Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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NOVEL POLYPEPTIDES HOMOLOGOUS TO THYMOSIN, EPHRIN A
RECEPTORS, AND FIBROMODULIN, AND POLYNUCLEOTIDES
ENCODING 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 membrane
bound and secreted
polypeptides that are homologous to thymosin, fibromodulin and ephrin-type A
receptors, as well
as vectors, host cells, antibodies, and recombinant methods for producing
these nucleic acids and
polypeptides.
Beta-thymosins are a family of related peptides that were first isolated from
calf thymus,
but are known to be present in a wide variety of mammalian and other
vertebrate cells and tissues.
Thyrnosin-beta-4 (TMSB4) was the first member of the family to be
characterized, and was
proposed to be a thymic hormone acting at early stages of T-cell maturation.
However, the high
concentration of the protein and presence of its mRNA in a number of other
tissues and cells, in
addition to the lack of an identifiable secretory signal sequence, suggested a
more generalized
function in many cell types. This was confirmed by findings that TMSB4 forms a
1:1 complex
with G-actin in blood platelets (A. Weber et al., "Interaction of thymosin
beta 4 with muscle and
platelet actin: implications for actin sequestration in resting platelets," 31
(27) Biochemistry 6179-
85 (1992)).
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Thymosin-beta-10 is related closely to TMSB4 in sequence and is also an actin-
sequestering protein. mRNA species of similar molecular weights encoding TMSB
10 are found
in most tissues of rats, although Lin and Morrison-Bogorad (1991) identified
TMSB10 mRNA of
higher molecular weight in the testes of sexually mature rats. The latter
differs from the more
ubiquitous form only in its 5'-untranslated region, begimiing 14 nucleotides
upstream of the
translation initiation codon. This finding, together with primer extension
experiments, suggested
that the two mRNA types are transcribed from the same gene through a
combination of
differential promoter utilization and alternative splicing. Both mRNAs are
found in pachytene
spemnatocytes; only testes-specific mRNA is detected in postmeiotic haploid
spermatids.
Immunohistochemical analysis shows that the protein was present in
differentiating spermatids,
which suggests that testes-specific TMSB 10 mRNA is translated in haploid male
germ cells.
Tmmunoblot analysis using specific antibodies indicates that TMSB 10
synthesized in adult testes
is identical in size to that made in the brain (S.C. Lin et al., "Cloning and
characterization of a
testis-specific thymosin beta 10 cDNA. Expression in post-meiotic male germ
cells," 266(34) J.
Biol. Chem. 23347-53 (1991)).
Ephrin receptors comprise the largest known family of receptor protein
tyrosine lcinases.
They have been implicated in mediating developmental events, particularly in
the nervous system.
Receptors in the ephrin subfamily typically have a single kinase domain and an
extracellular
region containing a Cys-rich domain and two fibronectin type III repeats.
Along with their
ligands, called ephrins, they play important roles in neural development,
angiogenesis, and
vascular network assembly (9(4) Mol. Cells, 440-5 (1999 August 31)).
Fibromodulin is a member of a family of small interstitial proteoglycans that
also includes
decorin, biglycan and lumican. The proteoglycans bind to other matrix
macromolecules and
thereby help to stabilize the matrix. (Buckwalter et al., 47 Instr. Course
Lect 477-86 (1998)). It is
speculated that they may influence the function of chondrocytes and bind
growth factors.
Proteoglycan protein cores are structurally related and consist of a central
region of leucine-rich
repeats flanked by disulfide-bonded terminal domains. Fibromodulin has up to 4
lceratin sulfate
chains within its leucine-rich domain. It enjoys wide tissue distribution and
is most abundant in
articular cartilage, tendon and ligament. It has been suggested that
fibromodulin participates in
the assembly of the extracellular matrix by virtue of its ability to interact
with type I and type II
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collagen fibrils and to inhibit fribrillogenesis in uitYO. Sztrolovics et al.
cloned the 3'-untranslated
region of fibromodulin cDNA, and used it to map the gene by fluorescence in
situ hybridization
(FISH) to 1q32 (Sztrolovics et al., 23 Genomics 715-7 (1994)). This
localization to chromosome
1 has since been confirmed by PCR analysis of somatic cell hybrids.
SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of novel nucleic acid
sequences
encoding polypeptides. Nucleic acids encoding these polypeptides and
derivatives and fragments
thereof, will hereinafter be collectively designated as "NOV."
In one aspect, the invention provides an isolated NOV 1 nucleic acid molecule
encoding a
NOV 1 polypeptide that has identity to the polypeptide sequence for the small
actin-sequestering
peptide thymosin-beta-10. In another aspect, the invention provides an
isolated NOV2 nucleic
acid molecule encoding a NOV2 polypeptide that has identity to ephrin type-A
receptor 8. In yet
another aspect, the invention provides an isolated NOV3 nucleic acid molecule
encoding a NOV3
polypeptide that has homology to a family of proteoglycans. In still another
aspect, the invention
provides an isolated NOV4 nucleic acid molecule encoding a NOV4 polypeptide
that has identity
to mature extracellular ephrin type-A receptor 8. In still another aspect, the
invention provides an
isolated NOVS nucleic acid molecule encoding a NOVS polypeptide that has
homology to the
proteoglycan, fibromodulin.
In some embodiments, the NOV nucleic acid molecule can hybridize under
stringent
conditions to a nucleic acid sequence complementary to a nucleic acid molecule
that includes a
protein-coding sequence of the nucleic acid sequence. A preferred embodiment
of the invention
is an oligonucleotide, e.g., an oligonucleotide which includes at least 6
contiguous nucleotides of
a NOV nucleic acid (e.g., SEQ ID NO: 1, 4, 6, 65, or 67) or a complement of
said oligonucleotide.
Also included in the invention are substantially purified NOV polypeptides
(SEQ ID NO:
2, 5, 7, 66 or 68), and polypeptides having conservative amino acid
substitutions to these NOV
polypeptides. The invention also features antibodies that innmnoselectively-
bind to NOV
polypeptides.
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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 NOV nucleic acid, a NOV
polypeptide, or an
antibody specific for a NOV polypeptide. W 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 NOV nucleic acid, under conditions allowing
for expression of the
NOV polypeptide encoded by the DNA. If desired, the NOV polypeptide can then
be recovered.
In another aspect, the invention includes a method of detecting the presence
of a NOV
polypeptide in a sample. In the method, a sample is contacted with a compound
that selectively
binds to the polypeptide under conditions allowing for formation of a complex
between the
polypeptide and the compound. The complex is detected, if present, thereby
identifying the NOV
polypeptide within the sample.
Also included in the invention is a method of detecting the presence of a NOV
nucleic
acid molecule in a sample by contacting the sample with a NOV nucleic acid
probe or primer, and
detecting whether the nucleic acid probe or primer bound to a NOV nucleic acid
molecule in the
sample.
In a fuxther aspect, the invention provides a method for modulating the
activity of a NOV
polypeptide by contacting a cell sample that includes the NOV polypeptide with
a compound that
binds to the NOV 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 outlined in the
preferred
embodiment below. The therapeutic can be, e.g., a NOV nucleic acid, a NOV
polypeptide, or a
NOV-specific antibody, or biologically-active derivatives or fragments
thereof.
In the preferred embodiments, the invention further includes methods for
screening for a
modulator of disorders or syndromes including, e.g., those involving
development, differentiation,
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and activation of thymic immune cells; methods for diagnosing and treating
disorders, and/or for
screening for a modulator of disorders or syndromes including, e.g., diagnosis
of several human
neoplasias; rheumatoid arthritis; congenital muscular dystrophies; various
muscle disorders; fixed
deformities (arthrogryposis); small cell lung cancer NCI-H23; prostate cancer;
atopy;
dysprothrombinemia; hypoprothrombinemia; Smith-Lemli-Opitz syndrome, type I;
Smith-Lemli-
Opitz syndrome, type II; xeroderma pigmentosum, group E, subtype 2; high bone
mass; Bardet-
Biedl syndrome 1; CPT deficiency, hepatic, type I; carcinoid tumor of lung;
centrocytic
lynphoma; cervical carcinoma; hyperparathyroidism, AD; hypokalemic periodic
paralysis Leigh
syndrome; acute promyelocytic leukemia, NLTMAlRARA
type; macular dystrophy, vitelliform type; McArdle disease; Meckel syndrome,
type 2; multiple
endocrine neoplasia I; multiple myeloma; parathyroid adenomatosis 1;
prolactinoma;
hyperparathyroidism; carcinoid syndrome; digenic retinitis pigmentosa;
somatotrophinoma;
neovascular inflammatory vitreoretinopathy; arthritis; and tendonitis, in
addition to other diseases,
disorders and conditions associated with fibromodulin deficiency or disorder;
in pathologies
related to spermatogenesis and male infertility; diagnosis of several human
neoplasias; in diseases
or pathologies of cells in blood circulation such as red blood cells and
platelets; neurological,
cardiac and vascular pathologies; rheumatoid arthritis; congenital muscular
dystrophies; various
muscle disorders; fixed deformities (arthrogryposis); small cell lung cancer
NCI-H23; prostate
cancer; and abnormal white matter. The method includes contacting a test
compound with a NOV
polypeptide and determining if the test compound binds to said NOV
polypeptide. Binding of the
test compound to the NOV polypeptide indicates the test compound is a
modulator of activity, or
of latency or predisposition to the aforementioned disorders or syndromes. In
addition, these
materials are further useful in the generation of antibodies that bind
immunospecifically to the
novel substances of the invention for use in diagnostic and/or therapeutic
methods.
Also within the scope of the invention is a method for screening for a
modulator of
activity, or of latency or predisposition to disorders or syndromes listed
above 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 NOV nucleic
acid. Expression
or activity of NOV polypeptide is then measured in the test animal, as is
expression or activity of
the protein in a control animal which recombinantly-expresses NOV polypeptide
and is not at
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increased risk for the disorder or syndrome. Next, the expression of NOV
polypeptide in both the
test animal and the control animal is compared. A change in the activity of
NOV 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 NOV
polypeptide, a NOV nucleic
acid, or both, in a subject (e.g., a human subject). The method includes
measuring the amount of
the NOV polypeptide in a test sample from the subject and comparing the amount
of the
polypeptide in the test sample to the amount of the NOV polypeptide present in
a control sample.
An alteration in the level of the NOV 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 including those listed in the preferred embodiment above.
In 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 NOV
polypeptide, a NOV nucleic acid, or a NOV-specific antibody to a subject
(e.g., a human subject),
in an amount sufficient to alleviate or prevent the pathological condition.
Unless otherwise defined, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary shill in the art to which
the invention
belongs. Although methods and materials similar or equivalent to those
described herein can be
used in the practice or testing of the 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 case of conflict, the present
specification, including
definitions, will control. In addition, the materials, methods, and examples
are illustrative
purposes only, and not intended to be limiting in any mamzer. Other features
and advantages of
the invention will be apparent from the following detailed description and
claims.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Western blot of a NOV2 polypeptide secreted by 293 cells.
Figure 2. Western blot of a NOV3 polypeptide secreted by 293 cells.
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Figure 3. Monoclonal antibody proliferation assay of T47D cells in response to
NOV4.
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 "NOV nucleic acids" or "NOV
polynucleotides" and the
corresponding encoded polypeptides are referred to as "NOV polypeptides" or
"NOV proteins."
Unless indicated otherwise, "NOV" is meant to refer to any of the novel
sequences disclosed
herein. Table 14 provides a summary of the NOV nucleic acids and their encoded
~polypeptides.
NOV nucleic acids and their encoded polypeptides are useful in a variety of
applications
and contexts. The various NOV 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.
For example, NOV1 is homologous to members of the thyrnosin beta 10 family of
proteins. As a result, NOV 1 has various marker utilities as described herein.
Also, NOV 1 has
efficacy in treatment of conditions involving development, differentiation,
and activation of
thymic immune cells; in pathologies related to spermatogenesis and male
infertility; diagnosis of
several human neoplasias; in diseases or pathologies of cells in blood
circulation such as red
blood cells and platelets; and detection of small cell lung cancer.
NOV2 and NOV4 are homologous to members of the ephrin A receptor family. As a
result, NOV2 and NOV4 have various marker utilities as described herein. NOV2
and NOV4 also
have roles in the treatment of conditions involving neurological, cardiac and
vascular pathologies,
as well as in the detection of prostate and breast cancers. For example, tests
for clone NOV2 on a
panel of tissue lines show highest percentage of expression in prostate cancer
with bone
metastasis and a prostate cancer tissue line (tissue 87073, OD04720-O1), small
cell lung cancer
and certain breast cancer tissue lines (GENPAK 064006 and Clontech 9100266),
but negligible
expression in prostate cancer tissue, benign prostate tissue (tissue 84141,
OD04410), large cell
and non-small cell lung cancers, and non-cancerous breast tissue immediately
adjacent to cancer
tissue (see Tables 16 and 17). Likewise, tests for clone NOV4 on panels of
tissue lines show high
percentage levels of expression in testis, certain breast cancers, single-
stranded central nervous
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system tissue 94909-XF-498 and dermal fibroblast line 93771-IL-4; however,
normal prostate and
prostate cancer tissue, other breast cancer cell lines (MDA-N, BT-249 and MCF-
7, for example),
spinal cord and certain other dermal fibroblast tissue lines (IFN-gamma,
CCD1070-IL-1-beta and
CCD1070-TNF alpha, as examples) show little or no level of expression.
NOV3 and NOVS are homologous to members of the proteoglycan family. As a
result,
NOV3 and NOVS have various marker utilities as described herein, and function
in the treatment
of conditions involving rheumatoid arthritis; congenital muscular dystrophies;
various muscle
disorders; fixed deformities (arthrogryposis); and abnormal white matter.
Multiple sequence and
BLAST alignment of NOV3 polypeptide and human fibronectin leucine-rich repeat
transmembrane protein FLRTl shows approximately 99% identity. This indicates
that NOV3
functions in the same manner as the FLRT1 transmembrane protein. Multiple
sequence and
BLAST alignments for NOVS homo sapiens leucine-rich repeat transmembrane
protein
messenger RNA and polypeptide show 89% and 97% respectively. As with NOV3,
this degree of
sequence identity with fibronectin, a known proteoglycan, indicates that NOVS
has utility both in
targeting and treating connective tissue disorders. Additional utilities for
NOV nucleic acids and
polypeptides according to the invention are also discussed herein.
NOV1
A NOV 1 nucleic acid sequence according to the invention includes nucleic
acids encoding
a polypeptide related to the small actin-sequestering peptide thyrnosin-beta-
10. An example of
this nucleic acid and its encoded polypeptide is presented in Table 1. The
disclosed nucleic acid
(SEQ ID NO: 1) is 430 nucleotides in length and contains an open reading frame
(ORF) that
begins with an ATG initiation codon at nucleotides 61-63 and ends with a TAG
stop codon at
nucleotides 235-237.
The representative ORF includes a 58 amino acid polypeptide (SEQ ID NO: 2) and
is
flanlced by putative upstream and downstream untranslated regions that are
underlined in Table 1.
The encoded polypeptide has a high degree of homology (approximately 85
percent identity) with
thyrnosin beta 10 from human (Table 2). A search of the PROSITE database of
protein families
and domains confirmed that a NOV 1 polypeptide is a member of the thymosin
beta family, which
is defined by polypeptides containing a stretch of 11 highly conserved amino
acid residues
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K-L-K-K-T-[E or N]-T-[Q or E]-E-K-N (SEQ ID NO: 3)
located in the central part of the thymosin beta proteins (Table 2). The
PROSITE database
consists of biologically significant sites, patterns and profiles that help to
reliably identify to
which known protein family a new sequence belongs.
Furthermore, a search of the PFAM database reveals that a NOV1 polypeptide
conforms
to the sequence profile of thymosin beta family of proteins (Table 3). The
query sequence in the
table is a NOV1 polypeptide and the subject is a consensus sequence formed
from the thymosin
beta family of proteins. The presently disclosed NOV 1 polypeptide has 84
percent identity across
its entire length to the consensus thymosin beta sequence (Table 4). The NOVl
polypeptide bears
more homology to the consensus thymosin beta sequence than do many other
members of the
family. Also, this degree of homology between a NOV1 polypeptide and the
thymosin beta
consensus (both in terms of length and complexity) is very unlikely to have
occurred by change
alone (Expect value in Table 3 less than 1 in 5 *' 1 Ol° by chance).
Pfam is a large collection of
multiple sequence aligmnents and profile hidden Markov models covering many
common protein
domain families. It is designed to be both an accurate and comprehensive
method to determine
homology. A multiple alignment of the thymosin-beta family is presented in
Table 3. Based on its
relatedness to the thynosin-beta-10 protein, the NOV 1 protein is a novel
member of the actin-
sequestering protein family.
The thyrnosin-betas comprise a family of structurally related, highly
conserved acidic
polypeptides that sequester actin and regulate actin dynamics within cells.
During embryogenesis
the control of actin polymerization is essential in processes such as cell
migration, angiogenesis
and neurogenesis. Direct visualization and quantitation of actin filaments has
shown that
thymosin-betas, like agonists, induced actin depolymerization at the apical
membrane where
exocytosis occurs (S. Muallem et al., 128(4) J. Cell Biol 589-98 (1995)).
Thymosin-beta-10 is
widely distributed in mammalian tissues including the nervous system, and the
presence of this
transcript in different regions of the rat forebrain, including hippocampus,
neocortex ,and several
brain nuclei, provides evidence for the participation of thymosin-beta-10 in
the control of the actin
dynamics that talces place in neurons. Thymosin-beta-10 is expressed at
relatively high levels in
embryonic and developing tissues (A.K. Hall, 41(3) Cell Mol. Biol. Res.167-80
(1995)), and
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given that it is involved in the inhibition of actin polymerization, the
thymosin-beta-10 protein-
lilce proteins can play an impouant role in early development.
Messenger RNA species of similar molecular weights encoding thymosin beta-10
are
found in most tissues of the rat; however, Lin and Morrison-Bogorad identified
an additional
thymosin-beta-10 mRNA of higher molecular weight in the testis of sexually
mature rats (Lin et
al., "Cloning and characterization of a testis-specific thymosin beta 10 cDNA.
Expression in
post-meiotic male germ cells," 266(34) J. Biol. Chem., 23347-53 (1991)). The
latter mRNA
differs from the ubiquitous form only in its 5-prime untranslated region,
beginning 14 nucleotides
upstream of the translation initiation codon. This finding, together with
primer extension
experiments, suggested that the two mRNA types are transcribed from the same
gene through a
combination of differential promoter utilization and alternative splicing.
Both mRNAs were
present in pachytene spermatocytes; only the testis-specific mRNA was detected
in postmeiotic
haploid spermatids. Immunohistochemical analysis showed that the protein was
present in
differentiating spennatids, suggesting that the testis-specific thymosin-beta-
10 mRNA is
translated in haploid male germ cells. Immunoblot analysis using specific
antibodies showed that
the thymosin-beta-10 protein synthesized in adult testis was identical in size
to that synthesized in
brain.
Thyrnosin-beta-10-like proteins also influence several properties of
lymphocytes including
cyclic nucleotide levels, migration inhibitory factor production, T-dependent
antibody production,
as well as the expression of various cell surface maturation/differentiation
markers (Bodey et al.,
22(4) Int. J. Tmmunopharmacology 261-73 (2000)). These and other observations
suggest that
thymosin beta-10 (a) plays a significant and possibly obligatory role in
cellular processes
controlling apoptosis possibly by acting as an actin-mediated tumor
suppressor, (b) functions as a
neoapoptotic influence during embryogenesis, and (c) can mediate some of the
pro-apoptotic
anticancer actions of retinoids. Thymosin-beta-10 mRNA is also abundant in a
variety of tumors
and tumor cell lines.
Thymosin-beta-10 gene overexpression is a general event in human
carcinogenesis.
Analysis of thymosin-beta-10 mRNA levels in human colon carcinomas, germ cell
tumors of
different histological types, breast carcinomas, ovarian carcinomas, uterine
carcinomas, colon and
esophageal carcinoma cell lines all indicated thymosin-beta-10 was over
expressed in all of the
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neoplastic tissues and cell lines compared to the respective normal tissues.
Therefore, detection of
thymosin-beta-10-lilee expression can be considered a potential tool for the
diagnosis of several
human neoplasias. (Santelli et al., 155(3) Annals of Am. J. Pathol. 799-804
(1999)). Not only can
thymosins lilce thymosin beta-10 be used for early detection and diagnosis of
neoplasms, but also
in recent clinical trials derivatives of thymic hormones, mostly of thymosins,
have been used to
help treat neoplasms (Bodey et al., 22(4) Int. J. Immunopharmacol. 261-73
(2000)). Thyrnic
hormones strengthen the effects of immunomodulators in immunodeficiencies,
autoimmune
diseases, and neoplastic malignancies. Combined chemo-immunotherapeutical anti-
cancer
treatment seems to be more efficacious than chemotherapy alone, and the
significant
hematopoietic toxicity associated with most chemotherapeutical clinical trials
can be reduced
significantly by the addition of immunotherapy.
Based on its relatedness to the thymosin-beta-10 protein, the NOV 1 protein is
a novel
member of the actin-sequestering protein family. The discovery of molecules
related to thymosin-
beta-10 satisfies a need in the art by providing new diagnostic or therapeutic
compositions useful
in the treatment of disorders associated with alterations in the expression of
members of
thymosin-beta-10- like proteins. Nucleic acids, polypeptides, antibodies, and
other compositions
of the present invention are useful in a variety of diseases and pathologies,
including by way of
nonlimiting example, those involving development, differentiation and
activation of thymic
immune cells, pathologies related to spermatogenesis and male infertility,
diagnosis of several
human neoplasias, and diseases or pathologies of cells in blood circulation
such as red blood cells
and platelets.
A NOV 1 nucleic acid is useful for detecting specific cell-types. For example
a variant
splice form of a NOV 1 nucleic acid according to the invention can be present
in different levels in
postmeiotic haploid spermatids. Also, according to the invention the
expression of a NOV 1
nucleic acid has utility in identifying developing and embryonic tissues from
other tissue types.
Thymosin-beta-10 mRNA is overexpressed in a variety of tumors and tumor cell
lines. Expression
levels of thymosin-beta-10 like nucleic acids such as NOV1 are also useful in
distinguishing T
cell types given that expression of various cell surface/differentiation
markers is influenced by
thymosin-beta-10 like proteins such as a NOV 1 polypeptide. A NOV 1 nucleic
acid has enhanced
expression in certain cancer cell lines, especially non-small cell lung cancer
NCI-H23, but not in
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cell lines from the corresponding normal tissue; therefore, NOV 1 nucleic
acids are useful as a
cancer specific marker in such tissues (Example 1).
Given that thymosin-beta-10 related proteins can sequester actin and regulate
actin
dynamics within cells, proteins related to the NOV 1 polypeptide are useful in
screens for test
compounds that can modulate actin polymerization or the formation or stability
of actin-thylnosin
beta-10 complexes. Finally, since thymic hormones strengthen the effects of
immunomodulators
in imrnunodeficiencies, autoimmune diseases, and neoplastic malignancies, NOV
1 related
proteins can be used in combined chemo-immunotherapeutical anti-cancer
treatments.
Table 1.
A representative cDNA sequence encoding the thymosin-beta-10-like protein
according to
the invention
Putative untranslated regions are underlined. The start and stop codons are in
bold type.
GCCAGCAGGAGTGCCATGGTGAGAGGCACTGGCAGGGAATGCTAGGATTGTTTTAAGAAAATGGCAGACAAACCAGACA
TAGGGGAA
ATCGCCAGCTTCAATAAGGCCAAGCTGAAGAAAACAGAGATGCAGGAGAACACCCTGCTGACCAAAGAGGCCATTGAGC
AGGAGAAG
CGGGTGAAATTTCCTAAGAGCCTGGAGGATTCCCTACCCCTGTCATCTTCGAGACCCCAGTAGTAATGTGGAGGAAGAA
TCACCACA
AGATGGACACAAGCCACAAACTGTGACGTGAACCTGGGCACTCCGTGCTGATGCCACCAGCCTGAGGGTCCCTATGGGT
CCAATCAG
2O
ACTGCCAAATTCTCTGGTTTGCCCTGGGATATTATAGAAAATTATTTGCGTGAATAATGAA.AACACAGCTCATGGCAA
AAAA (SEQ
ID N0:1)
A representative amino acid sequence of the thymosin-beta-10-like protein
according to the
invention
MADKPDIGEIASFNKAKLKKTEMQENTLLTKEAIEQEKRVKFPKSLEDSLPLSSSRPQ (SEQ ID N0:2)
Table 2.
Comparison between a NOVl polypeptide and thymosin beta-10 from human
>gbIAAA36746.1) (M92383) thymosin beta-ZO [Homo Sapiens]
Length = 49
3S Score = 84.5 bits (192), Expect = 3e-16
Identities = 34/40 (85%), Positives = 36/40 (90%), Gaps = 1/40 (2%)
NOV1 : 1 MADKPDIGEIASFNKAKLKKTEMQE-NTLLTKEAIEQEKR 39 (SEQ ID NO: 2)
111111+111111+IIIIIIII II IIl III 111111
4O Sbjct: 6 MADKPDMGEIASFDKAKLKKTETQEKNTLPTKETIEQEKR 45 (SEQ ID NO: 34)
Table 3.
Multiple Sequence alignment of a NOV1 polypeptide and the thymosin beta family
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(Black outlined amino acids indicate potential regions of conserved sequence;
grayed amino acids
represent amino acids conservatively substituted; and non-highlighted amino
acids indicate
positions in which mutations to a broad range of alternative amino acid
residues occurs.
Sequences may be referenced by the SWISSPROT or TREMBL ID.)
10 20 30 40
thymosin beta family motif .(SEQ TD N0:35)
NOV1 (2-38) (SEQ ID N0:2)
TYBO_HUMAN(1-38) (SEQ ID N0:36)
TYB9 (SEQ ID N0:37)
BOVIN(1-40)
_ (SEQ TD N0:38)
TYB9_PIG(1-40)
TYB4 (SEQ ID N0:39)
HUMAN(1-40)
_ (SEQ ID N0:40)
TYB4
MOUSE(7-47)
_ (SEQ ID N0:41)
TYB4
RABIT(1-40)
_ (SEQ ID N0:42)
TYB4
XENLA(1-39)
_ (SEQ ID N0:43)
TYBY_HUMAN(1-40)
TYBA (SEQ TD N0:44)
ONCMY(1-40)
_ (SEQ ID N0:45)
TYBB
ONCMY(1-40)
_ (SEQ ID N0:46)
TYBB
LATJA(1-40
_ (SEQ ID N0:47)
P97563
RAT(1-39)
_ (SEQ ID N0:48)
TYBN
HUMAN(1-38)
_ (SEQ ID N0:49)
097428 DROME(95-129)
097428 DROME(59-89) (SEQ ID N0:50)
Table 4.
PFAM alignment of a NOVl polypeptide to the consensus sequence of the thymosin
beta
family
>PD005116 (Closest domain: TYBO_HUMAN 1-38)
Number of sequences in family: 16
Most frequent protein names: TYB4(4) TYB9(2) TYBB(2)
Commentary (automatic):
THYMOSIN ACETYLATION T-CELL DIFFERENTIATION
TMMUNOPOTENTIATION THYMUS BETA-4 ACTIN-BINDING PROTEIN
BETA
Length = 38
Score = 145 (60.9 bits), Eacpect = 5e-10
Identities = 32/38 (84%), Positives = 34/38 (89%), Gaps = 1/38 (2%)
NOV1: 2 ADKPDIGEIASFNKAKLKKTEMQE-NTLLTKEAIEQEK 38 (SEQ ID N0: 2)
IIIII+111111+Illlllll II 111 III IIIII
Sbjct: 1 ADKPDMGEIASFDKAKLKKTETQEKNTLPTKETIEQEK 38 (SEQ ID NO: 51)
NOV2
A NOV2 nucleic acid according to the invention includes nucleic acids encoding
a
polypeptide related to ephrin type-A receptors. An example of nucleic acid and
its encoded
polypeptide is presented in Table 5. The disclosed nucleic acid (SEQ m NO: 4)
is 301
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nucleotides in length and contains an open reading frame that begins with an
ATG initiation
codon at nucleotide 1-3 and ends at nucleotides 2974-2976.
The representative ORF includes a 992 amino acid polypeptide (SEQ ID NO: 5).
The
encoded polypeptide has a high degree of homology (approximately 95 percent
identity) with
mouse ephrin type-A receptor 8 precursor (Table 6) (SWISSPROT ACC: 009127, 956
out of
1005 residues). The NOV2 polypeptide also has an even higher degree of
homology (100 percent
identity) to a human eph- and elk-related kinase known as ephrin receptor
EphA8 (Table 6A,
partial sequence disclosed in Chan et al., 6 Onco ene 1057-1061 (1991); the
full length human
ephrin receptor EphA8 full length sequence was deposited in Genbank September
14, 2000 as
accession number NP_065387.1.) A multiple aligmnent with similar proteins
showed comparable
degrees of similarity to ephrin receptors from mouse (EPA8 mouse), human (EPAS
human), and
chicken (EPAS chick) (Table 7). In the predicted extracellular domain, a
cysteine-rich region and
tandem fibronectin type III repeats are present while a catalytic domain is
present in the
intracellular domain. These features are consistent with other members of the
Eph family. Based
on its relatedness, the NOV2 protein is a member of the ephrin type-A receptor
tyrosine-protein
kinase family.
The Eph receptors constitute the largest known family of receptor protein
tyrosine kinases.
They have been implicated in mediating developmental events, particularly in
the nervous system.
Receptors in the Eph subfamily typically have a single kinase domain and an
extracellular region
containing a Cys-rich domain and two fibronectin type III repeats. These
receptors play important
roles along with their ligands, called ephrins, in neural development,
angiogenesis, and vascular
networlc assembly. (S. Choi et al., 9(4) Mol. Cells 440-45 (1999)).
The ephrin type-A receptor 8 (EC 2.7.1.112) (tyrosine-protein kinase receptor
eek) (eph-
and elk-related kinase) (fragment) is designated as the gene product of the
gene: epha~ o~ eek. It
is a Type I membrane bound receptor, and its function is to serve as a
receptor for members of the
ephrin-a family. Its catalytic activity is as a protein tyrosine kinase,
phosphorylating tyrosine in
appropriate target proteins. It is similar to other protein-tyrosine kinases
in the catalytic domain
and belongs to the ephrin receptor family.
Eph receptors have tyrosine-kinase activity, and, together with their ephrin
ligands,
mediate contact-dependent cell interactions that are implicated in the
repulsion mechanisms that
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guide migrating cells and neuronal growth cones to specific destinations.
Since Eph receptors and
ephrins have complementary expression in many tissues during embryogenesis,
bidirectional
activation of Eph receptors may occur at interfaces of their expression
domains, for example, at
segment boundaries in the vertebrate hindbrain. Indeed, Eph receptors play key
roles in
S development of the nervous system and angiogenesis. In the nervous system,
they provide
positional information by empolying mechanisms that involve repulsion of
migrating cells and
growing axons (Frisen et al., 18(19)EMBO J. S1S9-S16S (1999)). Also, an
important function of
Eph receptors and ephrins is to mediate cell-contact-dependent repulsion.
A NOV2 sequence according to the invention is useful for detecting cells that
express
GPI-anchored ephrin-A ligands. For example, cells expressing either a NOV2
nucleic acid or a
NOV2 protein have utility in screening for other cells that express GPI-
anchored ephrin-A ligands
or mimics therefore. As a result, a NOV2 sequence is useful for screening for
new ephrin-A
ligands expressed on cells. NOV2 is lughly expressed in many surgical tumor
samples, especially
prostate cancer, but minimally or not detectably in the immediate normal
adjacent tissue;
1 S therefore, the NOV2 expression can be used as a marker for certain
cancers, especially prostate
cancer (Example 1).
Also, a NOV2 sequence according to the invention is useful to direct the
development of
the nervous system and angiogenesis by modulating the boundaries between
arteries and veins.
For example, mice expressing defective Eph receptors similar to a NOV2
sequence have been
shown to be defective in angiogensis and die in mid-gestation (Wang et al., 93
Cell 741-7S3
(1998)). The protein of the present invention will be useful in a variety of
diseases and
pathologies, including by way of nonlimiting example, those involving
neurological, cardiac and
vascular pathologies.
2S Table 5.
A representative DNA sequence encoding the ephrin type-A receptor 8-like
protein of the
invention
ATGGCCCCCGCCCGGGGCCGCCTGCCCCCTGCGCTCTGGGTCGTCACGGCCGCGGCGGCGGCGGCCACCT
3O GCGTGTCCGCGGCGCGCGGCGAAGTGAATTTGCTGGACACGTCGACCATCCACGGGGACTGGGGCTGGCT
CACGTATCCGGCTCATGGGTGGGACTCCATCAACGAGGTGGACGAGTCCTTCCAGCCCATCCACACGTAC
CAGGTTTGCAACGTCATGAGCCCCAACCAGAACAACTGGCTGCGCACGAGCTGGGTCCCCCGAGACGGCG
CCCGGCGCGTCTATGCTGAGATCAAGTTTACCCTGCGCGACTGCAACAGCATGCCTGGTGTGCTGGGCAC
CTGCAAGGAGACCTTCAACCTCTACTACCTGGAGTCGGACCGCGACCTGGGGGCCAGCACACAAGAAAGC
3S CAGTTCCTCAAAATCGACACCATTGCGGCCGACGAGAGCTTCACAGGTGCCGACCTTGGTGTGCGGCGTC
TCAAGCTCAACACGGAGGTGCGCAGTGTGGGTCCCCTCAGCAAGCGCGGCTTCTACCTGGCCTTCCAGGA
1S
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CATAGGTGCCTGCCTGGCCATCCTCTCTCTCCGCATCTACTATAAGAAGTGCCCTGCCATGGTGCGCAAT
CTGGCTGCCTTCTCGGAGGCAGTGACGGGGGCCGACTCGTCCTCACTGGTGGAGGTGAGGGGCCAGTGCG
TGCGGCACTCAGAGGAGCGGGACACACCCAAGATGTACTGCAGCGCGGAGGGCGAGTGGCTCGTGCCCAT
CGGCAAATGCGTGTGCAGTGCCGGCTACGAGGAGCGGCGGGATGCCTGTGTGGCCTGTGAGCTGGGCTTC
S TACAAGTCAGCCCCTGGGGACCAGCTGTGTGCCCGCTGCCCTCCCCACAGCCACTCCGCAGCTCCAGCCG
CCCAAGCCTGCCACTGTGACCTCAGCTACTACCGTGCAGCCCTGGACCCGCCGTCCTCAGCCTGCACCCG
GCCACCCTCGGCACCAGTGAACCTGATCTCCAGTGTGAATGGGACATCAGTGACTCTGGAGTGGGCCCCT
CCCCTGGACCCAGGTGGCCGCAGTGACATCACCTACAATGCCGTGTGCCGCCGCTGCCCCTGGGCACTGA
GCCGCTGCGAGGCATGTGGGAGCGGCACCCGCTTTGTGCCCCAGCAGACAAGCCTGGTGCAGGCCAGCCT
1O GCTGGTGGCCAACCTGCTGGCCCACATGAACTACTCCTTCTGGATCGAGGCCGTCAATGGCGTGTCCGAC
CTGAGCCCCGAGCCCCGCCGGGCCGCTGTGGTCAACATCACCACGAACCAGGCAGCCCCGTCCCAGGTGG
TGGTGATCCGTCAAGAGCGGGCGGGGCAGACCAGCGTCTCGCTGCTGTGGCAGGAGCCCGAGCAGCCGAA
CGGCATCATCCTGGAGTATGAGATCAAGTACTACGAGAAGGACAAGGAGATGCAGAGCTACTCCACCCTC
AAGGCCGTCACCACCAGAGCCACCGTCTCCGGCCTCAAGCCGGGCACCCGCTACGTGTTCCAGGTCCGAG
IS CCCGCACCTCAGCAGGCTGTGGCCGCTTCAGCCAGGCCATGGAGGTGGAGACCGGGAAACCCCGGCCCCG
CTATGACACCAGGACCATTGTCTGGATCTGCCTGACGCTCATCACGGGCCTGGTGGTGCTTCTGCTCCTG
CTCATCTGCAAGAAGAGGCACTGTGGCTACAGCAAGGCCTTCCAGGACTCGGACGAGGAGAAGATGCACT
ATCAGAATGGACAGGCACCCCCACCTGTCTTCCTGCCTCTGCATCACCCCCCGGGAAAGCTCCCAGAGCC
CCAGTTCTATGCGGAACCCCACACCTACGAGGAGCCAGGCCGGGCGGGCCGCAGTTTCACTCGGGAGATC
ZO GAGGCCTCTAGGATCCACATCGAGAAAATCATCGGCTCTGGAGACTCCGGGGAAGTCTGCTACGGGAGGC
TGCGGGTGCCAGGGCAGCGGGATGTGCCCGTGGCCATCAAGGCCCTCAAAGCCGGCTACACGGAGAGACA
GAGGCGGGACTTCCTGAGCGAGGCGTCCATCATGGGGCAATTCGACCATCCCAACATCATCCGCCTCGAG
GGTGTCGTCACCCGTGGCCGCCTGGCAATGATTGTGACTGAGTACATGGAGAACGGCTCTCTGGACACCT
TCCTGAGGACCCACGACGGGCAGTTCACCATCATGCAGCTGGTGGGCATGCTGAGAGGAGTGGGTGCCGG
ZS CATGCGCTACCTCTCAGACCTGGGCTATGTCCACCGAGACCTGGCCGCCCGCAACGTCCTGGTTGACAGC
AACCTGGTCTGCAAGGTGTCTGACTTCGGGCTCTCACGGGTGCTGGAGGACGACCCGGATGCTGCCTACA
CCACCACGGGCGGGAAGATCCCCATCCGCTGGACGGCCCCAGAGGCCATCGCCTTCCGCACCTTCTCCTC
GGCCAGCGACGTGTGGAGCTTCGGCGTGGTCATGTGGGAGGTGCTGGCCTATGGGGAGCGGCCCTACTGG
AACATGACCAACCGGGATGTGATCAGCTCTGTGGAGGAGGGGTACCGCCTGCCCGCACCCATGGGCTGCC
3O CCCACGCCCTGCACCAGCTCATGCTCGACTGTTGGCACAAGGACCGGGCGCAGCGGCCTCGCTTCTCCCA
GATTGTCAGTGTCCTCGATGCGCTCATCCGCAGCCCTGAGAGTCTCAGGGCCACCGCCACAGTCAGCAGG
TGCCCACCCCCTGCCTTCGTCCGGAGCTGCTTTGACCTCCGAGGGGGCAGCGGTGGCGGTGGGGGCCTCA
CCGTGGGGGACTGGCTGGACTCCATCCGCATGGGCCGGTACCGAGACCACTTCGCTGCGGGCGGATACTC
CTCTCTGGGCATGGTGCTACGCATGAACGCCCAGGACGTGCGCGCCCTGGGCATCACCCTCATGGGCCAC
3S CAGAAGAAGATCCTGGGCAGCATTCAGACCATGCGGGCCCAGCTGACCAGCACCCAGGGGCCCCGCCGGC
ACCTCTGA (SEQ ID N0:4)
A representative amino acid sequence of the ephrin type-A receptor 8-like
protein of the
invention.
MAPARGRLPPALWWTAAAAAATCVSAARGEVNLLDTSTIHGDWGWLTYPAHGWDSINEVDESFQPIHTYQVCNVMSPNQ
NNWLRTS
WPRDGARRVYAEIKFTLRDCNSMPGVLGTCKETFNLYYLESDRDLGASTQESQFLKIDTIAADESFTGADLGVRRLKLN
TEVRSVG
PLSKRGFYLAFQDIGACLAILSLRIYYKKCPAMVRNLAAFSEAVTGADSSSLVEVRGQCVRHSEERDTPKMYCSAEGEW
LVPIGKCV
CSAGYEERRDACVACELGFYKSAPGDQLCARCPPHSHSAAPAAQACHCDLSYYRAALDPPSSACTRPPSAPVNLISSVN
GTSVTLEW
4S
APPLDPGGRSDITYNAVCRRCPWALSRCEACGSGTRFVPQQTSLVQASLLVANLLAHMNYSFWIEAVNGVSDLSPEPRR
AAVVNITT
NQAAPSQVWIRQERAGQTSVSLLWQEPEQPNGITLEYEIKYYEKDKEMQSYSTLKAVTTRATVSGLKPGTRYVFQVRAR
TSAGCGR
FSQAMEVETGKPRPRYDTRTIWICLTLITGLWLLLLLICKKRHCGYSKAFQDSDEEKMHYQNGQAPPPVFLPLHHPPGK
LPEPQF
YAEPHTYEEPGRAGRSFTREIEASRIHTEKIIGSGDSGEVCYGRLRVPGQRDVPVAIKALKAGYTERQRRDFLSEASIM
GQFDHPNI
IRLEGWTRGRLAMIVTEYMENGSLDTFLRTHDGQFTIMQLVGMLRGVGAGMRYLSDLGYVHRDLAARNVLVDSNLVCKV
SDFGLSR
SO
VLEDDPDAAYTTTGGKIPIRWTAPEAIAFRTFSSASDWSFGWMWEVLAYGERPYWNMTNRDVISSVEEGYRLPAPMGCP
HALHQL
MLDCWHKDRAQRPRFSQIVSVLDALIRSPESLRATATVSRCPPPAFVRSCFDLRGGSGGGGGLTVGDWLDSIRMGRYRD
HFAAGGYS
SLGMVLRMNAQDVRALGITLMGHQKKILGSIQTMR (SEQ ID N0:5)
Table 6.
SS Comparison between a NOV2 polypeptide and mouse ephrin type-A receptor 8
precursor
>refINP 031965.11 Eph receptor A8
sp~009127~EPA8_MOUSE EPHRIN TYPE-A RECEPTOR 8 PRECURSOR (TYROSINE-PROTEIN
KINASE
RECEPTOR EEK) (EPH-AND ELK-RELATED KINASE)
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gbIAAB39218.11 (U72207) Eph-and Elk-related kinase [Mus musculus]
Length = 1004
Score = 3036 bits (7128), Expect = 0.0
S Identities = 945/992 (95%), Positives = 964/992 (96%), Gaps = 1/992 (0%)
NOV2: 1 MAPARGRLPPALWWTAAAAAATCVSAARGEVNLLDTSTIHGDWGWLTYPAHGWDSINEV 60
Illll Il Illlllllllll IIIII IIIIllllllllllllllllllllllllllll
Sbjct: 1 MAPARARLSPALWWTAAAAA-TCVSAGRGEVNLLDTSTIHGDWGWLTYPAHGWDSINEV 59
NOV2: 61 DESFQPIHTYQVCNVMSPNQNNWLRTSWPRDGARRVYAEIKFTLRDCNSMPGVLGTCKE 120
IIII+IIIIlllllllllllllllll+IIIIIIIIIIIIIIIIIIIIIII+IIIIlllll
Sbjct: 60 DESFRPIHTYQVCNVMSPNQNNWLRTNWPRDGARRVYAEIKFTLRDCNSIPGVLGTCKE 119
IS NOV2: 121 TFNLYYLESDRDLGASTQESQFLKTDTTAADESFTGADLGVRRLKLNTEVRSVGPLSKRG 180
IIII+IIIIllllllllllllllllllllllllllllllllllllllllll IIIIIIII
Sbjct: 120 TFNLHYLESDRDLGASTQESQFLKIDTIAADESFTGADLGVRRLKLNTEVRGVGPLSKRG 179
NOV2: 181 FYLAFQDIGACLAILSLRIYYKKCPAMVRNLAAFSEAVTGADSSSLVEVRGQCVRHSEER 240
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct: 180 FYLAFQDTGACLAILSLRIYYKKCPAMVRNLAAFSEAVTGADSSSLVEVRGQCVRHSEER 239
NOV2: 241 DTPKMYCSAEGEWLVPIGKCVCSAGYEERRDACVACELGFYKSAPGDQLCARCPPHSHSA 300
Ilillllllllllllllllllllllllllllll+Illlllllllllllllllllllllll
2S SbjCt: 240 DTPKMYCSAEGEWLVPIGKCVCSAGYEERRDACMACELGFYKSAPGDQLCARCPPHSHSA 299
NOV2: 301 APAAQACHCDLSYYRAALDPPSSACTRPPSAPVNLISSVNGTSVTLEWAPPLDPGGRSDI 360
IIII I IIIIIIIIIIIIII+IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct: 300 TPAAQTCRCDLSYYRAALDPPSAACTRPPSAPWLISSVNGTSVTLEWAPPLDPGGRSDI 359
NOV2: 361 TYNAVCRRCPWALSRCEACGSGTRFVPQQTSLVQASLLVANLLAHMNYSFWIEAVNGVSD 420
IIIIIIIIIII111 11111111111111111 IIII1111111111111111111111+
Sbjct: 360 TYNAVCRRCPWALSHCEACGSGTRFVPQQTSLAQASLLVANLLAHMNYSFWIEAVNGVSN 419
3S NOV2: 421 LSPEPRRAAVVNITTNQAAPSQWVIRQERAGQTSVSLLWQEPEQPNGIILEYEIKYYEK 480
111111 Illllllllllllllllllllllllllllllllllllllllllllllllllll
Sbjct: 420 LSPEPRSAAVVNITTNQAAPSQVWIRQERAGQTSVSLLWQEPEQPNGIILEYEIKYYEK 479
NOV2: 481 DKEMQSYSTLKAVTTRATVSGLKPGTRWFQVRARTSAGCGRFSQAMEVETGKPRPRYDT 540
4o Illlllllllllllllllllllllllllllllllllllllllllllllllllllllllll
SbjCt: 480 DKEMQSYSTLKAVTTRATVSGLKPGTRWFQVRARTSAGCGRFSQAMEVETGKPRPRYDT 539
NOV2: 541 RTIVWICLTLITGLWLLLLLICKKRHCGYSKAFQDSDEEKMHYQNGQAPPPVFLPLHHP 600
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII+II
4S SbjCt: 540 RTIVWICLTLTTGLVVLLLLLICKKRHCGYSKAFQDSDEEKMHYQNGQAPPPVFLPLNHP 599
NOV2: 601 PGKLPEPQFYAEPHTYEEPGRAGRSFTREIEASRIHIEKIIGSGDSGEVCYGRLRVPGQR 660
III II II Illlllllllllllllllllllllllllllllll+Illllllll+11111
Sbjct: 600 PGKFPETQFSAEPHTYEEPGRAGRSFTREIEASRIHIEKIIGSGESGEVCYGRLQVPGQR 659
S0
NOV2: 661 DVPVAIKALKAGYTERQRRDFLSEASIMGQFDHPNIIRLEGWTRGRLAMIVTEYMENGS 720
Illlllllllllllllll+111111+lllllllllllllllllllillllllllllllll
Sbjct: 660 DVPVAIKALKAGYTERQRQDFLSEAAIMGQFDHPNIIRLEGWTRGRLAMIVTEYMENGS 719
SS NOV2: 721 LDTFLRTHDGQFTIMQLVGMLRGVGAGMRYLSDLGWHRDLAARNVLWSNLVCKVSDFG 780
II 11111111111+111111111111111111111+111111111111 IIIIIIIII
Sbjct: 720 LDAFLRTHDGQFTIVQLVGMLRGVGAGMRYLSDLGYIHRDLAARNVLVDGRLVCKVSDFG 779
NOV2: 781 LSRVLEDDPDAAYTTTGGKIPIRWTAPEAIAFRTFSSASDVWSFGWMWEVLAYGERPW 840
60 III IIIII+11111 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct: 780 LSRALEDDPEAAYTTAGGKIPIRWTAPEATAFRTFSSASDVWSFGWMWEVLAYGERPW 839
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NOV2: 841 NMTNRDVISSVEEGYRLPAPMGCPHALHQLMLDCWHKDRAQRPRFSQIVSVLDALIRSPE 900
Illl+Illllllllllllllllll Illlllllllllllllllll+ +1111111+ III
Sbjct: 840 NMTNQDVISSVEEGYRLPAPMGCPRALHQLMLDCWHKDRAQRPRFAHWSVLDALVHSPE 899
S
NOV2: 901 SLRATATVSRCPPPAFVRSCFDLRGGSGGGGGLTVGDWLDSIRMGRYRDHFAAGGYSSLG 960
IIIIIIIIIIIIIIII 1111111 l I I IIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct: 900 SLRATATVSRCPPPAFARSCFDLRAGGSGNGDLTVGDWLDSTRMGRYRDHFAAGGYSSLG 959
1O NOV2: 961 MVLRMNAQDVRALGITLMGHQKKILGSIQTMR 992 (SEQ ID N0: 5)
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct: 960 MVLRMNAQDVRALGTTLMGHQKKILGSIQTMR 991 (SEQ ID NO: 52)
Table 6A.
1 S Comparison between a NOVZ polypeptide and human ephrin receptor EphA8
>refINP_065387.11 EphA8; Ephrin receptor EphAB (eph- and elk-related kinase);
Hek3;
eph-, elk-related tyrosine kinase; ephrin receptor EphAB
embICAB81612.11 (AL035703) dJ61A9.1 (tyrosine kinase ) [Homo sapiens]
20 Length = 1005
Score = 2054 bits (5262), Expect = 0.0
Identities = 992/992 (100%), Positives = 992/992 (100%)
2S NOV2 1 MAPARGRLPPALWWTAAAAAATCVSAARGEVNLLDTSTIHGDWGWLTYPAHGWDSINEV60
:
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct: 1 MAPARGRLPPALWWTAAAAAATCVSAARGEVNLLDTSTIHGDWGWLTYPAHGWDSINEV60
NOV2 61 DESFQPIHTYQVCNVMSPNQNNWLRTSWVPRDGARRVYAETKFTLRDCNSMPGVLGTCKE120
:
30 Illllllllllllllllllllllllllllllllllllllllillllllllllllllllll
SbjCt: 61 DESFQPIHTYQVCNVMSPNQNNWLRTSWVPRDGARRVYAEIKFTLRDCNSMPGVLGTCKE120
NOV2 121 TFNLYYLESDRDLGASTQESQFLKIDTIAADESFTGADLGVRRLKLNTEVRSVGPLSKRG180
:
TFNLYYLESDRDLGASTQESQFLKIDTIAADESFTGADLGVRRLKLNTEVRSVGPLSKRG
3S Sbjct: 121 TFNLYYLESDRDLGASTQESQFLKIDTIAADESFTGADLGVRRLKI~NTEVRSVGPLSKRG180
NOV2 181 FYLAFQDIGACLAILSLRIYYKKCPAMVRNLAAFSEAVTGADSSSLVEVRGQCVRHSEER240
:
Illlllllllllllllllllllllllllllllllllllllllllllllllllllllllll
Sbjct: 181 FYLAFQDIGACLAILSLRIYYKKCPAMVRNLAAFSEAVTGADSSSLVEVRGQCVRHSEER240
40
NOV2 241 DTPKMYCSAEGEWLVPIGKCVCSAGYEERRDACVACELGFYKSAPGDQLCARCPPHSHSA300
:
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct: 241 DTPKMYCSAEGEWLVPIGKCVCSAGYEERRDACVACELGFYKSAPGDQLCARCPPHSHSA300
4S NOV2 301 APAAQACHCDLSYYRAALDPPSSACTRPPSAPVNLISSVNGTSVTLEWAPPLDPGGRSDI360
:
APAAQACHCDLSYYRAALDPPSSACTRPPSAPVNLISSVNGTSVTLEWAPPLDPGGRSDI
SbjCt: 301 APAAQACHCDLSYYRAALDPPSSACTRPPSAPVNLISSVNGTSVTLEWAPPLDPGGRSDT360
NOV2 361 TYNAVCRRCPWALSRCEACGSGTRFVPQQTSLVQASLLVANLLAHMNYSFWIEAVNGVSD420
:
SO 111111111111111111111111111111111111111111111111111111111111
Sbjct: 361 TYNAVCRRCPWALSRCEACGSGTRFVPQQTSLVQASLLVANLLAHMNYSFWIEAVNGVSD420
NOV2 421 LSPEPRRAAVVNITTNQAAPSQVWIRQERAGQTSVSLLWQEPEQPNGIILEYEIKYYEK480
:
111111111111111111111111111111111111111111111111111111111111
SS Sbjct: 421 LSPEPRRAAVVNITTNQAAPSQVWIRQERAGQTSVSLLWQEPEQPNGIILEYEIKYYEK480
NOV2 481 DKEMQSYSTLKAVTTRATVSGLKPGTRWFQVRARTSAGCGRFSQAMEVETGKPRPRYDT540
:
DKEMQSYSTLKAVTTRATVSGLKPGTRWFQVRARTSAGCGRFSQAMEVETGKPRPRYDT
Sbjct: 481 DKEMQSYSTLKAVTTRATVSGLKPGTRWFQVRARTSAGCGRFSQAMEVETGKPRPRYDT540
60
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NOV2 : 541 RTIVWICLTLITGLWLLLLLICKKRHCGYSKAFQDSDEEKMHYQNGQAPPPVFLPLHHP 600
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
SbjCt: 541 RTIVWICLTLITGLWLLLLLICKKRHCGYSKAFQDSDEEKMHYQNGQAPPPVFLPLHHP 600
S NOV2 : 601 PGKLPEPQFYAEPHTYEEPGRAGRSFTREIEASRIHIEKIIGSGDSGEVCYGRLRVPGQR 660
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct: 601 PGKLPEPQFYAEPHTYEEPGRAGRSFTREIEASRIHIEKIIGSGDSGEVCYGRLRVPGQR 660
to
1S
NOV2 : 661 DVPVAIKALKAGYTERQRRDFLSEASTMGQFDHPNIIRLEGWTRGRLAMIVTEYMENGS 720
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct: 661 DVPVAIKALKAGYTERQRRDFLSEASIMGQFDHPNIIRLEGWTRGRLAMIVTEYMENGS 720
NOV2 : 721 LDTFLRTHDGQFTIMQLVGMLRGVGAGMRYLSDLGWHRDLAARNVLVDSNLVCKVSDFG 780
Illlllllllllllllllllllllllllllllllllllllllllllllllllllllllll
SbjCt: 721 LDTFLRTHDGQFTIMQLVGMLRGVGAGMRYLSDLGYVHRDLAARNVLVDSNLVCKVSDFG 780
NOV2 781LSRVLEDDPDAAYTTTGGKIPIRWTAPEAIAFRTFSSASDVWSFGVVMWEVLAYGERPYW840
: IIII
I
II
III
IIII
Sbjct: 781I 840
II
I
I
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
LSRVLEDDPDAAYTTTGGKIPIRWTAPEAIAFRTFSSASDWSFGWMWEVLAYGERPYW
20
NOV2 841NMTNRDVISSVEEGYRLPAPMGCPHALHQLMLDCWHKDRAQRPRFSQTVSVLDALIRSPE900
: lllllllll
1l
llllllll
SbjCt: 841l 900
l
Illllllllllllllllllllllllllllllllllllll
NMTNRDVISSVEEGYRLPAPMGCPHALHQLMLDCWHKDRAQRPRFSQIVSVLDALIRSPE
2S NOV2 901SLRATATVSRCPPPAFVRSCFDLRGGSGGGGGLTVGDWLDSIRMGRYRDHFAAGGYSSLG960
: llllllllllllllllllllll
SbjCt: 901Illlllllllllllllllllllllllllllllllllll 960
SLRATATVSRCPPPAFVRSCFDLRGGSGGGGGLTVGDWLDSIRMGRYRDHFAAGGYSSLG
NOV2 961MVLRMNAQDVRALGITLMGHQKKILGSIQTMR 992 (SEQ ID
: NO: 5)
30 Illlllllllllllllllllllllllllllll
Sbjct: 961MVLRMNAQDVRALGTTLMGHQKKTLGSIQTMR 992 (SEQ ID
NO: 53)
Table 7.
Multiple ignment of the NOV2 ephrin type-A receptor e invention,
al 8-like protein of th
3S shown as
AL035703
Spliced2,
with
similar
proteins
Table 7 shows multiple sequence alignment of the NOV2 ephrim type-A receptor 8-
like
protein of the invention, shown as AL03S703 Spliced2 , with similar proteins.
The various aligned
proteins are as follows: AL03S703 Spliced2 (SEQ ID NO:S) with several
proteins: EPA8 Mouse
40 (SEQ ID NO:S4), EPAS Human (SEQ ID NO:SS) and EPAS Chick (SEQ ID NO:S6).
(Black
outlined amino acids indicate potential regions of conserved sequence; greyed
amino acids
represent amino acids conservatively substituted; and non-highlighted amino
acids indicate
positions in which mutations to a broad range of alternative amino acid
residues occurs)
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WO 02/30979 PCT/USO1/31498
ALD35703 Spliced2 _ _ _ _ _i APR ' _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
_ PA VVTAAA AT~S AR~_
EPA8MOUSE -----i Ap RA 5-----------------------PA VUT-AA 'AT S'GR
EPASHUMAN MRCS PRG GH R'P5 GDTPITPASLAGCYSAPRRAP TCL LC 'LRT L PSN
EPASCHICK ---M LRGG'=G --A~---------------P---APG TCL~LC 'LRSL' P~S
ALD35703 Spliced2 T S IH L:T ' AH 'S ~ S FQ ~ N S Pi v 1PR
EPA8MOUSE STS IH ~~ LT 'AH S n SFR' ~ N SPi v N ~pR
EPAS HUMAN ~ 5 R V M ~ L I A:F ' K N EE " ~ N Y ~ K E Qi v L x 5 N
EPAS CHICK ~ ~ R VM L T A ' KN EE ~ N'Y ' ~ K EQ~ v L I~SN
AL035703 Spliced2 T? I I ' yT' U L L R S T 5 i FLi ~
EPA8 MOUSE ~ I i ' ~ I' ' V ' LH L ~ R ~ S T S ~ F L i ~
SPAS HUMAN E ' S ' L I?I L i ' ~ ' G F ~ D Q N N I K ~ Ia
EPAS CHICK E R P A S S F L i ' ~ ' G ,. F D E D N I R x i
AL035703 Spliced2 G A ~ V ~' R L i ' S ~ I= ' L T L ~:, ~ I ~Yi '
EPA8 MOUSE G A ~ V i R Lt ~ I It I L I> t I M '
EPAS~HUNIAN EL ~ Di UTUD ' ~ ' ~ U I LY yi S Y ' H
SPAS CHICK E L ~ Di VIuIi ~ ' ~ ~ ~ I, ~ L.~; IFy :, S ~ L1
AL035703 5pliced2 S~EAV ' ~ S V. R EERII 5 Ri
EPA8 MOUSE S~EA:T~T ' ~ 5 'SI R ~ EERTJ 5 ~R~
EPA HUMAN V FT,?.T,IL S S TD';EP ' K ,;NC
EFAS CHICK ' R PT~L°' ' ~ 1. 5 TD E',A ' Ly° K ' NN
AL0357D3 Spliced2 A V A E L Ire 5 A G ~L ' ' S A P A Q A H 'Y3' A A Lu ~; S
EPA8 MOUSE A A E L ~ ~ 5 A G ~ ~ L ' S ~T P ' A Q T R Y~' A A Lu ' ' S,A
EPAS HUMAN T ~ V RP F'i A S 'H I S _GK ' ' TH~~E S T V E;KD Fj RESu' ' n
EFAS CHICK T ~ V RP Fi A S H S P S 5~~ ' ' TLI~'E 5 T L' 'EH ~3 RESi ' i '
AL035703 Spliced2 V L S G A ' Lu P ' S a I T N A V R R P W A S R T H?~' ' ~ v
5
EPA8 MOUSE ' V~L S G A' ' Lnp 'Si iT NAV RR PWA~SH ~ Ti'pV' m S
EPAS_HUNIAN ' R A N E F I ' nT ' nV a Y I A K~ S HAGS' E GHUi L' R SG
EPAS CHICK ' R S A N E F I ' ' ~ T n V::S Y I A K T- S H S G L V i rI. ' "
AL035703 Spliced2 V i~ A I. L. 'F A N S ~ ' E P i R V I ~ i R'Q E~L.~A
EPA8 MOUSE AAA I. L'~;A N ' S'i ' N ' E P i S V I i R!QEIA
EPAS HUMAN N;T VMI~jIVD T T- E ' ~ GAi i YVS V ~ ' P N~k.'KGI~~I
EPAS CHICK LT"T YiVIIYIVD ' T TE ' QN GAi ~ FVS t~. ~ ' F SS~h~KG,T~3I
NOV3
A NOV3 nucleic acid sequence according to the invention includes nucleic acids
encoding
a polypeptide related to proteoglycans such as fibromodulin and fibronectin.
An example of this
nucleic acid and its encoded polypeptide is presented in Table ~. The
disclosed nucleic acid
sequence (SEQ ID N0:6) is 2025 nucleotides in length and contains an open
reading frame that
begins with an ATG initiation codon at nucleotides 1-3 and ends with a TGA
stop codon at
nucleotides 2023-2025.
The representative ORF includes a 674 amino acid polypeptide (SEQ ID NO: 7).
The
encoded polypeptide has a high degree of homology to several leucine-rich
repeat members of the
proteogylcan family found in the extracellular matrix including fibronectin
(Table 9 human
fibronectin like proteins, 99 percent to AAF2~459.1 (Lacy et al.,
"Identification of FLRTl,
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FLRT2, and FLRT3: a novel family of transmembrane leucine-rich repeat
proteins," 62(3)
Genomics 417-426 (1999)) and fibromodulin (Table 10, various fibromodulin or
fibromodulin-
like proteins).
The extracellular matrix (ECM) is composed of collagens, protoglycans, and
noncollagenous glycoproteins that provide cells and tissues with a mechanical
scaffold for
adhesion, migration, and signal transduction (Aumailley and Gayruad, 76(3-4)
J. Mol. Med. 253-
265 (1998)). These varied and complex functions depend on interactions between
ECM
components and cellular receptors such as protoglycans that are located on the
cell surface.
Fibronectins and fibromodulins are both protoglycans that comprise the
extracellular matrix.
Disruption of the cell-matrix interactions due to mutations in the genes of
the matrix proteins can
result in functional failures in all tissues (Bruclcner-Tudennan and Bruckner,
76(3-4) J. Mol. Med.
76(3-4) 226-237(1998)). Included in these disorders are the congenital
muscular dystrophies,
various muscle disorders, fixed deformities (arthrogryposis), and abnormal
white matter by
cranial MRI.
Fibronectins are glycoproteins with 2 chains each linked by disulphide bonds
that occur in
insoluble fibrillar form in the extracellular matrix of animal tissues and
soluble in plasma, the
latter previously known as cold insoluble globulin. The various slightly
different forms of
fibronectin appear to be generated by tissue specific differential splicing of
fibronectin mRNA,
transcribed from a single gene. Fibronectins have multiple domains that confer
the ability to
interact with many extracellular substances such as collagen, fibrin and
heparin and also with
specific membrane receptors on responsive cells. Notable is the RGD domain
recognized by
integrins and two repeats of the EGF like domain. Interaction of a cell's
fibronectin receptors
(members of the integrin family) with fibronectin adsorbed to a surface
results in adhesion and
spreading of the cell.
Fibromodulin is collagen-binding protein component of the proteoglycan found
in the
extracellular matrix. It is mainly expressed in articular cartilage, tendon,
and ligament, and is a
member of a group of proteins having leucine-rich repeat (LRR) domains;
fibromodulin includes
as many as ten such motifs. Other components of this family include decorin,
biglycan, and
lumican. Proteins of this family bind to other matrix macromolecules and
thereby help to
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stabilize the matrix. These proteins may also influence the function of the
chondrocytes and bind
growth factors.
The core proteins of these proteoglycans are structurally related, consisting
of a central
region composed of leucine-rich repeats flanked by disulfide-bonded terminal
domains.
Fibromodulin's central region possesses up to 4 lceratan sulfate chains within
its leucine-rich
domain. Fibromodulin exhibits a wide tissue distribution, with the highest
abundance observed in
articular cartilage, tendon, and ligament. It has been suggested that
fibromodulin participates in
the assembly of the extracellular matrix by virtue of its ability to interact
with type I and type II
collagen fibrils and to inhibit fibrillogenesis in vitro. The 3-prime
untranslated region of the
fibromodulin cDNA has previously been cloned and used to map the gene by
fluorescence in situ
hybridization to 1q32. (Sztrolovics et al., 23 Genomics 715-717 (1994)). In
that study, secondary
signals were detected at 9q34.1; however, PCR analysis of somatic cell hybrids
confirmed the
localization to chromosome 1.
Small proteoglycans, including decorin, biglycan, and fibromodulin, bind to
other matrix
macromolecules and thereby help to stabilize the matrix. They may also
influence the function of
the chondrocytes and bind growth factors.
In a bovine nasal-cartilage culture system, it was found that interleukin-1
stimulated
cartilage catabolism included the effect that the small leucine-rich repeat
proteoglycans decorin,
biglycan and lumican showed a resistance to both proteolytic cleavage and
release throughout the
culture period. In contrast, fibromodulin exhibited a marked decrease in size
after day 4,
presumably due to proteolytic modification (Sztrolovics R, et al., 339 (Pt 3)
Biochem. J., 571-577
(1999)).
The nucleic acids and proteins of the invention are useful in potential
therapeutic
applications implicated in various orthopedic disorders and/or injuries. They
are potentially of
use in aiding repair of damage to cartilage and ligaments, and in therapeutic
applications to joint
repair. Additionally they may be used in treatment of inflammatory diseases of
connective tissue,
including by way of nonlimiting example, rheumatoid arthritis, congenital
muscular dystrophies,
various muscle disorders, fixed deformities (arthrogryposis), and abnormal
white matter. For
example, a cDNA encoding the proteoglycan-like protein may be useful in gene
therapy, and the
proteoglycan -like protein may be useful when administered to a subject in
need thereof. The
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novel nucleic acid encoding proteoglycan -like protein, and the proteoglycan -
like protein of the
invention, or fragments thereof, may further be useful in diagnostic
applications, wherein the
presence or amount of the nucleic acid or the protein are to be assessed. For
example in
identifying tissue from kidney or brain. These materials are further useful in
the generation of
S antibodies that bind immunospecifically to the novel substances of the
invention for use in
therapeutic or diagnostic methods.
Table 8
A representative DNA sequence of the proteoglycan-like protein of the
invention
ATGGTGGTGGCACACCCCACCGCCACTGCCACCACCACGCCCACTGCCACTGTCACGGCCACCGTTGTGA
TGACCACGGCCACCATGGACCTGCGGGACTGGCTGTTCCTCTGCTACGGGCTCATCGCCTTCCTGACGGA
GGTCATCGACAGCACCACCTGCCCCTCGGTGTGCCGCTGCGACAACGGCTTCATCTACTGCAACGACCGG
GGACTCACATCCATCCCCGCAGATATCCCTGATGATGCCACCACCCTCTACCTGCAGAACAACCAGATCA
IS ACAACGCCGGCATCCCCCAGGACCTCAAGACCAAGGTCAACGTGCAGGTCATCTACCTATACGAGAATGA
CCTGGATGAGTTCCCCATCAACCTGCCCCGCTCCCTCCGGGAGCTGCACCTGCAGGACAACAATGTGCGC
ACCATTGCCAGGGACTCGCTGGCCCGCATCCCGCTGCTGGAGAAGCTGCACCTGGATGACAACTCCGTGT
CCACCGTCAGCATTGAGGAGGACGCCTTCGCCGACAGCAAACAGCTCAAGCTGCTCTTCCTGAGCCGGAA
CCACCTGAGCAGCATCCCCTCGGGGCTGCCGCACACGCTGGAGGAGCTGCGGCTGGATGACAACCGCATC
ZO TCCACCATCCCGCTGCATGCCTTCAAGGGCCTCAACAGCCTGCGGCGCCTGGTGCTGGACGGTAACCTGC
TGGCCAACCAGCGCATCGCCGACGACACCTTCAGCCGCCTACAGAACCTCACAGAGCTCTCGCTGGTGCG
CAATTCGCTGGCCGCGCCACCCCTCAACCTGCCCAGCGCCCACCTGCAGAAGCTCTACCTGCAGGACAAT
GCCATCAGCCACATCCCCTACAACACGCTGGCCAAGATGCGTGAGCTGGAGCGGCTGGACCTGTCCAACA
ACAACCTGACCACGCTGCCCCGCGGCCTGTTCGACGACCTGGGGAACCTGGCCCAGCTGCTGCTCAGGAA
ZS CAACCCTTGGTTTTGTGGCTGCAACCTCATGTGGCTGCGGGACTGGGTGAAGGCACGGGCGGCCGTGGTC
AACGTGCGGGGCCTCATGTGCCAGGGCCCTGAGAAGGTCCGGGGCATGGCCATCAAGGACATTACCAGCG
AGATGGACGAGTGTTTTGAGACGGGGCCGCAGGGCGGCGTGGCCAATGCGGCTGCCAAGACCACGGCCAG
CAACCACGCCTCTGCCACCACGCCCCAGGGTTCCCTGTTTACCCTCAAGGCCAAAAGGCCAGGGCTGCGC
CTCCCCGACTCCAACATTGACTACCCCATGGCCACGGGTGATGGCGCCAAGACCCTGGCCATCCACGTGA
3O AGGCCCTGACGGCAGACTCCATCCGCATCACGTGGAAGGCCACGCTCCCCGCCTCCTCTTTCCGGCTCAG
TTGGCTGCGCCTGGGCCACAGCCCAGCCGTGGGCTCCATCACGGAGACCTTGGTGCAGGGGGACAAGACA
GAGTACCTGCTGACAGCCCTGGAGCCCAAGTCCACCTACATCATCTGCATGGTCACCATGGAGACCAGCA
ATGCCTACGTAGCTGATGAGACACCCGTGTGTGCCAAGGCAGAGACAGCCGACAGCTATGGCCCTACCAC
CACACTCAACCAGGAGCAGAACGCTGGCCCCATGGCGAGCCTGCCCCTGGCGGGCATCATCGGCGGGGCA
3S GTGGCTCTGGTCTTCCTCTTCCTGGTCCTGGGGGCCATCTGCTGGTACGTGCACCAGGCTGGCGAGCTGC
TGACCCGGGAGAGGGCCTACAACCGGGGCAGCAGGAAAAAGGATGACTATATGGAGTCAGGGACCAAGAA
GGATAACTCCATCCTGGAAATCCGCGGCCCTGGGCTGCAGATGCTGCCCATCAACCCGTACCGCGCCAAA
GAGGAGTACGTGGTCCACACTATCTTCCCCTCCAACGGCAGCAGCCTCTGCAAGGCCACACACACCATTG
GCTACGGCACCACGCGGGGCTACCGGGACGGCGGCATCCCCGACATAGACTACTCCTACACATGA (SEQ ID
N0:6)
A representative amino acid sequence of the proteoglycan-like protein of the
invention
MWAHPTATATTTPTATVTATVVMTTATMDLRDWLFLCYGLIAFLTEVIDSTTCPSVCRCDNGFIYCNDR
GLTSIPADIPDDATTLYLQNNQINNAGIPQDLKTKVNVQVIYLYENDLDEFPINLPRSLRELHLQDNNVR
4S TIARDSLARIPLLEKLHLDDNSVSTVSIEEDAFADSKQLKLLFLSRNHLSSIPSGLPHTLEELRLDDNRI
STIPLHAFKGLNSLRRLVLDGNLLANQRIADDTFSRLQNLTELSLVRNSLAAPPLNLPSAHLQKLYLQDN
AISHIPYNTLAKMRELERLDLSNNNLTTLPRGLFDDLGNLAQLLLRNNPWFCGCNLMWLRDWVKARAAW
NVRGLMCQGPEKVRGMAIKDITSEMDECFETGPQGGVANAAAKTTASNHASATTPQGSLFTLKAKRPGLR
LPDSNIDYPMATGDGAKTLAIHVKALTADSIRITWKATLPASSFRLSWLRLGHSPAVGSITETLVQGDKT
SO EYLLTALEPKSTYIICMVTMETSNAWADETPVCAKAETADSYGPTTTLNQEQNAGPMASLPLAGIIGGA
VALVFLFLVLGAICWWHQAGELLTRERAYNRGSRKKDDYMESGTKKDNSILEIRGPGLQMLPINPYRAK
EEYVVHTIFPSNGSSLCKATHTIGYGTTRGYRDGGIPDIDYSYT (SEQ ID NO: 7)
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Table 9. Multiple sequence and BLAST alignment of a NOV3 polypeptide and the
human fibronectin leucine repeat transmembrane family.
Table 9 shows sequence alignment between a NOV3 polypeptide with several
members of the
S human fibronectin leucine repeat transmembrane family: AAF28461.1 ~AF169
(SEQ ID NO:S7),
AAF28460.1~AF169 (SEQ ID NO:SB) and AAF284S9.1~AF169 (SEQ ID NO:S9). (Black
outlined
amino acids indicate potential regions of conserved sequence; greyed amino
acids represent
amino acids conservatively substituted; and non-highlighted amino acids
indicate positions in
which mutations to a broad range of alternative amino acid residues occurs).
100
..
AAF28461.1~AF169 1 -----------------------
1 S 'I,.~.WSIFLIGTKIGLF'QVAPLS~M~'SK~ F T ~S 77
AAF28460.1IAF169 1 -- -----------MGLQT KWP3HG FF ACS ITS---
,GLY~Q,SKLLp~RN~V'E]S-t?~L-G.. F~ 82
A_AF28459.1~AF169 1
100
NOV3 1
100
110 120 130 140 150 160 170 180
2S l90 200
.,....,....,....i..~...~ .~, y...y.. i.....,....i....i....i....i....i
AAF28461.1~AF169 78 LL R IS T~YI TY~SIt-
~Y~7. Y R '.~ W R 17 6
AAF28460.1 ~ AF169 83 E QS ~~T~I Q I t1 -Q~Q-
~Lx-~~Gv/~ HIS v vn 181
AAF28459.1~AF169 101
199
NOV3 101
3S 199
210 220 230 240 250 260 270 280
290 300
AAF28461.1~AF169..177..~....~....~....~....I....I....1....I....~....I....~....I
....~....~....I....I
~~SP~L-~IH1~GI~IF~~?IR~~R~/P~Y~~IYI 2 7 6
AAF28460.1~AF169 182
~~TL~1/~~~E~'~mI~Y''.~ITKIKIFIx)~IP~~I~LT~K~ 281
AAF28459.1~AF169 200
4S 299
NOV3 200
299
310 320 330 340 350 360 370 380
S0 390 400
AAF28461.1IAF169 277 ~SN~,I~'IT~I~QSL~VK-A~.F~~KL3S,-------
__ItVS'I!IQI, 367 ...
S S AAF28460.1IAF169 282
-Q,RM'T~S~K~TA-D,ST-T~,F~-YT,3SL-F-Q_VItE~LS,PT,T,GLPLFTPAPS'T,9P~ 381
AAF28459.1 AF169 300 --
395
NOV3 300 --
60 - 395
410 420 430 440 450 460 470 480
490 500
6S ..
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AAF28461.11AF169 368
f~SIPN~VYPA~QWPA~V.'~'~,Q.D'~1~KLTK~T~~.",GS,SR-'~iT~S~S~T~L~MT~L-F-~~.5~ 467
AAF28460.1~AF169 382
QPPTLLPN,SR'YTP,TPTTSK,PTIPDWDGR~RV'P'I,,SER'QIaSI,HF~LTDT~QQV~L~LFT,VN~XK=T'~L
VG~I~1Q'R~S-QHLSrV 481
AAF28459.1~AF169 396
495
NOV3 396
495
1 0 510 520 530 540 550 560 570 580
590 600
1 5 LAF28461.1SAF169C~4656~D'P~L'LF~I~~PLRi~-~R~EKE,YK~P.. ~T~A~-
AAF28460.1IAF169 482 N~.LTD1F,-
,R~T,1~S~T~fiA,Yx.,NN,SN~S~FI.T'~'~SHSMGSPFL~L_IF,L~~-
VFILFC,RYT,QKWFC, 579
AAF28459.1 AF169 496
590
20 NOV3 496 - -
590
25 AAF28461.1~AF169 566 649
AAF28460.1~AF169 580 660
AAF28459.1~AF169 591 674
NOV3 591 674
TABLE
9.
continued
>refINP037412.11 fibronectin leucine rich transmembrane
protein 1
gbIAAF28459.11AF169675
1
(AF169675)
leucine-rich
repeat
transmembrane
protein
FLRT1
fHomo
sapiens]
Length = 674
Score = 1365 bits (3494), Expect = 0.0
Identities = 673/674 (99%), Positives = 674/674 (99%)
NOV3: 1 MVVAHPTATATTTPTATVTATVVMTTATMDLRDWLFLCYGLIAFLTEVIDSTTCPSVCRC60
l
l
l
l
Illllllllllllllllllllllllllllllll
Sbjct:lllllllll 60
l
lllllllll
llll
1 MWAHPTATATTTPTATVTATVVMTTATMDLRDWLFLCYGLIAFLTEVIDSTTCPSVCRC
NOV3: 61 DNGFIYCNDRGLTSTPADIPDDATTLYLQNNQINNAGIPQDLKTKVNVQVIYLYENDLDE120
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
SbjCt:IIIIIIIIIIIIIIIIIIIIIIIIIIIII 120
61 DNGFIYCNDRGLTSTPADIPDDATTLYLQNNQINNAGIPQDLKTKVNVQVIYLYENDLDE
SO NOV3: 121 FPINLPRSLRELHLQDNNVRTIARDSLARIPLLEKLHLDDNSVSTVSIEEDAFADSKQLK180
IIIII
I
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct:IIIII 180
IIIIIIIIII
121 FPTNLPRSLRELHLQDNNVRTIARDSLARIPLLEKLHLDDNSVSTVSIEEDAFADSKQLK
NOV3: 181 LLFLSRNHLSSIPSGLPHTLEELRLDDNRISTIPLHAFKGLNSLRRLVLDGNLLANQRIA240
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct:181 LLFLSRNHLSSIPSGLPHTLEELRLDDNRISTIPLHAFKGLNSLRRLVLDGNLLANQRIA240
NOV3: 241 DDTFSRLQNLTELSLVRNSLAAPPLNLPSAHLQKLYLQDNAISHIPYNTLAKMRELERLD300
Illlllllllllllllllllllllllllllllllllllllllllllllllllllllllll
6O Sbjct:241 DDTFSRLQNLTELSLVRNSLAAPPLNLPSAHLQKLYLQDNAISHIPYNTLAKMRELERLD300
NOV3: 301 LSNNNLTTLPRGLFDDLGNLAQLLLRNNPWFCGCNLMWLRDWVKARAAVVNVRGLMCQGP360
II
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
Sbjct:IIIIIIIIIIIIIIIIII 360
301 LSNNNLTTLPRGLFDDLGNLAQLLLRNNPWFCGCNLMWLRDWVKARAAVVNVRGLMCQGP
NOV3: 361 EKVRGMAIKDITSEMDECFETGPQGGVANAAAKTTASNHASATTPQGSLFTLKAKRPGLR420
Ill
Ill
lllllll
Sbjct:lllllllllllllllllllllllllllll 420
llllllllllllllllll
361 EKVRGMAIKDITSEMDECFETGPQGGVANAAAKTTASNHASATTPQGSLFTLKAKRPGLR
25
610 620 630 640 650 660 670 680
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NOV3: 421LPDSNIDYPMATGDGAKTLAIHVKALTADSTRITWKATLPASSFRLSWLRLGHSPAVGSI480
111111111
11
SbjCt: 421111111111111111111111111111111111111111111111111480
1
LPDSNIDYPMATGDGAKTLAIHVKALTADSIRITWKATLPASSFRLSWLRLGHSPAVGSI
NOV3: 481TETLVQGDKTEYLLTALEPKSTYIICMVTMETSNAYVADETPVCAKAETADSYGPTTTLN540
IIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
SbjCt: 481I 540
TETLVQGDKTEYLLTALEPKSTYIICMVTMETSNAYVADETPVCAKAETADSYGPTTTLN
1O NOV3: 541QEQNAGPMASLPLAGIIGGAVALVFLFLVLGAICWYVHQAGELLTRERAYNRGSRKKDDY600
IIIllllllllllllllllllllllllllllllllllllllllllllllllllll+1111
SbjCt: 541QEQNAGPMASLPLAGIIGGAVALVFLFLVLGAICWYVHQAGELLTRERAYNRGSREKDDY600
NOV3: 601MESGTKKDNSILEIRGPGLQMLPINPYRAKEEYWHTIFPSNGSSLCKATHTIGYGTTRG660
IIIIIIIIIIIIIII111111111111111111111111111111111111111111111
SbjCt: 601MESGTKKDNSILEIRGPGLQMLPINPYRAKEEYWHTIFPSNGSSLCKATHTIGYGTTRG660
NOV3: 661YRDGGIPDTDYSYT 674 (SEQ ID N0: 7)
Illlllllllllll
SbjCt: 661YRDGGIPDIDYSYT 674 (SEQ ID NO: 60)
Table 10. Multiple Sequence alignment between NOV3 polypeptide and various
fibromodulins
(NOV3 is denoted AP000597_GENSCAN 3)
Table 10 shows sequence alignment between a NOV3 polypeptide with various
fibromodulins:
ACC NO: 043408 (SEQ ID N0:61), ACC NO: AP000597 (SEQ ID N0:7), ACC NO: 043155
(SEQ m N0:62), ACC NO: 042235 (SEQ m N0:63) and FMOD Bovin FM (SEQ >I7 N0:64).
(Black outlined amino acids indicate potential regions of conserved sequence;
greyed amino acids
represent amino acids conservatively substituted; and non-highlighted amino
acids indicate
positions in which mutations to a broad range of alternative amino acid
residues occurs)
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WO 02/30979 PCT/USO1/31498
043408 HHypotltetncal _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
AP000597!GENSCAN3 I.~SNNNLT~L.'PR~LFL7~DLGNLA~LLLN F LMWLRDWVKARAAVVNyRGLMCQG'
043155 Humsn-KIA ISNNQLRML.TQ VF'pNLSNLK LTAiy~N F S I KWVTEWLKYI
PSSLNV~RGFMGQG'
042235 Chicken-KSP V'H S VWTR VRQVYN,E LDPEHWSH YTFEC ' Q E F PP S F~PNA
LYCDNKG~KE I'P - - - -AI '
FMODBOVIN-FM PYEP-YPBGEEBPAYAYGSPP~PEFiy~DC'QE PPN:~'jPTAMYCDNRNLKYL:P----FV
0434D8_Huxnan-Hypothetical _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
AP000597_GENSCAN_3 ET''VRGMAIKDITSEIt?IDECFETG' ----
QGGVANAAAKTTAS~HAS'ATTPQGSLFTLKAKR
043155HuxnanKIA EQ~TVR.GMAVRELhT,M LLSCPTTT~GLPL TP
PSTASPTTQPPTLS'IPNPSRSYTPPTPTT
042235 Chicken KSP ARIVK~T-=-YLYT.Q~N~1~IETISEK --
~VN~HLRWINLNK~IT,NNGIESGVLS'KLKRL
FMODBOVINFM - ,.S',R~vI ' -YVYF N QI'SSIQEGV---- DN GLLWIALHG
IT'SDKVGKK.QFaKLRHL
043408 Human Hypothetical - - - - - - - - - - - P fYyA DCxAKT-L. A' IH~'K A
TAD R~I "r: ~A,T PA S S ~R S L:'-RL PA
AP000597 GENSCAN 3 PG ROPDSN IDYP~v2A D't,~AKTT,AIHtrKA~TAD RIx a~T~PAS S~R S
LRL PA
043155Humsn-KIA SK PTIPDWDGRERV~P I,SER'IQ S~IfiFV:,NDT
Q:'~~"jLS,LFTV~uL'Af~K~T~'SfT4IyI~LaV
042235 Chicken KSP LY F EDN- - E~L.EEV PLP~V_'G EQLR ARNK SRI
PE_f°.zVFSN~L.ENLTIvILDLHQNNL,L
FMODBOVINFM ER YeDHN-- -NL;TRI-~SPLPRS~RELH~DHNQ SRVFNNA~E12~L;ENLTALYLHHNET;Q
043408 Human_Hypothetical V~ S ~I T I. W' E Y A IC T ~t=IE~ S A V ~ P K D - - -
APDOD597_GENSGAN 3 V ~S I T~L ~T E Y A~TCI.~ ~'tE~S ~A~V P K D - - -
043155-Human_KIA G ~ I V Q I; S E Q H 1L.'5 N I~. -R L ' L I2A F R T I-~ S E T
A LNN
042235 Chicken-KSP D S ALQ S DT F LN'S'L~~ITQ I AKN S LKKMP L S x AN~L f,~l-
LF LmNN S I~tEV I P NYF A- - I
FIvIODBOVIN-FIV E---VG'w.S~e~K L'RSLI LSYNHLRK~PDGL'SALE?Qi-
LYL~HNN~FSVP~T?'~SYFRG--S
043408 Huxnsn_Hypothetical ~P T T Q ~ G P MA S ' - ~I A L F T.F T~T~LGA I V A
E L'TiR E RA
AP000597-GENSCAN3 ~PT T~hTQ ~ GPMAS '--~I AL FLF C~~LGAIV A~EBL"F~RERA~
043155Huxnan-KIA ~5N ASSH TSHSM S'FL~T; ~ IF FV -VF It~I -K RYTSQKWK
042235 Chicken KSP P K V F ~R. L N Y~ L 5 D D I - - - - - P N F N S S ~It ,L;Q
- - - - - - Q T K I F P I NA
FMODBOVINFM PKLLY~,VRLSHi SLTNN ----SNTFNSSSh ELD ------YET;--QBQKIFPVST
043408 Huxnsn-H othetical S Y;, ~~ S ~ T RGPGL LPG. F RAi E~YiVtlHT FPS S S
LCi T
AP000597-GENSCAN 3 S I; ~ ~ H S ~ I RCP G L ~ L P I F ~R Ai E E I!V ~TJ:,H T
F:P S S S L C y.ST
043155 HKIA ~-~I~ m G~A ~ TETSF n.I VSL~NDQLLG~FRL<QP~"TP INYTDC
042235 Chicken KSP HLEHLHL HNRIK~SVNGTQ CPVS------IA'~'AED~GLYGNIPRLRYLRLD -
NEIQP
FMODBOVIN-FM NLENLYLQGNRINEFSIS FCTXX'VD----- --------VMNF=SKLQixQRLD -EI~S
043408 Huxnsn-Hypothetical T I G Y G T T R G D G G I~ ~ Y S Y
APOD0597 GENSCAN 3 ~ T I G Y G T T R G D G G I Y S Y
043155 Humsn KIA I.~- - -NNMR N S S V~~LE~HCH
042235 Chicken KSP P I ~~- - LD I MI F Q L>LQAVV I - - -
FMOD BOVIN -FM Ah~I - - AD A P L L R L~A S LSE I - - -
NOV4
A NOV4 nucleic acid according to the invention includes nucleic acids encoding
a
polypeptide related to ephrin type-A receptors. An example of nucleic acid and
its encoded
polypeptide is presented in Table 11. The disclosed nucleic acid (SEQ ID NO:
65) is 1545
nucleotides in length.
The representative ORF includes a 515 amino acid polypeptide (SEQ ID NO: 66).
The
encoded polypeptide has a high degree of homology (approximately 95 percent
identity) with
mouse ephrin type-A receptor 8. NOV4 is a sequence variant of NOV2 and, as
such, is a member
of the ephrin type-A receptor tyrosine-protein kinase family.
The Eph receptors constitute the largest known family of receptor protein
tyrosine kinases.
They have been implicated in mediating developmental events, particularly in
the nervous system.
Receptors in the Eph subfamily typically have a single kinase domain and an
extracellular region
containing a Cys-rich domain and two fibronectin type III repeats. These
receptors play important
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roles along with their ligands, called ephrins, in neural development,
angiogenesis, and vascular
networlc assembly. (S. Choi et al., 9(4) Mol. Cells 440-45 (1999)).
The ephrin type-A receptor 8 (EC 2.7.1.112) (tyrosine-protein kinase receptor
eek) (eph-
and ells-related kinase) (fragment) is designated as the gene product of the
gene: epha8 oy eek. It
is a Type I membrane bound receptor, and its function is to serve as a
receptor for members of the
ephrin-a family. Its catalytic activity is as a protein tyrosine kinase,
phosphorylating tyrosine in
appropriate target proteins. It is similar to other protein-tyrosine kinases
in the catalytic domain
and belongs to the ephrin receptor family.
Eph receptors have tyrosine-kinase activity, and, together with their ephrin
ligands,
mediate contact-dependent cell interactions that are implicated in the
repulsion mechanisms that
guide migrating cells and neuronal growth cones to specific destinations.
Since Eph receptors and
ephrins have complementary expression in many tissues during embryogenesis,
bidirectional
activation of Eph receptors may occur at interfaces of their expression
domains, for example, at
segment boundaries in the vertebrate hindbrain. Indeed, Eph receptors play key
roles in
development of the nervous system and angiogenesis. hi the nervous system,
they provide
positional information by empolying mechanisms that involve repulsion of
migrating cells and
growing axons (Frisen et al., 18(19) EMBO J. 5159-5165 (1999)). Also, an
important function of
Eph receptors and ephrins is to mediate cell-contact-dependent repulsion.
A NOV4 sequence according to the invention is useful for detecting cells that
express
GPI-anchored ephrin-A ligands. For example, cells expressing either a NOV4
nucleic acid or a
NOV4 protein have utility in screening for other cells that express GPI-
anchored ephrin-A ligands
or mimics therefore. As a result, a NOV4 sequence is useful for screening for
new ephrin-A
ligands expressed on cells. NOV4 is highly expressed in many surgical tumor
samples; therefore,
the NOV4 expression can be used as a marker for certain cancers.
Also, a NOV4 sequence according to the invention is useful to direct the
development of
the nervous system and angiogenesis by modulating the boundaries between
arteries and veins.
For example, mice expressing defective Eph receptors similar to a NOV4
sequence have been
shown to be defective in angiogensis and die in mid-gestation (Wang et al., 93
Cell 741-753
(1998)). The protein of the present invention will be useful in a variety of
diseases and
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pathologies, including by way of nonlimiting example, those involving
neurological, cardiac and
vascular pathologies.
NOV4 exhibited highest levels of expression in TAQMANR Reverse Transcription
runs in
testis (100%), 85976 Breast Cancer Mets (100%), breast cancer Res. Gen. (90%),
94909-XF-498
CNS-ssDNA (100%) and dermal fibroblast-IL-4 (100%) cell lines. Lower but
consistent levels of
expression were obtained in breast cancer (p1. effusion) T47D cells (15%),
melanoma UACC-62
(10%), breast cancer Clontech 9100266 (38%), 94925-NCI-H1155-large cell lung
cancer/neuroendocrine-ss cDNA (21 %), 94923-NCI-H82-small cell lung
cancer/neuroendocrine-
ss cDNA (18%), 94918-DMS-79-small cell lung cancer/neuroendocrine-ss cDNA
(19%) and
normal prostate clontech A+ 6546-1 (20%) cell lines. These results differ to
some degree from
the parent clone, NOV2, which did not exhibit high levels of expression in
central nervous
system, testis and fibroblast tissue, but did exhibit positive levels of
expression in cancer cell
lines. Such differences in expression reflect the distinction among variants.
It is noteworthy that
clone NOV4 is minimally or not detectably found in the immediate normal tissue
adjacent to
breast and lung tissue.
Table 11.
A representative DNA sequence encoding the ephrin type-A receptor 8-like
protein of the
invention.
GCGCGCGGCGAAGTGAATTTGCTGGACACGTCGACCATCCACGGGGACTGGGGCTGGCTC
ACGTATCCGGCTCATGGGTGGGACTCCATCAACGAGGTGGACGAGTCCTTCCAGCCCATC
CACACGTACCAGGTTTGCAACGTCATGAGCCCCAACCAGAACAACTGGCTGCGCACGAGC
TGGGTCCCCCGAGACGGCGCCCGGCGCGTCTATGCTGAGATCAAGTTTACCCTGCGCGAC
TGCAACAGCATGCCTGGTGTGCTGGGCACCTGCAAGGAGACCTTCAACCTCTACTACCTG
GAGTCGGACCGCGACCTGGGGGCCAGCACACAAGAAAGCCAGTTCCTCAAAATCGACACC
ATTGCGGCCGACGAGAGCTTCACAGGTGCCGACCTTGGTGTGCGGCGTCTCAAGCTCAAC
ACGGAGGTGCGCAGTGTGGGTCCCCTCAGCAAGCGCGGCTTCTACCTGGCCTTCCAGGAC
ATAGGTGCCTGCCTGGCCATCCTCTCTCTCCGCATCTACTATAAGA.AGTGCCCTGCCATG
GTGCGCAATCTGGCTGCCTTCTCGGAGGCAGTGACGGGGGCCGACTCGTCCTCACTGGTG
GAGGTGAGGGGCCAGTGCGTGCGGCACTCAGAGGAGCGGGACACACCCAAGATGTACTGC
AGCGCGGAGGGCGAGTGGCTCGTGCCCATCGGCAAATGCGTGTGCAGTGCCGGCTACGAG
GAGCGGCGGGATGCCTGTGTGGCCTGTGAGCTGGGCTTCTACAAGTCAGCCCCTGGGGAC
CAGCTGTGTGCCCGCTGCCCTCCCCACAGCCACTCCGCAGCTCCAGCCGCCCAAGCCTGC
CACTGTGACCTCAGCTACTACCGTGCAGCCCTGGACCCGCCGTCCTCAGCCTGCACCCGG
CCACCCTCGGCACCAGTGAACCTGATCTCCAGTGTGAATGGGACATCAGTGACTCTGGAG
TGGGCCCCTCCCCTGGACCCAGGTGGCCGCAGTGACATCACCTACAATGCCGTGTGCCGC(SEQID NO:
65)
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A representative amino acid sequence encoding the ephrin type-A receptor 8-
like protein of
the invention
ARGEVNLLDTSTIHGDWGWLTYPAHGWDSINEVDESFQPIHTYQVCNVMSPNQNNWLRTS
WVPRDGARRVYAEIKFTLRDCNSMPGVLGTCKETFNLYYLESDRDLGASTQESQFLKIDT
IAADESFTGADLGVRRLKLNTEVRSVGPLSKRGFYLAFQDIGACLAILSLRIYYKKCPAM
VRNLAAFSEAVTGADSSSLVEVRGQCVRHSEERDTPKMYCSAEGEWLVPIGKCVCSAGYE
ERRDACVACELGFYKSAPGDQLCARCPPHSHSAAPAAQACHCDLSYYRAALDPPSSACTR
PPSAPVNLISSVNGTSVTLEWAPPLDPGGRSDITYNAVCRRCPWALSRCEACGSGTRFVP
QQTSLVQASLLVANLLAHMNYSFWIEAVNGVSDLSPEPRRAAVVNITTNQAAPSQVVVIR
QERAGQTS V SLLW QEPEQPNGIILEYEIKYYEKDKEMQSYSTLKAVTTRATV SGLKP GTR
YVFQVRARTSAGCGRFSQAMEVETGKPRPRYDTRT (SEQ ID NO: 66)
NOVS
A NOVS nucleic acid sequence according to the invention includes nucleic acids
encoding
a polypeptide related to members of the proteoglycan family and specifically
to fibromodulin. An
example of this nucleic acid and its encoded polypeptide is presented in
Tables 12 and 13. The
disclosed nucleic acid sequence (SEQ ID NO: 67) is 2660 nucleotides in length
and contains an
open reading frame (ORF) that begins with an ATG initiation codon at
nucleotides 1-3 and ends
with an ATG stop codon at nucleotides 1993-1995. The representative ORF
includes a 664 amino
acid polypeptide (SEQ ID NO: 68) and is flanked by putative untranslated
regions, if any,
upstream from the initiation codon and downstream from the termination codon.
The encoded
polypeptide has a high degree of homology to several leucine-rich repeat
members of the
proteoglycan family found in the extracellular matrix including fibronectin.
The extracellular matrix (ECM) is composed of collagens, protoglycans, and
noncollagenous glycoproteins that provide cells and tissues with a mechanical
scaffold for
adhesion, migration, and signal transduction (Aumailley and Gayruad 76(3-4) J.
Mol. Med. 253-
265). These varied and complex functions depend on interactions between ECM
components and
cellular receptors such as protoglycans that are located on the cell surface.
The fibromodulin-like
gene disclosed in this invention is expressed in at least cartilage, tendon
and ligament tissues.
Fibronectins and fibromodulins are both protoglycans that comprise the
extracellular matrix.
Disruption of the cell-matrix interactions due to mutations in the genes of
the matrix proteins can
result in functional failures in all tissues (Bruckner-Tuderman and Bruckner
76(3-4) J. Mol. Med.
226-237(1998)). Included in these disorders are the congenital muscular
dystrophies, various
muscle disorders, fixed deformities (arthrogryposis), and abnormal white
matter by cranal MRI.
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Fibronectins are glycoproteins with 2 chains each linked by disulphide bonds
that occur in
insoluble fibrillar form in the extracellular matrix of animal tissues and
soluble in plasma, the
latter previously known as cold insoluble globulin. The various slightly
different forms of
fibronectin appear to be generated by tissue specific differential splicing of
fibronectin mRNA,
transcribed from a single gene. Fibronectins have multiple domains that confer
the ability to
interact with many extracellular substances such as collagen, fibrin and
heparin and also with
specific membrane receptors on responsive cells. Notable is the RGD domain
recognized by
integrins and two repeats of the EGF like domain. Interaction of a cell's
fibronectin receptors
(members of the integrin family) with fibronectin adsorbed to a surface
results in adhesion and
spreading of the cell.
Fibromodulin is collagen-binding protein component of the proteoglycan found
in the
extracellular matrix. It is mainly expressed in articular cartilage, tendon,
and ligament, and is a
member of a group of proteins having leucine-rich repeat (LRR) domains;
fibromodulin includes
as many as ten such motifs. Other components of this family include decorin,
biglycan, and
1 S lumican. Proteins of this family bind to other matrix macromolecules and
thereby help to
stabilize the matrix. These proteins may also influence the function of the
chondrocytes and bind
growth factors.
The core proteins of these proteoglycans are structurally related, consisting
of a central
region composed of leucine-rich repeats flanked by disulfide-bonded terminal
domains.
Fibromodulin's central region possesses up to 4 keratan sulfate chains within
its leucine-rich
domain. Fibromodulin exhibits a wide tissue distribution, with the highest
abundance observed in
articular cartilage, tendon, and ligament. It has been suggested that
fibromodulin participates in
the assembly of the extracellular matrix by virtue of its ability to interact
with type I and type II
collagen fibrils and to inhibit fibrillogenesis in vitro. The 3-prime
untranslated region of the
fibromodulin cDNA has previously been cloned and used to map the gene by
fluorescence in situ
hybridization to 1q32. (Sztrolovics et al., 23 Genomics 715-717(1994)). In
that study, secondary
signals were detected at 9q34.1; however, PCR analysis of somatic cell hybrids
confirmed the
localization to chromosome 1.
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Small proteoglycans, including decorin, biglycan, and fibromodulin, bind to
other matrix
macromolecules and thereby help to stabilize the matrix. They may also
influence the function of
the chondrocytes and bind growth factors.
In a bovine nasal-cartilage culture system, it was found that interleukin-1
stimulated
cartilage catabolism included the effect that the small leucine-rich repeat
proteoglycans decorin,
biglycan and lumican showed a resistance to both proteolytic cleavage and
release throughout the
culture period. In contrast, fibromodulin exhibited a marked decrease in size
after day 4,
presumably due to proteolytic modification (Sztrolovics et a1.,339(Pt3)
Biochem. J., 571-577,
(1999)).
The nucleic acids and proteins of the invention are useful in potential
therapeutic
applications implicated in various orthopedic disorders and/or injuries. They
are potentially of
use in aiding repair of damage to cartilage and ligaments, and in therapeutic
applications to joint
repair. Additionally they may be used in treatment of inflammatory diseases of
connective tissue,
including by way of nonlimiting example, rheumatoid arthritis, congenital
muscular dystrophies,
various muscle disorders, fixed deformities (arthrogryposis), and abnormal
white matter. NOVS
has utility similar to that of NOV3, and may be fouxld in skeletal tissue,
carcinoid lung tumors,
arthritis and tendonitis as examples. Thus, a cDNA encoding the proteoglycan-
like protein may
be useful in gene therapy, and the proteoglycan -like protein may be useful
when administered to
a subject in need thereof. The novel nucleic acid encoding proteoglycan -like
protein, and the
proteoglycan -like protein of the invention, or fragments thereof, may further
be useful in
diagnostic applications, wherein the presence or amount of the nucleic acid or
the protein are to be
assessed as fox example, in identifying tissue from kidney or brain. These
materials are further
useful in the generation of antibodies that bind immunospecifically to the
novel substances of the
invention for use in therapeutic or diagnostic methods.
NOVS maps to chromosome l 1q12-13. This assignment was made using mapping
information associated with genomic clones, public genes and ESTs sharing
sequence identity
with the disclosed sequence and CuraGen Corporation's Electronic Northern
bioinformatic tool.
NOVS is expressed in at least the following tissues: cartilage, tendon, and
ligament.
Expression information was derived from the tissue sources of the sequences
that were included
in the derivation of the sequence of NOVS.
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The disclosed NOVS nucleic acid of 1992 nucleotides encoding a novel
Fibromodulin-like
protein is shown in Table 12. An open reading frame was identified beginning
at nucleotides 1-3,
which results in a polypeptide that is a novel functional Fibromodulin-like
protein. The start
codon of the open reading frame is highlighted in bold type. Putative
untranslated regions
(underlined), if any, are found upstream from the initiation codon and
downstream from the
termination codon. The encoded protein having 664 amino acid residues is
presented using the
one-letter code in Table 13.
Table 12. A representative nucleotide sequence encoding the fibromodulin-like
protein of
the invention.
>CG54254 02
ATGGTGGTGGCACACCCCACCGCCACTGCCACCACCACGCCCACTGCCACTGTCACGGCCACCGTTGTG
ATGACCACGGCCACCATGGACCTGCGGGACTGGCTGTTCCTCTGCTACGGGCTCATCGCCTTCCTGACG
GAGGTCATCGACAGCACCACCTGCCCCTCGGTGTGCCGCTGCGACAACGGCTTCATCTACTGCAACGAC
CGGGGACTCACATCCATCCCCGCAGATATCCCTGATGATGCCACCACCCTCTACCTGCAGAACAACCAG
ATCAACAACGCCGGCATCCCCCAGGACCTGAAGACCAAGGTCAACGTGCAGGTCATCTACCTATACGA
GAATGACCTGGATGAGTTCCCCATCAACCTGCCCCGCTCCCTCCGGGAGCTGCACCTGCAGGACAACAA
TGTGCGCACCATTGCCAGGGACTCGCTGGCCCGCATCCCGCTGCTGGAGAAGCTGCACCTGGATGACAA
CTCCGTGTCCACCGTCAGCATTGAGGAGGACGCCTTCGCCGACAGCAAACAGCTCAAGCTGCTCTTCCT
GAGCCGGAACCACCTGAGCAGCATCCCCTCGGGGCTGCCGCACACGCTGGAGGAGCTGCGGCTGGATG
ACAACCGCATCTCCACCATCCCGCTGCATGCCTTCAAGGGCCTCAACAGCCTGCGGCGCCTGGTGCTGG
ACGGTAACCTGCTGGCCAACCAGCGCATCGCCGACGACACCTTCAGCCGCCTACAGAACCTCACAGAG
CTCTCGCTGGTGCGCAATTCGCTGGCCGCGCCACCCCTCTACCTGCAGGACAATGCCATCAGCCACATC
CCCTACAACACGCTGGCCAAGATGCGTGAGCTGGAGCGGCTGGACCTGTCCAACAACAACCTGACCAC
GCTGCCCCGCGGCCTGTTCGACGACCTGGGGAACCTGGCCCAGCTGCTGCTCAGGAACAACCCTTGGTT
TTGTGGCTGCAACCTCATGTGGCTGCGGGACTGGGTGAAGGCACGGGCGGCCGTGGTCAACGTGCGGG
GCCTCATGTGCCAGGGCCCTGAGAAGGTCCGGGGCATGGCCATCAAGGACATTACCAGCGAGGTGGAG
AGTGTTTTGAGACGGGCGCCGCAGGGCGGCGTGGCCAATGCGGCTGCCAAGACCACGGCCAGCAACCA
CGCCTCTGCCACCACGCCCCAGGGTTCCCTGTTTACCCTCAAGGCCAAAAGGCCAGGGCTGCGCCTCCC
CGACTCCAACATTGACTACCCCATGGCCACGGGTGATGGCGCCAAGACCCTGGCCATCCACGTGAAGG
CCCTGACGGCAGACTCCATCCGCATCACGTGGAAGGCCACGCTCCCCGCCTCCTCTTTCCGGCTCAGTT
GGCTGCGCCTGGGCCACAGCCCAGCCGTGGGCTCCATCACGGAGACCTTGGTGCAGGGGGACAAGACA
GAGTACCTGCTGACAGCCCTGGAGCCCAAGTCCACCTACATCATCTGCATGGTCACCATGGAGACCAGC
AATGCCTACGTAGCTGATGAGACACCCGTGTGTGCCAAGGCAGAGACAGCCGACAGCTATGGCCCTAC
CACCACACTCAACCAGGAGCAGAACGCTGGCCCCATGGCGAGCCTGCCCCTGGCGGGCATCATCGGCG
GGGCAGTGGCTCTGGTCTTCCTCTTCCTGGTCCTGGGGGCCATCTGCTGGTACGTGCACCAGGCTGGCG
AGCTGCTGACCCGGGAGAGGGCCTACAACCGGGGCAGCAGGAAAAAGGATGACTATATGGAGTCAGG
GACCAAGAAGGATAACTCCATCCTGGAAATCCGCGGCCCTGGGCTGCAGATGCTGCCCATCAACCCGT
ACCGCGCCAAAGAAGAGTACGTGGTCCACACTATCTTCCCCTCCAACGGCAGCAGCCTCTGCAAGGCCA
CACACACCATTGGCTACGGCACCACGCGGGGCTACCGGGACGGCGGCATCCCCGACATAGACTACTCC
TACACA (SEQ ID NO: 67)
Table 13. Representative amino acid sequence of the fibromodulin-like protein
of the
invention.
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>CG54254 02
MVVAHPTATATTTPTATVTATVVMTTATMDLRDWLFLCYGLIAFLTEVIDSTTCPSVCRC
DNGFIYCNDRGLTSIPADIPDDATTLYLQNNQINNAGIPQDLKTKVNVQVIYLYENDLDE
FPINLPRSLRELHLQDNNVRTIARDSLARIPLLEKLHLDDNSVSTVSIEEDAFADSKQLK
LLFLSRNHLSSIPSGLPHTLEELRLDDNRISTIPLHAFKGLNSLRRLVLDGNLLANQRIA
DDTFSRLQNLTELSLVRNSLAAPPLYLQDNAISHIPYNTLAKMRELERLDLSNNNLTTLP
RGLFDDLGNLAQLLLRNNPWFCGCNLMWLRDWVKARAAVVNVRGLMCQGPEKVRGMAIKD
ITSEVESVLRRAPQGGVANAAAKTTASNHASATTPQGSLFTLKAKRPGLRLPDSNIDYPM
ATGDGAKTLAIHVKALTADSIRITWKATLPASSFRLSWLRLGHSPAVGSITETLVQGDKT
EYLLTALEPKSTYIICMVTMETSNAYVADETPVCAKAETADSYGPTTTLNQEQNAGPMAS
LPLAGIIGGAVALVFLFLVLGAICWYVHQAGELLTRERAYNRGSRKKDDYMESGTKKDNS
ILEIRGPGLQMLPINPYRAKEEYVVHTIFPSNGSSLCKATHTIGYGTTRGYRDGGIPDID
YSYT (SEQ ID NO: 68)
PSORT predicts presence of the sequence within the plasma membrane with a
certainty of
0.46 and integral lil~elihood of transmembrane presence between positions 51
and 52 of SEQ 1D
NO: 68. Signal P Predicts that NOVS has a signal peptide that cleaves between
positions 51 and
52 of SEQ m NO: 68.
In a search of sequence databases, it was found, for example, that the nucleic
acid
sequence of this invention has 882 of 951 bases (92%) identical to a human
leucine-rich repeat
transmembrane protein FLRT1 (FLRT1) mRNA, (Accession No. gb: GENBANK-
m:AF169675~acc:AF169675.1) (FLRT1; Table 22). The full amino acid sequence of
the protein
of the invention was found to have 655 of 674 amino acid residues (97%)
identical to, and 658 of
674 amino acid residues (97%) similar to, the 674 amino acid protein from a
human leucine-rich
repeat transmembrane protein FLRT1 (Accession No: ptnr: SPTREMBL-ACC:Q9NZU1)
(FLRT1; Table 23).
Table 22. SLASTN search using NOVS (CuraGeu Acc. No. CG554254-02).
>gb:GENBANK-ID:AF1696751acc:AF169675.1 Homo Sapiens leucine-rich repeat
hansmembrane protein FLRT1 (FLRT1) mRNA, complete cds - Homo
Sapiens, 2660 bp.
Length = 2660
Score = 3978 (596.9 bits), Expect = 4.0e-174, P = 4.0e-174
Identities = 882/951 (92°l0), Positives = 882/951 (92%), Strand = Plus
l Plus
NOV5: 1 ATGGTGGTGGCACACCCCACCGCCACTGCCACCACCACGCCCACTGCCACTGTCACGGCC 60
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
4O FLRT1 1 ATGGTGGTGGCACACCCCACCGCCACTGCCACCACCACGCCCACTGCCACTGTCACGGCC 60
NOVS: 61 ACCGTTGTGATGACCACGGCCACCATGGACCTGCGGGACTGGCTGTTCCTCTGCTACGGG 120
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
FLRT1 61 ACCGTTGTGATGACCACGGCCACCATGGACCTGCGGGACTGGCTGTTCCTCTGCTACGGG 120
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NOV5: 121 CTCATCGCCTTCCTGACGGAGGTCATCGACAGCACCACCTGCCCCTCGGTGTGCCGCTGC 180
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
FLRT1 121 CTCATCGCCTTCCTGACGGAGGTCATCGACAGCACCACCTGCCCCTCGGTGTGCCGCTGC l80
NOV5: 181 GACAACGGCTTCATCTACTGCAACGACCGGGGACTCACATCCATCCCCGCAGATATCCCT 240
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
FLRT1 181 GACAACGGCTTCATCTACTGCAACGACCGGGGACTCACATCCATCCCCGCAGATATCCCT 240
1O NOV5: 241 GATGATGCCACCACCCTCTACCTGCAGAACAACCAGATCAACAACGCCGGCATCCCCCAG 300
IIIII IIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIII IIIIIII11111
FLRT1 241 GATGACGCCACCACCCTCTATCTGCAGAACAACCAGATCAACAACGCTGGCATCCCCCAG 300
NOV5: 301 GACCTCAAGACCAAGGTCAACGTGCAGGTCATCTACCTATACGAGAATGACCTGGATGAG 360
15 Illlllllllllllllllllllllllllllllllllllllllllllllllllllllllll
FLRT1 301 GACCTCAAGACCAAGGTCAACGTGCAGGTCATCTACCTATACGAGAATGACCTGGATGAG 360
NOVS: 361 TTCCCCATCAACCTGCCCCGCTCCCTCCGGGAGCTGCACCTGCAGGACAACAATGTGCGC 420
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIillllll
2O FLRT1 361 TTCCCCATCAACCTGCCCCGCTCCCTCCGGGAGCTGCACCTGCAGGACAACAATGTGCGC 420
NOV5: 421 ACCATTGCCAGGGACTCGCTGGCCCGCATCCCGCTGCTGGAGAAGCTGCACCTGGATGAC 480
llllllllllllllllllllllllllllllllllllllllllllllllllllllllllll
FL,RT1 421 ACCATTGCCAGGGACTCGCTGGCCCGCATCCCGCTGCTGGAGAAGCTGCACCTGGATGAC 480
NOVS: 481 AACTCCGTGTCCACCGTCAGCATTGAGGAGGACGCCTTCGCCGACAGCAAACAGCTCAAG 540
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
FLRT1 481 AACTCCGTGTCCACCGTCAGCATTGAGGAGGACGCCTTCGCCGACAGCAAACAGCTCAAG 540
3O NOVS: 54l CTGCTCTTCCTGAGCCGGAACCACCTGAGCAGCATCCCCTCGGGGCTGCCGCACACGCTG 600
llllllllllllllllllllllllllllllllllllllllllllllllllilllllllll
FLRT1 541 CTGCTCTTCCTGAGCCGGAACCACCTGAGCAGCATCCCCTCGGGGCTGCCGCACACGCTG 600
NOV5: 60l GAGGAGCTGCGGCTGGATGACAACCGCATCTCCACCATCCCGCTGCATGCCTTCAAGGGC 660
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII11111111111111111111111
FLRTl 601 GAGGAGCTGCGGCTGGATGACAACCGCATCTCCACCATCCCGCTGCATGCCTTCAAGGGC 660
NOV5: 661 CTCAACAGCCTGCGGCGCCTGGTGCTGGACGGTAACCTGCTGGCCAACCAGCGCATCGCC 720
111111111111111111111111111111111111111111111111111111111111
4O FLRTl 661 CTCAACAGCCTGCGGCGCCTGGTGCTGGACGGTAACCTGCTGGCCAACCAGCGCATCGCC 720
NOVS: 721 GACGACACCTTCAGCCGCCTACAGAACCTCACAGAGCTCTCGCTGGTGCGCAATTCGCTG 780
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
FL,RT1 721 GACGACACCTTCAGCCGCCTACAGAACCTCACAGAGCTCTCGCTGGTGCGCAATTCGCTG 780
NOV5: 781 GCCGCGCCACCCCTCTACCTGCAGGACAATGCC-ATCAGCCACATCCCCTACAACACGCT 839
IIIIIIIIIIIIIII 111111 II III I I II I I 1 IIII I II
FLRT1 781 GCCGCGCCACCCCTCAACCTGCC---CAGCGCCCACCTGC-AGAAACTCTAC--CT-GCA 833
SO NOV5: 840 GGCCAAGATGCG-TGAGCTGGAGCGGCTGGACCTGTCCAAC-AACAACC-TGACCACGCT 896
II 111 III I III I I II I I I I II I I III I I
FLRTl 834 GGACAA--TGCCATCAGCCACATCCCCTACAACACGCTGGCCAAGATGCGTGAGCTGGA- 890
NOV5: 897 GCCCCGCGGCCTGTTCGACGACCTGGGGA--AC-CTGGCCCAGCTGC-TGCTC-AGGAAC 951
(SEQ ID
NO: 67)
II I I IIIII 1 II II II II III III II II II II I II I
FLRT1 891 GCGGCTGGACCTGTCCAACAACAACCTGACCACGCTGCCCC-GCGGCCTGTTCGACGACC 949
(SEQ ID
N0: 74)
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Table 23. BLASTP search using the protein of NOVS (CuraGen Acc. No. CG554254-
02).
>ptnr:SPTREMBL-ACC:Q9NZU1 LEUCINE-RICH REPEAT TRANSMEMBRANE PROTEIN FLRT1 -
Homo sapiens (Human), 674 aa.
Length = 674
Score = 3370 (1186.3 bits), Expect = 0.0, P = 0.0
Identities = 655/674 (97%), Positives = 658/674 (97%)
IO NOV5: 1 MWAHPTATATTTPTATVTATVVMTTATMDLRDWLFLCYGLIAFLTEVIDSTTCPSVCRC 60
************************************************************
FLRTl 1 MWAHPTATATTTPTATVTATVVMTTATMDLRDWLFLCYGLIAFLTEVIDSTTCPSVCRC 60
NOVS: 61 DNGFTYCNDRGLTSIPADTPDDATTLYLQNNQINNAGIPQDLKTKVNVQVTYLYENDLDE 120
************************************************************
FLRT1 61 DNGFIYCNDRGLTSIPADIPDDATTLYLQNNQINNAGIPQDLKTKVNVQVTYLYENDLDE 120
NOVS: 121 FPINLPRSLRELHLQDNNVRTIARDSLARIPLLEKLHLDDNSVSTVSIEEDAFADSKQLK 180
************************************************************
2O FLRT1 121 FPINLPRSLRELHLQDNNVRTIARDSLARIPLLEKLHLDDNSVSTVSIEEDAFADSKQLK 180
NOV5: 181 LLFLSRNHLSSIPSGLPHTLEELRLDDNRISTIPLHAFKGLNSLRRLVLDGNLLANQRIA 240
************************************************************
FLRT1 181 LLFLSRNHLSSIPSGLPHTLEELRLDDNRISTIPLHAFKGLNSLRRLVLDGNLLANQRIA 240
NOVS: 24l DDTFSRLQNLTELSLVRNSLAAPPL----------YLQDNAISHTPYNTLAKMRELERLD 290
************************* *************************
FLRT1 241 DDTFSRLQNLTELSLVRNSLAAPPLNLPSAHLQKLYLQDNAISHIPYNTLAKMRELERLD 300
3O NOV5: 291 LSNNNLTTLPRGLFDDLGNLAQLLLRNNPWFCGCNLMWLRDWVKARAAWNVRGLMCQGP 350
************************************************************
FLRT1 301 LSNNNLTTLPRGLFDDLGNLAQLLLRNNPWFCGCNLMWLRDWVKARAAVVNVRGLMCQGP 360
NOV5: 351 EKVRGMAIKDITSEVESVLRRAPQGGVANAAAKTTASNHASATTPQGSLFTLKAKRPGLR 410
**************++ **************************************
FLRTl 361 EKVRGMAIKDITSEMDECFETGPQGGVANAAAKTTASNHASATTPQGSLFTLKAKRPGLR 420
NOV5: 411 LPDSNIDYPMATGDGAKTLAIHVKALTADSIRITWKATLPASSFRLSWLRLGHSPAVGSI 470
************************************************************
4O FLRT1 421 LPDSNIDYPMATGDGAKTLAIHVKALTADSIRITWKATLPASSFRLSWLRLGHSPAVGSI 480
NOV5: 471 TETLVQGDKTEYLLTALEPKSTYTTCMVTMETSNAYVADETPVCAKAETADSYGPTTTLN 530
************************************************************
FLRT1 481 TETLVQGDKTEYLLTALEPKSTYIICMVTMETSNAYVADETPVCAKAETADSYGPTTTLN 540
NOV5: 531 QEQNAGPMASLPLAGIIGGAVALVFLFLVLGAICWYVHQAGELLTRERAYNRGSRKKDDY 590
*******************************************************+****
FLRT1 541 QEQNAGPMASLPLAGIIGGAVALVFLFLVLGAICWYVHQAGELLTRERAYNRGSREKDDY 600
SO NOV5: 591 MESGTKKDNSILEIRGPGLQMLPTNPYRAKEEYVVHTTFPSNGSSLCKATHTIGYGTTRG 650
************************************************************
FLRT1 601 MESGTKKDNSILEIRGPGLQMLPINPYRAKEEYVVHTIFPSNGSSLCKATHTIGYGTTRG 660
NOV5: 651 YRDGGIPDIDYSYT 664 (SEQ ID NO: 68)
**************
FLRT1 661 YRDGGIPDIDYSYT 674 (SEQ ID NO: 75)
Where * indicates identity and + indicates similarity.
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The presence of identifiable domains in the protein disclosed herein was
determined by
searches versus domain databases such as PFAM, PROSITE, PRODOM, BLOCKS or
PRINTS
and then identified by the Interpro domain accession number. Significant
domains are
smnmarized in the Table 24 below:
Table 24. Significant domains of NOVS.
Scoresfor sequence includes
family classification all
(score domains):
Model Description Score E-value
N
LRR Leucine Rich 105.5 1e-27
Repeat 9
LRRCT Leucine rich C-terminaldomain 48.0 2.1e-10
repeat 1
LRRNT Leucine rich N-terminaldomain 33.4 5.3e-06
repeat 1
fn3 Fibronectin domain 13.8 0.12
type III 1
Parsedfor domains:
Model Domain seq-f hmm-f scoreE-value
seq-t hmm-t
LRRNT 1/1 53 80 .. 1 31 33.4 5.3e-06
[]
LRR 1/9 82 106 .. 1 23 5.4 1.7e+02
[]
LRR 2/9 107 126 .. 1 23 5.6 1.5e+02
[]
2 LRR 3/9 128 151 .. 1 23 16.2 0.81
0 []
LRR 4/9 152 177 .. 1 23 13.7 4.4
[]
LRR 5/9 178 197 .. 1 23 14.4 2.7
[]
LRR 6/9 199 222 .. 1 23 25.0 0.0018
[]
LRR 7/9 223 248 .. 1 23 12.8 8.5
[]
LRR 8/9 249 276 .. 1 23 9.7 38
[]
LRR 9/9 285 308 .. 1 23 28.1 0.00021
[]
LRRCT 1/1 318 369 .. 1 54 48.0 2.1e-10
[]
fn3 1/1 425 503 .. 1 84 13.8 0.12
[]
3 0 Alignments of top-scoring domains:
LRRNT: domain 1 of 1, from 53 to 80: score 33.4, E = 5.3e-06
*->aCpreCtCspfglvVdCsgrgLtlevPrdlP<-* (SEQ ID NO: 76)
Cp C+C ++C +rgLt ++P d+P
SEQ ID N0: 68 53 TCPSVCRCD--NGFIYCNDRGLT-STPADIP 80
LRR: domain 1 of 9, from 82 to 106: score 5.4, E = 1.7e+02 (SEQ ID NO: 77)
*->nLeeLdLsnN.Lt..slppglfsnLp<-
++L+L+nN++ + ++p++ ++
SEQ ID N0: 68 82 DATTLYLQNNqINnaGIPQD-LKTKV 106
LRR: domain 2 of 9, from 107 to 126: score 5.6, E = 1.5e+02
*->nLeeLdLsnN.LtslppglfsnLp<-* (SEQ ID N0: 78)
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n ++++L N+L ++p nLp
SEQ ID N0: 68 107 NVQVIYLYENdLDEFPI----NLP 126
LRR: domain 3 of 9, from 128 to 151: score 16.2, E = 0.81
*->nLeeLdLsnN.LtslppglfsnLp<-* (SEQ ID NO: 79)
+L+eL+L++N+ +++ ++++ p
SEQ ID NO: 68 128 SLRELHLQDNnVRTIARDSLARIP 151
LRR: domain 4 of 9, from 152 to 177: score 13.7, E = 4.4
*->nLeeLdLSnN.Lt..slppglfsnLp<-* (SEQ ID NO: 80)
Le+L+L++N+ ++ s+++++f++ +
SEQ ID NO: 68 152 LLEKLHLDDNsVStvSIEEDAFADSK 177
LRR: domain 5 of 9, from 178 to 197: score 14.4, E = 2.7
IS *->nLeeLdLSnN.LtslppglfsnLp<-* (SEQ ID NO: 81)
+L+ L Ls+N+L+s+p+ +Lp
SEQ 2D NO: 68 178 QLKLLFLSRNhLSSIPS----GLP 197
LRR: domain 6 of 9, from 199 to 222: score 25.0, E = 0.0018
*->nLeeLdLSnN.LtslppglfsnLp<-* (SEQ ID NO: 82)
LeeL+L++N+++++p ++f++L+
SEQ ID NO: 68 199 TLEELRLDDNrISTIPLHAFKGLN 222
LRR: domain 7 of 9, from 223 to 248: score 12.8, E = 8.5
25 *->nLeeLdLsnN.Lt..slppglfsnLp<-* (SEQ ID NO: 83)
+L++L L++N L ++ + +++fs L+
SEQ ID NO: 68 223 SLRRLVLDGNILAnqRIADDTFSRLQ 248
LRR: domain 8 of 9, from 249 to 276: score 9.7, E = 38
3~ *->nLeeLdLSnN.Lt....slppglfsnLp<-* (SEQ ID NO: 84)
nL+eL+L +N+L ++ 1 ++++s+ p
SEQ ID NO: 68 249 NLTELSLVRNsLAappIYLQDNAISHIP 276
LRR: domain 9 of 9, from 285 to 308: score 28.1, E = 0.00021
35 *->nLeeLdLsnN.LtslppglfsnLp<-* (SEQ ID NO: 85)
Le+LdLsnN+Lt+lp glf++L
SEQ ID NO: 68 285 ELERLDLSNNnLTTLPRGLFDDLG 308
LRRCT: domain 1 of 1, from 318 to 369: score 48.0, E = 2.1e-10
*->NPfnCDCeLrwLlrWlretnprrledqedlrCasPeslrGqp11e11psdfsCp<-* (SEQ ID NO: 86)
NP+ C C+L+wL++W+++ ++ +++ + 1+C++Pe++rG+ +++++ ++
SEQ ID NO: 68 318 NPWFCGCNLMWLRDWVKA-RAAVVNVR-GLMCQGPEKVRGMAIKDITSEVESVL 362
fn3: domain 1 of l, from 425 to 503: score 13.8, E = 0.12
45 *->PSaPtnltvtdvtstsltlsWspptgngpitgYevtyRqpkngge..
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+ ++v+++t++s++++W++ p +++++++ ++ +
SEQ ID N0: 68 425 GAKTLAIHVKALTADSIRITWKAT---LPASSFRLSW-LRLGHSPav 467
..wneltvpgtttsytltgLkPgteYevrVqAvnggGGpeS<-* (SEQ ID NO: 87)
++ +e v g++t+y It L+P ++Y ++ + + + S
SEQ ID NO: 68 468 gsITETLVQGDKTEYLLTALEPKSTYIICMV---TM--ETS 503
Leucine-rich repeats (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. In
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.
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 3-D structure of ribonuclease inhibitor, a protein
containing 15 LRRs, has
been determined, revealing LRRs to be a new class'of alpha/beta fold. LFRs
form elongated non-
globular structures and are often flanked by cysteine rich domains.
This indicates that the 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.
The protein similarity information, expression pattern, cellular localization,
and map
location for the NOVS protein and nucleic acid disclosed herein suggest that
this fibromodulin-
like protein may have important structural and/or physiological functions
characteristic of the
fibromodulin family. Therefore, NOVS nucleic acids and proteins of the
invention are useful in
potential diagnostic and therapeutic applications and as a research tool.
These include serving as a
specific or selective nucleic acid or protein diagnostic 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/cytotoxic
antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene
ablation), (v) an agent
promoting tissue regeneration ira vity~o and in vivo, and (vi) a biological
defense weapon.
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The nucleic acids and proteins of the invention have applications in the
diagnosis and/or
treatment of various diseases and disorders. For example, the compositions of
the present
invention will have efficacy for the treatment of patients suffering from:
atopy;
Dysprothrombinemia; Hypoprothrombinemia; Smith-Lemli-Opitz syndrome, type I;
Smith-Lemli-
Opitz syndrome, type II; Xeroderma pigmentosum, group E, subtype 2; High bone
mass; Bardet-
Biedl syndrome 1; CPT deficiency, hepatic, type I; Carcinoid tumor of lung;
Centrocytic
lymphoma; Cervical carcinoma; Hyperparathyroidism, AD; Hypokalemic periodic
paralysis Leigh
syndrome; Leukemia, acute promyelocytic, NLTMA/RARA type; Macular dystrophy,
vitelliform
type; McArdle disease; Meckel syndrome, type 2; Multiple endocrine neoplasia
I; Multiple
myeloma; Parathyroid adenomatosis 1; Prolactinoma, hyperparathyroidism,
carcinoid syndrome;
Retinitis pigmentosa, digenic; Somatotrophinoma; Vitreoretinopathy,
neovascular inflammatory;
arthritis, tendinitis; as well as other diseases, disorders and conditions.
These materials are further useful in the generation of antibodies that bind
immunospecifically to the novel substances of the invention for use in
diagnostic and/or
therapeutic methods.
Table 14.
Sequences and Corresponding SEQ ID Numbers
SEQ ID SEQ ID
NOV number number of O~ of
of
clone nucleic encoded homology
number nucleic
acid
acid amino acid
se uence se uence
1 AL109798 A 1 2 thymosin beta 61-237
10
2 AL035703 A 4 5 ephrin A receptor1-2976
3 AP000597 A 6 7 proteoglycan 1-2025
4 AL035703 A 65 66 ephrin A receptor1-1545
5 CG54254-02 67 68 fibromodulin 1-1995
NOV Nucleic Acids and Polypeptides
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One aspect of the invention pertains to isolated nucleic acid molecules that
encode NOV
polypeptides or biologically-active portions thereof. Also included in the
invention are nucleic
acid fragments sufficient for use as hybridization probes to identify NOV-
encoding nucleic acids
(e.g., NOV mRNAs) and fragments for use as PCR primers for the amplification
and/or mutation
of NOV nucleic acid molecules. As used herein, the term "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 can be single-stranded or double-stranded,
but preferably is
comprised double-stranded DNA.
A NOV nucleic acid can encode a mature NOV polypeptide. As used herein, a
"mature"
form of a polypeptide or protein disclosed in the present invention is the
product of a naturally
occurnng 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 can be defined as the
polypeptide, precursor
or proprotein encoded by an open reading frame 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 codon of an
open reading frame, 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.
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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 are generally
obtained from a
natural or recombinant source, are highly specific, and much slower to
hybridize than shorter-
length oligomer probes. Probes can be single- or double-stranded and designed
to have specificity
in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
The term "isolated" nucleic acid molecule, as utilized herein, is one which 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 NOV 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 molecules of the invention, e.g., a nucleic acid molecule
having the nucleotide
sequence of SEQ ID NO: l, 4, 6, 65 or 67, 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 NO:
l, 4, 6, 65 or 67 as a hybridization probe, NOV molecules can be isolated
using standard
hybridization and cloning techniques (e.g., as described in Sambrook, et al.,
(eds.),
MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, (1989); and Ausubel, et al., (eds.),
CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, (1993).
A nucleic acids 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
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and characterized by DNA sequence analysis. Furthermore, oligonucleotides
corresponding to
NOV 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 can 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 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 of
SEQ ID NO: l, 4, 6, 65 or 67 or a complement thereof. Oligonucleotides can 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 ID NO: 1,
4, 6, 65 or 67 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 a NOV
polypeptide). A nucleic
acid molecule that is complementary to the nucleotide sequence shown in SEQ ID
NO: 1, 4, 6, 65
or 67, is one that is sufficiently complementary to the nucleotide sequence
shown in SEQ ID NO:
l, 4, 6, 65 or 67 that it can hydrogen bond with little or no mismatches to
the nucleotide sequence
shown in SEQ ID NO: 1, 4, 6, 65 or 67, 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 can be through or due to the effects of
another polypeptide or
compound. Direct binding refer s 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.
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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 can 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 chains.
Analogs can 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 can 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 30%, 50%, 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 isoforms of NOV 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. hl the invention, homologous nucleotide
sequences include
nucleotide sequences encoding for a NOV polypeptide of species other than
humans, including,
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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 NOV protein. Homologous nucleic acid sequences include
those
nucleic acid sequences that encode conservative amino acid substitutions (see
below) in SEQ ID
NO: 2, 5, 7, 66 or 68, as well as a polypeptide possessing NOV biological
activity. Various
biological activities of the NOV proteins are described below.
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
"start" codon and
terminates with one of the three "stop" codons, namely, TAA, TAG, or TGA. For
the purposes of
this invention, an ORF can 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 fide
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 NOV genes allows
for the
generation of probes and primers designed for use in identifying and/or
cloning NOV homologues
in other cell types, e.g. from other tissues, as well as NOV 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, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive
sense strand nucleotide
sequence of SEQ ID NO: 1, 4, 6, 65 or 67; or an anti-sense strand nucleotide
sequence of SEQ ID
NO: l, 4, 6, 65 or 67; or of a naturally occurring mutant of SEQ ID NO: 1, 4,
6, 65 or 67.
Probes based on the NOV 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 a NOV
protein, such as by
measuring a level of a NOV-encoding nucleic acid in a sample of cells from a
subject e.g.,
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detecting NOV mRNA levels or determining whether a genomic NOV gene has been
mutated or
deleted.
"A polypeptide having a biologically-active portion of a NOV 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 NOV" can be prepared by isolating a portion of SEQ ID NO: 1, 4, 6,
65 or 67 that
encodes a polypeptide having a NOV biological activity (the biological
activities of the NOV
proteins are described below), expressing the encoded portion of NOV protein
(e.g., by
recombinant expression in vitro) and assessing the activity of the encoded
portion of NOV.
NOV Nucleic Acid and Polypeptide Variants
The invention further encompasses nucleic acid molecules that differ from the
nucleotide
sequences shown in SEQ m NO: 1, 4, 6, 65 or 67, due to degeneracy of the
genetic code and thus
encode the same NOV proteins as that encoded by the nucleotide sequences shown
in SEQ m
NO: 1, 4, 6, 65 or 67. 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 ID
NO: 2, 5, 7, 66 or 68.
In addition to the NOV nucleotide sequences shown in SEQ 1D NO: 1, 4, 6, 65 or
67, 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 NOV polypeptides may exist within a
population
(e.g., the human population). Such genetic polymorphism in the NOV 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 a NOV protein, preferably a vertebrate NOV protein. Such natural
allelic variations can
typically result in 1-5% variance in the nucleotide sequence of the NOV genes.
Any and all such
nucleotide variations and resulting amino acid polymorphisms in the NOV
polypeptides, which
are the result of natural allelic variation and that do not alter the
functional activity of the NOV
polypeptides, are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding NOV proteins from other species, and
thus
that have a nucleotide sequence that differs from the human sequence of SEQ m
NO: 1, 4, 6, 65
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or 67 are intended to be within the scope of the invention. Nucleic acid
molecules corresponding
to natural allelic variants and homologues of the NOV cDNAs of the invention
can be isolated
based on their homology to the human NOV 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 NO: 1, 4, 6, 65 or 67.
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 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 NOV 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 known 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 (Tin) 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, 50% 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
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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 formamide.
Stringent conditions are known to those skilled in the art and can be found in
Ausubel, et
al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John 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 of SEQ
ID NO: 1, 4, 6, 65 or
67, corresponds to a naturally-occurring nucleic acid molecule. As used
herein, a "naturally-
occurnng" nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
W a second embodiment, a nucleic acid sequence that can hybridize to the
nucleic acid
molecule comprising the nucleotide sequence of SEQ ID NO: 1, 4, 6, 65 or 67,
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,
5X 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 can be used are well-known within the art. See, e.g., Ausubel
,et al. (eds.),
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY (1993), and
I~riegler, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton
Press, NY (1990).
In a third embodiment, a nucleic acid that can hybridize to the nucleic acid
molecule
comprising the nucleotide sequences of SEQ ID NO: l, 4, 6, 65 or 67, 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, 5X
SSC, 50 mM
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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/vol) 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 can be used are well known in the art (e.g., as employed for
cross-species
hybridizations). See, e.g., Ausubel, et al. (eds.), CURRENT PROTOCOLS 1N
MOLECULAR
BIOLOGY, John Wiley ~ Sons, NY (1993), and Kriegler, GENE TRANSFER AND
EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY (1990); Shilo et al., 78
Proc.
Natl. Acad. Sci.USA 78: 6789-6792 (1981).
Conservative Mutations
In addition to naturally-occurnng allelic variants of NOV sequences that may
exist in the
population, the skilled artisan will further appreciate that changes can be
introduced by mutation
into the nucleotide sequences of SEQ ID NO: 1, 4, 6, 65 or 67 thereby leading
to changes in the
amino acid sequences of the encoded NOV proteins, without altering the
functional ability of said
NOV proteins. For example, nucleotide substitutions leading to amino acid
substitutions at "non-
essential" amino acid residues can be made in the sequence of SEQ ID NO: 2, 5,
7, 66 or 68. A
"non-essential" amino acid residue is a residue that can be altered from the
wild-type sequences of
the NOV 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 NOV 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
NOV proteins
that contain changes in amino acid residues that are not essential for
activity. Such NOV proteins
differ in amino acid sequence from SEQ ID NO: 2, 5, 7, 66 or 68 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 of SEQ ID NO: 2, 5, 7, 66 or 68. Preferably, the
protein encoded by
the nucleic acid molecule is at least about 60% homologous to SEQ TD NO: 2, 5,
7, 66 or 68;
more preferably at least about 70% homologous to SEQ ID NO: 2, 5, 7, 66 or 68;
still more
preferably at least about 80% homologous to SEQ ID NO: 2, 5, 7, 66 or 68; even
more preferably
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at least about 90% homologous to SEQ m NO: 2, 5, 7, 66 or 68; and most
preferably at least
about 95% homologous to SEQ m NO: 2, 5, 7, 66 or 68.
An isolated nucleic acid molecule encoding a NOV protein homologous to the
protein of
SEQ m NO: 2, 5, 7, 66 or 68, can be created by introducing one or more
nucleotide substitutions,
additions or deletions into the nucleotide sequence of SEQ m NO: 1, 4, 6, 65
or 67, such that one
or more amino acid substitutions, additions or deletions are introduced into
the encoded protein.
Mutations can be introduced into the coding region of amino acid SEQ m NOS: 2,
5, 7, 9
or 11 by standard techniques, such as site-directed mutagenesis and PCR-
mediated mutagenesis.
Preferably, these mutations result in conservative amino acid substitutions
which 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 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 NOV 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
a NOV coding
sequence, such as by saturation mutagenesis, and the resultant mutants can be
screened for NOV
biological activity to identify mutants that retain activity. Following
mutagenesis of SEQ m NO:
2, 5, 7, 66 or 68, the encoded protein can be expressed by any recombinant
technology known in
the art and the activity of the protein can be determined.
In one embodiment, a mutant NOV protein can be assayed for (i) the ability to
form
protein:protein interactions with other NOV proteins, other cell-surface
proteins, or biologically-
active portions thereof, (ii) complex fonnation between a mutant NOV protein
and a NOV ligand;
or (iii) the ability of a mutant NOV protein to bind to an intracellular
target protein or
biologically-active portion thereof; (e.g. avidin proteins). In yet another
embodiment, a mutant
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NOV 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
can hybridize to or complementary to the nucleic acid molecule comprising the
nucleotide
sequence of SEQ 1D NO: 1, 4, 6, 65 or 67 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 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 NOV coding strand, or to only a
portion thereof. Nucleic
acid molecules encoding fragments, homologs, derivatives and analogs of a NOV
protein of SEQ
ID NO: 2, 5, 7, 66 or 68; or antisense nucleic acids complementary to a NOV
nucleic acid
sequence of SEQ ID NO: 1, 4, 6, 65 or 67, are additionally provided.
1 S In one embodiment, an antisense nucleic acid molecule is antisense to a
"coding region" of the
coding strand of a nucleotide sequence encoding a NOV protein. The term
"coding region" refers
to the region of the nucleotide sequence comprising codons that 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 NOV
protein. The teen
"noncoding region" refers to 5' and 3' sequences that 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 NOV 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 NOV mRNA, but more preferably is an oligonucleotide that is
antisense to only
a portion of the coding or noncoding region of NOV mRNA. For example, the
antisense
oligonucleotide can be complementary to the region surrounding the translation
start site of NOV
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
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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-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-
thiouridine, 5-
carboxynethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine, N6-
isopentenyladenine, 1-methylgua~iine, 1-methylinosine, 2,2-dimethylguanine, 2-
methyladenine, 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 in situ such that they hybridize with or bind to cellular
mRNA and/or
genomic DNA encoding a NOV 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
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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
alpha-anomeric nucleic acid molecule. An alpha-anomeric nucleic acid molecule
forms specific
double-stranded hybrids with complementary RNA in which, contrary to the usual
alpha-units, the
strands run parallel to each other. See, e.g., Gaultier, et al., 1987. Nucl.
Acids Res. 15: 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. FEBS Lett. 215: 327-330.
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 can be used, for example, as antisense binding nucleic acids in
therapeutic applications
in a subj ect.
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 NOV mRNA transcripts to thereby
inlubit translation
of NOV mRNA. A ribozyme having specificity for a NOV-encoding nucleic acid can
be
designed based upon the nucleotide sequence of a NOV cDNA disclosed herein
(i.e., SEQ ID NO:
1, 4, 6, 8, 9 or 10). For example, a derivative of a Tetrahymena 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 a NOV-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. NOV mRNA can also be used to
select a catalytic
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RNA having a specific ribonuclease activity from a pool of RNA molecules. See,
e.g., Bartel et
al., (1993) Science 261:1411-1418.
Alternatively, NOV gene expression can be inhibited by targeting nucleotide
sequences
complementary to the regulatory region of the NOV nucleic acid (e.g., the NOV
promoter and/or
enhancers) to form triple helical structures that prevent transcription of the
NOV 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 NOV 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 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 NOV 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 NOV 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 NOV 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 clumeras of NOV can be generated that may combine the
advantageous
properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g.,
RNase H and
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DNA polymerases) 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 staclcing, number of bonds between the
nucleobases, and
orientation (see, Hyrup, etal., 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. WO88/09810) or
the blood-brain barner (see, e.g., PCT Publication No. WO 89/10134). In
addition,
oligonucleotides can be modified with hybridization triggered cleavage agents
(see, e.g., Krol, et
al., 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon,
1988. Pharm. Res. 5:
539-549). To this end, the oligonucleotide can 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.
NOV Polypeptides
A polypeptide according to the invention includes a polypeptide including the
amino acid
sequence of NOV polypeptides whose sequences are provided in SEQ ID NO: 2, 5,
7, 66 or 68.
The invention also includes a mutant or variant protein any of whose residues
can be changed
from the corresponding residues shown in SEQ ID NO: 2, 5, 7, 66 or 68 while
still encoding a
protein that maintains its NOV activities and physiological functions, or a
functional fragment
thereof.
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In general, a NOV variant that preserves NOV-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. W
favorable circumstances, the substitution is a conservative substitution as
defined above.
One aspect of the invention pertains to isolated NOV proteins, and
biologically-active
poutions thereof, or derivatives, fragments, analogs or homologs thereof. Also
provided are
polypeptide fragments suitable for use as immunogens to raise anti-NOV
antibodies. In one
embodiment, native NOV proteins can be isolated from cells or tissue sources
by an appropriate
purification scheme using standard protein purification techniques. In another
embodiment, NOV
proteins are produced by recombinant DNA techniques. Alternative to
recombinant expression, a
NOV 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 cellular material'or other contaminating proteins from
the cell or tissue source
from which the NOV protein is derived, or substantially free from chemical
precursors ox other
chemicals when chemically synthesized. The language "substantially free of
cellular material"
includes preparations of NOV proteins in which the protein is separated 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
NOV proteins having
less than about 30% (by dry weight) of non-NOV proteins (also referred to
herein as a
"contaminating protein"), more preferably less than about 20% of non-NOV
proteins, still more
preferably less than about 10% of non-NOV proteins, and most preferably less
than about 5% of
non-NOV proteins. When the NOV 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 NOV protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes
preparations of NOV proteins in which the protein is separated from chemical
precursors or other
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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 NOV
proteins having less than about 30% (by dry weight) of chemical precursors or
non-NOV
chemicals, more preferably less than about 20% chemical precursors or non-NOV
chemicals, still
more preferably less than about 10% chemical precursors or non-NOV chemicals,
and most
preferably less than about 5% chemical precursors or non-NOV chemicals.
Biologically-active portions of NOV proteins include peptides comprising amino
acid
sequences sufficiently homologous to or derived from the amino acid sequences
of the NOV
proteins (e.g., the amino acid sequence shown in SEQ 1D NO: 2, 5, 7, 66 or 68)
that include
fewer amino acids than the full-length NOV proteins, and exhibit at least one
activity of a NOV
protein. Typically, biologically-active portions comprise a domain or motif
with at least one
activity of the NOV protein. A biologically-active portion of a NOV 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 NOV
protein.
In an embodiment, the NOV protein has an amino acid sequence shown in SEQ m
NO: 2,
5, 7, 66 or 68. In other embodiments, the NOV protein is substantially
homologous to SEQ 1D
NO: 2, 5, 7, 66 or 68, and retains the functional activity of the protein of
SEQ ID NO: 2, 5, 7,
9 or 11, yet differs in amino acid sequence due to natural allelic variation
or mutagenesis, as
described in detail, below. Accordingly, in another embodiment, the NOV
protein is a protein
that comprises an amino acid sequence at least about 45% homologous to the
amino acid
sequence of SEQ ID NO: 2, 5, 7, 66 or 68 and retains the functional activity
of the NOV proteins
of SEQ ID NO: 2, 5, 7, 66 or 68.
Determining Homology Between Two or More Sequences
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 alignment
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
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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 can be determined as the degree of identity
between
two sequences. The homology can be determined using computer programs known in
the art,
such as GAP software provided in the GCG program package. See, Needleman and
Wunsch,
1970. J Mol Biol 48: 443-453. Using GCG GAP software with the following
settings for nucleic
acid sequence comparison: GAP creation penalty of 5.0 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 NO: 1, 4, 6, 65 or 67.
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 I, 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 numbex of positions in the region of comparison
(i.e., the window
size), and multiplying the result by 100 to yield the percentage of sequence
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 NOV chimeric or fusion proteins. As used herein, a
NOV
"chimeric protein" or "fusion protein" comprises a NOV polypeptide operatively-
linked to a non-
NOV polypeptide. An "NOV polypeptide" refers to a polypeptide having an amino
acid sequence
corresponding to a NOV protein (SEQ ID NO: 2, 5, 7, 66 or 68), whereas a "non-
NOV
polypeptide" refers to a polypeptide having an amino acid sequence
corresponding to a protein
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that is not substantially homologous to the NOV protein, e.g., a protein that
is different from the
NOV protein and that is derived from the same or a different organism. Within
a NOV fusion
protein the NOV polypeptide can correspond to all or a portion of a NOV
protein. In one
embodiment, a NOV fusion protein comprises at least one biologically-active
portion of a NOV
protein. In another embodiment, a NOV fusion protein comprises at least two
biologically-active
portions of a NOV protein. In yet another embodiment, a NOV fusion protein
comprises at least
three biologically-active portions of a NOV protein. Within the fusion
protein, the term
"operatively-linked" is intended to indicate that the NOV polypeptide and the
non-NOV
polypeptide are fused in-frame with one another. The non-NOV polypeptide can
be fused to the
N-terminus or C-terminus of the NOV polypeptide.
In one embodiment, the fusion protein is a GST-NOV fusion protein in which the
NOV
sequences are fused to the C-terminus of the GST (glutathione S-transferase)
sequences. Such
fusion proteins can facilitate the purification of recombinant NOV
polypeptides.
In another embodiment, the fusion protein is a NOV protein containing a
heterologous signal
sequence at its N-terminus. In certain host cells (e.g., mammalian host
cells), expression and/or
secretion of NOV can be increased through use of a heterologous signal
sequence.
In yet another embodiment, the fusion protein is a NOV-immunoglobulin fusion
protein in
which the NOV sequences are fused to sequences derived from a member of the
immunoglobulin
protein family. The NOV-immunoglobulin fusion proteins of the invention can be
incorporated
into pharmaceutical compositions and administered to a subject to inhibit an
interaction between a
NOV ligand and a NOV protein on the surface of a cell, to thereby suppress NOV-
mediated signal
transduction in vivo. The NOV-immunoglobulin fusion proteins can be used to
affect the
bioavailability of a NOV cognate ligand. Inhibition of the NOV ligand/NOV
interaction can 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 NOV-
immunoglobulin
fusion proteins of the invention can be used as immmlogens to produce anti-NOV
antibodies in a
subject, to purify NOV ligands, and in screening assays to identify molecules
that inlubit the
interaction of NOV with a NOV ligand.
A NOV chimeric or fusion protein of the invention can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for the
different polypeptide
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sequences are ligated 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 caxried 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 1N MOLECULAR BIOLOGY, John Wiley &
Sons, 1992). Moreover, many expression vectors are commercially available that
already encode
a fusion moiety (e.g., a GST polypeptide). A NOV-encoding nucleic acid can be
cloned into such
an expression vector such that the fusion moiety is linked in-frame to the NOV
protein.
NOV Agonists and Antagonists
The invention also pertains to variants of the NOV proteins that function as
either NOV
agonists (i.e., mimetics) or as NOV antagonists. Variants of the NOV protein
can be generated by
mutagenesis (e.g., discrete point mutation or truncation of the NOV protein).
An agonist of the
NOV protein can retain substantially the same, or a subset of, the biological
activities of the
naturally occurring form of the NOV protein. An antagonist of the NOV protein
can inhibit one
or more of the activities of the naturally occurring form of the NOV protein
by, for example,
competitively binding to a downstream or upstream member of a cellular
signaling cascade which
includes the NOV protein. Thus, specific biological effects can be elicited by
treahnent with a
variant of limited function. In one embodiment, treatment of a subject with a
variant having a
subset of the biological activities of the naturally occurring form of the
protein has fewer side
effects in a subject relative to treatment with the naturally occurring form
of the NOV proteins.
Variants of the NOV proteins that function as either NOV agonists (i.e.,
mimetics) or as
NOV antagonists can be identified by screening combinatorial libraries of
mutants (e.g.,
truncation mutants) of the NOV proteins for NOV protein agonist or antagonist
activity. In one
embodiment, a variegated library of NOV variants is generated by combinatorial
mutagenesis at
the nucleic acid level and is encoded by a variegated gene library. A
variegated library of NOV
variants can be produced by, for example, enzylnatically ligating a mixture of
synthetic
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oligonucleotides into gene sequences such that a degenerate set of potential
NOV sequences is
expressible as individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for
phage display) containing the set of NOV sequences therein. There are a
variety of methods
which can be used to produce libraries of potential NOV 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 NOV sequences. Methods for
synthesizing
degenerate oligonucleotides are well-known within the art. See, e.g., Narang,
1983. Tetrahedron
39: 3; Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al.,
1984. Science 198: 1056;
Ike, et al., 1983. Nucl. Acids Res. 1 l: 477.
Polypeptide Libraries
In addition, libraries of fragments of the NOV protein coding sequences can be
used to
generate a variegated population of NOV fragments for screening and subsequent
selection of
variants of a NOV protein. In one embodiment, a library of coding sequence
fragments can be
generated by treating a double stranded PGR fragment of a NOV 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 NOV proteins.
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 NOV 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
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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 NOV variants. See, e.g.,
Arkin and Yourvan,
1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al., 1993.
Protein Engineering
6:327-331.
Anti-NOV Antibodies
The invention encompasses antibodies and antibody fragments, such as Fab or
(Fab)2, that
bind immunospecifically to any of the NOV polypeptides of said invention.
An isolated NOV protein, or a portion or fragment thereof, can be used as an
irninunogen
to generate antibodies that bind to NOV polypeptides using standard techniques
for polyclonal
and monoclonal antibody preparation. The full-length NOV proteins can be used
or, alternatively,
the invention provides antigenic peptide fragments of NOV proteins for use as
immunogens. The
antigenic NOV peptides comprises at least 4 amino acid residues of the amino
acid sequence
shown in SEQ m NO: 2, 5, 7, 66 or 68, and encompasses an epitope of NOV such
that an
antibody raised against the peptide forms a specific immune complex with NOV.
Preferably, the
antigenic peptide comprises at least 6, 8, 10, 15, 20, or 30 amino acid
residues. Longer antigenic
peptides are sometimes preferable over shorter antigenic peptides, depending
on use and
according to methods well known to someone skilled in the art.
In certain embodiments of the invention, at least one epitope encompassed by
the
antigenic peptide is a region of NOV that is located on the surface of the
protein (e.g., a
hydrophilic region). As a means for targeting antibody production, hydropathy
plots showing
regions of hydrophilicity and hydrophobicity can 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. Proc. Nat. Acad. Sci.
USA 78: 3824-
3828; Kyte and Doolittle, 1982. J. Mol. Biol. 157: 105-142, each incorporated
herein by reference
in their entirety).
As disclosed herein, NOV protein sequences of SEQ m NO: 2, 5, 7, 66 or 68, or
derivatives, fragments, analogs or homologs thereof, can be utilized as
immunogens in the
generation of antibodies that immunospecifically-bind these protein
components. The term
"antibody" as used herein refers to imrnunoglobulin molecules and
immunologically-active
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portions of immunoglobulin molecules, i.e., molecules that contain an antigen
binding site that
specifically-binds (immunoreacts with) an antigen, such as NOV. Such
antibodies include, but
are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab and
F(ab')Z fragments, and
an Fab expression library. In a specific embodiment, antibodies to human NOV
proteins are
disclosed. Various procedures lcnown within the art can be used for the
production of polyclonal
or monoclonal antibodies to a NOV protein sequence of SEQ )17 NO: 2, 5, 7, 66
or 68, or a
derivative, fragment, analog or homolog thereof. Some of these proteins are
discussed below.
For the production of polyclonal antibodies, various suitable host animals
(e.g., rabbit,
goat, mouse or other mammal) can be immunized by injection with the native
protein, or a
synthetic variant thereof, or a derivative of the foregoing. An appropriate
immunogenic
preparation can contain, for example, recombinantly-expressed NOV protein or a
chemically-
synthesized NOV polypeptide. 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.),
human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or
similar
immunostimulatory agents. If desired, the antibody molecules directed against
NOV can be
isolated from the mammal (e.g., from the blood) and further purified by well
known techniques,
such as protein A chromatography to obtain the IgG fraction.
The teen "monoclonal antibody" or "monoclonal antibody composition", as used
herein,
refers to a population of antibody molecules that contain only one species of
an antigen binding
site capable of immunoreacting with a particular epitope of NOV. A monoclonal
antibody
composition thus typically displays a single binding affinity for a particular
NOV protein with
which it immunoreacts. For preparation of monoclonal antibodies directed
towards a particular
NOV protein, or derivatives, fragments, analogs or homologs thereof, any
technique that provides
for the production of antibody molecules by continuous cell line culture can
be utilized. Such
techniques include, but are not limited to, the hybridoma technique (see,
e.g., Kohler & Milstein,
1975. Nature 256: 495-497); the trioma technique; the human B-cell hybridoma
technique (see,
e.g., Kozbor, et al., 1983. Immunol. Today 4: 72) and the EBV hybridoma
technique to produce
human monoclonal antibodies (see, e.g., Cole, et al., 1985. In: MONOCLONAL
ANTIBODIES
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AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal
antibodies can be
utilized in the practice of the invention and can be produced by using human
hybi-idomas (see,
e.g., Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by
transfonning human B-
cells with Epstein Barr Virus in vitro (see, e.g., Cole, et al., 1985. In:
MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Each of the
above
citations is incorporated herein by reference in their entirety.
According to the invention, techniques can be adapted for the production of
single-chain
antibodies specific to a NOV protein (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 NOV protein or derivatives, fragments,
analogs or homologs
thereof. Non-human antibodies can be "humanized" by techniques well known in
the art. See,
e.g., U.S. Patent No. 5,225,539. Antibody fragments that contain the idiotypes
to a NOV protein
can 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(ab')2 fragment; (iii) an Fab
fragment generated by the
treatment of the antibody molecule with papain and a reducing agent; and (iv)
Fv fragments.
Additionally, recombinant anti-NOV antibodies, such as chimeric and humanized
monoclonal antibodies, comprising both human and non-human portions, which can
be made
using standard recombinant DNA techniques, are within the scope of the
invention. Such
chimeric and humanized monoclonal antibodies can be produced by recombinant
DNA tecluuques
known in the art, for example using methods described in International
Application No.
PCT/LTS86/02269; European Patent Application No. 184,187; European Patent
Application No.
171,496; European Patent Application No. 173,494; PCT International
Publication No. WO
86/01533; U.S. Patent No. 4,816,567; U.S. Pat. Na. 5,225,539; European Patent
Application No.
125,023; Better, et al., 1988. Science 240: 1041-1043; Liu, et al., 1987.
Proc. Natl. Acad. Sci.
USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139: 3521-3526; Sun, et al.,
1987. Proc. Natl.
Acad. Sci. USA 84: 214-218; Nishimura, et al., 1987. Cancer Res. 47: 999-1005;
Wood, et al.,
1985. Nature 314 :446-449; Shaw, et al., 1988. J. Natl. Cancer Inst. 80: 1553-
1559);
Morrison(1985) Science 229:1202-1207; Oi, et al. (1986) BioTechniques 4:214;
Jones, et al.,
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1986. Nature 321: 552-525; Verhoeyan, et al., 1988. Science 239: 1534; and
Beidler, et al., 1988.
J. Tinmunol. 141: 4053-4060. Each of the above citations are incorporated
herein by reference in
their entirety.
In one embodiment, methods for the screening of antibodies that possess the
desired
specificity include, but are not limited to, enzyme-liu~ed immunosorbent assay
(ELISA) and other
irrununologically-mediated techniques known within the art. In a specific
embodiment, selection
of antibodies that are specific to a particular domain of a NOV protein is
facilitated by generation
of hybridomas that bind to the fragment of a NOV protein possessing such a
domain. Thus,
antibodies that are specific for a desired domain within a NOV protein, or
derivatives, fragments,
analogs or homologs thereof, are also provided herein.
Anti-NOV antibodies can be used in methods lmown within the art relating to
the
localization and/or quantitation of a NOV protein (e.g., for use in measuring
levels of the NOV
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 NOV
proteins, or
derivatives, fragments, analogs or homologs thereof, that contain the antibody
derived binding
domain, are utilized as pharmacologically-active compounds (hereinafter
"Therapeutics").
An anti-NOV antibody (e.g., monoclonal antibody) can be used to isolate a NOV
polypeptide by
standard techniques, such as affinity chromatography or immunoprecipitation.
An anti-NOV
antibody can facilitate the purification of natural NOV polypeptide from cells
and of
recombinantly-produced NOV polypeptide expressed in host cells. Moreover, an
anti-NOV
antibody can be used to detect NOV protein (e.g., in a cellular lysate or cell
supernatant) in order
to evaluate the abundance and pattern of expression of the NOV protein. Anti-
NOV 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. Detection
can be facilitated by coupling (i.e., physically linking) the antibody to a
detectable substance.
Examples of detectable substances include various enzymes, prosthetic groups,
fluorescent
materials, luminescent materials, bioluminescent materials, and radioactive
materials. Examples
of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-
galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group complexes include
streptavidin/biotin
and avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein,
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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 131I, ssS or 3H.
NOV Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors,
containing a nucleic acid encoding a NOV 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) are
integrated into the genome of a host cell upon introduction into the host
cell, and thereby are
replicated along with the host genome. Moreover, certain vectors are capable
of directing the
expression of genes to which they are operatively-lined. 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 connnonly 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 lined to the regulatory sequences) in a
manner that allows
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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., NOV proteins,
mutant forms of NOV
proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for
expression of
NOV proteins in prokaryotic or eulcaryotic cells. For example, NOV proteins
can be expressed in
bacterial cells such as Escherichia 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
Diego, Calif. (1990). Alternatively, the recombinant expression vector can be
transcribed and
translated in vitro, for example using T7 promoter regulatory sequences and T7
polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia
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.
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Such enzymes, and their cognate recognition sequences, include Factor Xa,
thrombin and
enterolcinase. Typical fusion expression vectors include pGEX (Pharmacia
Biotech Inc; Smith
and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.)
and pRITS
(Phannacia, 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) Gene 69:301-315) and pET l 1d (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: 2111-2118). Such alteration of
nucleic acid sequences of
the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the NOV expression vector is a yeast expression vector.
Examples of
vectors for expression in yeast Saccharomyces cerivisae include pYepSecl
(Baldari, et al., 1987.
EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943),
pJRY88 (Schultz
et al., 1987. Gene 54: 113-123), pYES2 (W vitrogen Corporation, San Diego,
Calif.), and picZ
(InVitrogen Corp, San Diego, Calif.). Alternatively, NOV can be expressed in
insect cells using
baculovirus expression vectors. Baculovirus vectors available for expression
of proteins in
cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al.,
1983. Mol. Cell. Biol. 3:
2156-2165) and the pVL series (Luclclow 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
pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufinan, 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
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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. Immunol. 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. Science
230: 912-916), and
marmnary 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 a-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 fox
expression (by transcription of the DNA molecule) of an RNA molecule that is
antisense to NOV
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 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
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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, NOV
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 terms
"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 6418, hygromycin and methotrexate. Nucleic acid
encoding a
selectable marker can be introduced into a host cell on the same vector as
that encoding NOV or
can be introduced on a separate vector. Cells stably transfected with the
introduced nucleic acid
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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) NOV protein. Accordingly, the invention
further provides methods
for producing NOV 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 NOV protein has been introduced) in a suitable medium such that NOV
protein is
produced. In another embodiment, the method further comprises isolating NOV
protein from the
medium or the host cell.
Transgenic NOV 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 NOV protein-coding sequences have been
introduced. Such host
cells can then be used to create non-human transgenic animals in which
exogenous NOV
sequences have been introduced into their genome or homologous recombinant
animals in which
endogenous NOV sequences have been altered. Such annals are useful for
studying the function
andlor activity of NOV protein and for identifying and/or evaluating
modulators of NOV 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, chiclcens, 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 NOV 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 NOV-
encoding nucleic acid
into the male pronuclei of a fertilized oocyte (e.g., by microinjection,
retroviral infection) and
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allowing the oocyte to develop in a pseudopregnant female foster animal. The
human NOV
cDNA sequences of SEQ ID NO:1, 4, 6, 65 or 67 can be introduced as a transgene
into the
genome of a non-human animal. Alternatively, a non-human homologue of the
human NOV
gene, such as a mouse NOV gene, can be isolated based on hybridization to the
human NOV
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 NOV transgene
to direct expression of NOV 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 THE 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 NOV transgene in its genome and/or expression of NOV 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 NOV protein
can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains
at least a
portion of a NOV gene into which a deletion, addition or substitution has been
introduced to
thereby alter, e.g., functionally disrupt, the NOV gene. The NOV gene can be a
human gene (e.g.,
the cDNA of SEQ ID NO: 1, 4, 6, 65 or 67), but more preferably, is a non-human
homologue of a
human NOV gene. For example, a mouse homologue of human NOV gene of SEQ ID NO:
1, 4,
6, 8 or 10 can be used to construct a homologous recombination vector suitable
for altering an
endogenous NOV gene in the mouse genome. W one embodiment, the vector is
designed such
that, upon homologous recombination, the endogenous NOV 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 NOV 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
NOV protein). In the homologous recombination vector, the altered portion of
the NOV gene is
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flanlced at its 5'- and 3'-termini by additional nucleic acid of the NOV gene
to allow for
homologous recombination to occur between the exogenous NOV gene carried by
the vector and
an endogenous NOV gene in an embryonic stem cell. The additional flanking NOV
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 then introduced into an embryonic stem
cell line (e.g., by
electroporation) and cells in which the introduced NOV gene has homologously-
recombined with
the endogenous NOV 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 further in Bradley, 1991. Curr. Opin. Biotechnol. 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 P1. For a
description of the cre/loxP
recombinase system, See, e.g., Lakso, et al., 1992. Proc. Natl. Acad. 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.
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Clones of the non-human transgenic animals described herein can also be
produced
according to the methods described in Wilinut, 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 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 NOV nucleic acid molecules, NOV proteins, and anti-NOV 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
carrier" 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 carriers 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. Liposomes
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
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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 bisulfate; 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 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 exaanple,
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., a
NOV protein or anti-NOV 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
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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 for use
as a mouthwash, wherein the compound in the fluid carrier 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
life 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.
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 admiiustration can also be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate to the
burner 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 transdennal administration, 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,
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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 for preparation of such
formulations will be
apparent to those slcilled in the art. The materials can also be obtained
commercially from Alza
Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including
liposomes
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.
It is especially advantageous to formulate oral or parenteral compositions in
dosage unit
form for ease of administration and uniformity of dosage. Dosage uut 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 earner. 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 an 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. 5,328,470) or by
stereotactic injection
(see, e.g., Chen, et al., 1994. Proc. 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
NOV protein
(e.g., via a recombinant expression vector in a host cell in gene therapy
applications), to detect
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NOV mRNA (e.g., in a biological sample) or a genetic lesion in a NOV gene, and
to modulate
NOV activity, as described further, below. In addition, the NOV proteins can
be used to screen
drugs or compounds that modulate the NOV protein activity or expression as
well as to treat
disorders characterized by insufficient or excessive production of NOV protein
or production of
NOV protein forms that have decreased or aberrant activity compared to NOV
wild-type protein.
In addition, the anti-NOV antibodies of the invention can be used to detect
and isolate NOV
proteins and modulate NOV activity.
The invention further pertains to novel agents identified by the screening
assays described
herein and uses thereof for treatments as described, supra.
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 NOV proteins or
have a stimulatory
or inhibitory effect on, e.g., NOV protein expression or NOV protein activity.
The invention also
includes compounds identified in the screening assays described herein.
W 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 a NOV
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, wlule the other
four approaches are
applicable to peptide, non-peptide oligomer or small molecule libraries of
compounds. See, e.g.,
Lam, 1997. Anticancer 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,
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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 call be found in
the art, for
example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb,
et al., 1994. Proc.
Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37:
2678; Cho, et al.,
1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl.
33: 2.059; Carell, et
al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al., 1994. J.
Med. Chem. 37:
1233.
Libraries of compounds can be presented in solution (e.g., Houghten, 1992.
Biotechniques
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.
Proc. Natl. Acad. Sci. U.S.A. 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 NOV 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 a NOV
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 NOV 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 NOV 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 NOV protein, or a biologically-active portion thereof, on the cell
surface with a known
compound which binds NOV to form an assay mixture, contacting the assay
mixture with a test
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compound, and determining the ability of the test compound to interact with a
NOV protein,
wherein determining the ability of the test compound to interact with a NOV
protein comprises
determining the ability of the test compound to preferentially bind to NOV
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 NOV 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 NOV protein or biologically-active
portion thereof.
Determining the ability of the test compound to modulate the activity of NOV
or a
biologically-active portion thereof can be accomplished, for example, by
determining the ability
of the NOV protein to bind to or interact with a NOV target molecule. As used
herein, a "target
molecule" is a molecule with which a NOV protein binds or interacts in nature,
for example, a
molecule on the surface of a cell which expresses a NOV 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. A NOV target
molecule can be a
non-NOV molecule or a NOV protein or polypeptide of the invention . In one
embodiment, a
NOV 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
NOV 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 NOV.
Determining the ability of the NOV protein to bind to or interact with a NOV
target
molecule can be accomplished by one of the methods described above for
determining direct
binding. In one embodiment, determining the ability of the NOV protein to bind
to or interact
with a NOV 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 catalytic/enzyrnatic activity of the target an appropriate
substrate, detecting the
induction of a reporter gene (comprising a NOV-responsive regulatory element
operatively linked
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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 a NOV protein or biologically-active portion thereof with a test
compound and
determining the ability of the test compound to bind to the NOV protein or
biologically-active
portion thereof. Binding of the test compound to the NOV protein can be
determined either
directly or indirectly as described above. hi one such embodiment, the assay
comprises
contacting the NOV protein or biologically-active portion thereof with a known
compound which
binds NOV to form an assay mixture, contacting the assay mixture with a test
compound, and
determining the ability of the test compound to interact with a NOV protein,
wherein determining
the ability of the test compound to interact with a NOV protein comprises
determining the ability
of the test compound to preferentially bind to NOV or biologically-active
portion thereof as
compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising
contacting NOV
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
NOV protein or
biologically-active portion thereof. Determining the ability of the test
compound to modulate the
activity of NOV can be accomplished, for example, by determining the ability
of the NOV protein
to bind to a NOV 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 NOV protein can be accomplished by determining the ability of
the NOV protein to
further modulate a NOV target molecule. For example, the catalytic/enzymatic
activity of the
target molecule on an appropriate substrate can be determined as described,
supra.
In yet another embodiment, the cell-free assay comprises contacting the NOV
protein or
biologically-active portion thereof with a lmown compound which binds NOV
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 a NOV protein, wherein determining the
ability of the test
compound to interact with a NOV protein comprises determining the ability of
the NOV protein
to preferentially bind to or modulate the activity of a NOV target molecule.
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The cell-free assays of the invention are amenable to use of both the soluble
form or the
membrane-bound form of NOV protein. In the case of cell-free assays comprising
the membrane-
bound form of NOV protein, it can be desirable to utilize a solubilizing agent
such that the
membrane-bound form of NOV protein is maintained in solution. Examples of such
solubilizing
agents include non-ionic detergents such as n-octylglucoside, n-
dodecylglucoside, n-
dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide,
Triton~ X-100,
Triton~ X-114, Thesit0, Isotridecypoly(ethylene glycol ether)n, 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
can be
desirable to immobilize either NOV protein or 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 NOV protein, or
interaction of NOV
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 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-NOV 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 NOV 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 NOV 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 NOV protein or its target
molecule can be
immobilized utilizing conjugation of biotin and streptavidin. Biotinylated NOV
protein or target
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molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using
techniques well-
lcnown within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,
Ill.), and inunobilized in
the wells of streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies
reactive with NOV protein or target molecules, but which do not interfere with
binding of the
NOV protein to its target molecule, can be derivatized to the wells of the
plate, and unbound
target or NOV 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 NOV protein or
target
molecule, as well as enzyme-linked assays that rely on detecting an enzymatic
activity associated
with the NOV protein or target molecule.
In another embodiment, modulators of NOV protein expression are identified in
a method
wherein a cell is contacted with a candidate compound and the expression of
NOV mRNA or
protein in the cell is determined. The level of expression of NOV mRNA or
protein in the
presence of the candidate compound is compared to the level of expression of
NOV mRNA or
protein in the absence of the candidate compound. The candidate compound can
then be
identified as a modulator of NOV mRNA or protein expression based upon this
comparison. For
example, when expression of NOV mRNA or protein is greater (i.e.,
statistically significantly
greater) in the presence of the candidate compound than in its absence, the
candidate compound is
identified as a stimulator of NOV mRNA or protein expression. Alternatively,
when expression
of NOV mRNA 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 NOV
mRNA or protein expression. The level of NOV mRNA or protein expression in the
cells can be
determined by methods described herein for detecting NOV mRNA or protein.
In yet another aspect of the invention, the NOV 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. Chem. 268: 12046-12054; Bartel, et
al., 1993.
Biotechniques 14: 920-924; Iwabuchi, et al., 1993. Oncogene 8: 1693-1696; and
Brent WO
94/10300), to identify other proteins that bind to or interact with NOV ("NOV-
binding proteins"
or "NOV-by") and modulate NOV activity. Such NOV-binding proteins are also
likely to be
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involved in the propagation of signals by the NOV proteins as, for example,
upstream or
downstream elements of the NOV 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 NOV 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 a
NOV-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 NOV.
The invention further 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 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 NOV sequences, SEQ ID NO:
1, 4, 6, 65 or
67, or fragments or derivatives thereof, can be used to map the location of
the NOV genes,
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respectively, on a chromosome. The mapping of the NOV sequences to chromosomes
is an
important first step in correlating these sequences with genes associated with
disease.
Briefly, NOV genes can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 by in length) from the NOV sequences. Computer analysis of
the NOV,
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 NOV 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 caimot 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 a
single thermal cycler. Using the NOV 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
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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
Yorlc 1988).
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 McI~usick, 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 NOV 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 sequencing of genes from
several individuals
can be performed to confirm the presence of a mutation and to distinguish
mutations from
polymorphisms.
Tissue Typing
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The NOV 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 NOV 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 NOV 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
N0:1, 4, 6, 65 or
67 are used, a more appropriate number of primers for positive individual
identification would be
500-2,000.
Predictive Medicine
The invention also pertains to the field of predictive medicine in which
diagnostic assays,
prognostic assays, phannacogenomics, and monitoring clinical trials are used
for prognostic
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(predictive) purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of
the invention relates to diagnostic assays for determining NOV protein and/or
nucleic acid
expression as well as NOV 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 NOV expression
or activity. The
invention also provides for prognostic (or predictive) assays for determining
whether an
individual is at risk of developing a disorder associated with NOV protein,
nucleic acid expression
or activity. For example, mutations in a NOV gene can be assayed in a
biological sample. Such
assays can be used for prognostic or predictive purpose to thereby
prophylactically treat am
individual prior to the onset of a disorder characterized by or associated
with NOV protein,
nucleic acid expression, or biological activity.
Another aspect of the invention provides methods for determining NOV 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 NOV in clinical trials.
These and other agents are described in further detail in the following
sections.
Diagnostic Assays
An exemplary method for detecting the presence or absence of NOV 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 NOV protein or nucleic
acid (e.g.,
mRNA, genomic DNA) that encodes NOV protein such that the presence of NOV is
detected in
the biological sample. An agent for detecting NOV mRNA or genomic DNA is a
labeled nucleic
acid probe capable of hybridizing to NOV mRNA or genomic DNA. The nucleic acid
probe can
be, for example, a full-length NOV nucleic acid, such as the nucleic acid of
SEQ m NO: 1, 4, 6,
65 or 67 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 NOV
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mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of
the invention
are described herein.
An agent for detecting NOV protein is an antibody capable of binding to NOV
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')2)
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 NOV mRNA, protein, or genomic DNA in a biological sample in
vitro as well as in
vivo. For example, in vitro techniques for detection of NOV mRNA include
Northern
hybridizations and in situ hybridizations. In vitro techniques for detection
of NOV protein
include enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations,
and immunofluorescence. In vitro techniques for detection of NOV genomic DNA
include
Southern hybridizations. Furthermore, in vivo techniques for detection of NOV
protein include
introducing into a subject a labeled anti-NOV 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
subject or genomic DNA molecules from the test subject. A preferred biological
sample is a
peripheral blood leukocyte sample isolated by conventional means from a
subject.
In another embodiment, the methods further involve obtaining a control
biological sample
from a control subject, contacting the control sample with a compound or agent
capable of
detecting NOV protein, mRNA, or genomic DNA, such that the presence of NOV
protein, mRNA
or genomic DNA is detected in the biological sample, and comparing the
presence of NOV
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protein, mRNA or genomic DNA in the control sample with the presence of NOV
protein, mRNA
or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of NOV in a
biological
sample. For example, the lcit can comprise: a labeled compound or agent
capable of detecting
NOV protein or mRNA in a biological sample; means for determining the amount
of NOV in the
sample; and means for comparing the amount of NOV 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 NOV 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
NOV 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 NOV protein, nucleic acid expression or activity. For example,
those involviilg
development, differentiation, and activation of thymic immune cells; in
pathologies related to
spermatogenesis and male infertility; diagnosis of several human neoplasias;
in diseases or
pathologies of cells in blood circulation such as red blood cells and
platelets; neurological, cardiac
and vascular pathologies; rheumatoid arthritis; congenital muscular
dystrophies; various muscle
disorders; fixed deformities (arthrogryposis); small cell lung cancer NCI-H23;
prostate cancer;
and abnormal white matter. 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 NOV
expression or activity
in which a test sample is obtained from a subject and NOV protein or nucleic
acid (e.g., mRNA,
genomic DNA) is detected, wherein the presence of NOV protein or nucleic acid
is diagnostic for
a subject having or at risk of developing a disease or disorder associated
with aberrant NOV
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,
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peptide, nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder
associated with aberrant NOV 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 NOV expression or activity in
which a test sample is
obtained and NOV protein or nucleic acid is detected (e.g., wherein the
presence of NOV protein
or nucleic acid is diagnostic for a subject that can be administered the agent
to treat a disorder
associated with aberrant NOV expression or activity).
The methods of the invention can also be used to detect genetic lesions in a
NOV 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 a NOV-
protein, or the misexpression of the NOV 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 a
NOV gene; (ii) an addition of one or more nucleotides to a NOV gene; (iii) a
substitution of one
or more nucleotides of a NOV gene, (iv) a chromosomal rearrangement of a NOV
gene; (v) an
alteration in the level of a messenger RNA transcript of a NOV gene, (vi)
aberrant modification of
a NOV 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 a NOV gene, (viii)
a non-wild-type
level of a NOV protein, (ix) allelic loss of a NOV gene, and (x) inappropriate
post-translational
modification of a NOV protein. As described herein, there are a large number
of assay techniques
known in the art which can be used for detecting lesions in a NOV 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 can 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.
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Sci. USA 91: 360-364), the latter of which can be particularly useful for
detecting point mutations
in the NOV-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 a NOV gene under conditions
such that
hybridization and amplification of the NOV 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 that PCR and/or
LCR can be 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~
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 a NOV 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 loss of
a ribozyme
cleavage site.
In other embodiments, genetic mutations in NOV 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 NOV can be
identified in two dimensional arrays containing light-generated DNA probes as
described in
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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
malting 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.
hi yet another embodiment, any of a variety of sequencing reactions known in
the art can
be used to directly sequence the NOV gene and detect mutations by comparing
the sequence of
the sample NOV with the corresponding wild-type (control) sequence. Examples
of sequencing
reactions include those based on techniques developed by Maxim and 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 l9: 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 NOV 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 NOV 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, RNA/DNA duplexes can be treated with
RNase and
DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the
mismatched regions.
In other embodiments, either DNA/DNA or RNAIDNA 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.
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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 NOV
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 a NOV sequence, e.g., a wild-type NOV 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.
W other embodiments, alterations in electrophoretic mobility will be used to
identify
mutations in NOV genes. For example, single strand conformation polymorphism
(SSCP) can be
used 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 NOV 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
can be labeled or detected with labeled probes. The sensitivity of the assay
can 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.
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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 teclmiques for detecting point mutations include, but are
not limited to,
selective oligonucleotide hybridization, selective amplification, or selective
primer extension. For
example, oligonucleotide primers can be prepared in which the known 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 al., 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 can 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,
under appropriate
conditions, mismatch can prevent, or reduce polymerase extension (see, e.g.,
Prossner, 1993.
Tibtech. 11: 238). In addition it can 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. Acad.
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 can be performed, for example, by utilizing pre-
packaged
diagnostic bits comprising at least one probe nucleic acid or antibody reagent
described herein,
which can be conveniently used, e.g., in clinical settings to diagnose
patients exlubiting symptoms
or family history of a disease or illness involving a NOV gene. Furthermore,
any cell type or
tissue, preferably peripheral blood leukocytes, in which NOV is expressed can
be utilized in the
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prognostic assays described herein. However, any biological sample containing
nucleated cells
can be used, including, for example, buccal mucosal cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on NOV
activity (e.g.,
NOV gene expression), as identified by a screening assay described herein can
be administered to
individuals to treat (prophylactically or therapeutically) disorders (e.g.,
cancer or immune
disorders associated with aberrant NOV activity. 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 can 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 NOV protein, expression of
NOV nucleic acid,
or mutation content of NOV 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. Pharnacol. Physiol., 23: 983-985; Linder, 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
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polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT
2) and
cytochrome P450 enzymes CYP2D6 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 taping 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 CYP2D6 gene amplification.
Thus, the activity of NOV protein, expression of NOV nucleic acid, or mutation
content of
NOV 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 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 a NOV
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 NOV (e.g., the ability to modulate aberrant cell proliferation and/or
differentiation) can be
applied not only in basic drug screenng, but also in clinical trials. For
example, the effectiveness
of an agent determined by a screening assay as described herein to increase
NOV gene
expression, protein levels, or upregulate NOV activity, can be monitored in
clinical trails of
subjects exhibiting decreased NOV gene expression, protein levels, or
downregulated NOV
activity. Alternatively, the effectiveness of an agent determined by a
screening assay to decrease
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NOV gene expression, protein levels, or downregulate NOV activity, can be
monitored in clinical
trails of subj ects exhibiting increased NOV gene expression, protein levels,
or upregulated NOV
activity. In such clinical trials, the expression or activity of NOV 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 NOV, that are
modulated in
cells by treatment with an agent (e.g., compound, drug or small molecule) that
modulates NOV
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 NOV 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 NOV 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 can 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
screening assays described
herein) comprising the steps of (i) obtaining a pre-administration sample from
a subject prior to
administration of the agent; (ii) detecting the level of expression of a NOV
protein, mRNA, or
genomic DNA in the preadministration sample; (iii) obtaining one or more post-
administration
samples from the subject; (iv) detecting the level of expression or activity
of the NOV protein,
mRNA, or genomic DNA in the post-administration samples; (v) comparing the
level of
expression or activity of the NOV protein, mRNA, or genomic DNA in the pre-
administration
sample with the NOV protein, mRNA, or genomic DNA in the post administration
sample or
samples; and (vi) altering the administration of the agent to the subj ect
accordingly. For example,
increased administration of the agent can be desirable to increase the
expression or activity of
NOV to higher levels than detected, i.e., to increase the effectiveness of the
agent. Alternatively,
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decreased administration of the agent can be desirable to decrease expression
or activity of NOV
to lower levels than detected, i.e., to decrease the effectiveness of the
agent.
Methods of Treatment
The invention provides for both prophylactic and therapeutic methods of
treating a subj ect
at rislc of (or susceptible to) a disorder or having a disorder associated
with aberrant NOV
expression or activity. Such related diseases or disorders include for NOV 1
for example, those
involving development, differentiation, and activation of thymic immune cells;
in pathologies
related to spermatogenesis and male infertility; diagnosis of several human
neoplasias; in diseases
or pathologies of cells in blood circulation such as red blood cells and
platelets; and small cell
lung cancer NCI-H23; for NOV2 and NOV4, for example, neurological, cardiac and
vascular
pathologies; for NOV3 and NOVS, for example, rheumatoid arthritis; congenital
muscular
dystrophies; various muscle disorders; fixed deformities (arthrogryposis);
prostate cancer; and
abnormal white matter. These methods of treatment will be discussed more
fully, below.
Disease and Disorders
Diseases and disorders that are characterized by increased (relative to a
subject not
suffering from the disease or disorder) levels or biological activity can be
treated with
Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics
that antagonize
activity can be administered in a therapeutic or prophylactic manner.
Therapeutics that can be
utilized include, but are not limited to: (i) an aforementioned peptide, or
analogs, derivatives,
fragments or homologs thereof; (ii) antibodies to an aforementioned peptide;
(iii) nucleic acids
encoding an aforementioned peptide; (iv) administration of antisense nucleic
acid and nucleic
acids that are "dysfunctional" (i.e., due to a heterologous insertion within
the coding sequences of
coding sequences to an aforementioned peptide) that are utilized to "knockout"
endoggenous
function of an aforementioned peptide by homologous recombination (see, e.g.,
Capecchi, 1989.
Science 244: 1288-1292); or (v) modulators ( i.e., inhibitors, agonists and
antagonists, including
additional peptide mimetic of the invention or antibodies specific to a
peptide of the invention)
that alter the interaction between an aforementioned peptide and its binding
partner.
Diseases and disorders that are characterized by decreased (relative to a subj
ect not
suffering from the disease or disorder) levels or biological activity can be
treated with
Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that
upregulate activity can
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be administered in a therapeutic or prophylactic manner. Therapeutics that can
be utilized
include, but are not limited to, an aforementioned peptide, or analogs,
derivatives, fragments or
homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide
and/or RNA,
by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying
it in vitro for RNA or
peptide levels, structure and/or activity of the expressed peptides (or mRNAs
of an
aforementioned peptide). Methods that are well-known within the art include,
but are not limited
to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed
by sodium
dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry,
etc.) and/or
hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot
blots, in situ
hybridization, and the lilce).
Prophylactic Methods
In one aspect, the invention provides a method for preventing, in a subject, a
disease or
condition associated with an aberrant NOV expression or activity, by
administering to the subject
an agent that modulates NOV expression or at least one NOV activity. These
conditions include
for NOV 1, for example, those involving development; differentiation, and
activation of thymic
immune cells; in pathologies related to spermatogenesis and male infertility;
diagnosis of several
human neoplasias; in diseases or pathologies of cells in blood circulation
such as red blood cells
and platelets; and small cell ltmg cancer NCI-H23; for NOV2 and NOV4, for
example,
neurological, cardiac and vascular pathologies; for NOV3 and NOVS, for
example, rheumatoid
arthritis; congenital muscular dystrophies; various muscle disorders; fixed
deformities
(arthrogryposis); prostate cancer; and abnormal white matter. Subjects at risk
for a disease that is
caused or contributed to by aberrant NOV expression or activity can be
identified by, for example,
any or a combination of diagnostic or prognostic assays as described herein.
Administration of a
prophylactic agent can occur prior to the manifestation of symptoms
characteristic of the NOV
aberrancy, such that a disease or disorder is prevented or, alternatively,
delayed in its progression.
Depending upon the type of NOV aberrancy, for example, a NOV agonist or NOV
antagonist
agent can be used for treating the subject. The appropriate agent can be
determined based on
screening assays described herein. The prophylactic methods of the invention
are further
discussed in the following subsections.
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Therapeutic Methods
Another aspect of the invention pertains to methods of modulating NOV
expression or
activity for therapeutic purposes. The modulatory method of the invention
involves contacting a
cell with an agent that modulates one or more of the activities of NOV protein
activity associated
with the cell. An agent that modulates NOV protein activity can be an agent as
described herein,
such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a
NOV protein, a
peptide, a NOV peptidomimetic, or other small molecule. In one embodiment, the
agent
stimulates one or more NOV protein activity. Examples of such stimulatory
agents include active
NOV protein and a nucleic acid molecule encoding NOV that has been introduced
into the cell.
In another embodiment, the agent inhibits one or more NOV protein activity.
Examples of such
inhibitory agents include antisense NOV nucleic acid molecules and anti-NOV
antibodies. These
modulatory methods can be performed in vitro (e.g., by culturing the cell with
the agent) or,
alternatively, in vivo (e.g., by administering the agent to a subject). As
such, the invention
provides methods of treating an individual afflicted with a disease or
disorder characterized by
aberrant expression or activity of a NOV protein or nucleic acid molecule. In
one embodiment,
the method involves administering an agent (e.g., an agent identified by a
screening assay
described herein), or combination of agents that modulates (e.g., up-regulates
or down-regulates)
NOV expression or activity. In another embodiment, the method involves
administering a NOV
protein or nucleic acid molecule as therapy to compensate for reduced or
aberrant NOV
expression or activity.
Stimulation of NOV activity is desirable in situations in wluch NOV is
abnormally
downregulated and/or in which increased NOV activity is likely to have a
beneficial effect. One
example of such a situation is where a subject has a disorder characterized by
aberrant cell
proliferation and/or differentiation (e.g., cancer or immune associated
disorders). Another
example of such a situation is where the subject has a gestational disease
(e.g., preclampsia).
Determination of the Biological Effect of the Therapeutic
In various embodiments of the invention, suitable in vitro or in vivo assays
are performed
to determine the effect of a specific Therapeutic and whether its
administration is indicated for
treatment of the affected tissue.
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In various specific embodiments, in vitro assays can be performed with
representative
cells of the types) involved in the patient's disorder, to determine if a
given Therapeutic exerts
the desired effect upon the cell type(s). Compounds for use in therapy can be
tested in suitable
animal model systems including, but not limited to rats, mice, chicken, cows,
monkeys, rabbits,
and the like, prior to testing in human subjects. Similarly, for in vivo
testing, any of the animal
model system known in the art can be used prior to administration to human
subjects.
Prophylactic and Therapeutic Uses of the Compositions of the Invention
The NOV nucleic acids and proteins of the invention are useful in potential
prophylactic
and therapeutic applications implicated in a variety of disorders including,
but not limited to: for
NOV 1 those involving development, differentiation, and activation of thymic
immune cells; in
pathologies related to spermatogenesis and male infertility; diagnosis of
several human
neoplasias; in diseases or pathologies of cells in blood circulation such as
red blood cells and
platelets; in blood circulation such as red blood cells and platelets; for
NOV2 and NOV4
neurological, cardiac and vascular pathologies; for NOV3 and NOVS rheumatoid
arthritis;
congenital muscular dystrophies; various muscle disorders; fixed deformities
(arthrogryposis);
and abnormal white matter.
As an example, a cDNA encoding the NOV protein of the invention can be useful
in gene
therapy, and the protein can be useful when administered to a subject in need
thereof. By way of
non-limiting example, the compositions of the invention will have efficacy for
treatment of
patients suffering from the above mentioned disorders.
Both the novel nucleic acid encoding the NOV protein, and the NOV protein of
the
invention, or fragments thereof, may also be useful in diagnostic
applications, wherein the
presence or amount of the nucleic acid or the protein are to be assessed. A
further use could be as
an anti-bacterial molecule (i.e., some peptides have been found to possess
anti-bacterial
properties). These materials are further useful in the generation of
antibodies which
immunospecifically-bind to the novel substances of the invention for use in
therapeutic or
diagnostic methods. Those involving development, differentiation, and
activation of thymic
immune cells; in pathologies related to spermatogenesis and male infertility;
diagnosis of several
human neoplasias; in diseases or pathologies of cells in blood circulation
such as red blood cells
and platelets; neurological, cardiac and vascular pathologies; rheumatoid
arthritis; congenital
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muscular dystrophies; various muscle disorders; fixed deformities
(arthrogryposis); and abnormal
white matter
EXAMPLES
The following examples illustrate by way of non-limiting example various
aspects of the
invention.
Example 1. Quantitative expression analysis of NOVl, NOV2 and NOV3 in various
cells and
tissues
The quantitative expression of various clones was assessed in about 41 normal
and about
55 tumor samples by real time quantitative PCR (TAQMAN°) performed on a
Perkin-Elmer
Biosystems ABI PRISM~ 7700 Sequence Detection System. In the following Tables
15-17, the
following abbreviations are used:
ca. = carcinoma,
* = established fiom metastasis,
met = metastasis,
s cell var= small cell variant,
non-s = non-sm =non-small,
squam = squasnous,
p1. eff = p1 effusion = pleural effusion,
glio = glioma,
astro = astrocytoma, and
neuro = neuroblastoma
NAT = normal adj acent tissue.
First, up to 96 RNA samples were normalized to (3-actin and GAPDH. RNA (~50 ng
total
or ~1 ng polyA+) was converted to cDNA using the TAQMAN° Reverse
Transcription Reagents
I~it (PE Biosystems, Foster City, CA; Catalog No. N808-0234) and random
hexamers according
to the manufacturer's protocol. Reactions were performed in 20 u1 and
incubated for 30 min. at
48°C. cDNA (S u1) was then transferred to a separate plate for the
TAQMAN~ reaction using [3-
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actin and GAPDH TAQMAN~ Assay Reagents (PE Biosystems; Catalog Nos. 4310881E
and
4310884E, respectively) and TAQMANOO universal PCR Master Mix (PE Biosystems;
Catalog
No. 4304447) according to the manufacturer's protocol. Reactions were
performed in 25 u1 using
the following parameters: 2 min. at 50°C; 10 min. at 95°C; 15
sec. at 95°C/1 min. at 60°C (40
cycles). Results were recorded as CT values (cycle at which a given sample
crosses a threshold
level of fluorescence) using a log scale, with the difference in RNA
concentration between a
given sample and the sample with the lowest CT value being represented as 2 to
the power of
delta CT. The percent relative expression is then obtained by taking the
reciprocal of this RNA
difference and multiplying by 100. The relative expression percent is in
reference to the 13-actin
and GAPDH levels. Higher relative expression in a normal versus a cancerous
tissues indicates an
increased expression of gene in cancerous tissues and that the gene is a
marker for a type of
cancer. The average CT values obtained for 13-actin and GAPDH were used to
normalize RNA
samples. The RNA sample generating the highest CT value required no further
diluting, while all
other samples were diluted relative to this sample according to their (3-actin
/GAPDH average CT
values.
Normalized RNA (5 u1) was converted to cDNA and analyzed via TAQMAN~ using One
Step RT-PCR Master Mix Reagents (PE Biosystems; Catalog No. 4309169) and gene-
specific
primers according to the manufacturer's instructions. Probes and primers were
designed for each
assay according to Perkin Elmer Biosystem's Primes Express Software package
(version I for
Apple Computer's Macintosh Power PC) or a similar algorithm using the target
sequence as input.
Default settings were used for reaction conditions and the following
parameters were set before
selecting primers: primer concentration = 250 nM, primer melting temperature
(Tn,) range = 58°-
60° C, primer optimal Tm = 59° C, maximum primer difference =
2° C, probe does not have 5' G,
probe Tm must be 10° C greater than primer Tm, amplicon size 75 by to
100 bp. The probes and
primers selected (see below) were synthesized by Synthegen (Houston, TX, USA).
Probes were
double purified by HPLC to remove uncoupled dye and evaluated by mass
spectroscopy to verify
coupling of reporter and quencher dyes to the 5' and 3' ends of the probe,
respectively. Their
final concentrations were: forward and reverse primers, 900 nM each, and
probe, 200nM.
PCR conditions: Normalized RNA from each tissue and each cell line was spotted
in each well of a 96
well PCR plate (Perlcin Elmer Biosystems). PCR cocktails including two probes
(SEQX-specific and
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another gene-specific probe multiplexed with the SEQX probe) were set up using
1X TaqManTM PCR
Master Mix for the PE Biosystems 7700, with 5 mM MgCl2, dNTPs (dA, G, C, U at
1:1:1:2 ratios), 0.25
U/ml AmpliTaq GoIdTM (PE Biosystems), and 0.4 U/~1 RNase inhibitor, and 0.25
U/O1 reverse
transcriptase. Reverse transcription was performed at 48° C for 30
minutes followed by amplification/PCR
cycles as follows: 95° C 10 min, then 40 cycles of 95° C for 15
seconds, 60° C for 1 minute.
A. NOV1
Probe Name: Ag190
PrimersSequences Length Start SEQ
PositionID
NO:
Forward5'-TGGAGGAAGAATCACCACAAGA-3' 22 243 8
Probe TET-5'-CAAGCCACAAACTGTGACGTGAACCTG-3'-27 271 9
TAMRA
Reverse5'-GTGGCATCAGCACGGAGTG-3' 19 300 10
The results obtained for clone NOV 1 using primer-probe set Ag190 are shown in
Table 15.
Table 15.
Tissue_Name Relative Tissue Name Relative
~
Expression % Expression
Endothelial cells 0.0 Renal ca. 786-0 0.0
Endothelial cells 0.0 Renal ca. A498 0.0
(treated)
Pancreas 0.0 Renal ca. RXF 393 0.0
Pancreatic ca. CAPAN 0.0 Renal ca. ACHN 0.9
2
Adrenal Gland (new 92.0 Renal ca. U0-31 0.0
lot*)
Thyroid 0.0 Renal ca. TK-10 0.6
Salavary gland 23.8 Liver 0.0
Pituitary gland 0.0 Liver (fetal) 0.0
Brain (fetal) 0.0 Liver ca. (hepatoblast)32.1
HepG2
Brain (whole) 0.0 Lung 0.0
Brain (amygdala) 0.0 Lung (fetal) 0.0
Brain (cerebellum) 0.0 Lung ca. (small 0.0
cell) LX-1
Brain (hippocampus) 0.0 Lung ca. (small 22.9
cell) NCI-
H69
Brain (thalamus) 0.0 Lung ca. (s.cell 0.0
var.) SHP-77
Cerebral Cortex 1.2 Lung ca. (large 4.0
cell)NCI-
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H460
Spinal cord 0.0 Lung ca. (non-sm. 8.4
cell) A549
CNS ca. (glio/astro) U87-MG2.5 Lung ca. (non-s.cell)100.0
NCI-
H23
CNS ca. (glio/astro) U-118-MG0.0 Lung ca (non-s.cell) 0.8
HOP-62
CNS ca. (astro) SW1783 0.0 Lung ca. (non-s.cl) 15.4
NCI-H522
CNS ca.* (neuro; met ) 7.8 Lung ca. (squam.) 0.0
SK-N-AS SW 900
CNS ca. (astro) SF-539 0,0 Lung ca. (squam.) 42.3
NCI-H596
CNS ca. (astro) SNB-75 0.0 Mammary gland 0.0
CNS ca. (glio) SNB-19 2.1 Breast ca.* (p1. effusion)2.1
MCF-7
CNS ca. (glio) U251 0.0 Breast ca.* (pl.ef) 0.0
MDA-MB-
231
CNS ca. (glio) SF-295 0.3 Breast ca.* (p1. effusion)21.0
T47D
Heart 0.0 Breast ca. BT-549 0.0
Skeletal Muscle (new lot*)0.0 Breast ca. MDA-N 16.5
Bone marrow 0.0 Ovary 0.0
Thymus 0.0 Ovarian ca. OVCAR-3 1.0
Spleen 0.0 Ovarian ca. OVCAR-4 0.2
Lymph node 0.0 Ovarian ca. OVCAR-5 1.8
Colorectal 0.0 Ovarian ca. OVCAR-8 7.5
Stomach 0.0 Ovarian ca. IGROV-1 3.9
Small intestine 7.3 Ovarian ca.* (ascites)0.5
SK-
OV-3
Colon ca. SW480 0.0 Uterus 0.0
Colon ca.* (SW480 met)SW6200.4 Plancenta 0.0
Colon ca. HT29 0.0 Prostate 2.1
Colon ca. HCT-116 14.6 Prostate ca.* (bone 11.5
met)PC-3
Colon ca. CaCo-2 14.5 Testis 0.0
.
83219 CC Well to Mod Diff 0.0 Melanoma Hs688(A).T 0.0
(0D03866)
Colon ca. HCC-2998 55.1 Melanoma* (met) 0.0
Hs688(B).T
Gastric ca.* (liver met) 0.0 Melanoma UACC-62 0.0
NCI-N87
Bladder 0.0 Melanoma M14 0.0
Trachea 0.0 Melanoma LOX IMVI 0.0
Kidney 4.4 Melanoma* (met) SK-MEL-50.0
Kidney (fetal) 6.1
Table 15.A.
Tissue Name Rel. Expr., % 2dtm2376t
ag190
Normal Colon GENPAiC 061003 27
83219 CC Well to Mod Diff (0D03866)8.9
83220 CC NAT (0D03866) 15.8
83221 CC Gr.2 rectosigmoid (0D03868)5.1
83222 CC NAT (0D03868) 0
83235 CC Mod Diff (0D03920) 35.1
83236 CC NAT (0D03920) 7
83237 CC Gr.2 ascend colon (0D039219
)
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83238 CC NAT (0D03921 ) 0
83241 CC from Partiai Hepatectomy (0D04309) 7.9
83242 Liver NAT (0D04309) 4.1
87472 Colon mets to lung (0D04451-01) 37.9
87473 Lung NAT (0D04451-02) 0
Normal Prostate Clontech A+ 6546-1 23.7
84140 Prostate Cancer (0D04410) 12.5
84141 Prostate NAT (0D04410) 19.2
87073 Prostate Cancer (0D04720-01 ) 35.6
87074 Prostate NAT (0D04720-02) 4.5
Normal Lung GENPAK 061010 3.6
83239 Lung Met to Muscle (0D04286) 0
83240 Muscle NAT (0D04286) 8.1
84136 Lung Malignant Cancer (0D03126) 0
84137 Lung NAT (0D03126) 4.6
84871 Lung Cancer (0D04404) 0
84872 Lung NAT (0D04404) 10.3
84875 Lung Cancer (0D04565) 0
84876 Lung NAT (0D04565) 0
85950 Lung Cancer (0D04237-01 ) 70.2
85970 Lung NAT (0D04237-02) 0
83255 Ocular Mel Met to Liver (0D04310) 9.3
83256 Liver NAT (0D04310) 0
84139 Meianoma Mets to Lung (0D04321 ) 28.3
84138 Lung NAT (0D04321 ) 0
Normal Kidney GENPAK 061008 40.6
83786 Kidney Ca, Nuclear grade 2 (0D04338) 0
83787 Kidney NAT (0D04338) 17.8
83788 Kidney Ca Nuclear grade 1/2 (0D04339) 0
83789 Kidney NAT (0D04339) 4
83790 Kidney Ca, Clear cell type (0D04340) 4.1
83791 Kidney NAT (0D04340) 4.5
83792 Kidney Ca, Nuclear grade 3 (0D04348) 0
83793 Kidney NAT (0D04348) 4
87474 Kidney Cancer (0D04622-01 ) 0
87475 Kidney NAT (0D04622-03) 0
85973 Kidney Cancer (0D04450-01 ) 4.9
85974 Kidney NAT (0D04450-03) 10.5
Kidney Cancer Clontech 8120607 0
Kidney NAT Clontech 8120608 4.8
Kidney Cancer Clontech 8120613 0
Kidney NAT Clontech 8120614 4.1
Kidney Cancer Clontech 9010320 4.2
Kidney NAT Clontech 9010321 0
Normal Uterus GENPAK 061018 . 4
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Uterus Cancer GENPAK 064011 33.2
Normal Thyroid Clontech A+ 6570-1 100
Thyroid Cancer GENPAK 064010 14.8
Thyroid Cancer INVITROGEN A302152 4.3
Thyroid NAT INVITROGEN A302153 34.2
Normal Breast GENPAK 061019 39.8
84877 Breast Cancer (0D04566) 4.9
85975 Breast Cancer (0D04590-01 ) 0
85976 Breast Cancer Mets (0D04590-03) 2.3
87070 Breast Cancer Metastasis (0D04655-05) 19.1
GENPAK Breast Cancer 064006 0
Breast Cancer Res. Gen. 1024 8.2
Breast Cancer Clontech 9100266 15.9
Breast NAT Clontech 9100265 0
Breast Cancer INVITROGEN A209073 22.1
Breast NAT INVITROGEN A2090734 34.4
Normal Liver GENPAK 061009 0
Liver Cancer GENPAK 064003 13.4
Liver Cancer Research Genetics RNA 1025 0
Liver Cancer Research Genetics RNA 1026 0
Paired Liver Cancer Tissue Research Genetics RNA 6004-T 5.6
Paired Liver Tissue Research Genetics RNA 6004-N 30.6
Paired Liver Cancer Tissue Research Genetics RNA 6005-T 0
Paired Liver Tissue Research Genetics RNA 6005-N 0
Normal Bladder GENPAK 061001 0
Bladder Cancer Research Genetics RNA 1023 5.1
Bladder Cancer INVITROGEN A302173 33.2
87071 Bladder Cancer (0D04718-01 ) 3.8
87072 Bladder Normal Adjacent (0D04718-03) 7.6
Normal Ovary Res. Gen. 4
Ovarian Cancer GENPAK 064008 8.9
87492 Ovary Cancer (0D04768-07) 29.9
87493 Ovary NAT (0D04768-08) 4.4
Normal Stomach GENPAK 061017 4.2
Gastric Cancer Clontech 9060358 9.5
NAT Stomach Clontech 9060359 0
Gastric Cancer Clontech 9060395 0
NAT Stomach Clontech 9060394 0
Gastric Cancer Clontech 9060397 15.4
NAT Stomach Clontech 9060396 0
Gastric Cancer GENPAK 064005 5.4
Table 15.B.
Rel. Expr.,
Tissue Name 2Dtm2390t ag190
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Normal Colon GENPAK 061003 63.7
83219 CC Well to Mod Diff (0D03866) 32.3
83220 CC NAT (0D03866) 13
83221 CC Gr.2 rectosigmoid (0D03868) 0
83222 CC NAT (0D03868) 3.1
83235 CC Mod Diff (0D03920) 42
83236 CC NAT (0D03920) 0
83237 CC Gr.2 ascend colon (0D03921 ) ~ 8.1
83238 CC NAT (0D03921 ) 0
83241 CC from Partial Hepatectomy (0D04309) 7.1
83242 Liver NAT (0D04309) 0
87472 Colon mets to lung (0D04451-01 ) 11.8
87473 Lung NAT (0D04451-02) 0
Normal Prostate Clontech A+ 6546-1 63.7
84140 Prostate Cancer (0D04410) 35.4
84141 Prostate NAT (0D04410) 8.7
87073 Prostate Cancer (0D04720-01 ) 0
87074 Prostate NAT (0D04720-02) 44.1
Normal Lung GENPAK 061010 15.8
83239 Lung Met to Muscle (0D04286) 8.4
83240 Muscle NAT (ODO4286) 0
84136 Lung Malignant Cancer (0D03126) 0
84137 Lung NAT (0D03126) 0
84871 Lung Cancer (0D04404) 0
84872 Lung NAT (0D04404) 11.1
84875 Lung Cancer (0D04565) 0
84876 Lung NAT (0D04565) 0
85950 Lung Cancer (0D04237-01 ) 83.5
85970 Lung NAT (0D04237-02) 0
83255 Ocular Mel Met to Liver (0D04310) 0
83256 Liver NAT (0D04310) 0
84139 Melanoma Mets to Lung (0D04321 ) 47
84138 Lung NAT (0D04321 ) 0
Normal Kidney GENPAK 061008 51.4
83786 Kidney Ca, Nuclear grade 2 (0D04338) 0
83787 Kidney NAT (0D04338) 5.6
83788 Kidney Ca Nuclear grade 1/2 (0D04339) 0
83789 Kidney NAT (0D04339) 0
83790 Kidney Ca, Clear cell type (0D04340) 10.7
83791 Kidney NAT (0D04340) 15.6
83792 Kidney Ca, Nuclear grade 3 (0D04348) 0
83793 Kidney NAT (0D04348) 0
87474 Kidney Cancer (0D04622-01 ) 0
87475 Kidney NAT (0D04622-03) 8.9
85973 Kidney Cancer (0D04450-01 ) 3.6
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85974 Kidney NAT ~ (0D04450-03) 4.3
Kidney Cancer Clontech 8120607 0
Kidney NAT Clontech 8120608 6.6
Kidney Cancer Clontech 8120613 0
Kidney NAT Clontech 8120614 9
Kidney Cancer Clontech 9010320 0
Kidney NAT Clontech 9010321 0
Normal Uterus GENPAK 061018 13
Uterus Cancer GENPAK 064011 11.7
Normal Thyroid Clontech A+ 6570-1 100
Thyroid Cancer GENPAK 064010 2.9
Thyroid Cancer INVITROGEN A302152 8
Thyroid NAT INVITROGEN A302153 29.7
Normal Breast GENPAK 061019 24.3
84877 Breast Cancer (0D04566) 0
85975 Breast Cancer (0D04590-01 ) 0
85976 Breast Cancer Mets (0D04590-03) 7.6
87070 Breast Cancer Metastasis (0D04655-05) 6.3
GENPAK Breast Cancer 064006 7.5
Breast Cancer Res. Gen. 1024 12.1
Breast Cancer Clontech 9100266 16.8
Breast NAT Clontech 9100265 0
Breast Cancer INVITROGEN A209073 8.5
Breast NAT INVITROGEN A2090734 25.3
Normal Liver GENPAK 061009 5.5
Liver Cancer GENPAK 064003 13.5
Liver Cancer Research Genetics RNA 1025 0
Liver Cancer Research Genetics RNA 1026 0
Paired Liver Cancer Tissue Research Genetics0
RNA 6004-T
Paired Liver Tissue Research Genetics RNA 0
6004-N
Paired Liver Cancer Tissue Research Genetics0
RNA 6005-T
Paired Liver Tissue Research Genetics RNA 0
6005-N
Normal Bladder GENPAK 061001 8.1
Bladder Cancer Research Genetics RNA 1023 0
Bladder Cancer INVITROGEN A302173 34.9
87071 Bladder Cancer (0D04718-01 ) 0
87072 Bladder Normal Adjacent (0D04718-03) 0
Normal Ovary Res. Gen. 4.3
Ovarian Cancer GENPAK 064008 4.5
87492 Ovary Cancer (0D04768-07) 28.1
87493 Ovary NAT (0D04768-08) 0
Normal Stomach GENPAK 061017 0
Gastric Cancer Clontech 9060358 0
NAT Stomach Clontech 9060359 0
Gastric Cancer Clontech 9060395 0
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NAT Stomach Clontech 9060394 5.4
Gastric Cancer Clontech 9060397 2.3
NAT Stomach Clontech 9060396 0
Gastric Cancer GENPAK 064005 0
Table 15.C.
Tissue Name Rel. Expression % Rel. Expression
1.3Dtm3336t ag190 1.3dx4tm5422t ag190_b1
Liver adenocarcinoma 34.9 17
Pancreas 9.3 7.4
Pancreatic ca. CAPAN 2 9,9 6
Adrenal gland 66.4 34
Thyroid 28.5 29.8
Salivary gland 12.2 15.7
Pituitary gland 32.5 25.3
Brain (fetal) 6.1 45.8
Brain (whole) 19.8 50.8
Brain (amygdala) 16.7 31.1
Brain (cerebellum) 3.2 24.7
Brain (hippocampus) 56.6 9.4
Brain (substantia nigra) 20.4 10.7
Brain (thalamus) 27.9 18.6
Cerebral Cortex 13.2 0
Spinal cord 9 0
CNS ca. (glio/astro) U87-MG 8.1 22
CNS ca. (glio/astro) U-118-MG58.6 100
CNS ca. (astro) SW1783 34.9 0
CNS ca.* (neuro; met ) Si<-N-AS32.1 24.1
CNS ca. (astro) SF-539 14.8 9.1
CNS ca. (astro) SNB-75 20 0
CNS ca. (glio) SNB-19 23.8 22.4
CNS ca. (glio) U251 0 20.6
CNS ca. (glio) SF-295 7.9 7.8
Heart (fetal) 0 0
Heart 4.9 0
Fetal Skeletal 92.7 0
Skeletal muscle 0 0
Bone marrow 7.6 38.1
Thymus 21.3 8
Spleen 12.8 15.1
Lymph node 13 12.9
Colorectal 5 10.3
Stomach 9.7 0
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Small intestine 13.5 20.1
Colon ca. SW480 12.7 17.8
Colon ca.* (SW480 met)SW620 53.2 6.6
Colon ca. HT29 11.3 0
Colon ca. HCT-116 9 0
Colon ca. CaCo-2 48.6 24.3
83219 CC Well to Mod Diff (0D03866)4 0
Colon ca. HCC-2998 12.7 24.5
Gastric ca.* (liver met) NCI-N870 0
Bladder 11 7.6
Trachea 3.5 0
Kidney 0 5.9
Kidney (fetal) 0 0
Renal ca. 786-0 15.3 8.9
Renal ca. A498 4.3 0
Renal ca. RXF 393 0 24
Renal ca. ACHN 23 0
Renal ca. U0-31 5.3 0
Renal ca. TK-10 0 0
Liver 0 0
Liver (fetal) 18.2 17.9
Liver ca. (hepatoblast) HepG2 30.8 18
Lung 0 0
Lung (fetal) 9.4 25.2
Lung ca. (small cell) LX-1 0 11.5
Lung ca. (small cell) NCI-H69 22.7 20.1
Lung ca. (s.cell var.) SHP-77 0 0
Lung ca. (large cell)NCI-H460 20.4 77.7
Lung ca. (non-sm. cell) A549 17.1 45.3
Lung ca. (non-s.cell) NCI-H23 100 26
Lung ca (non-s.cell) HOP-62 0 0
Lung ca. (non-s.cl) NCI-H522 33.7 28
Lung ca. (squam.) SW 900 0 0
Lung ca. (squam.) NCI-H596 29.9 56.2
Mammary gland 0 0
Breast ca.* (p1. effusion) 41.2 54.8
MCF-7
Breast ca.* (pl.ef) MDA-MB-23116.3 2.9
Breast ca.* (p1. effusion) 35.6 34.7
T47D
Breast ca. BT-549 33 70.1
Breast ca. MDA-N 32.5 0
Ovary 4.2 0
Ovarian ca. OVCAR-3 12.5 10
Ovarian ca. OVCAR-4 4.4 35
Ovarian ca. OVCAR-5 7.6 8.1
Ovarian ca. OVCAR-8 22.8 37.9
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Ovarian ca. IGROV-1 2.6 0
Ovarian ca.* (ascites) SK-OV-327.5 37.1
Uterus 18.2 34.2
Placenta 11.7 0
Prostate 19.8 9.5
Prostate ca.* (bone met)PC-333.2 0
Testis 26.2 1.7
Melanoma Hs688(A).T 0 0
Melanoma* (met) Hs688(B).T 0 0
Melanoma UACC-62 2.6 0
Melanoma M14 4.3 8
Melanoma LOX IMVI 5.7 0
Melanoma* (met) SK-MEL-5 10.4 5
Adipose 0 0
Table 15.D.
Rel. Expr., % Rel. Expr.,
Tissue Name l.2tm1980t ag190 1.2tm2173t ag190
Endothelial cells 0 1.1
Heart (fetal) 0 0.4
Pancreas 0 0.6
Pancreatic ca. CAPAN 2 0 0.6
Adrenal Gland (new lot*) 92 27.2
Thyroid 0 0.2
Salavary gland 23.8 10.4
Pituitary gland 0 0
Brain (fetal) 0 0.7
Brain (whole) 0 1
Brain (amygdala) 0 1.9
Brain (cerebellum) 0 0.7
Brain (hippocampus) 0 3.3
Brain (thalamus) 0 1.9
Cerebral Cortex . 1.2 4.6
Spinal cord 0 0.2
CNS ca. (glio/astro) U87-MG 2.5 4.6
CNS ca. (glio/astro) U-118-MG 0 3.5
CNS ca. (astro) SW1783 0 2
CNS ca.* (neuro; met ) SK-N-AS 7.8 2.9
CNS ca. (astro) SF-539 0 1.5
CNS ca. (astro) SNB-75 0 1.2
CNS ca. (glio) SNB-19 2.1 3.1
CNS ca. (glio) U251 0 1.6
CNS ca. (glio) SF-295 0.3 3.3
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Heart 0 4.8
Skeletal Muscle (new lot*) 0 0.1
Bone marrow 0 3.3
Thymus 0 0.7
Spleen 0 0.9
Lymph node 0 0.3
Colorectal 0 1
Stomach 0 0.3
Small intestine 7.3 3.9
Colon ca. SW480 0 1.8
Colon ca.* (SW480 met)SW620 0.4 4.7
Colon ca. HT29 0 2.1
Colon ca. HCT-116 14.6 4.9
Colon ca. CaCo-2 14.5 6.2
83219 CC Well to Mod Diff (0D03866)0 0.7
Colon ca. HCC-2998 55.1 12.2
Gastric ca.* (liver met) NCI-N87 0 0
Bladder 0 4.4
Trachea 0 0.2
Kidney 4.4 6.2
Kidney (fetal) 6.1 1.5
Renal ca. 786-0 0 1
Renal ca. A498 0 0.7
Renal ca. RXF 393 0 0.1
Renal ca. ACHN 0.9 5.6
Renal ca. U0-31 0 0.5
Renal ca. TK-10 0.6 2
Liver 0 1.1
Liver (fetal) 0 2.8
Liver ca. (hepatoblast) HepG2 32.1 7.7
Lung 0 0.1
Lung (fetal) 0 0.2
Lung ca. (small cell) LX-1 0 0.6
Lung ca. (small cell) NCI-H69 22.8 8.9
Lung ca. (s.cell var.) SHP-77 0 0
Lung ca. (large cell)NCI-H460 4 14.8
Lung ca. (non-sm. cell) A549 8.4 5.1
Lung ca. (non-s.cell) NCI-H23 100 15.3
Lung ca (non-s.cell) HOP-62 0.8 2.5
Lung ca. (non-s.cl) NCI-H522 15.4 37.6
Lung ca. (squam.) SW 900 0 2.8
Lung ca. (squam.) NCI-H596 42.3 18.6
Mammary gland 0 0.7
Breast ca.* (p1. effusion) MCF-7 2.1 ~ 3.6
Breast ca.* (pl.ef) MDA-MB-231 0 0.5
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Breast ca.* (p1. effusion) T47D21 9.6
Breast ca. BT-549 0 2.2
Breast ca. MDA-N 16.5 9.2
Ovary 0 1.5
Ovarian ca. OVCAR-3 1 4.9
Ovarian ca. OVCAR-4 0.2 6
Ovarian ca. OVCAR-5 1.8 4.5
Ovarian ca. OVCAR-8 7.5 6.4
Ovarian ca. IGROV-1 3.9 3.3
Ovarian ca.* (ascites) SK-OV-3 0.5 11.3
Uterus 0 1.6
Placenta 0 0.5
Prostate 2.1 4
Prostate ca.* (bone met)PC-3 11.5 5.3
Testis 0 1
Melanoma Hs688(A).T 0 0.4
Melanoma* (met) Hs688(B).T 0 0.4
Melanoma UACC-62 0 0.7
Melanoma M14 0 2.7
Melanoma LOX IMVI 0 1.1
Melanoma* (met) SK-MEL-5 0 4.5
Adipose 94.6 100
Table 15.E.
Rel. Expr.,
Tissue Name 4Dtm3337t ag190
93768 Secondary Th1 anti-CD28/anti-CD3 4
93769 Secondary Th2 anti-CD28/anti-CD3 20
93770 Secondary Tr1 anti-CD28/anti-CD3 9.7
93573 Secondary Th1 resting day 4-6 in IL-2 0
93572_Secondary Th2_resting day 4-6 in IL-2 2.8
93571 Secondary Tr1 resting day 4-6 in IL-2 0
93568_primary Th1 anti-CD28/anti-CD3 19.8
93569_primary Th2 anti-CD28/anti-CD3 8.5
93570_primary Tr1 anti-CD28/anti-CD3 24.5
93565_primary Th1 resting dy 4-6 in IL-2 8.8
93566_primary Th2_resting dy 4-6 in IL-2 0
93567_primary Tr1 resting dy 4-6 in IL-2 5.5
93351 CD45RA CD4 lymphocyte_anti-CD28/anti-CD3 6.5
93352 CD45R0 CD4 lymphocyte anti-CD28/anti-CD3 9.9
93251 CD8 Lymphocytes anti-CD28/anti-CD3 9
93353 chronic CD8 Lymphocytes 2ry_resting dy 4-6 in IL-2 3.8
93574 chronic CD8 Lymphocytes 2ry_activated CD3/CD28 0
93354 CD4 none 1.9
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93252 Secondary Th1/Th2/Tr1 anti-CD95 CH11 0
93103 LAK cells resting 0
93788 LAK cells_IL-2 8
93787 LAK cells_IL-2+IL-12 1.8
93789 LAK cells_IL-2+IFN gamma 1.9
93790 LAK cells IL-2+ IL-18 17.2
93104 LAK cells PMA/ionomycin and IL-18 0
93578 NK Cells IL-2 resting 11.6
93109 Mixed Lymphocyte Reaction Two Way MLR 3
93110 Mixed Lymphocyte Reaction Two Way MLR 0
93111_Mixed Lymphocyte Reaction Two Way MLR 0
93112 Mononuclear Cells (PBMCs) resting 1.8
93113 Mononuclear Cells (PBMCs) PWM 11.9
93114 Mononuclear Cells (PBMCs) PHA-L 3.8
93249_Ramos (B cell) none 74.2
93250 Ramos (B cell) ionomycin 100
93349_B lymphocytes_PWM 2.8
93350 B lymphoytes CD40L and IL-4 3.6
92665_EOL-1 (Eosinophil) dbcAMP differentiated 16.2
93248 EOL-1 (Eosinophil) dbcAMP/PMAionomycin 12.9
93356 Dendritic Cells none 1.9
93355 Dendritic Cells LPS 100 ng/ml 0
93775 Dendritic Cells_anti-CD40 0
93774 Monocytes resting 3.1
93776_Monocytes LPS 50 ng/ml 0
93581_Macrophages resting 0
93582 Macrophages LPS 100 ng/ml 2
93098 HUVEC (Endothelial) none 1.9
93099 HUVEC (Endothelial) starved 7.1
93100 HUVEC (Endothelial) IL-1 b 0
93779_HUVEC (Endothelial)~IFN gamma 3.9
93102_HUVEC (Endothelial)_TNF alpha + IFN gamma 1.9
93101 HUVEC (Endothelial) TNF alpha + IL4 1.8
93781 HUVEC (Endothelial)_IL-11 0
93583 Lung Microvascular Endothelial Cells none 3.6
93584 Lung Microvascular Endothelial Cells TNFa (4 ng/ml) and IL1 b (1 ng/ml)
3.9
92662 Microvascular Dermal endothelium none 2
92663 Microsvasular Dermal endothelium TNFa (4 ng/ml) and IL1b (1 ng/ml) 3.6
93773 Bronchial epithelium TNFa (4 ng/ml) and IL1b (1 ng/ml) ** 0
93347 Small Airway Epithelium none 0
93348 Small Airway Epithelium TNFa (4 ng/ml) and IL1b (1 ng/ml) 3.8
92668 Coronery Artery SMC_resting 1.5
92669 Coronery Artery SMC_TNFa (4 ng/ml) and IL1 b (1 ng/ml) 0
93107 astrocytes_resting 0
93108 astrocytes_TNFa (4 ng/ml) and IL1b (1 ng/ml) 0
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92666 KU-812 (Basophil) resting 13.9
92667 KU-812 (Basophil) PMA/ionoycin 44.1
93579 CCD1106 (Keratinocytes) none 5.3
93580 CCD1106 (Keratinocytes) TNFa and IFNg ** 0
93791 Liver Cirrhosis 8.3
93792 Lupus Kidney 0
93577 NC1-H292 0
93358 NCI-H292_IL-4 0
93360 NC1-H292_IL-9 0
93359 NCI-H292 IL-13 0
93357 NCI-H292 IFN gamma 0
93777 HPAEC - 0
93778 HPAEC_IL-1 beta/TNA alpha 2.1
93254 Normal Human Lung Fibroblast_none 7.8
93253 Normal Human Lung Fibroblast_TNFa (4 ngiml) and IL-1b (1 nglml) 8
93257 Normal Human Lung Fibroblast IL-4 0
93256 Normal Human Lung Fibroblast IL-9 2.1
93255 Normal Human Lung Fibroblast_IL-13 2.1
93258 Normal Human Lung Fibroblast_IFN gamma 5.8
93106 Dermal Fibroblasts CCD1070 resting 14
93361 Dermal Fibroblasts CCD1070 TNF alpha 4 ng/ml 18.4
93105 Dermal Fibroblasts CCD1070 IL-1 beta 1 ng/ml 7.4
93772 dermal fibroblast_IFN gamma 6.1
93771 dermal fibroblast_IL-4 10.2
93259 1BD Colitis 1** 0
93260 1BD Colitis 2 1.7
93261 1BD Crohns 1.8
735010 Colon_normal 7.9
735019 Lung none 3.9
64028-1 Thymus none 15.7
64030-1 Kidney_none 17.1
It is seen that clone NOV 1 expression is enhanced in certain cancer cell
lines, especially
non-small cell lung cancer NCI-H23, breast cancer , and colon cancer, but not
in cell lines from
the corresponding normal tissue. For example, Table 15 shows greatest relative
% expression for
lung carcinoma (100% and 42.3%), normal adrenal (92%) and colon cancer (55.1%)
cell lines.
Table 15.A. shows significant relative expression levels for lung cancer
(83.5% and 70.2%),
prostate (35.6%) and colon cancer metastatic to lung (37.9%). Thus, Table
15.A. provides
support for the results found in Table 15.
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Similarly, Table 15.B. shows high % expression levels for lung cancer (83.5%),
normal
colon (63.7%) and normal prostate (63.7%); Table 15.C. indicates high %
expression levels for
glial/astrocytomal carcinoma (58.6% and 100%), fetal skeletal (92.7%), large
cell lung (77.7%)
and breast (70.1%) cancers; Table 15.D. shows overall lower % expression
levels but still 55.1%
for colon carcinoma, 42.3% for lung squamous cell carcinoma, and 94.6% and
100% for adipose
tissue; and Table 15.E. shows 74.2% for Ramos (B cell) and 100% for Ramos (B
cell)-ionomycin
cell lines.
All these results are consistent with a showing that NOV 1 can be used as a
cancer-
specific marker in such tissues. Moreover, the differences in levels of
expression may be used to
distinguish between fetal and adult same-type tissues.
B. NOV2
Probe Name: Ag087
Primers Sequences LengthStarl SEQ
DJ
PositionNO:
Forward 5'-CGCAGTTTCACTCGGGAGAT-3' 20 1870 11
Probe TET-5'- 1895 12
CCTCTAGGATCCACATCGAGAAAATCATCGG-3'- 31
TAMRA
Reverse 5'-AGCAGACTTCCCCGGAGTCT-3' 20 1932 13
The results obtained on a panel of cell lines for clone NOV2 using primer-
probe set Ag087 are shown in
Table 16, and those obtained on a second panel of surgical tissue samples are
shown in Table 17. 1n Table
17, "NAT"designates surgical tissues deemed not cancerous obtained by the
surgeon from the region
immediately adjacent to a tumor or cancer.
Table 16.
Tissue_Name Relative Tissue_Name Relative
Expression Expression
Endothelial cells 0.3 Kidney (fetal) 1.0
Endothelial cells ~ 0.6 Renal ca.786-0 0.6
(treated)
Pancreas 1.0 Renal ca.A498 0.3
Pancreatic ca. CAPAN2.5 Renal ca. RXF 0.2
2 393
Adipose 1.8 Renal ca. ACHN 0.4
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Adrenal gland 0.2 Renal ca. U0-31 0.3
Thyroid 0.1 Renal ca. TK-10 1,3
Salavary gland 0.2 Liver 0.3
Pituitary gland 0.2 Liver (fetal) 0.1
Brain (fetal) 0.9 Liver ca. (hepatoblast)1.0
HepG2
Brain (whole) 3.0 Lung 0.2
Brain (amygdala) 0.7 Lung (fetal) 0.8
Brain (cerebellum) 7.1 Lung ca. (small cell) 0.3
LX-1
Brain (hippocampus) 2.8 Lung ca. (small cell)NCI-H690.7
Brain (substantia ugra)2.7 Lung ca. (s.cell var.) 25.9
SHP-77
Brain (thalamus) 2.5 Lung ca. (large cell)NCI-H4600.7
Brain (hypothalamus) 0.3 Lung ca. (non-sm. cell)1.1
A549
Spinal cord 2.1 Lung ca. (non-s.cell) 0.6
NCI-H23
CNS ca. (glio/astro)U87-MG0.4 Lung ca (non-s.cell) 1.0
HOP-62
CNS ca. (glio/astro)U-118-MG0.3 Lung ca. (non-s.cl) 0.3
NCI-H522
CNS ca. (astro)SW1783 0.3 Lung ca. (squam.) SW 11.5
900
CNS ca.* (neuro; met 1.1 Lung ca. (squam.) NCI-H5960.8
) SK-N-AS
CNS ca. (astro) SF-539 0.0 Mammary gland 1.8
CNS ca. (astro) SNB-75 2.2 Breast ca.* (p1. effusion)0.3
MCF-7
CNS ca. (glio) SNB-19 2.0 Breast ca.* (pl.ef) 1.6
MDA-MB-231
CNS ca. (glio) U251 0.9 Breast ca.* (p1. effusion)T47D0.5
CNS ca. (glio) SF-295 0.0 Breast ca. BT-549 4.7
Heart 0.4 Breast ca. MDA-N 1.6
Skeletal muscle 0.1 Ovary 0.6
Bone marrow 0.1 Ovarian ca. OVCAR-3 0.6
Thymus 3.5 Ovarian ca. OVCAR-4 0.5
Spleen 0.4 Ovarian ca. OVCAR-5 4.6
Lymph node 0.4 Ovarian ca.OVCAR-8 0.3
Colon (ascending) 0.6 Ovarian ca. IGROV-1 0.6
Stomach 1.3 Ovarian ca.* (ascites) 1.0
SK-OV-3
Small intestine 0.5 Uterus 1.8
Colon ca. SW480 0.3 Plancenta 1.5
Colon ca.* (SW480 met)SW6200.2 Prostate 0.5
Colon ca. HT29 2.8 Prostate ca.* (bone 100.0
met)PC-3
Colon ca. HCT-116 8.0 Testis 4.6
Colon ca. CaCo-2 1.2 Melanoma Hs688(A).T 0.1
Colon ca. HCT-15 0.9 Melanoma* (met) Hs688(B).T0.1
Colon ca. HCC-2998 1.5 Melanoma UACC-62 0.8
Gastric ca.* (liver 2.8 Melanoma M14 0.3
met) NCI-N87
Bladder 0.4 Melanoma LOX IMVI 0.7
Trachea 1.3 Melanoma* (met)SK-MEL-50.2
Kidney 1.7 Melanoma SK-MEL-28 0.3
Table 16.A.
Rel.
Expr., % Rel. Expr.,
Tissue Name tm254t tm252t ag087
Endothelial cells 0.3 0.3
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Endothelial cells (treated)0.1 0.6
Pancreas 10.2 1
Pancreatic ca. CAPAN 2.6 2.5
2
Adipose 2 1.8
Adrenal gland 11.5 0.2
Thyroid 3.1 0.1
Salavary gland 3 0.2
Pituitary gland 7.3 0.2
Brain (fetal) 4.6 0.9
Brain (whole) 15.9 3
Brain (amygdala) 21 0.7
Brain (cerebellum) 98.6 7.1
Brain (hippocampus) 22.1 2.8
Brain (substantia nigra)24.1 2.7
Brain (thalamus) 62,4 2.5
Brain (hypothalamus) 11.3 0.3
Spinal cord 4 2.1
CNS ca. (glio/astro) 0.2 0.4
U87-MG
CNS ca. (glio/astro) 0.6 0.3
U-118-MG
CNS ca. (astro) SW17831.1 0.3
CNS ca.* (neuro; met 2.2 1.1
) SK-N-AS
CNS ca. (astro) SF-5390.5 0
CNS ca. (astro) SNB-750.5 2.2
CNS ca. (glio) SNB-19 2.8 2
CNS ca. (glio) U251 0 0.9
CNS ca. (glio) SF-295 3.5 0
Heart 6 0.4
Skeletal muscle 1.8 0
Bone marrow 0.8 0
Thymus 2.8 3.5
Spleen 10.8 0.4
Lymph node 1.8 0.4
Colon (ascending) 0.4 0.6
Stomach 1.1 1.3
Small intestine 2.8 0.5
Colon ca. SW480 0.6 0.3
Colon ca.* (SW480 met)SW6200.2 0.2
Colon ca. HT29 0.4 2.8
Colon ca. HCT-116 0 8
Colon ca. CaCo-2 0.3 1.2
Colon ca. HCT-15 0.6 0.8
Colon ca. HCC-2998 1.3 1.5
Gastric ca.* (liver 1.1 2.8
met) NCI-N87
Bladder 0.2 0.4
Trachea 0 1.3
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Kidney 2.8 1.7
Kidney (fetal) 11.3 1
Renal ca. 786-0 0.4 0.6
Renal ca. A498 0.3 0.3
Renal ca. RXF 393 0.2 0.2
Renal ca. ACHN 0 0.4
Renal ca. U0-31 0.4 0.3
Renal ca. TK-10 0.3 1.3
Liver 4.5 0.3
Liver (fetal) 2.6 0.1
Liver ca. (hepatoblast)0.3 1
HepG2
Lung 0.1 0.2
Lung (fetal) 4.9 0.8
Lung ca. (small cell) 0 0.3
LX-1
Lung ca. (small cell) 1.7 0.7
NCI-H69
Lung ca. (s.cell var.)0 25.9
SHP-77
Lung ca. (large cell)NCI-H4600 0.7
Lung ca. (non-sm. cell)0.5 1.1
A549
Lung ca. (non-s.cell) 1.4 0.6
NCI-H23
Lung ca (non-s.cell) 0.6 1
HOP-62
Lung ca. (non-s.cl) 7.7 0.3
NCI-H522
Lung ca. (squam.) SW 0.6 11.5
900
Lung ca. (squam.) NCI-H5962.7 0.8
Mammary gland 3.8 1.8
Breast ca.* (p1. effusion)1.6 0.3
MCF-7
Breast ca.* (pl.ef) 0.3 1.6
MDA-MB-231
Breast ca.* (p1. effusion)11 0.5
T47D
Breast ca. BT-549 0 4.7
Breast ca. MDA-N 3.1 1.6
Ovary 4.4 0.6
Ovarian ca. OVCAR-3 0.2 0.6
Ovarian ca. OVCAR-4 0 0.5
Ovarian ca. OVCAR-5 1 4.6
Ovarian ca. OVCAR-8 2 0.2
Ovarian ca. IGROV-1 0.6 0.6
Ovarian ca.* (ascites)1.2 1
SK-OV-3
Uterus 4.8 1.8
Placenta 2.9 1.5
Prostate 3.4 0.5
Prostate ca.* (bone 0 100
met)PC-3
Testis 100 4.6
Melanoma Hs688(A).T 1.3 0.1
Melanoma* (met) Hs688(B).T0.4 0
Melanoma UACC-62 0.2 0.8
Melanoma M14 2.4 0.3
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Melanoma LOX IMVI 4.5 0.7
Melanoma* (met) SK-MEL-5 2.3 0.2
Melanoma SK-MEL-28 4.7 0.3
Table 16.B.
Rel. Expr.,
Tissue Name ~ 1.2tm2217t ag87
Endothelial cells 0
Heart (fetal) 0
Pancreas 97.3
Pancreatic ca. CAPAN 2 0
Adrenal Gland (new lot*) 22.5
Thyroid 0
Salavary gland 0
Pituitary gland 0
Brain (fetal) 3.4
Brain (whole) 8
Brain (amygdala) 24.8
Brain (cerebellum) 8.3
Brain (hippocampus) 20.7
Brain (thalamus) 100
Cerebral Cortex 63.7
Spinal cord 0
CNS ca. (glio/astro) U87-MG 0
CNS ca. (glio/astro) U-118-MG 0
CNS ca. (astro) SW1783 0
CNS ca.* (neuro; met ) SK-N-AS 21.9
CNS ca. (astro) SF-539 0
CNS ca. (astro) SNB-75 0
CNS ca. (glio) SNB-19 0
CNS ca. (glio) 0251 0
CNS ca. (glio) SF-295 0
Heart 0
Skeletal Muscle (new lot*) 4.2
Bone marrow 0
Thymus 0
Spleen 12.2
Lymph node 0
Colorectal 0
Stomach 0
Small intestine 0
Colon ca. SW480 0
Colon ca.* (SW480 met)SW620 0
Colon ca. HT29 0
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Colon ca. HCT-116 0
Colon ca. CaCo-2 0
83219 CC Well to Mod Diff (0D03866) 0
Colon ca. HCC-2998 5.5
Gastric ca.* (liver met) NCI-N87 0
Bladder 2.9
Trachea 0
Kidney 0
Kidney (fetal) 0
Renal ca. 786-0 0
Renal ca. A498 0
Renal ca. RXF 393 0
Renal ca. ACHN 0
Renal ca. U0-31 0
Renal ca. TK-10 0
Liver 0
Liver (fetal) 0
Liver ca. (hepatoblast) HepG2 0
Lung 0
Lung (fetal) 0
Lung ca. (small cell) LX-1 0
Lung ca. (small cell) NCI-H69 33
Lung ca. (s.cell var.) SHP-77 3.3
Lung ca. (large cell)NCI-H460 0
Lung ca. (non-sm. cell) A549 0
Lung ca. (non-s.cell) NCI-H23 1.7
Lung ca (non-s.cell) HOP-62 0
Lung ca. (non-s.cl) NCI-H522 17.1
Lung ca. (squam.) SW 900 0
Lung ca. (squam.) NCI-H596 55.5
Mammary gland 0
Breast ca.* (p1. effusion) MCF-7 0
Breast ca.* (pl.ef) MDA-MB-231 0
Breast ca.* (p1. effusion) T47D 26.6
Breast ca. BT-549 0
Breast ca. MDA-N 0
Ovary 8.2
Ovarian ca. OVCAR-3 0
Ovarian ca. OVCAR-4 0
Ovarian ca. OVCAR-5 0
Ovarian ca. OVCAR-8 0
Ovarian ca. IGROV-1 6.6
Ovarian ca.* (ascites) SK-OV-3 0
Uterus 0
Placenta 0
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Prostate0
Prostate.* (bone met)PC-3 0
ca
Testis 41.5
MelanomaHs688(A).T 0
Melanoma*(met) Hs688(B).T 0
MelanomaUACC-62 0
MelanomaM14 0
MelanomaLOX IMVI 0
Melanoma*(met) SK-MEL-5 0
Adipose24.3
Table 16.C.
Rel. Expr.,
Tissue Name 1.3dtm4681t ag87
Liver adenocarcinoma 0
Pancreas 4.8
Pancreatic ca. CAPAN 2 0
Adrenal gland 0
Thyroid 0
Salivary gland 0
Pituitary gland 7.8
Brain (fetal) 15.2
Brain (whole) 0
Brain (amygdala) 15
Brain (cerebellum) 52.5
Brain (hippocampus) 33
Brain (substantia nigra) 19.9
Brain (thalamus) 88.9
Cerebral Cortex 8.1
Spinal cord 9
CNS ca. (glio/astro) U87-MG 0
CNS ca. (glio/astro) U-118-MG 0
CNS ca. (astro) SW1783 0
CNS ca.* (neuro; met ) SK-N-AS 0
CNS ca. (astro) SF-539 0
CNS ca. (astro) SNB-75 0
CNS ca. (glio) SNB-19 0
CNS ca. (glio) 0251 0
CNS ca. (glio) SF-295 0
Heart (fetal) 0
Heart 0
Fetal Skeletal 9
Skeletal muscle 0
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Bone marrow 0
Thymus 0
Spleen 34.2
0
Lymph node
Colorectal 0
Stomach 0
Small intestine 0
Colon ca. SW480 0
Colon ca.* (SW480 met)SW620 0
Colon ca. HT29 0
Colon ca. HCT-116 0
Colon ca. CaCo-2 0
83219 CC Well to Mod Diff (0D03866) 0
Colon ca. HCC-2998 0
Gastric ca.* (liver met) NCI-N87 13.5
Bladder 11.7
Trachea 0
Kidney 0
Kidney (fetal) 0
Renal ca. 786-0 0
Renal ca. A498 0
Renal ca. RXF 393 0
Renal ca. ACHN 0
Renal ca. U0-31 0
Renal ca. TK-10 0
Liver 0
Liver (fetal) 0
Liver ca. (hepatoblast) HepG2 0
Lung 0
Lung (fetal) 0
Lung ca. (small cell) LX-1 0
Lung ca. (small cell) NCI-H69 0
Lung ca. (s.cell var.) SHP-77 0
Lung ca. (large cell)NCI-H460 0
Lung ca. (non-sm. cell) A549 0
Lung ca. (non-s.cell) NCI-H23 0
Lung ca (non-s.cell) HOP-62 0
Lung ca. (non-s.cl) NCI-H522 0
Lung ca. (squam.) SW 900 0
Lung ca. (squam.) NCI-H596 0
Mammary gland 0
Breast ca.* (p1. effusion) MCF-7 0
Breast ca.* (pl.ef) MDA-MB-231 0
Breast ca.* (p1. effusion) T47D 14.9
Breast ca. BT-549 0
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Breast MDA-N 0
ca.
Ovary 0
OvarianOVCAR-3 0
ca.
OvarianOVCAR-4 0
ca.
OvarianOVCAR-5 0
ca.
OvarianOVCAR-8 0
ca.
OvarianIGROV-1 0
ca.
Ovarian* (ascites) SK-OV-3 0
ca.
Uterus 0
Placenta0
Prostate0
Prostate.* (bone met)PC-3 0
ca
Testis ~ 100
MelanomaHs688(A).T 0
Melanoma*(met) Hs688(B).T 0
MelanomaUACC-62 9.8
MelanomaM14 0
MelanomaLOX IMVI 0
Melanoma*(met) SK-MEL-5 0
Adipose0
TABLE 17.
Relative
Expr.
Tissue_Name/Run_Name 2tm723t 2tm819t
Normal Colon GENPAK 061003 0.0 0.0
83219 CC Well to Mod Diff 0.0 0.0
(0D03866)
83220 CC NAT (ODO3866) 0.0 0.0
83221 CC Gr.2 rectosigmoid 0.0 0.0
(0D03868)
83222 CC NAT (0D03868) 0.0 0.0
83235 CC Mod Diff (ODO3920) 0.0 0.0
83236 CC NAT (0D03920) 0.0 0.0
83237 CC Gr.2 ascend colon 0.0 0.0
(0D03921)
83238 CC NAT (0D03921) 0.0 0.0
83241 CC from Partial Hepatectomy0.0 0.0
(ODO4309)
83242 Liver NAT (0D04309) 27.0 3.0
87472 Colon mets to lung (0D04451-01)0.0 0.0
87473 Lung NAT (0D04451-02) 0.0 0.0
Normal Prostate Clontech A+ 0.0 0.0
6546-1
84140 Prostate Cancer (OD04410)5.6 6.8
84141 Prostate NAT (0D04410) 0.0 0.0
87073 Prostate Cancer (0D04720-01)100.0 100.0
87074 Prostate NAT (0D04720-02)0.6 0.0
Normal Lung GENPAK 061010 0.0 0.0
83239 Lung Met to Muscle (0D04286)0.0 0.0
83240 Muscle NAT (0D04286) 0.4 0.0
84136 Lung Malignant Cancer 1.2 0.0
(0D03126)
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84137 Lung NAT (0D03126) 0.0 0.0
84871 Lung Cancer (0D04404) 1.7 0.0
84872 Lung NAT (0D04404) 0.0 0.0
84875 Lung Cancer (0D04565) 0.0 0.4
85950 Lung Cancer (0D04237-01)0.0 0.0
85970 Lung NAT (0D04237-02) 0.0 0.0
83255 Ocular Mel Met to Liver 1.6 0.8
(0D04310)
83256 Liver NAT (0D04310) 48.6 1.8
84139 Melanoma Mets to Lung 0.0 0.0
(OD04321)
84138 Lung NAT (0D04321) 0.0 0.0
Normal Kidney GENPAK 061008 0.0 0.0
83786 Kidney Ca, Nuclear grade0.0 0.0
2 (0D04338)
83787 Kidney NAT (0D04338) 0.0 0.0
83788 Kidney Ca Nuclear grade 0.0 0.0
1/2 (0D04339)
83789 Kidney NAT (OD04339) 0.9 0.6
83790 Kidney Ca, Clear cell 0.0 0.0
type (0D04340)
83791 Kidney NAT (0D04340) 0.0 0.0
83792 Kidney Ca, Nuclear grade0.0 0.0
3 (OD04348)
83793 Kidney NAT (0D04348) 0.0 0.0
87474 Kidney Cancer (0D04622-01)0.0 0.0
87475 Kidney NAT (0D04622-03) 0.0 0.0
85973 Kidney Cancer (0D04450-01)0.0 0.0
85974 Kidney NAT (0D04450-03) 0.0 0.0
Kidney Cancer Clontech 81206070.0 0.0
Relative Expr.
Tissue_Name/Run_Name 2tm723t 2tm819t
Kidney NAT Clontech 8120608 0.0 0.0
Kidney Cancer Clontech 81206130.0 0.0
Kidney NAT Clontech 8120614 0.0 0.0
Kidney Cancer Clontech 90103200.0 0.0
Kidney NAT Clontech 9010321 0.0 0.0
Normal Uterus GENPAK 061018 0.0 0.0
Uterus Cancer GENPAK 064011 4.2 0.0
Normal Thyroid Clontech A+ 0.0 0.0
6570-1**
Thyroid Cancer GENPAK 064010 0.0 0.0
Thyroid Cancer INVITROGEN 3.3 1.3
A302152
Thyroid NAT INVITROGEN A3021530.0 0.0
Normal Breast GENPAK 061019 3.8 2.9
84877 Breast Cancer (0D04566)42.6 56.6
85975 Breast Cancer (0D04590-01)7.6 6.0
85976 Breast Cancer Mets (OD04590-03)39.2 38.4
87070 Breast Cancer Metastasis35.4 45.7
(0D04655-05)
GENPAK Breast Cancer 064006 50.7 55.1
Breast Cancer Clontech 910026646.0 28.5
Breast NAT Clontech 9100265 2.1 0.0
Breast Cancer 1NVITROGEN A2090738.5 0.0
Breast NAT INVITROGEN A20907344.2 1.3
Normal Liver GENPAK 061009 0.6 0.0
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Liver Cancer GENPAK 064003 0.0 0.0
Liver Cancer Research Genetics RNA 0.5 0.0
1025
Liver Cancer Research Genetics RNA 0.0 0.0
1026
Paired Liver Cancer Tissue Research0.0 0.0
Genetics RNA
6004-T
Paired Liver Tissue Research Genetics0.0 0.0
RNA 6004-N
Paired Liver Cancer Tissue Research0.9 0.0
Genetics RNA
6005-T
Paired Liver Tissue Research Genetics2.7 0.0
RNA 6005-N
Normal Bladder GENPAK 061001 1.4 0.0
Bladder Cancer Research Genetics 0.0 0.0
RNA 1023
Bladder Cancer INVITROGEN A302173 0.0 0.0
87071 Bladder Cancer (0D04718-01) 6.0 1.0
87072 Bladder Normal Adjacent (0D04718-03)2.7 0.0
Normal Ovary Res. Gen. 6.5 0.0
Ovarian Cancer GENPAI~ 064008 0.0 0.0
87492 Ovary Cancer (0D04768-07) 25.9 11.7
87493 Ovary NAT (0D04768-08) 0.0 0.0
Normal Stomach GENPAK 061017 0.0 0.0
NAT Stomach Clontech 9060359 0.0 0.0
Gastric Cancer Clontech 9060395 20.7 27.9
NAT Stomach Clontech 9060394 1.7 0.0
Gastric Cancer Clontech 9060397 0.0 0.0
NAT Stomach Clontech 9060396 0.0 0.0
Gastric Cancer GENPAI~ 064005 0.0 0.0
Table 17.A.
Rel. Expr.,
Rel. Expr.,
2Dtm2323t 2Dtm2361t ago
Tissue Name ag87 87
Normal Colon GENPAfC 061003 0 0
83219 CC Well to Mod Diff (0D03866)0 0
83220 CC NAT (0D03866) 0 , 0
83221 CC Gr.2 rectosigmoid (0D03868)0 0
83222 CC NAT (0D03868) 0 0
83235 CC Mod Diff (0D03920) 0 0
83236 CC NAT (0D03920) 0 0
83237 CC Gr.2 ascend colon (0D039210 0
)
83238 CC NAT (0D03921 ) 0 0
83241 CC from Partial Hepatectomy 0 0
(0D04309)
83242 Liver NAT (0D04309) 0 0
87472 Colon mets to lung (0D04451-01)0 0
87473 Lung NAT (0D04451-02) 0 0
Normal Prostate Clontech A+ 6546-1 0 19.8
84140 Prostate Cancer (0D04410) 0 0
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84141 Prostate NAT (0D04410) 0 0
87073 Prostate Cancer (0D04720-01 0 0
)
87074 Prostate NAT (0D04720-02) 0 0
Normal Lung GENPAK 061010 0 0
83239 Lung Met to Muscle (0D04286) 0 0
83240 Muscle NAT (0D04286) 0 0
84136 Lung Malignant Cancer (0D03126)0 0
84137 Lung NAT (0D03126) 0 0
84871 Lung Cancer (0D04404) 0 0
84872 Lung NAT (0D04404) 0 0
84875 Lung Cancer (0D04565) 0 0
84876 Lung NAT (0D04565) 0 0
85950 Lung Cancer (0D04237-01 ) 0 0
85970 Lung NAT (0D04237-02) 0 0
83255 Ocular Mel Met to Liver (0D04310)0 0
83256 Liver NAT (0D04310) 0 0
84139 Melanoma Mets to Lung (0D043210 0
)
84138 Lung NAT (0D04321 ) 0 0
Normal Kidney GENPAK 061008 0 0
83786 Kidney Ca, Nuclear grade 2 0 0
(0D04338)
83787 Kidney NAT (0D04338) 0 0
83788 Kidney Ca Nuclear grade 1/2 0 0
(0D04339)
83789 Kidney NAT (OD04339) 0 18.7
83790 Kidney Ca, Clear cell type 0 0
(0D04340)
83791 Kidney NAT (0D04340) 0 0
83792 Kidney Ca, Nuclear grade 3 0 0
(0D04348)
83793 Kidney NAT (0D04348) 0 0
87474 Kidney Cancer (0D04622-01 0 0
)
87475 Kidney NAT (0D04622-03) 0 0
85973 Kidney Cancer (0D04450-01 0 0
)
85974 Kidney NAT (0D04450-03) 0 0
Kidney Cancer Clontech 8120607 0 0
Kidney NAT Clontech 8120608 0 0
Kidney Cancer Clontech 8120613 0 0
Kidney NAT Clontech 8120614 0 0
Kidney Cancer Clontech 9010320 0 0
Kidney NAT Clontech 9010321 0 0
Normal Uterus GENPAK 061018 0 0
Uterus Cancer GENPAK 064011 0 0
Normal Thyroid Clontech A+ 6570-1 0 0
Thyroid Cancer GENPAK 064010 0 0
Thyroid Cancer INVITROGEN A302152 0 0
Thyroid NAT INVITROGEN A302153 0 0
Normal Breast GENPAK 061019 0 0
84877 Breast Cancer (0D04566) 0 0
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85975 Breast Cancer (0D04590-01 ) 0 0
85976 Breast Cancer Mets (0D04590-03)46.7 100
87070 Breast Cancer Metastasis (0D04655-05)30.1 0
GENPAK Breast Cancer 064006 0 0
Breast Cancer Res. Gen. 1024 100 90.8
Breast Cancer Clontech 9100266 0 35.8
Breast NAT Clontech 9100265 0 0
Breast Cancer INVITROGEN A209073 0 0
Breast NAT iNVITROGEN A2090734 21.5 0
Normal Liver GENPAK 061009 0 0
Liver Cancer GENPAK 064003 0 0
Liver Cancer Research Genetics RNA 0 0
1025
Liver Cancer Research Genetics RNA 0 0
1026
Paired Liver Cancer Tissue Research
Genetics RNA
6004-T 0 0
Paired Liver Tissue Research Genetics0 0
RNA 6004-N
Paired Liver Cancer Tissue Research
Genetics RNA
6005-T 0 0
Paired Liver Tissue Research Genetics0 0
RNA 6005-N
Normal Bladder GENPAK 061001 13.6 0
Bladder Cancer Research Genetics 0 0
RNA 1023
Bladder Cancer INVITROGEN A302173 0 0
87071 Bladder Cancer (0D04718-01 0 0
)
87072 Bladder Normal Adjacent (0D04718-03)0 0
Normal Ovary Res. Gen. 0 0
Ovarian Cancer GENPAK 064008 0 0
87492 Ovary Cancer (0D04768-07) 0 0
87493 Ovary NAT (0D04768-08) 0 0
Normal Stomach GENPAK 061017 0 0
Gastric Cancer Clontech 9060358 0 0
NAT Stomach Clontech 9060359 0 0
Gastric Cancer Clontech 9060395 15.3 0
NAT Stomach Clontech 9060394 0 0
Gastric Cancer Clontech 9060397 0 0
NAT Stomach Clontech 9060396 0 0
Gastric Cancer GENPAK 064005 0 0
The results in Tables 16 and 17 demonstrate that clone NOV2 is highly
expressed in
certain tumors, especially prostate cancer metastasis, but not in
corresponding normal cell lines,
and that this clone is highly expressed in many surgical tumor samples,
especially prostate cancer,
but minimally or not detestably in the immediate normal adjacent tissue. For
example, Table 16
shows 100% relative expression levels for prostate cancer and 25.9% for lung
carcinoma; Table
16.A. shows expression levels of 98.6% and 62.4% for brain cerebellum and
thalamus
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respectively, and 25.9% for lung carcinoma. Table 16.B. shows expression
levels of 97.3% for
pancreas, 100% for brain thalamus, 55.5% for lung carcinoma and 41.5% for
normal testis, while
Table 16C resulted in expression levels of 52.5% and 88.9% for brain
cerebellum and thalamus
respectively, and 100% for normal testis tissue. The results in Tables 16-16C
are substantiated by
the results in Tables 17-17A where expression levels were 100% for prostate
and breast cancers,
48.6% for normal liver, and 46.7% and 100% for metastatic breast cancer. These
results indicate
that clone NOV2 may be used as a marker for certain cancers, especially
prostate cancer.
NOV3
Probe Name: Ag148
Primers Sequences LengthStart SEQ
m
PositionNO:
Forward 5'-CGCAGTTTCACTCGGGAGAT-3' 20 1870 11
Probe TET-5'- 1895 12
CCTCTAGGATCCACATCGAGAAAATCATCGG-3'- 31
TAMRA
Reverse 5'-AGCAGACTTCCCCGGAGTCT-3' ~ 20 ~ 1932 13
~
The results obtained on a panel of cell lines for clone NOV3 using primer-
probe set Ag148 are shown in
Table 18.
TABLE 18.
Rel. Expr.,
Tissue Name 1.2tm1981f ag148
Endothelial cells 1.4
Heart (fetal) 1.7
Pancreas 1.6
Pancreatic ca. CAPAN 2 0.8
Adrenal Gland (new lot*) 14.1
Thyroid 2
Salavary gland 7.8
Pituitary gland 0.6
Brain (fetal) 2.9
Brain (whole) 6.7
Brain (amygdala) 10.7
Brain (cerebellum) 3
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Brain (hippocampus) 42.9
Brain (thalamus) 29.9
Cerebral Cortex 81.8
Spinal cord 3.1
CNS ca. (glio/astro) U87-MG 3.1
CNS ca. (glio/astro) U-118-MG 2.9
CNS ca. (astro) SW1783 0.7
CNS ca.* (neuro; met ) SK-N-AS4.8
CNS ca. (astro) SF-539 2
CNS ca. (astro) SNB-75 10.5
CNS ca. (glio) SNB-19 8.9
CNS ca. (glio) U251 3.9
CNS ca. (glio) SF-295 7.9
Heart 17.8
Skeletal Muscle (new lot*) 12.7
Bone marrow 1.3
Thymus 0.6
Spleen 0.6
Lymph node
0.1
Colorectal 1.2
Stomach 2.6
Small intestine 12.8
Colon ca. SW480 2.2
Colon ca.* (SW480 met)SW620 6.2
Colon ca. HT29 7.5
Colon ca. HCT-116 3.9
Colon ca. CaCo-2 5.5
83219 CC Well to Mod Diff (0D03866)1.1
Colon ca. HCC-2998 13
Gastric ca.* (liver met) NCI-N879.2
Bladder 4.9
Trachea 0.2
Kidney 64.6
Kidney (fetal) 4.8
Renal ca. 786-0 0.4
Renal ca. A498 4.4
Renal ca. RXF 393 1.6
Renal ca. ACHN 1.6
Renal ca. U0-31 1.6
Renal ca. TK-10 6.1
Liver 2.4
Liver (fetal) 1.3
Liver ca. (hepatoblast) HepG2 3.7
Lung 0.3
Lung (fetal) 0.6
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Lung ca. (small cell) LX-1 9.8
Lung ca. (small cell) NCI-H6921.2
Lung ca. (s.cell var.) SHP-772
Lung ca. (large cell)NCI-H46010.3
Lung ca. (non-sm. cell) A5496.4
Lung ca. (non-s.cell) NCI-H239.1
Lung ca (non-s.cell) HOP-62 5.6
Lung ca. (non-s.cl) NCI-H52212.5
Lung ca. (squam.) SW 900 3.6
Lung ca. (squam.) NCI-H596 21.8
Mammary gland 3.5
Breast ca.* (p1. effusion) 8.1
MCF-7
Breast ca.* (pl.ef) MDA-MB-2311.4
Breast ca.* (p1. effusion) 10.4
T47D
Breast ca. BT-549 2.6
Breast ca. MDA-N 25.5
Ovary 1.6
Ovarian ca. OVCAR-3 8.7
Ovarian ca. OVCAR-4 5.3
Ovarian ca. OVCAR-5 12.6
Ovarian ca. OVCAR-8 5.2
Ovarian ca. IGROV-1 4.4
Ovarian ca.* (ascites) SK-OV-36.3
Uterus 1.7
Placenta 0.4
Prostate 8.2
Prostate ca.* (bone met)PC-35.4
Testis 0.1
Melanoma Hs688(A).T 0.7
Melanoma* (met) Hs688(B).T 1
Melanoma UACC-62 1.3
Melanoma M14 2
Melanoma LOX IMVI 1.3
Melanoma* (met) SK-MEL-5 2.4
Adipose 100
TABLE 18.A.
Rel. Expr.,
Rel. Expr.,
2dtm2779f ag1 2Dtm3038f
Tissue Name 48 ag148
Normal Colon GENPAK 061003 40.1 59.9
83219 CC Well to Mod Diff (0D03866) 16.2 11.6
83220 CC NAT (0D03866) 14.8 25.9
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83221 CC Gr.2 rectosigmoid (0D03868) 2.3 6.4
83222 CC NAT (0D03868) 14.3 16.8
83235 CC Mod Diff (0D03920) 11.5 42
83236 CC NAT (0D03920) 13.3 25.5
83237 CC Gr.2 ascend colon (0D03921 6.7 30.6
)
83238 CC NAT (0D03921 ) 7.6 17.4
83241 CC from Partial Hepatectomy 2.4 15.8
(0D04309)
83242 Liver NAT (0D04309) 0.3 2
87472 Colon mets to lung (0D04451-01)2 0
87473 Lung NAT (0D04451-02) 1 3.7
Normal Prostate Clontech A+ 6546-1 20.3 44.1
84140 Prostate Cancer (0D04410) 18.7 47.6
84141 Prostate NAT (0D04410) 9.8 25.2
87073 Prostate Cancer (0D04720-01 21 35.6
)
87074 Prostate NAT (0D04720-02) 10.4 23
Normal Lung GENPAK 061010 9.6 27.2
83239 Lung Met to Muscle (ODO4286) 14 17.6
83240 Muscle NAT (0D04286) 9.5 21.2
84136 Lung Malignant Cancer (0D03126)0.3 5.8
84137 Lung NAT (0D03126) 1.2 6.2
84871 Lung Cancer (0D04404) 1.2 1.5
84872 Lung NAT (0D04404) 4.5 5.5
84875 Lung Cancer (0D04565) 1.6 3
84876 Lung NAT (0D04565) 1.9 2.9
85950 Lung Cancer (0D04237-01 ) 5.6 16.6
85970 Lung NAT (0D04237-02) 0.8 3.1
83255 Ocular Mel Met to Liver
(ODO4310) 8 7.1
83256 Liver NAT (0D04310) 2.6 3.1
84139 Melanoma Mets to Lung (0D04321 10 18.6
)
84138 Lung NAT (0D04321 ) 0.3 5.6
Normal Kidney GENPAK 061008 29.3 75.3
83786 Kidney Ca, Nuclear grade 2 (0D04338)2.7 7
83787 Kidney NAT (0D04338) 39 44.4
83788 Kidney Ca Nuclear grade 1/2 6.3 9.2
(OD04339)
83789 Kidney NAT (0D04339) 28.9 51
83790 Kidney Ca, Clear cell type (0D04340)11.4 9.2
83791 Kidney NAT (0D04340) 73.2 90.1
83792 Kidney Ca, Nuclear grade 3 (0D04348)3.7 4.3
83793 Kidney NAT (0D04348) 25.5 63.7
87474 Kidney Cancer (0D04622-01 ) 0.5 3
87475 Kidney NAT (0D04622-03) 19.2 21
85973 Kidney Cancer (0D04450-01 ) 1.8 3.3
85974 Kidney NAT (0D04450-03) 34.6 46.7
Kidney Cancer Clontech 8120607 0.8 2.6
Kidney NAT Clontech 8120608 23.8 25.2
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Kidney Cancer Clontech 8120613 7 25.9
Kidney NAT Clontech 8120614 12.4 39
Kidney Cancer Clontech 9010320 2 4.4
Kidney NAT Clontech 9010321 26.4 26.1
Normal Uterus GENPAK 061018 3 7.7
Uterus Cancer GENPAK 064011 8.3 16.3
Normal Thyroid Clontech A+ 6570-1 68.3 100
Thyroid Cancer GENPAK 064010 1.4 8.8
Thyroid Cancer INVITROGEN A302152 6 7.4
Thyroid NAT INVITROGEN A302153 15.2 36.6
Normal Breast GENPAK 061019 8.3 23.7
84877 Breast Cancer (0D04566) 4.1 5.6
85975 Breast Cancer (0D04590-01 ) 17.2 13.6
85976 Breast Cancer Mets (0D04590-03) 20 26.8
87070 Breast Cancer Metastasis (0D04655-05)18.9 7.7
GENPAK Breast Cancer 064006 8.1 14.7
Breast Cancer Res. Gen. 1024 100 100
Breast Cancer Clontech 9100266 8.7 17.9
Breast NAT Clontech 9100265 4.3 16
Breast Cancer INVITROGEN A209073 4 20.2
Breast NAT INVITROGEN A2090734 4.1 18
Normal Liver GENPAK 061009 4.4 10.3
Liver Cancer GENPAK 064003 3.9 1.6
Liver Cancer Research Genetics RNA 5.1 10.6
1025
Liver Cancer Research Genetics RNA 0 0.3
1026
Paired Liver Cancer Tissue Research
Genetics RNA
6004-T 4.9 11
Paired Liver Tissue Research Genetics 24.5 26.4
RNA 6004-N
Paired Liver Cancer Tissue Research
Genetics RNA
6005-T 2.9 3.1
Paired Liver Tissue Research Genetics 0 2
RNA 6005-N
Normal Bladder GENPAK 061001 4.7 19.1
Bladder Cancer Research Genetics RNA 1.9 9.1
1023
Bladder Cancer INVITROGEN A302173 0.3 13.6
Table 18.B.
Tissue Name Rel. Expr., % 1.3Dtm3037f
ag148
Liver adenocarcinoma 3.7
Pancreas 2.4
Pancreatic ca. CAPAN 4.7
2
Adrenal gland 4.8
Thyroid 20.6
Salivary gland 1.0
Pituitary gland 4.5
Brain (fetal) 8.4
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Brain (whole) 28.5
Brain (amygdala) 48.3
Brain (cerebellum) 7.5
Brain (hippocampus) 97.9
Brain (substantia nigra) 5.8
Brain (thalamus) 30.4
Cerebral Cortex 33.4
Spinal cord 6.4
CNS ca. (glio/astro) U87-MG 0.9
CNS ca. (glio/astro) U-118-MG 1.5
CNS ca. (astro) SW1783 1.0
CNS ca.* (neuro; met ) SK-N-AS 4.9
CNS ca. (astro) SF-539 3.4
CNS ca. (astro) SNB-75 5.8
CNS ca. (glio) SNB-19 3.4
CNS ca. (glio) 0251 3.3
CNS ca. (glio) SF-295 9.3
Heart (fetal) 2.5
Heart 0.8
Fetal Skeletal 100.0
Skeletal muscle 0.3
Bone marrow 1.2
Thymus 3.9
Spleen 0.9
Lymph node 1.5
Colorectal 4.8
Stomach 4.7
Small intestine 5.8
Colon ca. SW480 7.7
Colon ca.* (SW480 met)SW620 3.3
Colon ca. HT29 0.6
Colon ca. HCT-116 2.7
Colon ca. CaCo-2 3.2
83219 CC Well to Mod Diff (0D03866)0.5
Colon ca. HCC-2998 2.4
Gastric ca.* (liver met) NCI-N87 3.4
Bladder 1.8
Trachea 1.4
Kidney 2.8
Kidney (fetal) 4.6
Renal ca. 786-0 1.5
Renal ca. A498 7.3
Renal ca. RXF 393 0.4
Renal ca. ACHN 0.9
Renal ca. U0-31 2.0
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Renal ca. TK-10 3.3
Liver 0.9
Liver (fetal) 0.7
Liver ca. (hepatoblast) HepG2 4.0
Lung 0.6
Lung (fetal) 0.7
Lung ca. (small cell) LX-1 4.5
Lung ca. (small cell) NCI-H69 19.1
Lung ca. (s.cell var.) SHP-77 3.0
Lung ca. (large cell)NCI-H460 3.6
Lung ca. (non-sm. cell) A549 1.7
Lung ca. (non-s.cell) NCI-H23 6.7
Lung ca (non-s.cell) HOP-62 5.4
Lung ca. (non-s.cl) NCI-H522 4.8
Lung ca. (squam.) SW 900 1.0
Lung ca. (squam.) NCI-H596 4.6
Mammary gland 2.2
Breast ca.* (p1. effusion) MCF-73.1
Breast ca.* (pl.ef) MDA-MB-231 1.8
Breast ca.* (p1. effusion) T47D 1.6
Breast ca. BT-549 1.7
Breast ca. MDA-N 4.4
Ovary 2.8
Ovarian ca. OVCAR-3 0.7
Ovarian ca. OVCAR-4 0.3
Ovarian ca. OVCAR-5 4.1
Ovarian ca. OVCAR-8 4.0
Ovarian ca. IGROV-1 0.6
Ovarian ca.* (ascites) SK-OV-3 1.6
Uterus 4.2
Placenta 3.5
Prostate 4.7
Prostate ca.* (bone met)PC-3 1.8
Testis 1.8
Melanoma Hs688(A).T 1.5
Melanoma* (met) Hs688(B).T 4.9
Melanoma UACC-62 1.4
Melanoma M14 0.4
Melanoma LOX IMVI 1.4
Melanoma* (met) SK-MEL-5 1.8
Adipose 0.0
Table 18.C.
Rel. Expr.,
Tissue Name 4Dtm3075f ag148
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93768Secondary Th1 anti-CD28/anti-CD3 9.3
93769Secondary Th2_anti-CD28/anti-CD3 12.5
93770Secondary Tr1 anti-CD28/anti-CD3 16.7
93573Secondary Th1 resting day 4-6 in IL-2 8.9
93572Secondary Th2_resting day 4-6 in IL-2 6.7
93571Secondary Tr1 resting day 4-6 in IL-2 10.5
93568_primary Th1 anti-CD28/anti-CD3 26.8
93569_primary Th2 anti-CD28/anti-CD3 17.2
93570_primary Tr1 anti-CD28/anti-CD3 32.8
93565_primary Th1 resting dy 4-6 in IL-2 17.7
93566_primary Th2 resting dy 4-6 in IL-2 8.8
93567_primary Tr1 resting dy 4-6 in IL-2 22.8
93351CD45RA CD4 lymphocyte anti-CD28/anti-CD37.9
93352CD45R0 CD4 lymphocyte anti-CD28/anti-CD315.5
93251CD8 Lymphocytes anti-CD28/anti-CD3 22.5
93353chronic CD8 Lymphocytes 2ry_resting 8.7
dy 4-6 in IL-2
93574chronic CD8 Lymphocytes 2ry_activated 4.9
CD3/CD28
93354CD4_none 3.7
93252Secondary Th1/Th2/Tr1 anti-CD95 CH11 3.3
93103LAK cells resting 9.0
93788LAK cells_IL-2 8.4
93787LAK cells_IL-2+IL-12 10.0
93789LAK cells_IL-2+IFN gamma 6.3
93790LAK cells_IL-2+ IL-18 11.3
93104LAK cells_PMA/ionomycin and IL-18 0.0
93578NK Cells IL-2 resting 2.0
93109_Mixed Lymphocyte Reaction Two Way MLR 12.4
93110Mixed Lymphocyte Reaction Two Way MLR 2.0
93111Mixed Lymphocyte Reaction Two Way MLR 1.8
93112Mononuclear Cells (PBMCs) resting 1.8
93113Mononuclear Cells (PBMCs) PWM 32.3
93114Mononuclear Cells (PBMCs) PHA-L 8.2
93249Ramos (B cell) none 12.6
93250Ramos (B cell) ionomycin 59.0
93349B lymphocytes PWM 18.2
93350B lymphoytes CD40L and IL-4 11.5
92665EOL-1 (Eosinophil) dbcAMP differentiated46.3
93248EOL-1 (Eosinophil) dbcAMP/PMAionomycin 5.6
93356Dendritic Cells none 2.0
93355Dendritic Cells LPS 100 ng/ml 0.0
93775Dendritic Cells_anti-CD40 1.5
93774Monocytes resting 0.0
93776_Monocytes_LPS 50 ng/ml 0.0
93581_Macrophages resting 7.2
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93582_Macrophages_LPS 100 nglml 0.0
93098_HUVEC (Endothelial) none 18.2
93099HUVEC (Endothelial) starved 17.4
93100HUVEC (Endothelial) IL-1 b 7.2
93779HUVEC (Endothelial) IFN gamma 7.7
93102_HUVEC (Endothelial) TNF alpha + IFN gamma 5.3
93101_HUVEC (Endothelial) TNF alpha + IL4 15.0
93781_HUVEC (Endothelial)_IL-11 5.6
93583Lung Microvascular Endothelial Cells none 16.0
93584Lung Microvascular Endothelial Cells TNFa
(4 nglml) and
IL1 ng/ml) 12.9
b
(1
92662Microvascular Dermal endothelium_none 7.0
92663Microsvasular Dermal endothelium_TNFa (4
ng/ml) and IL1 b
(1 1.0
ng/ml)
93773Bronchial epithelium TNFa (4 ng/ml) and 10.3
IL1 b (1 ng/ml) **
93347Small Airway Epithelium none 4.6
93348Small Airway Epithelium TNFa (4 ng/ml) 8.1
and IL1b (1 ng/ml)
92668Coronery Artery SMC resting 0.0
92669Coronery Artery SMC TNFa (4 ng/ml) and 2.3
IL1 b (1 ng/ml)
93107astrocytes resting 7.6
93108astrocytes TNFa (4 ng/ml) and IL1b (1 ng/ml)4.0
92666_KU-812 (Basophil) resting 68.8
92667KU-812 (Basophil) PMA/ionoycin ,47.6
93579CCD1106 (Keratinocytes) none 16.2
93580CCD1106 (Keratinocytes) TNFa and IFNg ** 3.4
93791Liver Cirrhosis 4.3
93792Lupus Kidney 11.3
93577NCI-H292 43.8
~
93358NCI-H292_IL-4 95.3
93360NC1-H292_IL-9 95.9
93359NC1-H292 IL-13 41.8
93357NCI-H292_fFN gamma 52.5
93777HPAEC_- 9.5
93778HPAEC IL-1 beta/TNA alpha 12.0
93254Normal Human Lung Fibroblast_none 4.0
93253Normal Human Lung Fibroblast_TNFa (4 ng/ml)
and IL-1b (1
ng/ml) 1.7
93257Normal Human Lung Fibroblast_IL-4 14.0
93256Normal Human Lung Fibroblast_IL-9 12.6
93255Normal Human Lung Fibroblast_IL-13 4.3
93258Normal Human Lung Fibroblast_IFN gamma 8.2
93106Dermal Fibroblasts CCD1070 resting 25.0
93361Dermal Fibroblasts CCD1070 TNF alpha 4 44.4
ng/ml
93105Dermal Fibroblasts CCD1070_IL-1 beta 1 5.8
ng/ml
93772dermal fibroblast_IFN gamma 11.3
93771dermal fibroblast IL-4 5.7
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93259 1BD Colitis 1** 13.8
93260 1BD Colitis 2 0.0
93261 1BD Crohns 1.9
735010 Colon_normal 40.6
735019 Lung none 9.0
64028-1 Thymus none 100.0
64030-1_Kidney_none 9.7
Table 18.D.
Rel. Expr.,
Tissue Name 4dtm3351f ag148
93768Secondary Th1 anti-CD28/anti-CD3 20.2
93769Secondary Th2 anti-CD28/anti-CD3 12.2
93770Secondary Tr1 anti-CD28/anti-CD3 11.4
93573Secondary Th1 resting day 4-6 in IL-2 3.5
93572_Secondary Th2_resting day 4-6 in IL-2 3.5
93571Secondary Tr1 resting day 4-6 in IL-2 6.6
93568_primary Th1 anti-CD28/anti-CD3 11.7
93569_primary Th2_anti-CD28/anti-CD3 5.6
93570_primary Tr1 anti-CD28/anti-CD3 7.6
93565_primary Th1 resting dy 4-6 in IL-2 5.8
93566_primary Th2_resting dy 4-6 in IL-2 1.8
93567_primary Tr1 resting dy 4-6 in IL-2 5.6
93351CD45RA CD4 lymphocyte anti-CD28/anti-CD3 5.8
93352CD45R0 CD4 lymphocyte anti-CD28/anti-CD3 24.3
93251CD8 Lymphocytes anti-CD28/anti-CD3 9.1
93353chronic CD8 Lymphocytes 2ry_resting dy 4-6 16.0
in IL-2
93574 1.6
chronic
CD8
Lymphocytes
2ry_activated
CD3/CD28
93354CD4 none 0.0
93252Secondary Th1/Th2/Tr1 anti-CD95 CH11 2.7
93103LAK cells resting 4.9
93788LAK cells_IL-2 2.4
93787LAK cells_IL-2+IL-12 6.3
93789LAK cells_IL-2+IFN gamma 7.0
93790LAK cells_IL-2+ IL-18 10.3
93104LAK cells PMA/ionomycin and IL-18 7.7
93578NK Cells IL-2 resting 2.1
93109Mixed Lymphocyte Reaction Two Way MLR 17.3
93110Mixed Lymphocyte Reaction Two Way MLR 7.6
93111Mixed Lymphocyte Reaction Two Way MLR 1.5
93112Mononuclear Cells (PBMCs) resting 1.3
93113Mononuclear Cells (PBMCs) PWM 12.6
93114_Mononuclear Cells (PBMCs) PHA-L 1.6
93249_Ramos (B cell) none 10.8
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93250Ramos (B cell) ionomycin 13.7
93349B lymphocytes_PWM 5.6
93350B lymphoytes CD40L and IL-4 6.2
92665_EOL-1 (Eosinophil) dbcAMP differentiated 58.6
93248_EOL-1 (Eosinophil) dbcAMP/PMAionomycin 14.7
93356Dendritic Cells_none 4.0
93355Dendritic Cells LPS 100 ng/ml 5.4
93775Dendritic Cells_anti-CD40 7.1
93774Monocytes resting 0.0
93776Monocytes LPS 50 ng/ml 3.2
93581Macrophages resting 1.8
93582Macrophages LPS 100 ng/ml 4.4
93098_HUVEC (Endothelial) none 17.3
93099_HUVEC (Endothelial) starved 0.4
93100_HUVEC (Endothelial)_IL-1 b 1.3
93779_HUVEC (Endothelial)_IFN gamma 9.1
93102HUVEC (Endothelial) TNF alpha + IFN gamma 2.8
93101HUVEC (Endothelial) TNF alpha + IL4 14.2
93781HUVEC (Endothelial)_IL-11 7.1
93583Lung Microvascular Endothelial Cells none 11.3
93584Lung Microvascular Endothelial Cells TNFa (4 ng/ml)9.8
and ILIb (1 ng/ml)
92662Microvascular Dermal endothelium none 5.8
92663_Microsvasular Dermal endothelium TNFa (4 ng/ml) 8.8
and IL1b (1 ng/ml)
93773Bronchial epithelium TNFa (4 ng/ml) and IL1b (1 0.2
ng/ml) **
93347Small Airway Epithelium none 3.8
93348Small Airway Epithelium TNFa (4 ng/ml) and IL1b 4.2
(1 ng/ml)
92668Coronery Artery SMC_resting 1.1
92669Coronery Artery SMC TNFa (4 ng/ml) and IL1 b (1 1.4
ng/ml)
93107astrocytes resting 7.3
93108astrocytes_TNFa (4 ng/ml) and IL1b (1 ng/ml) 13.2
92666KU-812 (Basophil) resting 63.7
92667KU-812 (Basophil) PMA/ionoycin 55.1
93579CCD1106 (Keratinocytes) none 11.3
93580CCD1106 (Keratinocytes) TNFa and IFNg ** 0.0
93791Liver Cirrhosis 9.5
93792Lupus Kidney 1.6
93577NC1-H292 16.0
93358NC1-H292_IL-4 28.5
93360NC1-H292_IL-9 23.2
93359NC1-H292 IL-13 100.0
93357NCI-H292_IFN gamma 52.5
93777HPAEC_- 8.9
93778HPAEC_IL-1 beta/TNA alpha 12.9
93254Normal Human Lung Fibroblast none 8.9
93253Normal Human Lung Fibroblast TNFa (4 ng/ml) and 1.0
IL-1b (1 ng/ml)
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93257_Normal Human Lung Fibroblast_IL-4 5.2
93256_Normal Human Lung Fibroblast_IL-9 7.9
93255Normal Human Lung Fibroblast_IL-13 5.3
93258Normal Human Lung Fibroblast_IFN gamma 4.0
93106Dermal Fibroblasts CCD1070 resting 7.5
93361Dermal Fibroblasts CCD1070 TNF alpha 4 ng/ml2.5
93105Dermal Fibroblasts CCD1070 IL-1 beta 1 nglml20.6
93772dermal fibroblast_IFN gamma 10.2
93771dermal fibroblast_IL-4 6.7
y
93259IBD Colitis 1** 5.8
'
93260IBD Colitis 2 1.3
'
93261IBD Crohns 0.0
735010Colon normal 26.2
735019_Lung_none 9.1
64028-1 Thymus none 55.9
64030-1 Kidney_none 8.7
Table 18.E.
Rel. Expr.,
Tissue Name cns_1x4tm6548f ag148 a1
102633BA4 Control 93.8
102634BA7 Control 60.7
102632BA9 Control 42.7
102635BA17 Control 58.2
102636Glob Palladus Control 14.7
102637_Sub Nigra Control 53.4
102638Temp Pole Control 9.2
102639_Cing Gyr Control 49.1
102641BA4 Control2 67.7
102642BA7 Control2 79.8
102640BA9 Control2 73.8
102643BA17 Control2 61.4
102644Glob Palladus Control2 34.6
102645Sub Nigra Control2 25.6
102646 94.8
Temp
Pole
Control2
102647Cing Gyr Control2 83.8
102617BA9 Alzheimer's 6.5
102620Glob Palladus Alzheimer's 18.8
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102622 6.8
Temp
Pole
Alzheimer's
102623Cing Gyr Alzheimer's 32.2
102625BA4 Alzheimer's2 11.5
102626BA7 Alzheimer's2 15.1
102624BA9 Alzheimer's2 21.3
102627BA17 Alzheimer's2 11.5
102628Glob Palladus Alzheimer's2 13.1
102629Sub Nigra Alzheimer's2 22.4
102630 12.9
Temp
Pole
Alzheimer's2
102631Cing Gyr Alzheimer's2 26.0
102649BA4 Parkinson's 47.5
102650BA7 Parkinson's 19.4
102648BA9 Parkinson's 19.5
102651BA17 Parkinson's 57.0
102652Glob Palladus Parkinson's 100.0
102653Temp Pole Parkinson's 20.7
102654Cing Gyr Parkinson's 37.0
102656BA4 Parkinson's2 55.0
102657BA7 Parkinson's2 17.0
102655BA9 Parkinson's2 49.6
102658BA17 Parkinson's2 56.1
102659Glob Palladus Parkinson's2 41.9
102660Sub Nigra Parkinson's2 30.8
102661Temp Pole Parkinson's2 36.4
102662Cing Gyr Parkinson's2 44.2
102664BA4 Huntington's 24.2
102665BA7 Huntington's 33.0
102663_BA9 Huntington's 37.8
102666BA17 Huntington's 46.4
102667Sub Nigra Huntington's 47.8
102668 58.8
Temp
Pole
Huntington's
102669Cing Gyr Huntington's 71.2
102671BA4 Huntington's2 13.6
102672BA7 Huntington's2 34.0
102670BA9 Huntington's2 19.8
102673BA17 Huntington's2 18.6
102674Sub Nigra Huntington's2 20.2
102676Cing Gyr Huntington's2 41.0
102603BA4 PSP 13.3
102604BA7 PSP 27.1
102602BA9 PSP 24.1
102605BA17 PSP 23.2
102606Glob Palladus PSP 16.6
102607 7.1
Temp
Pole
PSP
102608Cing Gyr PSP 23.9
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102610BA4 PSP2 37.4
102611BA7 PSP2 24.1
102609BA9 PSP2 9.3
102612BA17 PSP2 8.6
102613Glob Palladus PSP2 15.7
102614Sub Nigra PSP2 11.2
102615 3.9
Temp
Pole
PSP2
102616Cing Gyr PSP2 15.6
102588BA4 Depression 28.8
102589BA7 Depression 9.3
102587BA9 Depression 26.0
102590BA17 Depression 24.3
102591Glob Palladus Depression 12.0
102592Sub Nigra Depression 32.3
102594Cing Gyr Depression 49.9
102596BA4 Depression2 21.5
102595BA9 Depression2 23.7
102597BA17 Depression2 48.6
102599Sub Nigra Depression2 22.0
102600Temp Pole Depression2 26.1
102601Cing Gyr Depression2 30.7
The results in Tables 18-18.E. demonstrate that clone NOV3 is highly expressed
in normal
brain, kidney, fetal skeletal, colon, thyroid and adipose tissues (Tables 18,
18.A, and 18.B); in
certain tumors, especially breast cancer (Table 18.A); in IL-13 (Table 18.C);
and in numerous
areas of the central nervous system (Table 18.E), but not in corresponding
normal cell lines.
These results indicate that clone NOV3 may be used as a marker for certain
cancers, especially
breast cancer.
1 O EXAMPLE 2: Radiation Hybrid Mapping Provides the Chromosomal Location of
NOV2
and NOV3.
Radiation hybrid mapping using human chromosome markers was carried out for
NOVZ
and NOV3 in the present invention. The procedure used to obtain these results
is analogous to
that described in Steen, RG et al. (A High-Density Integrated Genetic Linkage
and Radiation
Hybrid Map of the Laboratory Rat, Genome Research 1999 (Published Online on
May 21, 1999)
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Vol. 9, APl-APB, 1999). A panel of 93 cell clones containing randomized
radiation-induced
human chromosomal fragments was screened in 96 well plates using PCR primers
designed to
identify the sought clones in a unique fashion. Table 19 provides the results
obtained for two of
the three clones of the present invention, showing the markers straddling the
gene of the
invention, and the distance in cR separating them.
Table 19
Clone Chromosome Distance from
Marker, cR
NOV2 1 AFMA129ZB5,
0.0
NOV3 11 D11S913, 5.5
LNOV3 ~ 11 WI-1409, 4.7
Example 3. Molecular Cloning of NOV2
The open reading frame of clone NOV2 codes for a Type I membrane protein with
a
transmembrane domain, predicted by PSORT, to be between residues 540-566. In
addition,
SIGNALP predicts that a signal peptidase cleavage site occurs between residues
27 and 28.
Accordingly the mature form of the predicted extracellular domain of clone
NOV2 was targeted
for cloning, from residue 28 to 538. Oligonucleotide primers were designed to
PCR amplify a
DNA segment coding for this mature domain of NOV2. The forward primer includes
an in frame
BamHI site. The reverse primer contains an in frame XhoI restriction site. The
sequences of the
primers are the following:
NOV2 Forward: GGATCCGCGCGCGGCGAAGTGAATTTGCTGG (SEQ ID N0:14)
and
NOV2 Reverse: CTCGAGGGTCCTGGTGTCATAGCGGGGCC (SEQ ID N0:15).
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PCR reactions were set up using 5 ng human hypothalamus cDNA as a template, 1
microM of each of the NOV2 Forward and NOV2 Reverse primers, 5 micromoles dNTP
(Clontech Laboratories, Palo Alto CA) and 1 microliter of SOxAdvantage-HF 2
polymerase
(Clontech Laboratories, Palo Alto CA) in 50 microliter volume. The following
reaction conditions
were used:
a) 96°C 3 minutes
b) 96°C 30 seconds denaturation
c) 70°C 30 seconds, primer annealing. This temperature was gradually
decreased by
1 °C/cycle
d) 72°C 3 minutes extension.
Repeat steps b-d 10 times
e) 96°C 30 seconds denaturation
f) 60°C 30 seconds annealing
g) 72°C 3 minutes extension
Repeat steps e-g 25 times
h) 72°C 5 minutes final extension
A single amplified product having a size of approximately 1500 by was detected
by
agarose gel electrophoresis. The product was isolated and ligated into the
pCR2.1 vector
(Invitrogen Corp, Carlsbad CA).
The construct was sequenced using the following gene-specific primers:
NOV2 S1: TACCTGGAGTCGGACCGC (SEQID NO: 16),
NOV2 S2: GCGGTCCGACTCCAGGTA (SEQID NO: 17),
NOV2 S3: CAGTGCGTGCGGCACTCAG (SEQID NO: 18),
NOV2 S4: TGAGTGCCGCACGCACTGG (SEQID NO: 19),
NOV2 S5: CTGGACCCAGGTGGCCGC (SEQID NO: 20),
NOV2 S6: GCGGCCACCTGGGTCCAG (SEQID NO: 21),
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NOV2 S7: CCCGAGCAGCCGAACGGC (SEQ ID NO: 22), and
NOV2 S8: GCCGTTCGGCTGCTCGGG (SEQ ID NO: 23).
The cloned insert was verified to be 100% identical to the nucleotide sequence
of clone NOV2
(SEQ ID N0:4) from residues 28 to 538. The construct is called pCR2.1-cgNOV2-
5340-1C.
Example 4. Molecular Cloning of NOV3.
The open reading frame of clone NOV3 codes for a Type I membrane protein with
a
transmembrane domain, predicted by PSORT, between residues 547-580. SIGNALP
predicted
the signal peptidase cleavage site between residues 51 and 52. For these
reasons the mature form
of the extracellular domain was targeted for cloning, from residues 52 to 546.
Oligonucleotide
primers were designed to PCR amplify a DNA segment coding for this mature
extracellular
domain. The forward primer includes an in frame BamHI site. The reverse primer
contains an in
frame XhoI restriction site. The sequences of the primers are the following:
NOV3Forw: GGATCCACCACCTGCCCCTCGGTGTGC (SEQ ID N0:24) and
NOV3Rev: CTCGAGGCCAGCGTTCTGCTCCTGGTTGAGTGTGG (SEQ ID N0:25).
PCR reactions were set up using 5 ng human fetal brain cDNA template, 1 microM
of each of the
NOV3Forw and NOV3Rev primers, 5 micromoles dNTP (Clontech Laboratories, Palo
Alto CA)
and 1 microliter of SOxAdvantage-HF 2 polyrnerase (Clontech Laboratories, Palo
Alto CA) in 50
microliter volume. The reaction conditions used were the same as described in
Example A.
A single amplified product having a size of approximately 1500 by was detected
by
agarose gel electrophoresis. The product was isolated and ligated into the
pCR2.1 vector
(Invitrogen Corp, Carlsbad CA).
The construct was sequenced using the following gene-specific primers:
NOV3S1: CGCACCATTGCCAGGGAC (SEQ ID NO: 26),
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NOV3S2: GTCCCTGGCAATGGTGCG (SEQ ID NO: 27),
NOV3S3: CTGGTGCGCAATTCGCTGGCC (SEQ ID NO: 28),
NOV3S4: GGCCAGCGAATTGCGCACCAG (SEQ ID NO: 29),
NOV3S5: CACGCCTCTGCCACCACG (SEQ ID N0: 30), and
NOV3S6; CGTGGTGGCAGAGGCGTG (SEQ ID NO: 31).
The cloned insert was verified as being 100% identical to clone NOV3 (SEQ ID
NO: 6) from
residues 52 to 546. The construct is called pCR2.1-cgNOV3-5331-3A.
Example 5. Preparation of mammalian expression vector pCEP4/Sec
The oligonucleotide primers,
pSec-V5-His Forward: CTCGTCCTCGAGGGTAAGCCTATCCCTAAC (SEQ
ID N0:32) and
pSec-V5-His Reverse: CTCGTCGGGCCCCTGATCAGCGGGTTTAAAC (SEQ
ID N0:33),
were designed to amplify a fragment from the pcDNA3.1-VSHis (Invitrogen,
Carlsbad, CA)
expression vector that includes VS and His6. The PCR product was digested with
XhoI and ApaI
and ligated into the XhoI/ApaI digested pSecTag2 B vector harboring an Ig
kappa leader
sequence (Invitrogen, Carlsbad CA). The correct structure of the resulting
vector, pSecVSHis,
including an in-frame Ig-kappa leader and VS-His6 was verified by DNA sequence
analysis. The
vector pSecVSHis was digested with PmeI and MleI to provide a fragment
retaining the above
elements in the correct frame. The PmeI-NheI fragment was ligated into the
BamHI/Klenow and
NheI treated vector pCEP4 (Invitrogen, Carlsbad, CA). The resulting vector was
named
pCEP4/Sec and includes an in-frame Ig kappa leader, a site for insertion of a
clone of interest, VS
and His6 under control of the PCMV and/or the PT7 promoter. pCEP4/Sec is an
expression
vector that allows heterologous protein expression and secretion by fusing any
protein to the Ig
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Kappa chain signal peptide. Detection and purification of the expressed
protein are aided by the
presence of the VS epitope tag and 6xHis tag at the C-terminus (Invitrogen,
Carlsbad, CA).
Example 6. Expression of NOV2 in human embryonic kidney (HEIR 293 cells.
The BamHI-XhoI fragment containing the NOV2 sequence was isolated from pCR2.1-
cgNOV2-5340-1C (Example 3) and subcloned into the vector pCEP4/Sec (Example 5)
to
generate expression vector pCEP4/Sec-NOV2. The pCEP4/Sec-NOV2 vector was
transfected
into HEK293 cells using the LipofectaminePlus reagent following the
manufacturer's instructions
(Gibco/BRL/Life Technologies, Rockville, MD). The cell pellet and supernatant
were harvested
72 hours after transfection and examined for NOV2 expression by Western
blotting, after SDS-
PAGE run under reducing conditions, with an anti-VS antibody. Fig. 1 shows
that NOV2 is highly
expressed in the supernatant medium as a polypeptide having an apparent
molecular weight of
approximately 64 kDa protein that is secreted by the transfected 293 cells.
The molecular weight
standard used was SeeBlue Marker (Invitrogen, Carlsbad, CA). This result is in
reasonable
agreement with the predicted molecular weight of 56842.5 Da. The program
PROSITE predicts
that there are three N-glycosylation sites in this polypeptide. Glycosylation
of the polypeptide
expressed in the transfected cells may be responsible for the difference
between the predicted and
observed molecular weights.
Example 7. Expression of NOV3 in human embryonic kidney 293 cells.
The BamHI-XhoI fragment containing the NOV3 sequence was isolated from pCR2.1-
cgNOV3-5331-3A (Example 4) and subcloned into the vector pCEP4/Sec (Example 5)
to
generate expression vector pCEP4/Sec-NOV3. The pCEP4/Sec-NOV3 vector was
transfected
into HEK293 cells using the LipofectaminePlus reagent following the
manufacturer's instructions
(Gibco/BRL/Life Technologies). The cell pellet and supernatant were harvested
72 hours after
transfection and examined for NOV3 expression by Western blotting, after SDS-
PAGE run under
reducing conditions, with an anti-VS antibody. Fig. 2 shows that NOV3 is
highly expressed in the
supernatant as a polypeptide with an apparent molecular weight of
approximately 70 kDa,
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secreted by the transfected 293 cells. The molecular weight standard used was
SeeBlue Marker
(Invitrogen, Carlsbad, CA). This result is in reasonable agreement with the
predicted molecular
weight of 54572.3 Da. The program PROSITE predicts that there are three N-
glycosylation sites
in this polypeptide. Glycosylation of the polypeptide produced in the
transfected cells may be
responsible for the difference in the molecular weights. The program PROSITE
predicts that
there are two N-glycosylation sites in this polypeptide. Glycosylation of the
polypeptide
expressed in the transfected cells may be responsible for the difference
between the predicted and
observed molecular weights.
Example 8. Quantitative Expression Analysis of NOV4 in various cells and
tissues.
TAQMANR Reverse Transcription Reagents Kit was used as given above in Example
1.
NOV4 exhibited highest levels of expression in testis (100%), 85976 Breast
Cancer Mets (100%),
breast cancer Res. Gen. (90%), 94909-XF-498-CNS-ssDNA (100%) and dermal
fibroblast-IL-4
(100%), and lower but consistent levels of expression in breast cancer (p1.
effusion) T47D cells
(15%), melanoma UACC-62 (10%), breast cancer Clontech 9100266 (38%), 94925-NCI-
H1155-
large cell lung cancer/neuroendocrine-ss cDNA (21%), 94923-NCI-H82-small cell
lung
cancer/neuroendocrine-ss cDNA (18%), 94918-DMS-79-small cell lung
cancer/neuroendocrine-ss
cDNA (19%) and normal prostate clontech A+ 6546-1 (20%). These results differ
to some
degree from the parent clone, NOV2, which did not exhibit such high levels of
expression in
central nervous system tissue, testis and fibroblast tissue, but did exhibit
positive levels of expression in cancer cell lines. Such differences in
expression reflect the
distinction among variants. It is noteworthy that clone NOV4 is minimally or
not detectably
found in the immediate normal tissue adjacent to breast and lung tissue. Thus,
clones of NOV4
may be used as markers for certain types of cancers, especially breast and
lung cancer.
Probe Name: Ag087
Primers Sequences LengthStart SEQ
m
PositionNO:
Forward 5'-CGCAGTTTCACTCGGGAGAT-3' 20 1870 11
Probe TET-5'- 1895 12
CCTCTAGGATCCACATCGAGAA.AATCATCGG-3'-31
T~MRA
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Reverse 5'-AGCAGACTTCCCCGGAGTCT-3' ~ 20 1932 13
The results obtained for clone NOV4 using primer-probe set Ag87 are shown in
Tables 20.A. -
20.E.
Table 20.A. TAQMANR Results for clone NOV4.
Tissue Name Rel. Expr., % tm252t_ag087
Endothelial cells 0.3
Endothelial cells (treated)0.6
Pancreas 1
Pancreatic ca. CAPAN 2.5
2
Adipose 1.8
Adrenal gland 0.2
Thyroid 0.1
Salavary gland 0.2
Pituitary gland 0.2
Brain (fetal) 0.9
Brain (whole) 3
Brain (amygdala) 0.7
Brain (cerebellum) 7.1
Brain (hippocampus) 2.8
Brain (substantia nigra)2.7
Brain (thalamus) 2.5
Brain (hypothalamus) 0.3
Spinal cord 2.1
CNS ca. (glio/astro) 0.4
U87-MG
CNS ca. (glio/astro) 0.3
U-118-MG
CNS ca. (astro) SW1783 0.3
CNS ca.* (neuro; met 1.1
) SK-N-AS
CNS ca. (astro) SF-539 0
CNS ca. (astro) SNB-75 2.2
CNS ca. (glio) SNB-19 2
CNS ca. (glio) U251 0.9
CNS ca. (glio) SF-295 0
Heart 0.4
Skeletal muscle 0
Bone marrow 0
Thymus 3.5
Spleen 0.4
Lymph node 0.4
Colon (ascending) 0.6
Stomach 1.3
Small intestine 0.5
Colon ca. SW480 0.3
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Colon ca.* (SW480 met)SW620 0.2
Colon ca. HT29 2.8
Colon ca. HCT-116 8
Colon ca. CaCo-2 1.2
Colon ca. HCT-15 0.8
Colon ca. HCC-2998 1.5
Gastric ca.* (liver met) NCI-N872.8
Bladder 0.4
Trachea 1.3
Kidney 1.7
Kidney (fetal) 1
Renal ca. 786-0 0.6
Renal ca. A498 0.3
Renal ca. RXF 393 0.2
Renal ca. ACHN 0.4
Renal ca. U0-31 0.3
Renal ca. TK-10 1.3
Liver 0.3
Liver (fetal) 0.1
Liver ca. (hepatoblast) HepG2 1
Lung 0.2
Lung (fetal) 0.8
Lung ca. (small cell) LX-1 0.3
Lung ca. (small cell) NCI-H69 0.7
Lung ca. (s.cell var.) SHP-77 25.9
Lung ca. (large cell)NCI-H460 0.7
Lung ca. (non-sm. cell) A549 1.1
Lung ca. (non-s.cell) NCI-H23 0.6
Lung ca (non-s.cell) HOP-62 1
Lung ca. (non-s.cl) NCI-H522 0.3
Lung ca. (squam.) SW 900 11.5
Lung ca. (squam.) NCI-H596 0.8
Mammary gland 1.8
Breast ca.* (p1. effusion) 0.3
MCF-7
Breast ca.* (pl.ef) MDA-MB-2311.6
Breast ca.* (p1. effusion) 0.5
T47D
Breast ca. BT-549 4.7
Breast ca. MDA-N 1.6
Ovary 0.6
Ovarian ca. OVCAR-3 0.6
Ovarian ca. OVCAR-4 0.5
Ovarian ca. OVCAR-5 4.6
Ovarian ca. OVCAR-8 0.2
Ovarian ca. IGROV-1 0.6
Ovarian ca.* (ascites) SK-OV-31
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Uterus 1.8
Placenta 1.5
Prostate 0.5
Prostate.* (bone met)PC-3 100
ca
Testis 4.6
MelanomaHs688(A).T 0.1
Melanoma*(met) Hs688(B).T 0
MelanomaUACC-62 0.8
MelanomaM14 0.3
MelanomaLOX IMVI 0.7
Melanoma*(met) SK-MEL-5 0.2
MelanomaSK-MEL-28 0.3
Table 20.5. TAQMANR Results for clone NOV4.
Tissue Name Rel. Expr., % 1.2tm2217t
ag87
Endothelial cells 0
Heart (fetal) 0
Pancreas 97.3
Pancreatic ca. CAPAN 0
2
Adrenal Gland (new lot*)22.5
Thyroid 0
Salavary gland 0
Pituitary gland 0
Brain (fetal) 3.4
Brain (whole) 8
Brain (amygdala) 24.8
Brain (cerebellum) 8.3
Brain (hippocampus) 20.7
Brain (thalamus) 100
Cerebral Cortex 63.7
Spinal cord 0
CNS ca. (glio/astro) 0
U87-MG
CNS ca. (glio/astro) 0
U-118-MG
CNS ca. (astro) SW1783 0
CNS ca.* (neuro; met 21.9
) SK-N-AS
CNS ca. (astro) SF-539 0
CNS ca. (astro) SNB-75 0
CNS ca. (glio) SNB-19 0
CNS ca. (glio) U251 0
CNS ca. (glio) SF-295 0
Heart 0
Skeletal Muscle (new 4.2
lot*)
Bone marrow 0
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Thymus 0
Spleen
12.2
Lymph node
0
Colorectal 0
Stomach 0
Small intestine 0
Colon ca. SW480 0
Colon ca.* (SW480 met)SW620 0
Colon ca. HT29 0
Colon ca. HCT-116 0
Colon ca. CaCo-2 0
83219 CC Well to Mod Diff (0D03866) 0
Colon ca. HCC-2998 5.5
Gastric ca.* (liver met) NCI-N87 0
Bladder 2.9
Trachea 0
Kidney 0
Kidney (fetal) 0
Renal ca. 786-0 0
Renal ca. A498 0
Renal ca. RXF 393 0
Renal ca. ACHN 0
Renal ca. UO-31 0
Renal ca. TK-10 0
Liver 0
Liver (fetal) 0
Liver ca. (hepatoblast) HepG2 0
Lung 0
Lung (fetal) 0
Lung ca. (small cell) LX-1 0
Lung ca. (small cell) NCI-H69 33
Lung ca. (s.cell var.) SHP-77 3.3
Lung ca. (large cell)NCI-H460 0
Lung ca. (non-sm. cell) A549 0
Lung ca. (non-s.cell) NCI-H23 1.7
Lung ca (non-s.cell) HOP-62 0
Lung ca. (non-s.cl) NCI-H522 17.1
Lung ca. (squam.) SW 900 0
Lung ca. (squam.) NCI-H596 55.5
Mammary gland 0
Breast ca.* (p1. effusion) MCF-7 0
Breast ca.* (pl.ef) MDA-MB-231 0
Breast ca.* (p1. effusion) T47D 26.6
Breast ca. BT-549 0
Breast ca. MDA-N 0
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Ovary 8.2
Ovarian OVCAR-3 0
ca.
Ovarian OVCAR-4 0
ca.
Ovarian OVCAR-5 0
ca.
Ovarian OVCAR-8 0
ca.
Ovarian IGROV-1 6.6
ca.
Ovarian (ascites) SK-OV-3 0
ca.*
Uterus 0
Placenta0
Prostate0
Prostate(bone met)PC-3 0
ca.*
Testis 41.5
MelanomaHs688(A).T 0
Melanoma*met) Hs688(B).T 0
(
MelanomaUACC-62 0
MelanomaM14 0
MelanomaLOX IMVI 0
Melanoma*
(met)
SK-MEL-5
0
Adipose 24.3
Table 20.C. TAQMANR Results for clone IVOV4.
Rel. Expr.,
Tissue Name 1.3dtm4681t ag87
Liver adenocarcinoma 0.0
Pancreas 4.8
Pancreatic ca. CAPAN 0.0
2
Adrenal gland 0.0
Thyroid 0.0
Salivary gland 0.0
Pituitary gland 7.8
Brain (fetal) 15.2
Brain (whole) 0.0
Brain (amygdala) 15.0
Brain (cerebellum) 52.5
Brain (hippocampus) 33.0
Brain (substantia nigra)19.9
Brain (thalamus) 88.9
Cerebral Cortex 8.1
Spinal cord 9.0
CNS ca. (glio/astro) 0.0
U87-MG
CNS ca. (glio/astro) 0.0
U-118-MG
CNS ca. (astro) SW1783 0.0
CNS ca.* (neuro; met 0.0
) SK-N-AS
CNS ca. (astro) SF-539 0.0
CNS ca. (astro) SNB-75 0.0
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CNS ca. (glio) SNB-19 0.0
CNS ca. (glio) 0251 0.0
CNS ca. (glio) SF-295 0.0
Heart (fetal) 0.0
Heart 0.0
Fetal Skeletal 9.0
Skeletal muscle 0.0
Bone marrow 0.0
Thymus 0.0
Spleen 34.2
Lymph node 0.0
Colorectal 0.0
Stomach 0.0
Small intestine 0.0
Colon ca. SW480 0.0
Colon ca.* (SW480 met)SW620 0.0
Colon ca. HT29 0.0
Colon ca. HCT-116 0.0
Colon ca. CaCo-2 0.0
83219 CC Well to Mod Diff (0D03866)0.0
Colon ca. HCC-2998 0.0
Gastric ca.* (liver met) NCI-N8713.5
Bladder 11.7
Trachea 0.0
Kidney 0.0
Kidney (fetal) 0.0
Renal ca. 786-0 0.0
Renal ca. A498 0.0
Renal ca. RXF 393 0.0
Renal ca. ACHN 0.0
Renal ca. U0-31 0.0
Renal ca. TK-10 0.0
Liver 0.0
Liver (fetal) , 0.0
Liver ca. (hepatoblast) HepG2 0.0
Lung 0.0
Lung (fetal) 0.0
Lung ca. (small cell) LX-1 0.0
Lung ca. (small cell) NCI-H69 0.0
Lung ca. (s.cell var.) SHP-77 0.0
Lung ca. (large cell)NCI-H460 0.0
Lung ca. (non-sm. cell) A549 0.0
Lung ca. (non-s.cell) NCI-H23 0.0
Lung ca (non-s.cell) HOP-62 0.0
Lung ca. (non-s.cl) NCI-H522 0.0
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Lung ca. (squam.) SW 900 0.0
Lung ca. (squam.) NCI-H596 0.0
Mammary gland 0.0
Breast ca.* (p1. effusion) 0.0
MCF-7
Breast ca.* (pl.ef) MDA-MB-2310.0
Breast ca.* (p1. effusion) 14.9
T47D
Breast ca. BT-549 0.0
Breast ca. MDA-N 0.0
Ovary 0.0
Ovarian ca. OVCAR-3 0.0
Ovarian ca. OVCAR-4 0.0
Ovarian ca. OVCAR-5 0.0
Ovarian ca. OVCAR-8 0.0
Ovarian ca. IGROV-1 0.0
Ovarian ca.* (ascites) SK-OV-30.0
Uterus 0.0
Placenta 0.0
Prostate 0.0
Prostate ca.* (bone met)PC-3 0.0
Testis 100.0
Melanoma Hs688(A).T 0.0
Melanoma* (met) Hs688(B).T 0.0
Melanoma UACC-62 9.8
Melanoma M14 0.0
Melanoma LOX IMVI 0.0
Melanoma* (met) SK-MEL-5 0.0
Adipose 0.0
Table 20.D. TAQMANR Results for clone NOV4.
Tissue Name Rel. Expr., % 2Dtm2323t
ag87
Normal Colon GENPAK 061003 0
83219 CC Well to Mod Diff (0D03866)0
83220 CC NAT (0D03866) 0
83221 CC Gr.2 rectosigmoid (0D03868)0
83222 CC NAT (0D03868) 0
83235 CC Mod Diff (0D03920) 0
83236 CC NAT (ODO3920) 0
83237 CC Gr.2 ascend colon (0D039210
)
83238 CC NAT (0D03921 ) 0
83241 CC from Partial Hepatectomy 0
(0D04309)
83242 Liver NAT (0D04309) 0
87472 Colon mets to lung (0D04451-010
)
87473 Lung NAT (0D04451-02) 0
Normal Prostate Clontech A+ 6546-10
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84140 Prostate Cancer (0D04410) 0
84141 Prostate NAT (0D04410) 0
87073 Prostate Cancer (0D04720-01 ) 0
87074 Prostate NAT (0D04720-02) 0
Normal Lung GENPAK 061010 0
83239 Lung Met to Muscle (0D04286) 0
83240 Muscle NAT (0D04286) 0
84136 Lung Malignant Cancer (0D03126) 0
84137 Lung NAT (0D03126) 0
84871 Lung Cancer (0D04404) 0
84872 Lung NAT (0D04404) 0
84875 Lung Cancer (0D04565) 0
84876 Lung NAT (0D04565) 0
85950 Lung Cancer (0D04237-01 ) 0
85970 Lung NAT (0D04237-02) 0
83255 Ocular Mel Met to Liver (OD04310) 0
83256 Liver NAT (0D04310) 0
84139 Melanoma Mets to Lung (0D04321 ) 0
84138 Lung NAT (OD04321 ) 0
Normal Kidney GENPAK 061008 0
83786 Kidney Ca, Nuclear grade 2 (0D04338) 0
83787 Kidney NAT (0D04338) 0
83788 Kidney Ca Nuclear grade 1/2 (0D04339) 0
83789 Kidney NAT (0D04339) 0
83790 Kidney Ca, Clear cell type (0D04340) 0
83791 Kidney NAT (0D04340) 0
83792 Kidney Ca, Nuclear grade 3 (OD04348) . 0
83793 Kidney NAT (OD04348) 0
87474 Kidney Cancer (0D04622-01 ) 0
87475 Kidney NAT (0D04622-03) 0
85973 Kidney Cancer (0D04450-01 ) 0
85974 Kidney NAT (0D04450-03) 0
Kidney Cancer Clontech 8120607 0
Kidney NAT Clontech 8120608 0
Kidney Cancer Clontech 8120613 0
Kidney NAT Clontech 8120614 0
Kidney Cancer Clontech 9010320 0
Kidney NAT Clontech 9010321 0
Normal Uterus GENPAK 061018 0
Uterus Cancer GENPAK 064011 0
Normal Thyroid Clontech A+ 6570-1 0
Thyroid Cancer GENPAK 064010 0
Thyroid Cancer INVITROGEN A302152 0
Thyroid NAT INVITROGEN A302153 0
Normal Breast GENPAK 061019 0
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84877 Breast Cancer (0D04566) 0
85975 Breast Cancer (0D04590-01 ) 0
85976 Breast Cancer Mets (0D04590-03) 46.7
87070 Breast Cancer Metastasis (OD04655-05) 30.1
GENPAK Breast Cancer 064006 0
Breast Cancer Res. Gen. 1024 100
Breast Cancer Clontech 9100266 0
Breast NAT Clontech 9100265 0
Breast Cancer INVITROGEN A209073 0
Breast NAT INVITROGEN A2090734 21.5
Normal Liver GENPAK 061009 0
Liver Cancer GENPAK 064003 0
Liver Cancer Research Genetics RNA 1025 0
Liver Cancer Research Genetics RNA 1026 0
Paired Liver Cancer Tissue Research Genetics RNA 6004-T 0
Paired Liver Tissue Research Genetics RNA 6004-N 0
Paired Liver Cancer Tissue Research Genetics RNA 6005-T 0
Paired Liver Tissue Research Genetics RNA 6005-N 0
Normal Bladder GENPAK 061001 13.6
Bladder Cancer Research Genetics RNA 1023 0
Bladder Cancer INVITROGEN A302173 0
87071 Bladder Cancer (0D04718-01 ) 0
87072 Bladder Normal Adjacent (0D04718-03) 0
Normal Ovary Res. Gen. 0
Ovarian Cancer GENPAK 064008 0
87492 Ovary Cancer (0D04768-07) 0
87493 Ovary NAT (0D04768-08) 0
Normal Stomach GENPAK 061017 0
Gastric Cancer Clontech 9060358 0
NAT Stomach Clontech 9060359 0
Gastric Cancer Clontech 9060395 15.3
NAT Stomach Clontech 9060394 0
Gastric Cancer Clontech 9060397 0
NAT Stomach Clontech 9060396 0
Gastric Cancer GENPAK 064005 0
Table 20.E. TAQMANR Results for clone NOV4.
Rel. Expr.,
Tissue Name 2Dtm2361t ag087
Normal Colon GENPAK 061003 0.0
83219 CC Well to Mod Diff (0D03866) 0.0
83220 CC NAT (0D03866) 0.0
83221 CC Gr.2 rectosigmoid (0D03868) 0.0
83222 CC NAT (0D03868) 0.0
83235 CC Mod Diff (0D03920) 0.0
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83236 CC NAT (0D03920) 0.0
83237 CC Gr.2 ascend colon (0D03921 ) 0.0
83238 CC NAT (0D03921 ) 0.0
83241 CC from Partial Hepatectomy (0D04309)0.0
83242 Liver NAT (0D04309) 0.0
87472 Colon mets to lung (0D04451-01) 0.0
87473 Lung NAT (0D04451-02) 0.0
Normal Prostate Clontech A+ 6546-1 19.8
84140 Prostate Cancer (0D04410) 0.0
84141 Prostate NAT (0D04410) 0.0
87073 Prostate Cancer (0D04720-01 ) 0.0
87074 Prostate NAT (0D04720-02) 0.0
Normal Lung GENPAK 061010 0.0
83239 Lung Met to Muscle (0D04286) 0.0
83240 Muscle NAT (0D04286) 0.0
84136 Lung Malignant Cancer (0D03126) 0.0
84137 Lung NAT (0D03126) 0.0
84871 Lung Cancer (0D04404) 0.0
84872 Lung NAT (OD04404) 0.0
84875 Lung Cancer (0D04565) 0.0
84876 Lung NAT (OD04565) 0.0
85950 Lung Cancer (0D04237-01 ) 0.0
85970 Lung NAT (0D04237-02) 0.0
83255 Ocular Mel Met to Liver (0D04310) 0.0
83256 Liver NAT (0D04310) . 0.0
84139 Melanoma Mets to Lung (0D04321 0.0
)
84138 Lung NAT (0D04321 ) 0.0
Normal Kidney GENPAK 061008 0.0
83786 Kidney Ca, Nuclear grade 2 (0D04338)0.0
83787 Kidney NAT (0D04338) 0.0
83788 Kidney Ca Nuclear grade 1/2 (OD04339)0.0
83789 Kidney NAT (0D04339) 18.7
83790 Kidney Ca, Clear cell type (0D04340)0.0
83791 Kidney NAT (0D04340) 0.0
83792 Kidney Ca, Nuclear grade 3 (0D04348)0.0
83793 Kidney NAT (0D04348) 0.0
87474 Kidney Cancer (0D04622-01 ) 0.0
87475 Kidney NAT (0D04622-03) 0.0
85973 Kidney Cancer (0D04450-01 ) 0.0
85974 Kidney NAT (0D04450-03) 0.0
Kidney Cancer Clontech 8120607 0.0
Kidney NAT Clontech 8120608 0.0
Kidney Cancer Clontech 8120613 0.0
Kidney NAT Clontech 8120614 0.0
Kidney Cancer Clontech 9010320 0.0
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Kidney NAT Clontech 9010321 0.0
Normal Uterus GENPAK 061018 0.0
Uterus Cancer GENPAK 064011 0.0
Normal Thyroid Clontech A+ 6570-1 0.0
Thyroid Cancer GENPAK 064010 0.0
Thyroid Cancer INVITROGEN A302152 0.0
Thyroid NAT INVITROGEN A302153 0.0
Normal Breast GENPAK 061019 0.0
84877 Breast Cancer (0D04566) 0.0
85975 Breast Cancer (0D04590-01 ) 0.0
85976 Breast Cancer Mets (0D04590-03) 100.0
87070 Breast Cancer Metastasis (0D04655-05) 0.0
GENPAK Breast Cancer 064006 0.0
Breast Cancer Res. Gen. 1024 90.8
Breast Cancer Clontech 9100266 35.8
Breast NAT Clontech 9100265 . 0.0
Breast Cancer INVITROGEN A209073 0.0
Breast NAT INVITROGEN A2090734 0.0
Normal Liver GENPAK 061009 0.0
Liver Cancer GENPAK 064003 0.0
Liver Cancer Research Genetics RNA 1025 0.0
Liver Cancer Research Genetics RNA 1026 0.0
Paired Liver Cancer Tissue Research Genetios0.0
RNA 6004-T
Paired Liver Tissue Research Genetics RNA 0.0
6004-N
Paired Liver Cancer Tissue Research Genetics0.0
RNA 6005-T
Paired Liver Tissue Research Genetics RNA 0.0
6005-N
Normal Bladder GENPAK 061001 0.0
Bladder Cancer Research Genetics RNA 1023 0.0
Bladder Cancer INVITROGEN A302173 0.0
87071 Bladder Cancer (0D04718-01 ) 0.0
87072 Bladder Normal Adjacent (0D04718-03) 0.0
Normal Ovary Res. Gen. 0.0
Ovarian Cancer GENPAK 064008 0.0
87492 Ovary Cancer (0D04768-07) 0.0
87493 Ovary NAT (0D04768-08) 0.0
Normal Stomach GENPAK 061017 0.0
Gastric Cancer Clontech 9060358 0.0
NAT Stomach Clontech 9060359 0.0
Gastric Cancer Clontech 9060395 0.0
NAT Stomach Clontech 9060394 0.0
Gastric Cancer Clontech 9060397 0.0
NAT Stomach Clontech 9060396 0.0
Gastric Cancer GENPAK 064005 0.0
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Tables 20.A - 20.E show expressions levels of NOV4 for various tissues with
highest
levels of expression found for lung carcinoma (25.9%) and prostate cancer
(100%)in Table 20.A;
normal pancreas (97.3%), cerebral cortex (63.7%), testis (41.5%) and lung
carcinoma (55.5%) in
Table 20.B; brain cerebellum (52.5%), brain thalamus (88.9%), and testis
(100%) in Table 20.C;
and metastatic breast cancer (46.7%, 30.1%, 100% and 90.8%) in Tables 20.D and
20.E. The
results observed in these tables support one another and are further evidence
that clones of NOV4
may be used as markers for certain types of cancers, notably breast cancer.
Table 21. Epitope Mapping for NOV4
Epitope Type
1 11 111
3.10 3.113.14
3.11
3.13
3.15
Table 21 shows the results from an enzyme-linked immunosorbent assay (ELISA)
for the
NOV4 clone. Here, monoclonal antibodies specific to a particular domain of the
NOV4 protein
are generated that can bind to a fragment of the NOV4 protein having that
domain. Detection is
facilitated by the use of a luminescent material in the assay ("Luminex
Multiplexing Monoclonal
Antibody Assay"). The assay comprises incubating monoclonal antibodies
prepared against the
NOV4 clone with the NOV4 clone to effect immunospecific binding of the
monoclonal antibodies
to the NOV4 clone, and then incubating the monoclonal antibody-NOV4 clone
complexes with an
additional, luminescent-labeled monoclonal antibody directed against the
monoclonal antibody
within the complex. Binding of the labeled monoclonal antibodies to the
monoclonal antibody-
NOV4 complexes results in luminescence and indicates the presence and location
of epitopes
(antigenic determinants) within the complexes.
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Competition among detecting monoclonal antibodies 3.10, 3.11, 3.13 and 3.15
for the
single epitope I is indicative of the specificity of the assay, while the
detection of more than one
epitope, here 3, shows the selectivity of the assay.
As seen in Figure 3, proliferative activity is measured by treatment of serum-
starved
cultured cells with a given stimulating agent, and measurement of
bromodeoxyuridine
incorporation during DNA synthesis is measured. Here, breast epithelial T47D
cells (ATTC;
Catalog No. HTB-133; Manassas, VA) were cultured in DMEM supplemented with 10%
fetal
bovine serum, and the cells grown to confluence at 37° C. in 10% C02
lair. Cells were then
starved in DMEM for 48 hours. Monoclonal antibodies at the indicated
concentrations of 500 ng
and 1 microgram were added and incubated for 18 hours. Finally, BrdU
(deoxybromouridine)
was added to obtain a 10 microgram final concentration in the culture, and the
culture was
incubated for 5 hours. BrdU incorporation then was assayed according to the
manufacturer's
(Boehringer Mannheim, Indianapolis, IN) specifications.
The results in Figure 3 indicate that stimulation with Cura 10 3.10.3
monoclonal antibody
results in DNA proliferation nearly as great as that in complete medium, while
monoclonal
antibodies Cura 10.3.11.7 and Cura 10 3.13.1 as well as the Cura 10 control,
all produce DNA
proliferation comparable to that of the serum-starved cells in culture.
Additional studies carried
out with NIH3T3 murine fibroblast cells and CCD1070 dermal fibroblast cells
indicate that these
cells exhibit no increase in DNA activity upon exposure to the same monoclonal
antibodies and
controls at identical concentrations as indicated in Figure 3.
These results suggest that the CCD1070 and NIH 3T3 fibroblast cell lines do
not express
receptor activity for the monoclonal antibodies to NOV4 tested. However, the
Cura 10 3.10.3
monoclonal antibody appears to trigger the receptors in T47D cells. Thus, this
particular
monoclonal antibody may play a role in targeting NOV4 receptor types in breast
cancers and may
have utility as an adjunct with chemotherapy to induce cell death.
Example 9. Method of Identifying the Nucleic Acid Encoding a Fibromodulin-like
Protein.
The sequence of Acc. No. CG554254-02 was derived by laboratory cloning of cDNA
fragments through ira silico prediction of the sequence. cDNA fragments
covering either the full
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length of the DNA sequence, or part of the sequence, or both, were cloned. In
silico prediction
was based on sequences available in Curagen's proprietary sequence databases
or in the public
human sequence databases, and provided either the full length DNA sequence, or
some portion
thereof.
Exon Linking: The cDNA coding for the CG554254-OZ sequence was cloned by the
polylnerase chain reaction (PCR) using the primers: CAACGTGCAGGTCATCTACCTATACG
(SEQ m NO: 69) and GCCCGTCTCAAAACACTCTCCATCT (SEQ >D NO: 70). Primers were
designed based on in silico predictions of the full length or some portion
(one or more exons) of
the cDNA/protein sequence of the invention. These primers were used to amplify
a cDNA from a
pool containing expressed human sequences derived from the following tissues:
adrenal gland,
bone manow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain -
substantia nigra,
brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal
lung, heart, kidney,
lymphoma - Raji, marninary gland, pancreas, pituitary gland, placenta,
prostate, salivary gland,
skeletal muscle, small intestine, spinal cord, spleen, stomach, testis,
thyroid, trachea and uterus.
Multiple clones were sequenced and these fragments were assembled together,
sometimes
including public human sequences, using bioinfonnatic programs to produce a
consensus
sequence for each assembly. Each assembly is included in CuraGen Corporation's
database.
Sequences were included as components for assembly when the extent of identity
with another
component was at least 95% over 50 bp. Each assembly represents a gene or
portion thereof and
includes information on variants, such as splice forms single nucleotide
polymorphisms (SNPs),
insertions, deletions and other sequence variations.
Variant sequences are also included in this application. 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. A SNP can arise
in several ways. For example, a SNP may be due to a substitution of one
nucleotide for another at
the polymorphic site. Such a substitution can be either a transition or a
transversion. A SNP can
also arise from a deletion of a nucleotide or an insertion of a nucleotide,
relative to a reference
allele. In this case, the polymorphic site is a site at which one allele bears
a gap with respect to a
particular nucleotide in another allele. SNPs occurring within genes may
result in an alteration of
the amino acid encoded by the gene at the position of the SNP. Intragenic SNPs
may also be
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silent, when a codon including a SNP encodes the same amino acid as a result
of the redundancy
of the genetic code. SNPs occurnng outside the region of a gene, or in an
intron within a gene, do
not result in changes in any amino acid sequence of a protein but may result
in altered regulation
of the expression pattern. Examples include alteration in temporal expression,
physiological
response regulation, cell type expression regulation, intensity of expression,
and stability of
transcribed message.
SeqCalling assemblies produced by the exon linking process were selected and
extended
using the following criteria. Genomic clones having regions with 98% identity
to all or part of the
initial or extended sequence were identified by BLASTN searches using the
relevant sequence to
query human genomic databases. The genomic clones that resulted were selected
for further
analysis because this identity indicates that these clones contain the genomic
locus for these
SeqCalling assemblies. These sequences were analyzed for putative coding
regions as well as for
similarity to the known DNA and protein sequences. Programs used for these
analyses include
Grail, Genscan, BLAST, HMMER, FASTA, Hybrid and other relevant programs.
Some additional genomic regions may have also been identified because selected
SeqCalling assemblies map to those regions. Such SeqCalling sequences may have
overlapped
with regions defined by homology or exon prediction. They may also be included
because the
location of the fragment was in the vicinity of genomic regions identified by
similarity or exon
prediction that had been included in the original predicted sequence. The
sequence so identified
was manually assembled and then may have been extended using one or more
additional
sequences taken from CuraGen Corporation's human SeqCalling database.
SeqCaIling fragments
suitable for inclusion were identified by the CuraTools~ program SeqExtend or
by identifying
SeqCalling fragments mapping to the appropriate regions of the genomic clones
analyzed. Such
sequences were included in the derivation of Acc. No. CG554254-02 only when
the extent of
identity in the overlap region with one or more SeqCalling assemblies
148471786 146315781 was
high. The extent of identity may be, for example, about 90% or higher,
preferably about 95% or
higher, and even more preferably close to or equal to 100%. When necessary,
the process to
identify and analyze SeqCalling fragments and genomic clones was reiterated to
derive the full
length sequence.
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The regions defined by the procedures described above were then manually
integrated and
corrected for apparent inconsistencies that may have arisen, for example, from
miscalled bases in
the original fragments or from discrepancies between predicted exon junctions,
EST locations and
regions of sequence similarity, to derive the final sequence disclosed herein.
When necessary, the
process to identify and analyze SeqCalling assemblies and genomic clones was
reiterated to
derive the full length sequence. The following public components were thus
included in the
invention: gb: AC0789107. In addition, the following Curagen Corporation
SeqCalling Assembly
ID's were also included in the invention: 148471786 146315781.
The DNA and protein sequences for the novel Fibromodulin-like gene are
reported here as
NOVS.
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Other Embodiments
It is to be understood that while the invention has been described in
conjunction with the
detailed description thereof, the foregoing description is intended to
illustrate and not limit the
scope of the invention, which is defined by the scope of the appended claims.
Other aspects,
advantages, and modifications are within the scope of the following claims.
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