This proximity allows
transcription of a
reporter gene (e.g., LacZ) that is operably linked to a transcriptional
regulatory site responsive
to the transcription factor. Expression of the reporter gene can be detected
and cell colonies
containing the functional transcription factor can be isolated and used to
obtain the cloned
gene that encodes the protein which interacts with NOVX.
The invention fiu they pertains to novel agents identified by the
aforementioned
screening assays and uses thereof for treatments as described herein.
Detection Assays
Portions or fragments of the cDNA sequences identified herein (and the
corresponding
complete gene sequences) can be used in numerous ways as polynucleotide
reagents. By way
of example, and not of limitation, these sequences can be used to: (i) map
their respective
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genes on a chromosome; and, thus, locate gene regions associated with genetic
disease; (ii)
identify an individual from a minute biological sample (tissue typing); and
(iii) aid in forensic
identification of a biological sample. Some of these applications are
described in the
subsections, below.
Chromosome Mapping
Once the sequence (or a portion of the sequence) of a gene has been isolated,
this
sequence can be used to map the location of the gene on a chromosome. This
process is called
chromosome mapping. Accordingly, portions or fragments of the NOVX sequences,
SEQ ID
NOS:1, 3, S, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, or
fragments or derivatives
thereof, can be used to map the location of the NOVX genes, respectively, on a
chromosome.
The mapping of the NOVX sequences to chromosomes is an important first step in
correlating
these sequences with genes associated with disease.
Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers
1 S (preferably 15-25 by in length) from the NOVX sequences. Computer analysis
of the NOVX,
sequences can be used to rapidly select primers that do not span more than one
exon in the
genomic DNA, thus complicating the amplification process. These primers can
then be used
for PCR screening of somatic cell hybrids containing individual human
chromosomes. Only
those hybrids containing the human gene corresponding to the NOVX sequences
will yield an
amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different
mammals
(e.g., human and mouse cells). As hybrids of human and mouse cells grow and
divide, they
gradually lose human chromosomes in random order, but retain the mouse
chromosomes. By
using media in which mouse cells cannot grow, because they lack a particular
enzyme, but in
which human cells can, the one human chromosome that contains the gene
encoding the
needed enzyme will be retained. By using various media, panels of hybrid cell
lines can be
established. Each cell line in a panel contains either a single human
chromosome or a small
number of human chromosomes, and a full set of mouse chromosomes, allowing
easy
mapping of individual genes to specific human chromosomes. See, e.g.,
D'Eustachio, et al.,
1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of
human
chromosomes can also be produced by using human chromosomes with
translocations and
deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a
particular
sequence to a particular chromosome. Three or more sequences can be assigned
per day using
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a single thermal cycler. Using the NOVX sequences to design oligonucleotide
primers, sub-
localization can be achieved with panels of fragments from specific
chromosomes.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase
chromosomal spread can further be used to provide a precise chromosomal
location in one
step. Chromosome spreads can be made using cells whose division has been
blocked in
metaphase by a chemical like colcemid that disrupts the mitotic spindle. The
chromosomes
can be treated briefly with trypsin, and then stained with Giemsa. A pattern
of light and dark
bands develops on each chromosome, so that the chromosomes can be identified
individually.
The FISH technique can be used with a DNA sequence as short as 500 or 600
bases.
However, clones larger than 1,000 bases have a higher likelihood of binding to
a unique
chromosomal location with sufficient signal intensity for simple detection.
Preferably 1,000
bases, and more preferably 2,000 bases, will suffice to get good results at a
reasonable amount
of time. For a review of this technique, see, Verma, et al., HUMAN
CHROMOSOMES: A
MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).
1 S Reagents for chromosome mapping can be used individually to mark a single
chromosome or a single site on that chromosome, or panels of reagents can be
used for
marking multiple sites and/or multiple chromosomes. Reagents corresponding to
noncoding
regions of the genes actually are preferred for mapping purposes. Coding
sequences are more
likely to be conserved within gene families, thus increasing the chance of
cross hybridizations
during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the
physical
position of the sequence on the chromosome can be correlated with genetic map
data. Such
data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-
line
through Johns Hopkins University Welch Medical Library). The relationship
between genes
and disease, mapped to the same chromosomal region, can then be identified
through linkage
analysis (co-inheritance of physically adjacent genes), described in, e.g.,
Egeland, et al., 1987.
Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and
unaffected with a disease associated with the NOVX gene, can be determined. If
a mutation is
observed in some or all of the affected individuals but not in any unaffected
individuals, then
the mutation is likely to be the causative agent of the particular disease.
Comparison of
affected and unaffected individuals generally involves first looking for
structural alterations in
the chromosomes, such as deletions or translocations that are visible from
chromosome
spreads or detectable using PCR based on that DNA sequence. Ultimately,
complete
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sequencing of genes from several individuals can be performed to confirm the
presence of a
mutation and to distinguish mutations from polymorphisms.
Tissue Typing
The NOVX sequences of the invention can also be used to identify individuals
from
minute biological samples. In this technique, an individual's genomic DNA is
digested with
one or more restriction enzymes, and probed on a Southern blot to yield unique
bands for
identification. The sequences of the invention are useful as additional DNA
markers for RFLP
("restriction fragment length polymorphisms," described in U.S. Patent No.
5,272,057).
Furthermore, the sequences of the invention can be used to provide an
alternative technique
that determines the actual base-by-base DNA sequence of selected portions of
an individual's
genome. Thus, the NOVX sequences described herein can be used to prepare two
PCR
primers from the 5'- and 3'-termini of the sequences. These primers can then
be used to
amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this
manner,
can provide unique individual identifications, as each individual will have a
unique set of such
DNA sequences due to allelic differences. The sequences of the invention can
be used to
obtain such identification sequences from individuals and from tissue. The
NOVX sequences
of the invention uniquely represent portions of the human genome. Allelic
variation occurs to
some degree in the coding regions of these sequences, and to a greater degree
in the noncoding
regions. It is estimated that allelic variation between individual humans
occurs with a
frequency of about once per each 500 bases. Much of the allelic variation is
due to single
nucleotide polymorphisms (SNPs), which include restriction fragment length
polymorphisms
(RFLPs).
Each of the sequences described herein can, to some degree, be used as a
standard
against which DNA from an individual can be compared for identification
purposes. Because
greater numbers of polymorphisms occur in the noncoding regions, fewer
sequences are
necessary to differentiate individuals. The noncoding sequences can
comfortably provide
positive individual identification with a panel of perhaps 10 to 1,000 primers
that each yield a
noncoding amplified sequence of 100 bases. If predicted coding sequences, such
as those in
SEQ ID NOS:1, 3, S, 7, 9, 1 l, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33
are used, a more
appropriate number of primers for positive individual identification would be
S00-2,000.
Predictive Medicine
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The invention also pertains to the field of predictive medicine in which
diagnostic
assays, prognostic assays, pharmacogenomics, and monitoring clinical trials
are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. Accordingly,
one aspect of the invention relates to diagnostic assays for determining NOVX
protein andlor
nucleic acid expression as well as NOVX activity, in the context of a
biological sample (e.g.,
blood, serum, cells, tissue) to thereby determine whether an individual is
afflicted with a
disease or disorder, or is at risk of developing a disorder, associated with
aberrant NOVX
expression or activity. The disorders include metabolic disorders, diabetes,
obesity, infectious
disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative
disorders,
Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic
disorders,
and the various dyslipidemias, metabolic disturbances associated with obesity,
the metabolic
syndrome X and wasting disorders associated with chronic diseases and various
cancers. The
invention also provides for prognostic (or predictive) assays for determining
whether an
individual is at risk of developing a disorder associated with NOVX protein,
nucleic acid
expression or activity. For example, mutations in an NOVX gene can be assayed
in a
biological sample. Such assays can be used for prognostic or predictive
purpose to thereby
prophylactically treat an individual prior to the onset of a disorder
characterized by or
associated with NOVX protein, nucleic acid expression, or biological activity.
Another aspect of the invention provides methods for determining NOVX protein,
nucleic acid expression or activity in an individual to thereby select
appropriate therapeutic or
prophylactic agents for that individual (referred to herein as
"pharmacogenomics").
Pharmacogenomics allows for the selection of agents (e.g., drugs) for
therapeutic or
prophylactic treatment of an individual based on the genotype of the
individual (e.g., the
genotype of the individual examined to determine the ability of the individual
to respond to a
particular agent.)
Yet another aspect of the invention pertains to monitoring the influence of
agents (e.g.,
drugs, compounds) on the expression or activity of NOVX in clinical trials.
These and other agents are described in further detail in the following
sections.
Diagnostic Assavs
An exemplary method for detecting the presence or absence of NOVX in a
biological
sample involves obtaining a biological sample from a test subject and
contacting the biological
sample with a compound or an agent capable of detecting NOVX protein or
nucleic acid (e.g.,
mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is
detected in the biological sample. An agent for detecting NOVX mRNA or genomic
DNA is a
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labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA.
The
nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such
as the nucleic
acid of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,
and 33, or a
portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250
or 500 nucleotides
in length and sufficient to specifically hybridize under stringent conditions
to NOVX mRNA
or genomic DNA. Other suitable probes for use in the diagnostic assays of the
invention are
described herein.
An agent for detecting NOVX protein is an antibody capable of binding to NOVX
protein, preferably an antibody with a detectable label. Antibodies can be
polyclonal, or more
preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab
or F(ab')a) can be
used. The term "labeled", with regard to the probe or antibody, is intended to
encompass
direct labeling of the probe or antibody by coupling (i. e., physically
linking) a detectable
substance to the probe or antibody, as well as indirect labeling of the probe
or antibody by
reactivity with another reagent that is directly labeled. Examples of indirect
labeling include
detection of a primary antibody using a fluorescently-labeled secondary
antibody and
end-labeling of a DNA probe with biotin such that it can be detected with
fluorescently-
labeled streptavidin. The term "biological sample" is intended to include
tissues, cells and
biological fluids isolated from a subject, as well as tissues, cells and
fluids present within a
subject. That is, the detection method of the invention can be used to detect
NOVX mRNA,
protein, or genomic DNA in a biological sample in vitro as well as in vivo.
For example, in
vitro techniques for detection of NOVX mRNA include Northern hybridizations
and in situ
hybridizations. In vitro techniques for detection of NOVX protein include
enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and
immunofluorescence. In vitro techniques for detection of NOVX genomic DNA
include
Southern hybridizations. Furthermore, in vivo techniques for detection of NOVX
protein
include introducing into a subject a labeled anti-NOVX antibody. For example,
the antibody
can be labeled with a radioactive marker whose presence and location in a
subject can be
detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the
test
subject. Alternatively, the biological sample can contain mRNA molecules from
the test
subj ect or genomic DNA molecules from the test subj ect. A preferred
biological sample is a
peripheral blood leukocyte sample isolated by conventional means from a
subject.
Tn another embodiment, the methods further involve obtaining a control
biological
sample from a control subject, contacting the control sample with a compound
or agent
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capable of detecting NOVX protein, mItNA, or genomic DNA, such that the
presence of
NOVX protein, mRNA or genomic DNA is detected in the biological sample, and
comparing
the presence of NOVX protein, mRNA or genomic DNA in the control sample with
the
presence of NOVX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of NOVX in a
biological sample. For example, the kit can comprise: a labeled compound or
agent capable of
detecting NOVX protein or mRNA in a biological sample; means for determining
the amount
of NOVX in the sample; and means for comparing the amount of NOVX in the
sample with a
standard., The compound or agent can be packaged in a suitable container. The
kit can further
comprise instructions for using the kit to detect NOVX protein or nucleic
acid.
Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to
identify
subjects having or at risk of developing a disease or disorder associated with
aberrant NOVX
expression or activity. For example, the assays described herein, such as the
preceding
diagnostic assays or the following assays, can be utilized to identify a
subject having or at risk
of developing a disorder associated with NOVX protein, nucleic acid expression
or activity.
Alternatively, the prognostic assays can be utilized to identify a subject
having or at risk for
developing a disease or disorder. Thus, the invention provides a method for
identifying a
disease or disorder associated with aberrant NOVX expression or activity in
which a test
sample is obtained from a subject and NOVX protein or nucleic acid (e.g.,
mRNA, genomic
DNA) is detected, wherein the presence of NOVX protein or nucleic acid is
diagnostic for a
subject having or at risk of developing a disease or disorder associated with
aberrant NOVX
expression or activity. As used herein, a "test sample" refers to a biological
sample obtained
from a subject of interest. For example, a test sample can be a biological
fluid (e.g., serum),
cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine
whether
a subject can be administered an agent (e.g., an agonist, antagonist,
peptidomimetic, protein,
peptide, nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder
associated with aberrant NOVX expression or activity. For example, such
methods can be
used to determine whether a subject can be effectively treated with an agent
for a disorder.
Thus, the invention provides methods for determining whether a subject can be
effectively
treated with an agent for a disorder associated with aberrant NOVX expression
or activity in
which a test sample is obtained and NOVX protein or nucleic acid is detected
(e.g., wherein
the presence of NOVX protein or nucleic acid is diagnostic for a subj ect that
can be
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administered the agent to treat a disorder associated with aberrant NOVX
expression or
activity).
The methods of the invention can also be used to detect genetic lesions in an
NOVX
gene, thereby determining if a subject with the lesioned gene is at risk for a
disorder
characterized by aberrant cell proliferation and/or differentiation. In
various embodiments, the
methods include detecting, in a sample of cells from the subject, the presence
or absence of a
genetic lesion characterized by at least one of an alteration affecting the
integrity of a gene
encoding an NOVX-protein, or the misexpression of the NOVX gene. For example,
such
genetic lesions can be detected by ascertaining the existence of at least one
of (i) a deletion of
one or more nucleotides from an NOVX gene; (ii) an addition of one or more
nucleotides to an
NOVX gene; (iii) a substitution of one or more nucleotides of an NOVX gene,
(iv) a
chromosomal rearrangement of an NOVX gene; (v) an alteration in the level of a
messenger
RNA transcript of an NOVX gene, (vi) aberrant modification of an NOVX gene,
such as of the
methylation pattern of the genomic DNA, (vii) the presence of a non-wild-type
splicing pattern
of a messenger RNA transcript of an NOVX gene, (viii) a non-wild-type level of
an NOVX
protein, (ix) allelic loss of an NOVX gene, and (x) inappropriate post-
translational
modification of an NOVX protein. As described herein, there are a large number
of assay
techniques known in the art which can be used for detecting lesions in an NOVX
gene. A
preferred biological sample is a peripheral blood leukocyte sample isolated by
conventional
means from a subject. However, any biological sample containing nucleated
cells may be
used, including, for example, buccal mucosal cells.
In certain embodiments, detection of the lesion involves the use of a
probe/primer in a
polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and
4,683,202), such
as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g.,
Landegran, et al., 1988. Science 241: 1077-1080; and Nakazawa, et al., 1994.
Proc. Natl.
Acad. Sci. USA 91: 360-364), the latter of which can be particularly useful
for detecting point
mutations in the NOVX-gene (see, Abravaya, et al., 1995. Nucl. Acids Res. 23:
675-682).
This method can include the steps of collecting a sample of cells from a
patient, isolating
nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample,
contacting the
nucleic acid sample with one or more primers that specifically hybridize to an
NOVX gene
under conditions such that hybridization and amplification of the NOVX gene
(if present)
occurs, and detecting the presence or absence of an amplification product, or
detecting the size
of the amplification product and comparing the length to a control sample. It
is anticipated
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that PCR and/or LCR maybe desirable to use as a preliminary amplification step
in
conjunction with any of the techniques used for detecting mutations described
herein.
Alternative amplification methods include: self sustained sequence replication
(see, Guatelli,
et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional
amplification system
(see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Q(3
Replicase (see,
Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid
amplification method,
followed by the detection of the amplified molecules using techniques well
known to those of
skill in the art. These detection schemes are especially useful for the
detection of nucleic acid
molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in an NOVX gene from a sample cell can
be
identified by alterations in restriction enzyme cleavage patterns. For
example, sample and
control DNA is isolated, amplified (optionally), digested with one or more
restriction
endonucleases, and fragment length sizes are determined by gel electrophoresis
and compared.
Differences in fragment length sizes between sample and control DNA indicates
mutations in
the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g.,
U.S. Patent
No. 5,493,531) can be used to score for the presence of specific mutations by
development or
Ioss of a ribozyme cleavage site.
In other embodiments, genetic mutations in NOVX can be identified by
hybridizing a
sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays
containing
hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al.,
1996. Human
Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example,
genetic
mutations in NOVX can be identified in two dimensional arrays containing light-
generated
DNA probes as described in Cronin, et al., supra. Briefly, a first
hybridization array of probes
can be used to scan through long stretches of DNA in a sample and control to
identify base
changes between the sequences by making linear arrays of sequential
overlapping probes.
This step allows the identification of point mutations. This is followed by a
second
hybridization array that allows the characterization of specific mutations by
using smaller,
specialized probe arrays complementary to all variants or mutations detected.
Each mutation
array is composed of parallel probe sets, one complementary to the wild-type
gene and the
other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in
the art
can be used to directly sequence the NOVX gene and detect mutations by
comparing the
sequence of the sample NOVX with the corresponding wild-type (control)
sequence.
Examples of sequencing reactions include those based on techniques developed
by Maxim and
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Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl.
Acad. Sci. USA
74: 5463. It is also contemplated that any of a variety of automated
sequencing procedures
can be utilized when performing the diagnostic assays (see, e.g., Naeve, et
al., 1995.
Biotechniques 19: 448), including sequencing by mass spectrometry (see, e.g.,
PCT
International Publication No. WO 94/16101; Cohen, et al., 1996. Adv.
Chromatography 36:
127-162; and Griffin, et al., 1993. Appl. Biochem. Biotechnol. 38: 147-159).
Other methods for detecting mutations in the NOVX gene include methods in
which
protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or
RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In
general, the
art technique of "mismatch cleavage" starts by providing heteroduplexes of
formed by
hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with
potentially
mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes
are
treated with an agent that cleaves single-stranded regions of the duplex such
as which will
exist due to basepair mismatches between the control and sample strands. For
instance,
1 S RNA/DNA duplexes can.be treated with RNase and DNA/DNA hybrids treated
with Si
nuclease to enzyrnatically digesting the mismatched regions. In other
embodiments, either
DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium
tetroXide
and with piperidine in order to digest mismatched regions. After digestion of
the mismatched
regions, the resulting material is then separated by size on denaturing
polyacrylamide gels to
determine the site of mutation. See, e.g:, Cotton, et al., 1988. Proc. Natl.
Acad. Sci. USA 85:
4397; Saleeba, et al., 1992. Methods Enzymol. 217: 286-295. In an embodiment,
the control
DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or
more
proteins that recognize mismatched base pairs in double-stranded DNA (so
called "DNA
mismatch repair" enzymes) in defined systems for detecting and mapping point
mutations in
NOVX cDNAs obtained from samples of cells. For example, the mutt enzyme of E.
coli
cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells
cleaves T
at G/T mismatches. See, e.g., Hsu, et al., 1994. Carcinogenesis 15: 1657-1662.
According to
an exemplary embodiment, a probe based on an NOVX sequence, e.g., a wild-type
NOVX
sequence, is hybridized to a cDNA or other DNA product from a test cell(s).
The duplex is
treated with a DNA mismatch repair enzyme, and the cleavage products, if any,
can be
detected from electrophoresis protocols or the like. See, e.g., U.S. Patent
No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to
identify mutations
in NOVX genes. For example, single strand conformation polymorphism (SSCP) may
be used
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to detect differences in electrophoretic mobility between mutant and wild type
nucleic acids.
See, e.g., Orita, et al., 1989. Proc. Natl. Acad. Sci. USA: 86: 2766;
Cotton,1993. Mutat. Res.
285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79. Single-
stranded DNA
fragments of sample and control NOVX nucleic acids will be denatured and
allowed to
renature. The secondary structure of single-stranded nucleic acids varies
according to
sequence, the resulting alteration in electrophoretic mobility enables the
detection of even a
single base change. The DNA fragments may be labeled or detected with labeled
probes. The
sensitivity of the assay may be enhanced by using RNA (rather than DNA), in
which the
secondary structure is more sensitive to a change in sequence. In one
embodiment, the subject
method utilizes heteroduplex analysis to separate double stranded heteroduplex
molecules on
the basis of changes in electrophoretic mobility. See, e.g:, Keen, et al.,
1991. Trends Genet. 7:
S.
In yet another embodiment, the movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed using
denaturing gradient
gel electrophoresis (DGGE). See, e.g., Myers, et al., 1985. Nature 313: 495.
When DGGE is
used as the method of analysis, DNA will be modified to insure that it does
not completely
denature, for example by adding a GC clamp of approximately 40 by of high-
melting GC-rich
DNA by PCR. In a further embodiment, a temperature gradient is used in place
of a
denaturing gradient to identify differences in the mobility of control and
sample DNA. See,
e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.
Examples of other techniques for detecting point mutations include, but are
not limited
to, selective oligonucleotide hybridization, selective amplification, or
selective primer
extension. For example, oligonucleotide primers may be prepared in which the
laiown
mutation is placed centrally and then hybridized to target DNA under
conditions that permit
hybridization only if a perfect match is found. See, e.g., Saiki, et al.,
1986. Nature 324: 163;
Saiki, et a1.,1989. Proc. Natl. Acad. Sci. USA 86: 6230. Such allele specific
oligonucleotides
are hybridized to PCR amplified target DNA or a number of different mutations
when the
oligonucleotides are attached to the hybridizing membrane and hybridized with
labeled target
DNA.
Alternatively, allele specific amplification technology that depends on
selective PCR
amplification may be used in conjunction with the instant invention.
Oligonucleotides used as
primers for specific amplification may carry the mutation of interest in the
center of the
molecule (so that amplification depends on differential hybridization; see,
e.g., Gibbs, et al.,
1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one
primer where,
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under appropriate conditions, mismatch can prevent, or reduce polymerise
extension (see, e.g.,
Prossner, 1993. Tibtech. 11: 238). In addition it may be desirable to
introduce a novel
restriction site in the region of the mutation to create cleavage-based
detection. See, e.g.,
Gasparini, et al., 1992. Mol. Cell Probes 6: 1. It is anticipated that in
certain embodiments
amplification may also be performed using Taq ligase for amplification. See,
e.g., Barany,
1991. Proc. Natl. Acid. Sci. USA 88: 189. In such cases, ligation will occur
only if there is a
perfect match.at the 3'-terminus of the 5' sequence, making it possible to
detect the presence of
a known mutation at a specific site by looking for the presence or absence of
amplification.
The methods described herein may be performed, for example, by utilizing pre-
packaged
diagnostic kits comprising at least one probe nucleic acid or antibody reagent
described herein,
which may be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting
symptoms or family history of a disease or illness involving an NOVX gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes,
in which
NOVX is expressed may be utilized in the prognostic assays described herein.
However, any
biological sample containing nucleated cells may be used, including, for
example, buccal
mucosal cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on NOVX
activity
(e.g., NOVX gene expression), as identified by a screening assay described
herein can be
administered to individuals to treat (prophylactically or therapeutically)
disorders (The
disorders include metabolic disorders, diabetes, obesity, infectious disease,
anorexia, cancer-
associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease,
Parkinson's
Disorder, immune disorders, and hematopoietic disorders, and the various
dyslipidemias,
metabolic disturbances associated with obesity, the metabolic syndrome X and
wasting
disorders associated with chronic diseases and various cancers.) In
conjunction with such
treatment, the pharmacogenomics (i.e., the study of the relationship between
an individual's
genotype and that individual's response to a foreign compound or drug) of the
individual may
be considered. Differences in metabolism of therapeutics can lead to severe
toxicity or
therapeutic failure by altering the relation between dose and blood
concentration of the
pharmacologically active drug. Thus, the pharmacogenomics of the individual
permits the
selection of effective agents (e.g., drugs) for prophylactic or therapeutic
treatments based on a
consideration of the individual's genotype. Such pharmacogenomics can further
be used to
determine appropriate dosages and therapeutic regimens. Accordingly, the
activity of NOVX
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protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in
an
individual can be determined to thereby select appropriate agents) for
therapeutic or
prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary variations in
the
response to drugs due to altered drug disposition and abnormal action in
affected persons. See
e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985; Linden,
1997. Clin.
Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be
differentiated. Genetic conditions transmitted as a single factor altering the
way drugs act on
the body (altered drug action) or genetic conditions transmitted as single
factors altering the
way the body acts on drugs (altered drug metabolism). These pharmacogenetic
conditions can
occur either as rare defects or as polymorphisms. For example, glucose-6-
phosphate
dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the
main
clinical complication is hemolysis after ingestion of oxidant drugs (anti-
malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a
major
determinant of both the intensity and duration of drug action. The discovery
of genetic
polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2
(NAT~2) and
cytochrome P450 enzymes CI~P2D6 and CYP2C19) has provided an explanation as to
why
some patients do not obtain the expected drug effects or show exaggerated drug
response and
serious toxicity after taking the standard and safe dose of a drug. These
polymorphisms are
expressed in two phenotypes in the population, the extensive metabolizes (EM)
and poor
metabolizes (PM). The prevalence of PM is different among different
populations. For
example, the gene coding for CYP2D6 is highly polymorphic and several
mutations have been
identified in PM, which all lead to the absence of functional CYP2D6. Poor
metabolizers of
CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and
side
effects when they receive standard doses. If a metabolite is the active
therapeutic moiety, PM
show no therapeutic response, as demonstrated for the analgesic effect of
codeine mediated by
its CYP2D6-formed metabolite morphine. At the other extreme are the so called
ultra-rapid
metabolizers who do not respond to standard doses. Recently, the molecular
basis of
ultra-rapid metabolism has been identified to be due to CI'I'2D6 gene
amplification.
Thus, the activity of NOVX protein, expression of NOVX nucleic acid, or
mutation
content of NOVX genes in an individual can be determined to thereby select
appropriate
agents) for therapeutic or prophylactic treatment of the individual. In
addition,
pharmacogenetic studies can be used to apply genotyping of polymorphic alleles
encoding
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drug-metabolizing enzymes to the identification of an individual's drug
responsiveness
phenotype. This knowledge, when applied to dosing or drug selection, can avoid
adverse
reactions or therapeutic failure and thus enhance therapeutic or prophylactic
efficiency when
treating a subject with an NOVX modulator, such as a modulator identified by
one of the
exemplary screening assays described herein.
Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drugs, compounds) on the expression
or
activity of NOVX (e.g., the ability to modulate aberrant cell proliferation
and/or
differentiation) can be applied not only in basic drug screening, but also in
clinical trials. For
example, the effectiveness of an agent determined by a screening assay as
described herein to
increase NOVX gene expression, protein levels, or upregulate NOVX activity,
can be
monitored in clinical trails of subjects exhibiting decreased NOVX gene
expression, protein
levels, or downregulated NOVX activity. Alternatively, the effectiveness of an
agent
determined by a screening assay to decrease NOVX gene expression, protein
levels, or
downregulate NOVX activity, can be monitored in clinical trails of subjects
exhibiting
increased NOVX gene expression, protein levels, or upregulated NOVX activity.
In such
clinical trials, the expression or activity of NOVX and, preferably, other
genes that have been
implicated in, for example, a cellular proliferation or immune disorder can be
used as a "read
out" or markers of the immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including NOVX, that are
modulated
in cells by treatment with an agent (e.g., compound, drug or small molecule)
that modulates
NOVX activity (e.g., identified in a screening assay as described herein) can
be identified.
Thus, to study the effect of agents on cellular proliferation disorders, for
example, in a clinical
trial, cells can be isolated and RNA prepared and analyzed for the levels of
expression of
NOVX and other genes implicated in the disorder. The levels of gene expression
(i. e., a gene
expression pattern) can be quantified by Northern blot analysis or RT-PCR, as
described
herein, or alternatively by measuring the amount of protein produced, by one
of the methods
as described herein, or by measuring the levels of activity of NOVX or other
genes. In this
manner, the gene expression pattern can serve as a marker, indicative of the
physiological
response of the cells to the agent. Accordingly, this response state may be
determined before,
and at various points during, treatment of the individual with the agent.
In one embodiment, the invention provides a method for monitoring the
effectiveness
of treatment of a subject with an agent (e.g., an agonist, antagonist,
protein, peptide,
peptidomimetic, nucleic acid, small molecule, or other drug candidate
identified by the
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